Accepted Manuscript Title: CHITOSAN-ISONIAZID CONJUGATES: SYNTHESIS, EVALUATION OF TUBERCULOSTATIC ACTIVITY, BIODEGRADABILITY AND TOXICITY Author: Alexander S. Berezin Yury A. Skorik PII: DOI: Reference:

S0144-8617(15)00273-8 http://dx.doi.org/doi:10.1016/j.carbpol.2015.03.060 CARP 9802

To appear in: Received date: Revised date: Accepted date:

12-1-2015 2-3-2015 14-3-2015

Please cite this article as: Berezin, A. S., and Skorik, Y. A.,CHITOSAN-ISONIAZID CONJUGATES: SYNTHESIS, EVALUATION OF TUBERCULOSTATIC ACTIVITY, BIODEGRADABILITY AND TOXICITY, Carbohydrate Polymers (2015), http://dx.doi.org/10.1016/j.carbpol.2015.03.060 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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CHITOSAN-ISONIAZID CONJUGATES: SYNTHESIS, EVALUATION OF

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TUBERCULOSTATIC ACTIVITY, BIODEGRADABILITY AND TOXICITY

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Alexander S. Berezin1, Yury A. Skorik2,3,*

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St. Petersburg State Chemical Pharmaceutical Academy, 14 Prof. Popov St., St. Petersburg 197022, Russian Federation

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Institute of Macromolecular Compounds of the Russian Academy of Sciences, 31 Bolshoi pr. VO, St. Petersburg 199004, Russian Federation

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Institute of Chemistry, St. Petersburg State University, Universitetskii pr. 26, Petrodvorets, St. Petersburg 198504, Russian Federation

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*

Corresponding author.

Tel.: + 7 812 3283224; Fax: +7 812 3286869 E-mail address: [email protected] 1 Page 1 of 24

Abstract

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Novel water-soluble chitosan-isoniazid conjugates were synthesized by two methods: (1) the

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carbodiimide method using isoniazid (INH) and N-(2-carboxyethyl)chitosan (CEC), and (2)

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the reaction between INH and N-(3-chloro-2-hydroxypropyl)chitosan (CHPC). The solubility of

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the conjugates under physiological conditions was enhanced by phosphorylation. Method (1)

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is preferable in terms of obtaining conjugates with a high content of active substance;

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depending on reaction conditions, the degree of substitution in the INH-CEC conjugates

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varies from 0.08 tо 0.39. Ultrasound treatment increased the reaction rate by a factor of 1.3–

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1.5, but caused partial degradation of the polymer. Consecutive modification led to a

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considerable decrease in polymer biodegradability in the following order: chitosan > CEC or

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CHPC > conjugate. In vitro screening of the antimicrobial activity against Mycobacterium

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tuberculosis H37Rv demonstrated a comparable or slightly higher minimum inhibitory

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concentration for conjugates than for INH itself (0.20, 0.25, and 1.05 g INH/mL for INH,

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CEC-INH, and CHPC-INH, respectively). A slug mucosal irritation test employing Limax

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flavus revealed a lower toxicity for the conjugates than for INH by a factor of 3–4; the most

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noticeable toxicity decrease was observed for the conjugates obtained by method (1).

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Studies of acute toxicity in mice revealed a 3–4-fold increase in median lethal dose for the

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conjugates compared with INH (LD50 210, 850, and 650 mg INH/kg for INH, CEC-INH, and

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CHPC-INH, respectively).

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Keywords: chitosan, isoniazid, conjugate, carbodiimide activation, activated ester method,

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drug delivery, toxicity, Limax flavus.

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1. INTRODUCTION

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A wide variety of antituberculosis drugs is now available; nevertheless, tuberculosis treatment remains a challenge due to the emergence of multidrug-resistant Mycobacterium

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strains and due to the high toxicity of existing drugs. Isoniazid (INH) is one of the most

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commonly used first-line antituberculosis drugs; it possesses high activity against

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Mycobacterium tuberculosis, but is rapidly egested, thereby necessitating high doses of a

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highly toxic INH (Sharma, 2004). For this reason, the development of drug delivery systems

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intended for targeted and controlled release of INH is a significant research interest.

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Chitosan, a natural polyaminosaccharide, is a promising drug carrier. This polymer is being increasingly used in applications in various fields of medicine and pharmacology due to

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its unique properties, including biocompatibility, biodegradability, mucoadhesive properties,

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and low toxicity (Berezin, Lomkova, & Skorik, 2012; Park, Saravanakumar, Kim, & Kwon,

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2010; Muzzarelli, 2010). Chitosan and its derivatives have already been tested as carriers for

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encapsulated, dispergated, adsorbed, or conjugated INH (Banik, Hussain, Ramteke,

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Sharmac, & Maji, 2012; Buys, du Plessis, Marais, Kotze, & Hamman, 2013; Pandey &

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Khuller, 2004; Pourshahab et al., 2011; Rando, Brandt, & Ferreira, 2004; Slivkin, Lapenko, &

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Bolgov, 2010). Chitosan-based microcapsules and nanoparticles, used for prolonging action

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and raising INH bioaccessibility, provide controlled drug release as a result of desorption,

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diffusion of adsorbed INH, and polymer biodegradation. Parameters of the release process

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are governed by the pH of the medium, the mechanism of complex formation, and the

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molecular weight (MW) of chitosan. The in vivo tests of these delivery systems showed

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increases in therapeutic efficiency so that the duration of treatment could be reduced several-

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fold (Pandey & Khuller, 2004; Pourshahab et al., 2011).

