WO1994001106A1

WO1994001106A1 - Use of byakangelicin and its derivative, composition comprising same and method for treating cataract

Info

Publication number: WO1994001106A1
Application number: PCT/KR1993/000057
Authority: WO
Inventors: Kuk Hyun Sin
Original assignee: Yuhan Corporation
Priority date: 1992-07-13
Filing date: 1993-07-13
Publication date: 1994-01-20
Also published as: KR940001899A; KR0139769B1

Abstract

The present invention relates to a novel use of byakangelicin and tert-O-methyl byakangelicin for preventing or treating a diabetic or age-related cataract, and a pharmaceutical composition comprising one or both of the above active compounds and a physiologically suitable carrier. Also provided herein is a method for preventing or treating a cataract by administering an effective amount of said composition into a patient.

Description

USE OF BYAKANGELICIN AND ITS DERIVATIVE, COMPOSITION COMPRISING SAME AND METHOD FOR TREATING CATARACT

Field of the Invention

The present invention relates to a novel use of byakangelicin and its derivative, a pharmaceutical composition comprising an effective amount of one or both of the compounds and a physiologically acceptable vehicle, and a method for treating or preventing diabetic or age-related cataract by administering an effective amount of the composition into a patient.

Background of the Invention

The condition of a significant number of patients suffering from diabetes is often exacerbated due to a common serious complication accompanying same, namely, cataract. It has been reported that cataractal symptoms are developed due to a hindrance in the polyol metabolism system, i.e., an impediment in the metabolism pathway from glucose into fructose via sorbitol.

Van Heyningen recognized in 1959 the presence of sorbitol in crystalline lenses of a patient suffering from a diabetic cataract(see Van Heyningen, Nature 184, 194(1959) ) . J.H. Kinoshita published in 1965 an osmosis theory to explain the mechanism as to how a cataractal symptom is developed from diabetes due to a disruption in the patient's polyol metabolism(see J.H .Kinashita, Invest. Qpthalmol . _4 , 786 (1965)). This theory is based on the analysis that hyperglycemia increases the concentration of glucose in crystalline lenses, and sorbitol is produced by the action of aldose reductase . A hindrance in the metabolism pathway, then, triggers an accumulation of such sorbitol, which in turn increases the osmotic pressure in the cell and the high osmotic pressure necessarily entails a hemolysis of the cell to thereby cause a cataract. It has been, therefore, theorized that controlling the accumulation of polyol may help prevent or treat a cataract; and that this may be achieved by inhibiting the activities of said aldose reductase .

Accordingly, attempts have been made in search for the compounds capable of controlling the role of aldose reductase; however, no real progress has been made to date. Furthermore, since aldose reductase has a low specificity for a substrate, the pathological mechanism on the development of a cataract has not been fully understood.

Summary of the Invention

Accordingly, it is an object of the present invention to provide a novel use of byakangelicin and one of its derivatives to effectively prevent and/or treat a cataract with little side effects .

As another object of the present invention, there is provided a composition comprising one or both of the compounds as an active ingredient and a physiologically suitable carrier .

Still another object of the present invention lies in a method for preventing and/or treating diabetic or age- related cataract by administering the above composition into a patient.

Brief Description of the Drawings

Fig. 1 shows inhibitory activities of sample compounds on aldose reductase;

Fig. 2 illustrates a densitometer spectrum of crystalline lenses extracted from rats;

Fig. 3 demonstrates the preventive effect of sample compounds upon a galactose-induced cataract jn vivo when they are administered before the injection of galactose; and

Fig. 4 exemplifies the effect on the occurrence of a galactose-induced cataract in rats when samples of byakangelicin and galactose are administered simultaneously in vivo . Detailed Description of the Invention

In accordance with the present invention, it has been found that byakangelicin and its derivative, i.e., tert-O- methyl byakangelicin, have a remarkable ability to prevent or treat diabetic or age-related cataractal symptoms by way of inhibiting the activities of aldose reductase.

Byakangelicin is a compound which is of a pale yellow needle shaped crystal having a molecular weight of 334; and can be extracted from a root of Angelica glabra Makino(see Japanese Chemical Index 7, 516 (1987)). The molecular structure of byakangelicin and its derivative, tert-O-methyl byakangelicin, may be represented as :

wherein : in byakangelicin; and

tert-O-methyl byakangelicin.

