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polymyxin B, decamethoxine, dioxidine, trimecaine, or urea. EXPERIMENTAL PART. Experiments were performed with alkaline pmtease from. Bacillus subtilis ...

Pharmaceutical Chemistry Journal

Vol, 32, No. 5, 1998

S T U D Y OF P R O T E O L Y T I C ENZYMES IMMOBILIZED ON H Y D R O P H I L I C POLYMERS I. A. K r a v c h e n k o , 1 T. I. Davidenko, l and R. I. C h a l a n o v a 2

Translated from Khimiko-Farmatsevticheskii Zhumal, Vol. 32, No. 5, pp. 40-43, May, 1998. Original article submitted February 20, 1997.

Infected wounds and burns are frequently treated with drug preparations in the form of ointments on hydrophilic bases, which markedly increase and accelerate the therapeutic effect of the drugs. In this work we have studied the ability of alkaline protease entering into a hydrophilic polymer base to form ointment compositions and the possibility of including alkaline protease into these polymer-based compositions jointly with polymyxin B, decamethoxine, dioxidine, trimecaine, or urea.

buffer (pH 7.4). The mixture was thoroughly stirred and y-irradiated to a dose of 25 kGy. A similar procedure was used to provide for the inclusion of proteolytic enzymes into mother PEO-based composition, comprising a mixture of 3 g PEO-1500 and 7 g PEO-400. We have also studied a carbon-gel composition obtained by mixing 80 parts PEO-400, 20 parts PEO-1500, and 5 parts carbon powder. The optimum pH values for the enzyme preparations were determined by adding a substrate solution and buffers with different pH values (from 2.0 to 10.0) to the samples of enzyme preparations with otherwise equal activities. The activity o f the buffered samples was determined as described

EXPERIMENTAL PART Experiments were performed with alkaline pmtease from

Bacillus subtilis 72 (a proteolytic enzyme with broad substrate specificity), protease C from Aeremonium chrysogenum, terrilytin from Aspergillus terricola, and asperase from Asperg771us oryzae.

above.

The optimum temperature range of the enzyme preparations was determined by measuring the activities of samples of natural or immobilized enzymes at temperatures between 20 and 75~ in the corresponding buffer solutions. To determine the pH range of stability of the preparations, the enzyme samples with equal activities were incubated in the corresponding buffers with preset pH for 3 0 180 rain at 37~ after which the pH was adjusted at the optimum level and the retained activity was measured. The thermal stability range of the enzyme preparations was determined by incubating the samples having equal activities in the corresponding buffers at different preset tern-

The proteolytic activity was determined according to [1]. The proteolytic activity unit (PE / g) is defined as the amount of enzyme increasing the optical absorption ofa peptide solution at 280 nm by 1.0 optical density unit (OU) upon a 10min incubation with casein. The enzymes were embedded into a poly(vinyl alcohol) poly(ethylene oxide) - glycerol (PVA- PEO - glycerol) gel matrix by the following procedure. To 2.5 g PVA dissolved (on a water bath) in 7.5 ml of 0.066 M phosphate buffer (pH 7.4) was added 10 ml PEO-400 (PEO with a molecular weight of 400) and a calculated amount of a proteolytic enzyme dissolved in 40 ml of glycerol. The mixture was thoroughly stirred and "/-irradiated to a dose of 25 kGy. Inclusion of the proteolytic enzymes into a PEO-propylene glycol (PEO - PG) composition was performed as follows.3 To a mixture of 4 g PEO-1500, 3 g PEO-400, and 2.8 g 1,2-propylene glycol was added a calculated amount of a proteolytic enzyme dissolved in 0.2 ml 0.066 M phosphate

TABLE 1. Proteolytic Activity of Alkaline Protease Immobilized in Various Polymeric Compositions Enzyme activity Composition

before-/-sterilization PE / g

I Bogatskii Instutute of Physical Chemistry, National Academy of Sciences of Ukraine, Odessa, Ukraine. 2 Filatov Institute of Eye Diseases and Tissue Therapy, Academy of Medical Sciences of Ukraine, Odessa, Ukraine. 3 The polymeric composition was kindly provided by Prof. N. A. Lyapunov (State Scientific Center for Drug Chemisny).