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Very limited information on INH-chitosan conjugates have been described (Rando et al.,

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2004; Slivkin et al., 2010). The difficulties associated with pharmacokinetic parameters were

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overcome by binding INH to a chitosan oligomer (MW = 30,000) via a methylene spacer. The

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mass fraction of INH in this conjugate was 26–30% (Slivkin et al., 2010). An N-

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methylenephosphonic chitosan derivative was also used to obtain conjugates with INH

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(Rando et al., 2004); the conjugate was prepared by the reaction between chitosan and

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phosphoric acid in the presence of formaldehyde, followed by the reaction with succinyl-INH.

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The authors did not provide data on the INH content of the conjugate; therefore, the

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efficiency of this immobilization method cannot be evaluated.

Analysis of the published data indicates that the development of efficient chitosan-

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based delivery systems for INH remains important. The present work is focused on the

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synthesis of water-soluble INH-chitosan conjugates with different spacers as well as on

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studies of their tuberculostatic activity, toxicity using two in vivo tests, and enzymatic

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biodegradation.

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2. EXPERIMENTAL

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2.1. Materials

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The chitosan used in this work had the following characteristics: degree of deacetylation

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was 0.95 (determined by 1H NMR spectroscopy); MW was 37,000 (determined by viscometry

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(Gamzazade et al., 1985)); N content was 5.4 mmol/g (determined by elemental analysis).

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Chemically pure isoniazid, acrylic acid, epichlorohydrin, methanesulfonic acid, P2O5, and

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formaldehyde, as well as analytical grade solvents, were purchased from Sigma-Aldrich and

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used without further purification.

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2.2. General methods

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1

H NMR spectra of samples were obtained using a Bruker Nanobay instrument with an

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operating frequency of 400 MHz in D2O/DCl solutions; elemental analysis was conducted

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using a Vario Micro Cube Elemental СНNS analyzer; lyophilization of samples was carried

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out in a CoolSafe 110-4 freeze dryer; the viscosity of samples was measured using a

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Brookfield LV viscometer.

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The mass fractions of INH in the conjugates were determined by UV-spectroscopy using a Shimadzu UV mini 1240 spectrophotometer; calibration curves were obtained using

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INH solutions in 0.1 M HCl with concentrations in the range of (1 – 10)  10-5 M; solutions of

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CEC and CHPC with similar mass concentrations in 0.1 M HCl were used as references.

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The intrinsic viscosity [η] of the polymer solutions was determined using an Ostwald

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viscometer with a capillary diameter of 0.64 ± 0.02 mm at a temperature of 25.0 ± 0.1оС. The

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measurements were conducted so that solution flow time was not less than 100 s, thereby

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excluding corrections for kinetic energy. The flow time was measured at least three times;

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deviations did not exceed 0.2 s. An acetate buffer with pH 5.0 was used as a solvent.

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2.3. Synthesis of N-(2-carboxyethyl)chitosan

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CEC was prepared according a procedure that was slightly modified from one that had

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been previously published (Sashiwa, Yamamori, Ichinose, Sunamoto, & Aiba, 2003): 2.0 g of

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chitosan was dissolved in 18.8 mL of acrylic acid; then 100 mL of water was added. The

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reaction mixture was stirred for 24 h at 80°С. The product was precipitated with acetone, then

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dissolved in 0.1 М HCl and dialyzed against distilled water for 2 days using an Orange

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Scientific 8030T2 membrane (MWCO = 6000–8000). The degree of substitution (DSCE =

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1.28) was calculated from the 1Н NMR data as previously described (Skorik, Gomes,

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Vasconcelos, & Yatluk, 2003).

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2.4. Preparation of isoniazid conjugates with N-(2-carboxyethyl)chitosan

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The CEC-INH conjugates were obtained according to the general procedure, which

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includes varying the molar ratios of reactants and the pH of the reaction mixture. The

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synthesis conditions and some characteristics of the products are given in Table 1.

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For example, the 6b conjugate was prepared according to the following procedure: 500

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mg of CEC was dissolved in 100 mL of water, and then 740 mg of INH was added. The

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mixture was cooled down to 5oC, and then 100 mg of N-(3-dimethylaminopropyl)-N'-

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ethylcarbodiimide (EDAC) and 621 mg of N-hydroxysuccinimide (NHS) were added. The pH 5 Page 5 of 24

of the reaction mixture was adjusted to 4.0 using 1 M NaOH. The mixture was stirred at 5oC

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for 1 h and then heated slowly over 1 h until it reached the ambient temperature; stirring was

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continued for another 20 h. Then 2 mL of methanesulfonic acid and 300 mg of anhydrous

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P2O5 were added to the solution (Nishi, Nishimura, Ebina, Tsutsumi, & Tokura, 1984). The

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reaction mixture was stirred at 5oC for 18 h. The resulting product was precipitated with

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acetone, dissolved in 0.1 M HCl, and dialyzed against distilled water for 2 days. The mass

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fraction of INH was calculated by elemental analysis and UV-spectroscopy (Table 1); the

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purity of the product was evaluated by 1Н NMR spectroscopy.