OH

By administering, either orally or intra-peritoneally, an effective amount, preferably from 0.1 mg/kg to 3000 mg/kg, more preferably from 1.0 mg/kg to 100 mg/kg of a subject patient per day of said byakangelicin and/or the derivative into a patient, together with a physiologically acceptable carrier, a diabetic or age-related cataract can be effectively prevented and/or treated. Representative compounds useful as a physiologically suitable carrier in the present composition include: lactose, dextrose, sucrose, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, methylhydroxybenzoates, propylhydroxy-benzoates, talc, magnesium stearate, mineral oil and the like. The formulation may additionally include lubricating, wetting, sweetening, flavoring, emulsifying and suspending agents, preservatives and the like. The active ingredient present in the compositions may range from 0.1 % to 99.9 % by weight thereof.

The following Examples are given for the purpose of illustration only and are not intended to limit the scope of the invention. Unless otherwise specified, percentages or ratios given herein for solids in solids, liquids in liquids and solids in liquids are on a wt/wt, vol/vol and wt/vol basis, respectively . Preparation Example 1 : Preparation of sample compounds from Angelica dahurica Benth . et Hook

3 kg of root of Angelica dahurica Benth. et Hook was milled to obtain a crude powder, which was extracted three times for 3 hours each time with 95 % of methanol in a water bath under reflux.

The extracted solution was combined and distilled in a vacuum for concentration; and, then, fractionated with a mixture of n-hexane :methanol :water(10 :1 :9 ) . Further, the water layer therefrom was fractionated with ether to provide n-hexane and ether fractions which were pooled and chromatographed in a silica gel column. Elution of the column with a mixture of benzene/ether(5 :1 ) gave: Compound 1(4.0 g); with benzene/ether (20 :1 ), Compound 2; with hexane/ether(100 :6 ), Compound 3; and with hexane/ether (100:5), Compound 4, respectively.

Compound 1 was analyzed to be of a pale yellow crystalline substance having a molecular weight of 334; m.p. 118°C; IR vmax(KBr, cm^-1) of 3540, 3100, 3120, 1710, 1605, 1595, 1490, 1386, 1370, 820; MS(m/z) of 334(M⁺), 319(M⁺-Me), 245(M⁺-C₄H₉0₂), 232(M⁺-C₅H₁₀O₂), 217(232-Me), 203(231-C0), 189(217-CO), 175(203-CO), 160 (189-CHO) ; and ¹H-NMR(CDC1₃)δ of 6.27(1H, d, J=9.8, H-3 ), 8.11(1H, d, J=9.8, H-4), 7.00(1H, d, J=2.4, H-2 ' ) , 7.63(1H, d, J=2.4, H-3'), 4.14- 4.36(2H, m, H-a ) , 4.60(1H, dd, H-b) , 1.31(3H, s, H-C¹ ), - 8 - First, a bovine eyeball sclera within 5 hours after death was peeled and cut off, a vitreous body was removed therefrom and then a crystalline lens was extracted from the eyeball. 5 mM phosphate buffer solution(pH 7.4) was added thereto in a five-fold volume of the lens; and, then, the mixture was homogenized for 5 minutes using a blender.

The resulting homogenized solution was cehtrifuged at 18,000 x g at 4 °C for 15 minutes and then the supernatant was taken therefrom. The supernatant was saturated with an ammonium sulfate to reach a 40 % concentration and left for 15 minutes. The resulting solution was centrifuged at 12,000 x g for 30 minutes and the supernatant was made as an AR source .

The protein quantification of the enzyme source was carried out using Lowry 's method(see O.H. Lowry, N.J. Rosenbrough, A.L. Farr, R.J. Randall, "Protein Measurement with the Folin Phenol Reagent", J. Bio. Chem. 193, 265(1951)). Bovine serum albumin(Sigma Fr . V) was chosen as a standard material . Further, a similar procedure was carried out with respect to a rat to obtain a crystalline lens therefrom. 135 mM of Na-K phosphate buffer solution including 0.5 mM PMSF (phenylmethyl sulfonyl fluoride) was added to the crystalline lens in a twelve-fold volume of the lens; and, then, the mixture was homogenized for 5 minutes using a blender . --- 7 _ 1 . 27 ( 3H , s , H-C² ) , 4 . 17 ( 3H, s , OMe ) .