after't-sterilization

% of theoretical

PE / g

% of initial

% of immobilized

1

215.2+-0.1

179.3::1:0.1 279.2::t:0.4

129.7::t:0.4 232.6+-0.4

2

229.6+0.4

191.3+0.4

297.6+0.5

129.6_+0.5

247.9_+0.5

3

108.0+-0.1

254.7+-0.1

118.4_+0.1 226.8+-0.1

279.2_+0.1

4

156.8_+0.2

130.5+-0.2

196.0_+0.3

163.1 _+0.3

125.0_+0.3

269 0091-150X/98/3205-0269520.009

Kluwer Academic/PlenumPublishers

270

I.A. Kravchenko et al. A,%

TABLE 2. Effect of the Alkaline Protease Content in Hydrophilic Compositions on Their Proteolytic Activity

I00

Enzyme activity Enzyme content, %

Composition 2 % of theoretical

0.5

123.2+-0.1

205.0+-0.1

1.0

229.6+-0.4

161.0+-0.4

168.8•

1.5

276.0•

153.0+-0.3

-

2.0

3~.4+-0.3

3.0

-

115.0• -

80

Composition 4 % of PE / g theoretical

PE / g

-

6O 40

-

20

132.0+-0.2

0 3

-

2~.8+-0.3

~.0•

236.8+-0.2

~.0+-0.2

after y-sterilization,

% o f initial

% of immobilized

% of theoretical

Protease C

382.4 _+0.1

87.0 + 0.1

327.2 + 0.3

85.6 + 0.3

74.4 + 80.3

Terrilytin

54.0 + 0.2

67.4 + 0.2

49.0 + 0.1

90.8 _+0.1

6122 +_0.1

Aspcr~

69.6+0.2

70.0+0.2

127.2+0.3

182.7+0.3

127.9+_0.3

63.2+-0.4

20.0_+0.4

Composition 1

Composition 2 Proteasr C

186.0+-0.3

31.7+-0.3

Terrilytin

24.0 +- 0.2

18.8 _+0.2

Aspcrasr

I 17.6 +- 0.2

98.0 • 0.2

46.4 +- 0.3 Composition 4

39.5 _+0.3

38.7 +- 0.3

Protease C

178.4 • 0.3

89.4 +- 0.3

154.4 +- 0.3

86.5 + 0.3

77.4 +- 0.3

Terrilytin

132.0 +- 0.1

69.4 +- 0.1

92.0 +- 0.2

69.7 • 0.2

48.4 :t: 0.2

Asperase

152.0 + 0.2

84.8 + 0.2

128.0 • 0.4

84.2 + 0.4

71.4 + 0.4

0

7

8

I0

9

RESULTS AND DISCUSSION

PE / g

117.6+0.4

6

(4 : 1) containing 5% of carbon powder (composition 3); a mixture of 25% PVA, glycerol, and PEO-400 in the weight ratio 1 : I : 1 (composition 4). The selection of hydrophilic polymers is justified by the fact that these bases markedly improve the therapeutic effect of the drug compositions by increasing the antibacterial activity of the oinmaents and providing a pronounced dehydrating action. Moreover, taking into account modem requirements on medicinals used for the therapy of wounds and bums [2], the hydrophilic polymer matrices provide the possibility for the development of combined preparations including, besides enzymes, components with antibacterial activity (polymyxin B, decamethoxine, dioxidine), local anesthetic properties (trimecaine), and dehydration effect (e.g., urea ensuring accelerated cleaning of burned wounds from necrotic tissues [31).

Enzyme activity

PE / g

5

Fig. 1. Proteolytic activity of alkaline protease (.4, % o f maximum) versus pH of incubation medium: 1 ) natural enzyme; 2 ) immobilized in composition 1; 3 ) immobilized in composition 4.