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2.5. Preparation of isoniazid conjugates with N-(3-chloro-2hydroxypropyl)chitosan

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To prepare the CHPC-INH conjugates, 0.4 mL of 12 M HCl and 100 mL of water were added to 500 mg of chitosan. After the chitosan dissolved, 35 mL of epichlorohydrin was

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added, and the pH of the solution was brought to 5.5 using 1 M NaOH. The reaction mixture

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was stirred at 30oC for 8 h, and then (without isolation of CHPC) 100 mg of INH was added,

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and the pH of the mixture was brought up to 6.0. The solution was stirred for 20 h at 60 оС.

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The resulting product was precipitated with acetone, dissolved in 0.1 M HCl, and washed

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repeatedly with acetone. Then 480 g of the obtained conjugate was dissolved in 0.1 М HCl,

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and 2 mL of methanesulfonic acid and 300 mg of anhydrous P2O5 were added (Nishi et al.,

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1984). The reaction mixture was stirred at 5oC for 18 h, the product of which was precipitated

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with acetone, dissolved in 0.1 М HCl, and dialyzed against distilled water for 48 h. The mass

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fraction of INH was calculated using elemental analysis and UV-spectroscopy data (Table 1);

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the purity of the product was evaluated by 1Н NMR spectroscopy.

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6b 6c 6d 6e 6f

1

1



1.0

4.0 S

1

2

2

1.0

4.0 S

1

2

2

2.5

4.0 S

1

1

2

2.5

4.5 S

1

2

2

2.5

4.5

1

2

2

2.5

1

2

2

2.5

S at pH≤7 S at 5.0 pH≤7 5.5 I

4 7e 7f

see Experimental section

S at pH≤7

see Experimental section

S

see Experimental section

S

Ac

3

144 145 146 147 148 149

pH

ce pt

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Molar ratio [EDAC] CEC INH NHS (g/L)

see Experimental section a

S

ip t Conjugate formulab

Found Calculated (%) C

H

N

43.79 43.79 43.18 43.21 43.90 43.86 44.42 44.46 44.07 44.18 45.32 45.27 44.96 44.96 44.30 44.30 38.97 37.04 38.82 38.99 38.59 38.68

6.66 6.70 6.76 6.76 6.61 6.69 6.50 6.63 6.52 6.66 6.55 6.55 6.54 6.58 6.79 6.98 5.98 5.91 6.11 6.01 5.84 5.83

4.92 5.08 6.17 6.00 6.41 6.53 6.90 6.83 7.93 7.79 9.58 9.33 7.61 7.43 11.27 11.32 9.34 9.47 6.89 6.87 7.89 7.97

(INH)c (%)

(INH)d (%)

Cl

Ch0.05Cs0.95CE1.28INHx  yH2O x (DSINH) y



0.08

1.1

3.8

4.9



0.08

1.4

3.8

4.4



0.12

1.2

5.6

6.9



0.14

1.1

6.5

7.8



0.24

1.4

10.6

9.2



0.39

1.3

16.3

15.7



0.19

1.0

8.7



0 0

Ch0.05Cs0.95HP0.35INH0.231.3H2O

13.3

12.2



Ch0.05Cs0.95HP0.35INH0.23(PO3H2)0.631.1H2O 11.1

11.7



Ch0.05Cs0.95CE1.28INH0.24(PO3H2)0.521.4H2O 9.3

10.7



Ch0.05Cs0.95CE1.28INH0.39(PO3H2)0.671.4H2O 13.9

12.4

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Solubility in watera

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Compound Synthesis conditions

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Тable 1 Synthesis conditions and some characteristics of the conjugates.

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S – soluble, I – insoluble. Ch – chitin monomer unit, Cs – chitosan monomer unit, CHP – 3-chloro-2-hydroxypropyl group, HP – 2-hydroxy-1,3-propene spacer, CE – carboxyethyl group/spacer, INH – isoniazid. c Mass fraction of isoniazid determined from the elemental analysis. d Mass fraction of isoniazid determined from the UV spectroscopy. b

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2.6. Enzymatic biodegradation

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The biodegradation of the chitosan derivatives and INH conjugates was studied using the procedure described earlier (Kast & Bernkop-Schnuerch, 2001). A 300 mg sample

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(chitosan, CEC, CHPC, or conjugate) was dissolved in 2.4 mL of 1 М HCl, 7.3 mL of distilled

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water was added, and the pH was adjusted to 5.0 using 1 М NaOH. Then, 1.5 mg of chicken

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egg white lysozyme per 1 mL of the polymer was added to each solution. At specific times,

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the viscosities of the reaction mixtures were measured at 37.0 ± 0.1°C using a Brookfield LV

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viscometer. The viscometer was connected to a PC for the purpose of adjusting analysis

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parameters, processing, and recording the data with the help of the Rheo Win Pro 2.64

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program. Polymer solutions containing no lysozyme were used as reference solutions.

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2.7. Determination of minimum inhibitory concentration (MIC)

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The MIC values were determined using the M. tuberculosis H37Rv laboratory strain

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(which is sensitive to antituberculosis drugs). Tuberculostatic activity was studied using the

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vertical diffusion method (growth medium: 0.05% KH2PO4, 0.05% sodium citrate, 0.05%

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MgSO4, 0.2% sodium pyruvate, 3.6% glycerol, 0.036 malachite green, 50% chicken egg

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yolk). Parameters of mycobacterial growth were evaluated in the presence of the conjugates

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at various concentrations according to the standard procedure (Kudon & Kudon, 1973). The

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results were compared to those obtained for the strain growth in the same medium without

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the conjugates. The appearance of growth inhibition zones indicated tuberculostatic

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properties of the compounds at the given concentration. All experiments were carried out in

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triplicate.