Compound 2 was analyzed to be of a pale yellow crystalline product having a molecular weight of 348; m.p. 88°C; IRvmax (KBr, cm^-1) of 3400 (broad, OH), 3100, 3120(furan), 1705, 1605, 1590, 1480, 1430, 1350, 1145, 1065, 815; MS(m/z) of 348(M⁺), 333(M⁺-Me), 317(M⁺-OMe), 276(M⁺- C₄H₈0), 246(276-CH₂0), 245(M⁺-C₅H_1l0₂), 232(M⁺-C_feH₁₂0₂), 217(232- Me), 203(231-CO), 189(217-CO), 175(203-CO), 160 (189-CHO) , 89(C₇H₅), 73(Me₂C=0Me); and ¹H-NMR(CDCl₃)δ of 6.25 (1H, d, J=9.8, H-3), 8.10(1H, d, J=9.8, H-4) , 7.00(1H, d, J=2.4, H- 2"), 7.60(1H, d, J=2.4, H-3 ' ), 3.85-4.30 (2H, m, H-a ), 4.60(1H, dd, H-b), 3.23(3H, s, H-d), 1.23(6H, s, H-C¹ and H- C²), 4.15(3H, s, OMe).

From the above analytical results. Compound 1 was identified as byakangelicin and Compound 2 as tert-O-methyl byakangelicin. Further, Compound 3(m.p.: 106 °C) and

Compound 4(m.p. : 108 °C) were identified as imperatorin and isoimperatorin, respectively.

Preparation Example 2: Preparation of Aldose Reductase(AR)

Sources

AR sources were prepared in accordance with a modified Hayman 's method(see S. Hayman, J.H. Kinoshita, "Isolation and Properties of Lens Aldose Reductase", J. Bio. Chem . 240, 877 (1965)). The homogenized solution was centrifuged at 100,000 x g for 30 minutes and the resulting supernatant was used as another AR source .

Example 1 : Measurement of inhibitory activity on aldose reductase(in vitro)

The inhibitory activity of said Compounds 1 to 4 obtained in Preparation Example 1 on AR was measured using Abram's method(see N. Abram, Brubaker, "Synthesis and Rat Lens Aldose Reductase Inhibitory Activity of Some Benzopyran-2-ones", J. Med. Chem. 29, 1094(1986)), as follows .

The enzyme source including 0.02 μg of protein prepared from the bovine crystalline lens obtained in Preparation Example 2 was added to a 0.1 M phosphate buffer solution(pH 6.2) containing 10 mM DL-glyceraldehyde and 0.16 mM NADPH a total volume of 1 ml ("control solution"). This procedure was repeated to obtain sufficient quantities of the control solution for the tests described below. The resulting solution was added to a cell of UV spectrophotometer and a decreasing rate of light absorbance was measured at an interval of 1 minute for 5 minutes . The rate so obtained( "A" ) was used as the enzyme activity of the bovine AR source .

Except for substituting the buffer solution with glyceraldehyde, the above procedure was repeated to prepare a blank test solution; and its light absorbance decreasing rate("B") was measured .

To each of 1 ml of the above control solution was added each of Compounds 1 to 4 obtained from Preparation Example 1 to reach 100 μg/ml for each of the four samples; and the light absorbance decreasing rate("C") was measured for 5 minutes, respectively. The inhibitory percentage was calculated by using the following equation: * Inhibitory Percentage(% )

A - C x 100

A - B

The test solution containing each of Compounds 1 to 4 was diluted to a 1/10 concentration at each time with water at least three times and the enzyme inhibitory percentages at each time were measured. The enzyme inhibitory percentage against the concentration of the test solution is shown in a logit-log plot to determine a 50% enzyme inhibitory concentration for each Compound(see Fig. 1). From Fig. 1, it can be readily seen that tert-O-methyl byakangelicin shows the strongest activity having IC₅₀ of 2.8 x 10^_6M(represented by line 2) and byakangelicin also shows a satisfactory activity having IC₅₀ of 6.2 x 10^"6M(line 1). However, the respective IC₅₀ values for imperatorin(line 3) and isoimperatorin(line 4) are determined to be 8.0 x 10^_6M and 2.2 x 10^-5M, which are not considered sufficient for inhibiting the activities of aldose reductase.