T A B L E 3. Proteolytic Activity of Some Enzymes Immobilized in Various Polymeric Compositions

before y-sterilization,

4

pH

peratures for 3 0 - 180 rain, after which the temperature was adjusted at 37~ and the retained activity was measured. The model corneal bum in rabbits was induced by a 10see contact with 10% NaOH solution using a special applicator, followed by washing the rabbit eye with a 10 ml volume of the physiological solution. The epibulbar anesthesia was performed by doubly or triply dropping 0.5% dicaine solution with a l-min interval into the conjunctival sac. Treatment of the damaged surface within the first hour upon the bum induction was carried out using a special bath, containing 2 ml of the corresponding alkaline protease solution in 0.9% NaCI, mounted on the lid slit beneath eyelids. The alkaline protease preparation in the ointment form (composition 4) was put behind the eyelids with a glass rod. The following hydrophilic polymer compositions were selected as bases for the enzyme immobilization: a mixture of PEO-400 and PEO-1500 in the weight ratio 4: 1 (composition 1); a hydrophilic base prepared by smelting 40% PEO1500, 20% PEO-400, 30% 1,2-propylene glycol, and 2% water (composition 2); a mixture of PEO-400 and PEO-1500

Enzyme

1

0

0

Data presented in Table 1 show evidence of an increase in the proteolytic activity of the alkaline protease upon immobilization in all the polymer compositions studied and T-sterilization. It was found that various compositions have different "responses" to an increase in the concentration of alkaline protease (Table 2). In particular, compositions 2 and 4 retain the maximum activity for an enzyme content of 0.5 - 1%. Inclusion of some other proteolytic enzymes (e.g., protease C) into the same hydrophilic bases leads to no increase in the proteolytic activity. Moreover, composition 2 even shows evidence of some inhibition of the activity ofprotease C (31.7%) and terrilytin (18.8%) (see Table 3). Taking into account the fact that proteases and antibiotics do not usually inactivate each other (moreover, proteases may facilitate penetration of the antibiotic component to the inflammation locus [4]), we have studied the el-

Study of Proteolytic Enzymes Immobilized on Hydrophilic Polymers A,%

271 A,% 100

80 I

60 ~ 40

1

20. O" 30

40

50

60

0

70 Temperature, *C

15

30

60

90

120

150

180 Time, min

lrtg. 2. Proteolytie activity o f alkaline protease (A, % of maximum) versus temperature o f incubation medium: I ) natural enzyme; 2 ) immobilized in eomlx~ition 1; 3 ) immobilized in composition 4.

Fig. 3. Stability o f alkaline protease (,4, % o f maximum) in the incubation medium at 55"C: 1) natural enzyrae; 2 ) immobilized in composition !; 3 ) immobilized in composition 4.

feet ofpolymyxin B on the proteolytic activity of the alkaline pmtease. Our data confirm the absence of any inhibiting effect of the antibiotic upon the alkaline protease activity (Table 4). Neither decamethoxine and dioxidine nor the local anesthetic trimecaine changed the activity of this enzyme. There are data suggesting expediency of using urea at high concentrations in combination with a proteolytic enzyme. This factor increases the enzymatic activity both due to the preliminary decomposition of proteins in the bum crust into coarse fragments and due to additional activation of the enzyme [3]. This is probably related to the fact that urea leads to degradation of both primary and secondary structure of protein molecules (by rupturing the hydrogen and disulfide bonds) and initiates decomposition of the protein molecule into large fragments, thus providing access to active groups of the protein. In our case, this effect is observed for an alkaline protease/urea ratio ranging from 1:20 to 1:50 and manifested by some (- 20%) increase in the proteolytic activity of the alkaline protease (Table 4). The joint inclusion of alkaline protease, antibacterial preparations, trimecaine, and urea into the hydrophilic polymer based compositions also leads to an increase in the proteolytic activity of this enzyme (Table 5). Taking into account the positive results obtained for the compositions including alkaline protease immobilized in the polymer bases studied, we have also investigated some other properties of the new preparations necessary to explain the functioning of immobilized enzymes (optimum pH and temperature range, thermal stability, storage conditions). Data presented in Fig. 1 show that the alkaline protease immobilized in composition 4 exhibits virtually no change in the pH profile. There is only some increase in the protease activity at pH 5.0 - 6.0. The activity of the immobilized enzyme at pH 5.0 reaches 2 0 - 3 5 % (against 8.0% for the natural enzyme). An increase in the width of the optimum pH range is also observed for composition 1, where the activity exhibits an increase in the region of acid media (pH 6 . 0 -

natural enzymes virtually coincide. However, the composite preparations show a more stable proteolytic activity at 55~ For example, the activity of an alkaline protease in composi-