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2.8. Determination of median lethal dose (LD50)

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Acute toxicity of the conjugates was tested using white outbred CD-I mice weighing 18–

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25 g; various doses of INH or conjugates dissolved in physiological saline were introduced

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intraperitoneally. Observations were made for 2 weeks. Toxicity was evaluated using the

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median lethal doses (LD50) obtained for test and control groups. The experiments were

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conducted in agreement with the Rules of Animal Care and Use Committee of the St.

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Petersburg State Chemical Pharmaceutical Academy, which comply fully with European

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Community Council directives 86/609/EEC. Naive animals were used for all the experiments. 2.9. Slug mucosal irritation test using Limax flavus

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Toxicity of INH and the novel conjugates was additionally estimated using the previously

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described slug mucosal irritation test (Adriaens et al., 2001). Two days prior to the

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experiments, Limax flavus slugs (3–4 g in weight) were placed in beakers filled with tissue

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paper, moistened with a pH 7.4 phosphate buffer solution (PBS, 0.01 M phosphate buffer,

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0.0027 M KCl and 0.137 M NaCl), and kept at room temperature. Each slug was weighed

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and then placed in a Petri dish containing a paper filter treated with 1% solution of INH (or a

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conjugate with a similar INH content) in PBS. The positive control was 1% benzalkonium

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chloride in PBS, and PBS was used as a negative control. After 1 hour, slugs were removed

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from the Petri dishes, washed with PBS, dried with filter paper, and weighed again. Slug

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weight loss was used to estimate the mass fraction of liberated mucus (MP):

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where ma and mb are the masses of the slugs before and after the experiment,

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respectively. Each experiment was carried out in triplicate.

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3. RESULTS AND DISCUSSION

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3.1. Synthesis and characterization of chitosan-isoniazid conjugates

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The INH-chitosan conjugates can be synthesized in two possible ways: (1) by

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attachment of a INH-spacer block to a polymer or (2) by consecutive attachment of a spacer

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and INH to a polymer. In the present work, we used the second way; the first step involved

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modification of chitosan with epichlorohydrin (Scheme 1, 2). Intermolecular crosslinking was

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prevented by carrying out the reaction at pH 5.5 and at a temperature of 30°С for 8 h; the

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polymer concentration in the reaction mixture was low (0.25–0.50%) (Berezin, Ishmetova, 9 Page 9 of 24

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Rusinov, & Skorik, 2014). The degree of chlorohydroxypropylation of 2 (DSHP, Table 1) was

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determined by elemental analysis of the product isolated from a portion of the reaction

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mixture. The calculated value of DSHP was used to determine the composition of conjugate 3.

205 O

OH

OH O HO

O HO

NH2

R= O

NH2

OH O

O

O HO

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H N

O

N

O

6

O R''

OR' O

CH3SO3H, P2O5

N

HO

O

R''

, OH;

R''

OR' O O

O

NH

OR'

N

OR'

O

O R''

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O R''

R''

Scheme 1. Synthesis of chitosan-INH conjugates.

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NH

O

O OH HO

N H

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5

O

O HO

R''=

OH

O

, NHS, EDAC

N

O OH

N

;

R'= PO 3H2 , H.

M

NH

O

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O

O

N

1

OH O

HO

H N

H N

an

H2 N

N H

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O HO

cr

OH

HO

4

R

Cl

O

NH2

OR'

OR'

3

R O

O

NH

OR'

OH

2

OH

O

NH2

HO

OR' O

OR' O

CH3SO3H, P2O5

O

NH

OH Cl

O

O

N

O

NH

OH

OH O

O HO

H N

ip t

H2 N

In the second step, CHPC 2 was used for INH immobilization via the hydrazide group of INH and chlorohydroxypropyl moiety of CHPC. Conjugation of CHPC with a two-fold excess

211

of INH was carried out in aqueous solution at pH 6.0; the reaction mixture was kept at 70оС.

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Under these conditions, the maximum degree of INH substitution (DSINH) in conjugate 3 was

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reached in 20 h (Fig. 1). The composition of conjugate 3, calculated from the elemental

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analysis data, indicates that approximately 60% of chlorohydroxypropyl groups reacted with

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INH (DSHP = 0.35, DSINH = 0.23). However, no chlorine was detected by the elemental

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analysis of 3, indicating the complete conversion of chlorohydroxypropyl groups in CHPC.

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The rest of the chlorohydroxypropyl groups of CHPC may possibly have reacted with the

218

amino groups of the polymer, thus leading to intermolecular crosslinking.

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Note that every subsequent reaction of chitosan (first with epichlorohydrin, then with INH) led to an increase in the intrinsic viscosity of the polymer solutions in the order 1 < 2 < 3 10 Page 10 of 24

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(Table 2). This may be caused both by the introduction of bulky substituents into the polymer

222

and by partial intermolecular crosslinking.