Example 2: The effects of byakangelicin on galactose-induced cataract formation(in vivo)

<2-l> Induction of cataract in animals

A cataract induction test using galactose was performed as follows.

Galactose-induced cataract was triggered by employing Okuda's method(see J. Okuda, "Effect of an Aldose Reductase Inhibitor on Cataract Formation and Tissue Polyol Level in Galactosemic Rat", Chem. Pharm. Bull. 33, 2990(1985)).

One(l) g/ml galactose suspended in a 0.9 % saline solution was administered orally into twelve Sprague-Dawley rats weighing 50-60 g in an amount of 20-40 g galactose/kg of the rats twice a day(10 a.m. and 7 p.m.). The treatment was continued for 20 days in order to induce cataract.

The control group of the like kind of rats in the same number as above was administered with the same quantities of the saline solution, exclusive of galactose, as in the test group.

The development of cataract was examined by observing the crystalline lenses of the rats with naked eyes using a pen light at first. Thereafter, the subjects were killed with CHC1-, and their eyeballs was extracted with ophthalmic scissors to remove the crystalline lenses, which were then placed in a densitometer to determine the formation of cataract spectrum. The results are shown in Fig 2. Further, the results on cataract formation are summarized in Table 1 below.

Table 1: Effect of galactose on cataract formation

As can be seen from Table 1, the induction of cataract largely depends on the body weight.

<2-2> Effect of pretreatment on cataract

Two groups of the test subjects, each group consisting of six male rats weighing 40-50 g, were selected. One of the groups was administered intra-peritoneally with imperatiorin and the other was given and byakangelicin obtained in Preparation Example 1 in a 0.5 % CMC(carboxymethyl cellulose) solution at a concentration of 25 mg/ml once a day for 7 days before the administration of galactose in an amount of 40 g/kg/day, respectively; and then occurrence and development of cataract and in the two test groups and, in a control group consisting of the six male rats weighing 40-50 g, but not pretreated with imperatiorin or byakangelicin, was examined by using a pen light from the date of the injection of galactose to the 45th day thereafter. The results are shown in Fig. 3. In the control group(shown in the solid line), cataract was induced on both right and left lenses (12 lenses) in all of the six rats during the period of ll-15th days after the administration .

In the imperatorin-administered group(shown in the dotted line), cataract was induced in one lens of one rat on the 13th day and the induction rate of cataract on the 24th day reached a 50 % level.

In the byakangelicin-administered group(shown in the broken line), cataract was induced in one lens of one rat on the 28th day and the other lens of the same rat on the 29th day, but no further induction appeared by the last day of the experiment, i.e., the 45th day.

<2-3> Effect on cataract when byakangelicin and galactose are administered simultaneously

The effect of the test compound, i.e., byakangelicin obtained in Preparation Example 1, during the developmental stage of a cataract was examined by treating the control and the test groups each consisting of five rats, in accordance with the same procedure as in Example 2-2, with a simultaneous administration of the byakangelicin suspension and galactose into the test group. The results are shown in Fig. 4.

When galactose alone was administered to the control group in an amount of 20 g/kg/day, cataractal symptoms began to surface from the 13th day and the induction rate reached the 100 % level on the 26th day from the beginning of the administration shown in the solid line, while cataract was induced on the 19th day in both eyes of one rat in the test group when byakangelicin in an amount of 50 mg/kg/day was administered together with .the same amount of galactose as was given to the control group(shown in the dotted line).

<2-4> Determination of galactitol content

Each subject in the test and the control groups used in Examples 2-2 and 2-3 were killed to extract their crystalline lenses; and the contents of galactitol in the crystalline lenses were measured, using Tian's method(see Tian-Sheung Hu, "Reversal of Galactose Cataract with Sorbitol in Rats", Invest. Opthalmol, Vis. Sci. 24, 640(1983) ) . One(l) ml of 0.3 N ZnSO^, solution was added to the crystalline lenses taken from each subject; and the resulting mixture was homogenized with a blender. The homogenate was neutralized with 1 ml of 0.3 N Ba(OH)₂ and centrifuged at 10,000 x g for 20 minutes. The supernatant was concentrated under a reduced pressure to remove the solvent completely to obtain a dried pellet.