7.0). The optimum temperatures of the enzyme preparations were determined by varying the reaction temperature in the range 30-75~ (Fig. 2). As is seen, the curves showing the temperature dependence of the activity of immobilized and

T A B L E 4. Effect of Antibacterial Drugs, Trimecaine, and Urea on the Proteolytic Activity o f Alkaline Protease Drug Polym.yxin B

Protease / drug weight ratio 1: 0

96.6 -t- 0.2

! : 1.2

93.4_+0.1

1 : 1.6

97.8 + 0.3

! :0

85.0 4- 0.2

1:8.0

91.0-+0.3

I : 120

77.0 + 0.2

I : 16.0

72.0-+0.l 100.0

1:0.1

110.04"0.1

1:0.2

100.1 •

1 : 0.4

106.0 + 0.3

I :0.8

106.0-+0.1

1 : 1.2

96.0 + 0.2

1 : 1.6

91.0 -+ 0.2

83.0+0.1

1:0

100.0

1:6

103.1 •

1 : 12

Urea

76.0 _+0.2

1:0

1:2.0 Trimecaine

87.7_+0.1 100.0

1 : 4.0

I : 24.0 Decamethoxine

100.0

1 : 0.8

1:2.0 Dioxidine

Proteolytic activity

92.2-+0.1

1:24

100.6-+0.2

1:36

104.0_+0.3

1:48

106.2 • 0.2

1:0

100.0

I : 10

103.2+0.1

I : 20

120.3 • 0.2

1 : 30

122.6 • 0.2

1:40

124.1 + 0 . 1

1 : 50

126.2 • 0.2

272

I . A . K r a v c h e n k o et ai.

TABLE 5. Proteolytic Activity of Alkaline Protease Immobilized in Combination with Antibacterial Drags, Trimecaine, and Urea in Various Hydrophilic Polymers Enzyme activity Combination

before y-sterilization, % of PE / g theoretical

alter "/-sterilization, % of initial % of PE / g immobilized

Composition 1 157.3 • 0.3 332.8• 0.4 152.6 • 0.5 246.4• 0.3 141.9 • 0.3 314A• 0.6 101.6:t:0.4 154.1• 143.1 • 198.7• Composition2 Alkaline protease + 1% dioxidine 183.2 • 0.1 152.6 • 0.1 321.6• 0.2 Alkaline proteas• + 3% trimecaine 178.4 • 0.2 148.6 • 0.2 240.0• 0.3 Alkaline protease + 1% dioxidine + 3 % trimecaine 152.8• 0.2 127.3 • 0.2 296.8• 0.3 Composition3 Alkaline pmtease + decamethoxine 102.8:1:0.1 192.1 • 0.1 106.4 • 0.2 221.1 • 0.4 129.6• 0.3 Alkaline protease + polymyxin B 118.6• Composition4 228.8• Alkaline protease + i% dioxidine 196.0 + 0.2 163.3• 139.2 • 0.5 224.8 • 0.3 Alkaline protease + 3% trimecaine 167.2 • 0.5 Alkaline protease + 1% dioxidine + 3 % Irimecaine 182.4• 0.3 152.0 • 0.3 283.2• 0.4 Alkaline protease + 1% dioxidine Alkaline protease + 3% trimecaine Alkaline protease + I% dioxidine + 3% lrimecaine Alkaline protease + polymyxin B Alkaline protease + 200 urea