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Тable 2

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Viscosity of solutions of chitosan and its derivatives (pH 5.0, 25.0oC). 1

2

3

6a



[η] (dL/g)

2.37

4.40

6.15

2.55

2.98

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6f

3.90

5.43

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6e

cr

Compound

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Additionally, the reaction mixture was sonicated by an ultrasound of 20 kHz for 2–4 h. The ultrasound treatment accelerated the reaction between INH and CHPC, as confirmed by

229

the 1.3–1.5-fold increase of the INH mass fraction in the conjugates obtained under

230

ultrasound treatment. However, the activating effect of the ultrasound was accompanied by

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undesirable polymer degradation. Thus, after sonication of the reaction mixture for 2 h, the

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viscosity of the conjugate solutions (η, mPa∙s) was lower by a factor of 1.2–1.5 compared to

233

solutions of the conjugates obtained without ultrasound treatment. A similar influence of

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ultrasound sonication has been observed elsewhere (Berezin et al., 2014; Manaenkov,

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Kamenshchikov, Kislitsa, Stepanenko, & Sulman, 2011).

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Conjugate 3 demonstrated low solubility at near-neutral pH values. An additional

237

phosphorylation step was performed according to a previously reported procedure (Nishi et

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al., 1984) that increased solubility under physiological conditions (Scheme 1, 4).

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11 Page 11 of 24

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Fig. 1. The dependence of the degree of INH functionalization (DSINH) on the reaction time

242

between CHPC and INH (CHPC:INH 1:2, pH 6.0, 70оС).

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Another type of INH-chitosan conjugates was obtained using the procedure that

245

included carboxyethylation of chitosan with subsequent introduction of INH into CEC by the

246

carbodiimide method. CEC appears to be a promising drug carrier because it is known to

247

possess high biodegradability and pronounced antimutagenic and antioxidant properties

248

(Kogan et al., 2004; Pestov, Skorik, Kogan, & Yatluk, 2008). CEC was obtained by aza-

249

Michael reaction between chitosan and acrylic acid (Sashiwa et al., 2003). Increases in

250

reactant concentrations, temperature, and reaction time, as compared with those described

251

previously (Sashiwa et al., 2003), allowed us to prepare CEC with a degree of substitution

252

(DSCE) equal to 1.28.

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Immobilization of INH on CEC was performed by the carbodiimide method (Scheme 1,

254

derivatives 6a-g) in water solutions by using EDAC and NHS as activators. Conjugation

255

leads to the amide bond formation between the activated carboxylic group of CEC and the 12 Page 12 of 24

amino group of INH. The degree of substitution (DSINH) for CEC-INH conjugates was

257

determined by elemental analysis and UV-spectroscopy, and it varied from 0.08 to 0.39

258

depending on the reaction conditions (Table 1). The main parameter that affected this

259

reaction was found to be the pH of the reaction mixture (Fig. 2). Thus, carrying out the

260

reaction at pH 4.5–5.0 resulted in conjugates with DSINH 0.14–0.39, (6d, f). Further increases

261

in pH up to 5.5 led to the formation of an insoluble product with DSINH 0.19 (6g). Apparently,

262

under these conditions, the polymer acquires a conformation that facilitates the formation of

263

amide bridges as a result of the reaction between CEC amino groups and the activated

264

carboxylic groups (Kumar, Muzzarelli, Muzzarelli, Sashiwa, & Domb, 2004). Consequently,

265

the rate of the target reaction decreases. Increasing the INH : CEC ratio promotes an

266

increase in the DSINH only at 4.5 ≤ pH ≤ 5.0 (6d, e) and does not have the same effect in

267

more acidic media (6a, b). Changes in EDAC concentration at the same pH value have

268

virtually no influence on the DSINH (6b, c). The temperature rise (up to 40–50°С) increases

269

crosslinking in the CEC and leads to gel formation as early as 10 min after mixing of the

270

reactants. Therefore, no or low polymer crosslinking and the formation of highly substituted

271

CEC-INH conjugates was realized by using low polymer concentrations (0.25–0.50%),

272

relatively low temperatures (less than 35°С), and pH values ranging from 4.5 to 5.0.

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Although only 10 to 30% of CEC carboxylic groups reacted with INH, and the remaining

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carboxylic groups were expected to provide water solubility, the CEC-INH conjugates with

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high DSINH demonstrated low solubility at physiological conditions. Additional phosphorylation

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was applied to increase the solubility of the conjugates (Scheme 1, 7e, f).

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The introduction of INH into the CEC structure was confirmed by 1H NMR spectroscopy.

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The spectra of the conjugates showed slightly shifted signals attributed to the pyridine

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protons of INH (as compared with INH itself in Fig.3).

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Note that the viscosity is higher for the chitosan-INH conjugates (Table 2) than for nonmodified chitosan; the viscosity of conjugate solutions increases in the order 6a–f with

13 Page 13 of 24

increasing DSINH. This may be explained both by the introduction of bulky substituents into

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polymer chains and by intermolecular crosslinking.

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Fig. 2. The influence of the CEC:INH molar ratio and pH of the reaction mixture on DSINH in

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CEC-INH conjugates.