100 μl of pyridine and 200 μl of acetic anhydride were added per 1 mg of the pellet and allowed to stand at room temperature for 24 hours . To the mixture was added ice water and the resulting mixture was allowed to stand 30 minutes, and then extracted with 3 ml of CHC1₃ three times. The collected CHC1₃ phases were concentrated under a reduced pressure to dry. The resulting product was dissolved again in 100 μl of CHC1₃; and 1 μl of the resulting solution was subjected to a gas chromatography to produce a chromatogram of galactitol. The galactitol content was calculated with each standard calibration curve, which was prepared by repeating the above procedure with various amounts of galactitol and by plotting their concentrations corresponding to each chromatogram.

The galactitol concentration in the crystalline lenses taken from the rats in the control group and the byakangelicin administered group was measured as above . The results are shown in Table 2. Table 2 : Effect of byakangelicin on the accumulation of galactitol in galactose-induced cataract

treatment galactitol (μ mole/g in crystalline lens) 5 10 15 45(dav)

Example 2-2 control - - - 17.4 byakangelicin - - - 3.4

Example 2-3 control 0.061 2.10 33.1 byakangelicin 0.058 1.50 16.8 -

As shown in Table 2, the concentration of galactitol reached on the 45th day of the experiment was 17.4 μmoles per 1 g of crystalline lens in the control group, while 3.4 μmoles was detected in the byakangelicin-administered group of Example 2-2, amounting to a 85 % reduction of galactitol accumulation. Further, it was shown that the concentration of galactitol was increased from 0.061 μmole to 33.1 μmoles per

1 g of crystalline lens between the 5th day and the 15th day in the control group of Example 2-3 while the concentration of galactitol in the test group reached a 50 % level of that of the control group on the 15th day. Example 3: The effect on streptozotocin(STZ )-induced diabetic cataractdn vivo)

<3-l> Induction of cataract in rats with STZ and determination of blood sugar content

3 ml of a composition including streptozotocin dissolved in a 0.01 M citrate buffer solution(pH 4.5) was injected into a tail vein of each of six rats weighing 50-60 g in an amount of 50-100 mg/kg in order to induce a cataract. In all, four groups of six rats weighing 50-60 g were used for the test. The occurrence and development of cataractal symptoms was observed by employing the same method as in Example 2-1.

Changes of blood sugar content in the rats suffering from galactosemia were monitored. Blood samples were drawn from a tail vein of the subjects; and, then, serum was extracted by centrifuging(3, 000 rpm, 20 min . ) the samples, followed by measuring the glucose concentration in the serum with a glucose oxidase kit in accordance with an enzymatic method (see L.P. Cawleey, F.E. Spear, R. Kendall, "Ultra- microchemical Analysis of Blood Glucose with Glucose Oxidase", Am. J. Clin. Pathol . 32, 195(1959)). The results are given in Table 3.

All rats administered in an amount of 100 mg/kg of STZ were killed within 6 days after the administration. In the two groups injected with STZ in the respective amounts of 65 and 75 mg/kg, all of the rats developed cataract in 7-8 weeks after the administration. Further two rats were killed due to toxicity of STZ in the 75 mg/kg injection group; and no cataract was induced in the 50 mg/kg injection group and the level of glucose in blood stayed normal during the test period.

Table 3: Data on STZ-induced diabetic cataract

STZ(I.V.) Body Weights Glucose* Cataract Glucose

a: mean 1 standard deviation b: mean 1 standard error

* : glucose level measured on the 4th day after the administration of STZ ** : glucose level measured upon the detection of cataract formation <3-2> Determination of sorbitol content

First, blood was taken from the rats used in Example 3-1 on the 4th day after the administration of STZ once and the level of glucose in blood was measured.