189.6• 183.2• i~.4• 140.6• 176.0•

tion 1 reaches after a l - h incubation at this temperature 93% against 70.0% for the natural enzyme. The proteolytic activity o f the immobilized and natural enzyme drops further to 68 and 30% after a 2-h incubation, and to 48.6 and 5.6% after a 3-h exposure at 55~ respectively (Fig. 3). W e have monitored variations in the stability o f action o f the alkaline protease stored for 2 years at 3 - 4 ~

It is estab-

lished that neither the proteolytic activity nor the stability with respect to aggregation (as manifested by the degree o f homogeneity, the lack o f granular structure formation, and the absence o f phase separation), p H level, and coloration differ to any significant extent from the initial characteristics. This level o f stability is observed for preparations sterilized by -./-irradiation.

Hydrolysis of Necrotic Mass with Alkaline Protease Immobilized in Hydrophilie Polymers Amino acids passed into Preparation solution, lag

175.5+ 0.4 276.1+ 0.4 134.5• 0.3 205.2• 0.3 184.5+ 0.6 261.8• 0.6 t09.0• 111.4• 113.0• 161.6• 175.5• 0.2 134.5:i:0.3 194.2• 0.3

267.9+ 0.2 200.0• 0.3 247.3• 0.3

103.5• 0.2 109.3• 0.3

198.8• 0.2 241.6• 0.3

i16.7• 190.6• 134.4• 0.3 187.2• 0.3 155.3+ 0.4 236.0• 0.4

Taking into account the necrolytic action o f the alkaline protease immobilized in hydrophilic ointment compositions, we have studied the hydrolysis o f a human b u m crust upon dissection. The results presented in Table 6 indicate that the alkaline protease immobilized in the polymeric compositions studied is more effective as compared to the natural enzyme. Experiments performed in cooperation with the staff o f the b u m therapy department o f the Filatov Institute o f Eye Diseases and Tissue Therapy) showed high efficacy o f using the alkaline protease immobilized in cornposition 4 for the lysis o f burn crust on the model o f

corneal b u m in rabbits. Indeed, it k,~s found that the level o f p r o t e o l y t i e activity o f the alkaline protease, which is necessary to provide for a noticeable necrolytic action on the burned cornea, is 2 0 0 216 PE for the natural preparation and 2 4 0 - 2 8 0 PE for the enzyme in composition. A band bounding the necrosis region appears after a 2 0 - 22 min (240 PE) action o f the natural alkaline protease preparation, and after 15 min for the same enzyme immobilized in a hydrophilie comtmsition. Accordingly, the complete necrolysis o f the corneal b u m crust is observed after a 35-min treatment with the natural alkaline protease and alter 23 - 25-rain, for the immobilized enzyme. The experimental data obtained in this w o r k show that immobilization o f the pmteolytic enzymes studied (especially o f the alkaline protease) in hydrophilic p o l y m e r matrices leads to the formation o f active stable preparations. This method can be used for development o f various enzyme-containing ointments offering a complex o f useful effects.

TABLE 6.

Alkaline proteasr (A = 28.8 PE/cm 3) Alkaline protease + composition I Alkaline protease + composition 1 + dioxidine + trimecaine Alkaline protease + composition2 Alkaline protease + composition 2 + dioxidine + trimecaine

23.3• 28.0• 29.1• 50.6• 41.2•

REFERENCES I. I. S. Petrova and M. M. Vintsyugonaite, PriM. Biokhira. MikrobioL, 2(3), 322 - 3 2 7 (1966). 2. A. M. Gonchar, A. S. Kogan, and 1L I. Salganik (eds.), Wound

Healing Process and Immobilized Proteolytic Enzymes. A Collection of Papers [in Russian], Nauka (Sib. Otdel.), Novosibirsk (1986). 3. T. L. Zaets and S. K. Zav'yalov, Izv. Akad. Med. Nauk SSSR, No. 8, 12 - 16 (1961). 4. S. N. Murashko, A. F. Moroz, and N. S. Brodina, Antibiot. Med. Biotekhnol., 31(5), 381 - 3 8 5 (1986).

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