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
46
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
62
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
84
was 0.95 (determined by 1H NMR spectroscopy); MW was 37,000 (determined by viscometry
85
(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
6g
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
Ch0.05Cs0.95HP0.35INH0.231.3H2O
13.3
12.2
–
Ch0.05Cs0.95HP0.35INH0.23(PO3H2)0.631.1H2O 11.1
11.7
–
Ch0.05Cs0.95CE1.28INH0.24(PO3H2)0.521.4H2O 9.3
10.7
–
Ch0.05Cs0.95CE1.28INH0.39(PO3H2)0.671.4H2O 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
153
(chitosan, CEC, CHPC, or conjugate) was dissolved in 2.4 mL of 1 М HCl, 7.3 mL of distilled
154
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
157
viscometer. The viscometer was connected to a PC for the purpose of adjusting analysis
158
parameters, processing, and recording the data with the help of the Rheo Win Pro 2.64
159
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%
164
MgSO4, 0.2% sodium pyruvate, 3.6% glycerol, 0.036 malachite green, 50% chicken egg
165
yolk). Parameters of mycobacterial growth were evaluated in the presence of the conjugates
166
at various concentrations according to the standard procedure (Kudon & Kudon, 1973). The
167
results were compared to those obtained for the strain growth in the same medium without
168
the conjugates. The appearance of growth inhibition zones indicated tuberculostatic
169
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
175
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
178
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
182
experiments, Limax flavus slugs (3–4 g in weight) were placed in beakers filled with tissue
183
paper, moistened with a pH 7.4 phosphate buffer solution (PBS, 0.01 M phosphate buffer,
184
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
186
conjugate with a similar INH content) in PBS. The positive control was 1% benzalkonium
187
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):
192 193
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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
195
3.1. Synthesis and characterization of chitosan-isoniazid conjugates
196
The INH-chitosan conjugates can be synthesized in two possible ways: (1) by
197
attachment of a INH-spacer block to a polymer or (2) by consecutive attachment of a spacer
198
and INH to a polymer. In the present work, we used the second way; the first step involved
199
modification of chitosan with epichlorohydrin (Scheme 1, 2). Intermolecular crosslinking was
200
prevented by carrying out the reaction at pH 5.5 and at a temperature of 30°С for 8 h; the
201
polymer concentration in the reaction mixture was low (0.25–0.50%) (Berezin, Ishmetova, 9 Page 9 of 24
202
Rusinov, & Skorik, 2014). The degree of chlorohydroxypropylation of 2 (DSHP, Table 1) was
203
determined by elemental analysis of the product isolated from a portion of the reaction
204
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
209
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''
7
O R''
R''
Scheme 1. Synthesis of chitosan-INH conjugates.
d
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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
206
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.
212
Under these conditions, the maximum degree of INH substitution (DSINH) in conjugate 3 was
213
reached in 20 h (Fig. 1). The composition of conjugate 3, calculated from the elemental
214
analysis data, indicates that approximately 60% of chlorohydroxypropyl groups reacted with
215
INH (DSHP = 0.35, DSINH = 0.23). However, no chlorine was detected by the elemental
216
analysis of 3, indicating the complete conversion of chlorohydroxypropyl groups in CHPC.
217
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.
219 220
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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
221
(Table 2). This may be caused both by the introduction of bulky substituents into the polymer
222
and by partial intermolecular crosslinking.
224
Тable 2
225
Viscosity of solutions of chitosan and its derivatives (pH 5.0, 25.0oC). 1
2
3
6a
6с
[η] (dL/g)
2.37
4.40
6.15
2.55
2.98
227
6f
3.90
5.43
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226
6e
cr
Compound
ip t
223
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
231
undesirable polymer degradation. Thus, after sonication of the reaction mixture for 2 h, the
232
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
234
ultrasound sonication has been observed elsewhere (Berezin et al., 2014; Manaenkov,
235
Kamenshchikov, Kislitsa, Stepanenko, & Sulman, 2011).
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236
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
238
al., 1984) that increased solubility under physiological conditions (Scheme 1, 4).