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Fig. 3. 1H NMR spectra of CEC (a), CEC-INH conjugate 7e (b), and INH (с); (400 MHz, D2O,

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50°C). 14 Page 14 of 24

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3.2. Enzymatic biodegradation

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The biodegradation study of chitosan, CEC, CHPC, and the chitosan-INH conjugates under the action of lysozyme revealed considerable differences in the biodegradation of

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these polymers. As described earlier (Ferrari, Rossi, Bonferoni, & Caramella, 1997; Lee, Ha,

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& Park, 1995), cleavage of polymer chains causes a decrease in the viscosity of their

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solutions. The time dependence of a relative decrease in viscosity of a polymer solution upon

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enzymatic hydrolysis is given in Fig. 4. The viscosity of chitosan in the absence of lysozyme

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remained constant at a given temperature of 37C; addition of the enzyme led to an almost

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70% decrease in solution viscosity after 2 h. These results confirm the capability of chitosan

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to degrade in the human organism, because lysozyme is present in different biological fluids

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and tissues (e.g., blood serum, mucus, and mucous membranes).

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Fig. 4. The dependence of the relative decrease in viscosity of polymer solutions of 1 (1), 5

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(2), 7f (3), 2 (4), 4 (5) on the duration of enzymatic hydrolysis by lysozyme (pH 5.0,

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37.0±0.1°C).

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15 Page 15 of 24

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Two aspects should be considered in the biodegradation of the chitosan-INH conjugates. First, elimination of INH from the carrier is apparently responsible for the

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tuberculostatic activity. The second aspect is the decrease in MW of the conjugates caused

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by the chemical or enzymatic hydrolysis of glycosidic bonds in chitosan chains. This process

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facilitates removal of the macromolecules from the organism, because the conjugates cannot

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be filtered by the kidney prior to biodegradation due to their large size. Both types of

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biodegradation can occur simultaneously (e.g., in lysosomes), but can also take place at

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different times. In this case, INH is first cleaved from the polymer carrier in the blood vessels,

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and then the polymer is transported to cells where biodegradation occurs (Labarre, Ponchel,

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& Vauthier, 2011).

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The viscosity of CEC, CHPC, and chitosan-INH conjugate solutions in the absence of the enzyme did not change during the experiment within the error of measurement. The

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experiments indicate that CEC is more prone to enzyme-induced biodegradation when

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compared to CHPC, as viscosity decreases by 47% and 21% after 2 h for the CEC and

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CHPC solutions, respectively. The addition of lysozyme to the conjugate solutions causes an

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even slower degradation compared to that of the chitosan, CEC, and CHPC. The

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biodegradation ability is lower for CHPC-INH than for the CEC-INH conjugate (36% vs. 13%

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decrease after 2 h, respectively). This decrease in biodegradability may be caused both by

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steric hindrances (caused by introducing bulky INH) and by the enzyme-inhibiting effect of

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the conjugates. Chitosan with immobilized thiol functional groups has previously been shown

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to have the same effect on various enzymes (Kast & Bernkop-Schnuerch, 2001; Varum,

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Holme, Izume, Stokke, & Smidsrod, 1996).

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3.3. Antimycobacterial activity of chitosan-INH conjugates

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The conjugates were studied in vitro by testing their antituberculosis activity against M.

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tuberculosis H37Rv (Table 3). The conjugates demonstrated similar (for CEC-INH

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conjugates) or higher (for CHPC-INH conjugates) MIC values in the screening tests when

16 Page 16 of 24

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compared with INH (taking the INH mass fraction in the conjugates into account). The

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decrease in the activity of INH-chitosan conjugates containing hydroxypropyl spacers is

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probably caused by limited availability of the bound INH for the target and/or incomplete

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cleavage of the strong С–N bond between INH and the polymer.

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Таble 3

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Acute toxicity and tuberculostatic activity of INH and its conjugates against M. tuberculosis

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H37Rv.

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MIC (μg INH/mL) 0.20 0.25 1.05

LD50 (mg INH/kg) 210 850 650

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Compound INH CEC-INH (7f) CHPC-INH (4)

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LD50/MIC 1050 3400 620

3.4. Toxicity of chitosan-INH conjugates

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The studies of acute toxicity in laboratory animals demonstrated a considerable

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increase (by a factor of 3–4) in the LD50 value for the conjugates when compared to INH

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(Table 3). Additionally, we applied a slug mucosal irritation test using Limax flavus terrestrial

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slugs (Adriaens et al., 2001; Adriaens & Remon, 1999; Adriaens & Remon, 2002). Chemical

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irritation causes the slugs to release mucus, which facilitates their departure from an

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aggressive medium. This test proved to be a reliable and promising method for the evaluation

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of chemical toxicity.

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Experiments with the positive control showed that the slugs experienced acute irritation

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and released about 33.1% yellow mucus, while tests with the negative control showed that

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the slugs released 3.6% colorless mucus. The amount of mucus produced by the slugs in the

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experiments with the positive and negative controls differed slightly from the data given in a

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previous experiment (Adriaens et al., 2001) (26.6% and 2.2%, respectively). These variations

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may be caused by differences in size (mass) of the slugs used by different research groups.

17 Page 17 of 24

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The results presented in Fig. 5 show that the capability of the chitosan derivatives (CEC and CHPC) to irritate slug mucous membranes did not exceed the parameters obtained with

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the negative control (3.3 and 3.8%, respectively) within the error of measurement. This

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indicates good biocompatibility and low toxicity of these polymers.

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Fig. 5. The toxicity of INH and its conjugates performed in Limax flavus slugs; MP is the

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mucus production estimated as a slug body weight loss; error bars represent one standard

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deviation from the mean.