Rats having at least 350 mg/dl of glucose were divided into a test and a control groups. 50 mg/kg of byakangelicin was injected intra-peritoneally to each subject in the test group once a day for 10 days. Crystalline lenses were excised from the test rats and the concentration of sorbitol was measured by employing the method described in Example 2-4. On the other hand, the concentration of sorbitol was measured as above for the rats in the control group without any further treatment after the division. The results are shown in Table 4.

Table 4: Effect of byakangelicin on accumulation of sorbitol

Treatment sorbitoKμ mole/g) control(STZ) 1.3910.05^a byakangelicin 1.0610.12° a: means 1 S.E.M. for five animals b: significantly different from the control: p < 0.05

As can be seen in Table 4, the concentration of glucose in the crystalline lenses of the control group(STZ alone administered) and the test group(byakangelicin further administered) are 1.39 μmoles/g lens and 1.06 μmoles/g lens, respectively, which shows that the j-n vivo administration of byakangelicin incurs a 24 % reduction of sorbitol accumulation as compared with that of the control group.

Example 4: Toxicity Test

The toxicity test for byakangelicin was conducted in two different ways as follows.

<4-l>: Per Os(P.O.) Test

The compound, byakangelicin, was orally administered to about five week-old mice in the form of a suspension in a 0.5 % CMC-Na solution of various dosages: i.e., 5000 mg/kg, 3125 mg/kg, 1953 mg/kg, 1221 mg/kg and 763 mg/kg.

Six pairs (each pair consisting of a male and a female; and the male having a body weight of 23.7 - 29.8 g and the female having a body weight of 20.4 - 24.5 g) of mice were used in the test .

After the administration, enervation, gasping, muscular rigidity, convulsion, motor disturbance, rotation, paralysis, etc. of the mice were observed. The number of mice which died over a period of three days was reported. The results of the test are shown in Table 5.

Table 5: Lethal Rate in P. 0. Test

Dose (mg/kg) Lethal Rate(%) ^LDso^* Male Female Male Female

5,000 67 50

3,125 67 33

1,953 50 50 >3083 >3242 1,221 33 50 763 33 0

* LD₅₀ (Median Lethal Dose) represents the amount of the test compound which causes a 50 % lethal rate measured under the Litchfield-Wikoxon method(see : J. Pharmacol. Exptl . Ther . 96, 99(1949)).

As shown above, the compound of the present invention is considered to be safe for use, especially at the level of an amount to be administered.

<4-2>: Intra-Peritoneal (I ,P . ) Test

The compound, byakangelicin, was intra-peritoneally administered into about five week-old mice in the form of a suspension in a 0.5 % CMC-Na solution of various dosages: 1,000 mg/kg, 833 mg/kg, 694 mg/kg, 579 mg/kg and 482 mg/kg. Ten pairs (each pair consisting of a male and a female; and a male having a body weight of 21.1-25.6 g and a female having a body weight of 21.1-25.4 g) of mice were used in the test . After the administration, enervation, urinal cibi, urinal cruenta, gasping, convulsion, motor disturbance, paralysis, etc. of the mice were observed. The number of mice which died over a period of two days was reported; and the results of the test are shown in Table 6.

Table 6: Lethal Rate in I.P. Test

Dose (mg/kg) LD₅₀ Male Female 1,000 833

694 > 453 > 680

579

4J2 40 10

As further shown in Table 6, the compound of the present invention is safe for use, especially at the level of an amount to be administered.

In view of the above test results on the efficacy and toxicity, byakangelicin and its derivative, tert-O-methyl byakangelicin, are believed to be useful for preventing and/or treating a cataract without incurring serious adverse effects .

While the invention has been described with respect to the above specific embodiments, it should be recognized that various modifications and changes may be made within the scope of the invention as defined by the claims that follow.

Claims

What is claimed is

1. A use, for preventing or treating a cataract, of byakangelicin and tertiary-O-methyl byakangelicin having the respective formulas of

in said tert-O-methyl byakangelicin

OH

2. A pharmaceutical composition for preventing or treating a diabetic or age-related cataract comprising an effective amount of byakangelicin and/or tert-O-methyl byakangelicin defined in claim 1 and a pharmacologically acceptable carrier.

3. A method for preventing or treating a diabetic or age- related cataract in a subject by administering an effective amount of the composition defined in claim 2 into the subject.