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In the experiments with the chitosan-INH conjugates, slugs produced colorless mucus,

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thereby indicating a relatively high biocompatibility and relatively low toxicity. The amount of

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mucus produced by slugs after contact with the conjugates was lower by a factor of 2.8 for

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CHPC-INH and by a factor of 3.7 for CEC-INH than that obtained from contact with INH itself. 18 Page 18 of 24

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Although we have not found any statistically significant difference in toxicity between CEC-

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INH (7f) and CHPC-INH (4) conjugates, the toxicity of the latter was somewhat higher in all

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cases.

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4. CONCLUSION

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The present work describes the preparation of novel water-soluble chitosan-INH

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conjugates via two different spacers (–CH2–CH(OH)–CH2– and –CH2–CH2–C(O)–). We have

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found reaction conditions that do not cause destruction or crosslinking of the chitosan matrix

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and provide a relatively high INH content ((INH) is up to 16%). Ultrasound treatment

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activated the conjugation processes, but caused partial degradation of the polymer chains.

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The chitosan-INH conjugates were prone to enzymatic biodegradation, but consecutive

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modification reduced biodegradability in the following order: chitosan > CEC and CHPC >

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chitosan-INH conjugate.

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Evaluation of the tuberculostatic activity of the chitosan-INH conjugates against M.

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tuberculosis H37Rv demonstrated that the MIC values of the conjugates were close to (for

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CEC-INH conjugates) or higher (for CHPC-INH conjugates) than that of INH itself. Toxicity

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tests with Limax flavus slugs and with mice showed that immobilization of chitosan

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decreased INH toxicity by a factor of 3–4.

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CEC is preferred as a carrier over CHPC because the CEC-INH conjugate contained a

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higher amount of the active substance, showed lower toxicity, and demonstrated a higher

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therapeutic index (LD50/MIC) than CHPC-INH. The CEC-INH conjugate is therefore a

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promising substance for further study, with possible applications in antituberculosis therapy.

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ACKNOWLEDGEMENTS

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This work was partially supported by the Russian Foundation for Basic Research (grant

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15-04-06664) and St Petersburg State University (grant 12.38.220.2015). ASB is also

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grateful for the Russian President scholarship (grant SP-249.2012.4).

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REFERENCES Adriaens, E., Dierckens, K., Bauters, T. G. M., Nelis, H. J., Van Goethem, F., Vanparys, P.,

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Remon, J. P. (2001). The mucosal toxicity of different benzalkonium chloride analogues

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evaluated with an alternative test using slugs. Pharmaceutical Research, 18, 937-942. Adriaens, E., Remon, J. P. (1999). Gastropods as an evaluation tool for screening the

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irritating potency of absorption enhancers and drugs. Pharmaceutical Research, 16,

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1240-1244.

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Adriaens, E., Remon, J. P. (2002). Evaluation of an alternative mucosal irritation test using

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slugs. Toxicology and Applied Pharmacology, 182, 169-175.

Banik, N., Hussain, A., Ramteke, A., Sharmac, H. K., Maji, T. K. (2012). Preparation and

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evaluation of the effect of particle size on the properties of chitosan-montmorillonite

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nanoparticles loaded with isoniazid. RSC Advances, 2, 10519-10528.

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Berezin, А. S., Ishmetova, R. I., Rusinov, G. L., Skorik, Y. А. (2014). Tetrazole derivatives of chitosan: synthetic approaches and evaluation of toxicity. Russian Chemical Bulletin,

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63, (in press).

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Berezin, A. S., Lomkova, E. A., Skorik, Y. A. (2012). Chitosan conjugates with biologically

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active compounds: design strategies, properties, targeted drug delivery. Russian

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Chemical Bulletin, 61, 781-795.

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Buys, G. M., du Plessis, L. H., Marais, A. F., Kotze, A. F., Hamman, J. H. (2013). Direct

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compression of chitosan: process and formulation factors to improve powder flow and

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tablet performance. Current Drug Delivery, 10, 348-356.

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Ferrari, F., Rossi, S., Bonferoni, M. C., Caramella, C. (1997). Characterization of rheological

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and mucoadhesive properties of three grades of chitosan-HCl. II Farmaco, 52, 493-497.

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Gamzazade, A. I., Slimak, V. M., Skljar, A. M., Stykova, E. V., Pavlova, S. A., Rogozin, S. V.

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(1985). Investigation of the hydrodynamic properties of chitosan solutions. Acta

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Polimerica, 36, 420-424.

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Kast, C. E., Bernkop-Schnürch, A. (2001). Thiolated polymers–thiomers: Development and in vitro evaluation of chitosan–thioglycolic acid conjugates, Biomaterials, 22, 2345-2352. Kogan, G., Skorik, Y. A., Žitnanová, I., Križková, L., Ďuračková, Z., Gomes, C. A. R., Yatluk, Y. G., Krajčovič, J. (2004). Antioxidant and antimutagenic activity of N-(2-

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carboxyethyl)chitosan. Toxicology and Applied Pharmacology, 201, 303-310.

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Kudon, S., Kudon, T. (1973). Study on the isolation culture technique of tubercle bacilli

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applicable in remote areas. Kekkaku (Tuberculosis), 48, 453 – 462.

Kumar, M. N. V., Muzzarelli, R. A. A., Muzzarelli, C., Sashiwa, H., Domb, A. J. (2004).

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Chitosan chemistry and pharmaceutical perspectives. Chemical Reviews, 104,

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6017−6084.

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Chicago: Pharmaceutical Press, (Chapter 4).

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Labarre, D., Ponchel, G., Vauthier, C. (2011). Biomedical, pharmaceutical polymers. London,

Lee, K. Y., Ha, W. S., Park, W. H. (1995). Blood compatibility and biodegradability of partially N-acylated chitosan derivatives. Biomaterials, 16, 1211-1216.

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Manaenkov, O. V., Kamenshchikov, A. A., Kislitsa, O. V., Stepanenko, Y. V., Sulman, M. G.

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(2011). Possibility of obtaining chitosan oligomers using low-frequency ultrasound.

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Izvestiya vuzov. Khimiya i khimicheskaya tekhnologiya, 54, 114-117. (In Russian).

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Muzzarelli, R. A. A. (2010) Chitins and chitosans as immunoadjuvants and non-allergenic drug carriers. Marine Drugs, 8, 292-312. Nishi, N., Nishimura, S. I., Ebina, A., Tsutsumi, A., Tokura, S. (1984). Preparation and

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characterization of water-soluble chitin phosphate. International Journal of Biological

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Macromolecules, 6, 53-54.

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Pandey, R., Khuller, G. K. (2004). Chemotherapeutic potential of alginate-chitosan

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microspheres as anti-tubercular drug carriers. Journal of Antimicrobial Chemotherapy,

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53, 635-640.

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Park, J. H., Saravanakumar, G., Kim, K., Kwon, I. C. (2010). Targeted delivery of low

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molecular drugs using chitosan and its derivatives. Advanced Drug Delivery Reviews,

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62, 28–41.

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Pestov, A. V., Skorik, Y. A., Kogan, G., Yatluk, Y. G. (2008). Alkylation of chitosan by βhalopropionic acids in the presence of various acceptors. Journal of Applied Polymer

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Science, 108, 119-127.

Pourshahab, P. S., Gilani, K., Moazeni, E., Eslahi, H., Fazeli, M. R., Jamalifar, H. (2011).

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Preparation and characterization of spray dried inhalable powders containing chitosan

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nanoparticles for pulmonary delivery of isoniazid. Journal of Microencapsulation, 28,

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605-613.

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Rando, D. G., Brandt, C. A., Ferreira, E. I. (2004). Use of N-methylene phosphonic chitosan to obtain an isoniazid prodrug. Brazilian Journal of Pharmaceutical Sciences, 40, 335-

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chitosan with acrylic acid water. Macromolecular Bioscience, 3, 231-233.

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Sashiwa, H., Yamamori, N., Ichinose, Y., Sunamoto, J., Aiba, S. (2003). Michael reaction of

Sharma, S. K. (2004). Anti-tuberculosis drugs and hepatotoxicity. Infection, Genetics and

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Evolution, 4, 167–170.

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Skorik, Y. A., Gomes, C. A. R., Vasconcelos, M. T. S. D., Yatluk, Y. G. (2003). N-(2-

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carboxyethyl)chitosans: regioselective synthesis, characterization and protolytic

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equilibria. Carbohydrate Research, 338, 271-276.

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Slivkin, A. I., Lapenko, V. L., Bolgov, A. A. (2010). Russian Federation Patent №2454226.

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Vårum, K. M., Holme, H. K., Izume, M., Stokke, B. T., Smidsrød, O. (1996). Determination of

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enzymatic hydrolysis specificity of partially N-acetylated chitosans. Biochimica et

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Biophysica Acta, 1291, 5-15.

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FIGURE CAPTIONS

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Scheme 1 Synthesis of chitosan-INH conjugates.

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Fig. 1. The dependence of the degree of INH functionalization (DSINH) on the reaction time

473

between CHPC and INH (CHPC:INH 1:2, pH 6.0, 70оС).

474

Fig. 2. The influence of the CEC:INH molar ratio and pH of the reaction mixture on DSINH in

475

CEC-INH conjugates.

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Fig. 3. 1H NMR spectra of CEC (a), CEC-INH conjugate 7e (b), and INH (с); (400 MHz, D2O,

477

50°C).

478

Fig. 4. The dependence of the relative decrease in viscosity of polymer solutions of 1 (1), 5

479

(2), 7f (3), 2 (4), 4 (5) on the duration of enzymatic hydrolysis by lysozyme (pH 5.0,

480

37.0±0.1°C).

481

Fig. 5. The toxicity of INH and its conjugates performed in Limax flavus slugs; MP is the

482

mucus production estimated as a slug body weight loss; error bars represent one standard

483

deviation from the mean.HIGHLIGHTS

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 Novel water-soluble chitosan-isoniazid conjugates were prepared and characterized

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 Conjugates exhibit high mycobacterial activity against Mycobacterium tuberculosis

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 Toxicity tests revealed a lower toxicity for the conjugates compared with isoniazid

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Chitosan-isoniazid conjugates: Synthesis, evaluation of tuberculostatic activity, biodegradability and toxicity.

Novel water-soluble chitosan-isoniazid conjugates were synthesized by two methods: (1) the carbodiimide method using isoniazid (INH) and N-(2-carboxye...
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