239
11 Page 11 of 24
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240
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).
d
M
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243
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.
253
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244
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
275
high DSINH demonstrated low solubility at physiological conditions. Additional phosphorylation
276
was applied to increase the solubility of the conjugates (Scheme 1, 7e, f).
277
The introduction of INH into the CEC structure was confirmed by 1H NMR spectroscopy.
278
The spectra of the conjugates showed slightly shifted signals attributed to the pyridine
279
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,
294
& Park, 1995), cleavage of polymer chains causes a decrease in the viscosity of their
295
solutions. The time dependence of a relative decrease in viscosity of a polymer solution upon
296
enzymatic hydrolysis is given in Fig. 4. The viscosity of chitosan in the absence of lysozyme
297
remained constant at a given temperature of 37C; addition of the enzyme led to an almost
298
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
300
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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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
309
by the chemical or enzymatic hydrolysis of glycosidic bonds in chitosan chains. This process
310
facilitates removal of the macromolecules from the organism, because the conjugates cannot
311
be filtered by the kidney prior to biodegradation due to their large size. Both types of
312
biodegradation can occur simultaneously (e.g., in lysosomes), but can also take place at
313
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
318
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
320
CHPC solutions, respectively. The addition of lysozyme to the conjugate solutions causes an
321
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%
323
decrease after 2 h, respectively). This decrease in biodegradability may be caused both by
324
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
331
conjugates) or higher (for CHPC-INH conjugates) MIC values in the screening tests when
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compared with INH (taking the INH mass fraction in the conjugates into account). The
333
decrease in the activity of INH-chitosan conjugates containing hydroxypropyl spacers is
334
probably caused by limited availability of the bound INH for the target and/or incomplete
335
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
339
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
344
(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
347
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
352
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
354
may be caused by differences in size (mass) of the slugs used by different research groups.
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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
362
deviation from the mean.
363 364
In the experiments with the chitosan-INH conjugates, slugs produced colorless mucus,
365
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
367
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
373
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
375
found reaction conditions that do not cause destruction or crosslinking of the chitosan matrix
376
and provide a relatively high INH content ((INH) is up to 16%). Ultrasound treatment
377
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
379
modification reduced biodegradability in the following order: chitosan > CEC and CHPC >
380
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
384
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
388
therapeutic index (LD50/MIC) than CHPC-INH. The CEC-INH conjugate is therefore a
389
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
394
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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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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344.
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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
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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
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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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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.
1 2 3 4
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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
23
carbodiimide method using isoniazid (INH) and N-(2-carboxyethyl)chitosan (CEC), and (2)
24
the reaction between INH and N-(3-chloro-2-hydroxypropyl)chitosan (CHPC). The solubility of
25
the conjugates under physiological conditions was enhanced by phosphorylation. Method (1)
26
is preferable in terms of obtaining conjugates with a high content of active substance;
27
depending on reaction conditions, the degree of substitution in the INH-CEC conjugates
28
varies from 0.08 tо 0.39. Ultrasound treatment increased the reaction rate by a factor of 1.3–
29
1.5, but caused partial degradation of the polymer. Consecutive modification led to a
30
considerable decrease in polymer biodegradability in the following order: chitosan > CEC or
31
CHPC > conjugate. In vitro screening of the antimicrobial activity against Mycobacterium
32
tuberculosis H37Rv demonstrated a comparable or slightly higher minimum inhibitory
33
concentration for conjugates than for INH itself (0.20, 0.25, and 1.05 g INH/mL for INH,
34
CEC-INH, and CHPC-INH, respectively). A slug mucosal irritation test employing Limax
35
flavus revealed a lower toxicity for the conjugates than for INH by a factor of 3–4; the most
36
noticeable toxicity decrease was observed for the conjugates obtained by method (1).
37
Studies of acute toxicity in mice revealed a 3–4-fold increase in median lethal dose for the
38
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
46
strains and due to the high toxicity of existing drugs. Isoniazid (INH) is one of the most
47
commonly used first-line antituberculosis drugs; it possesses high activity against
48
Mycobacterium tuberculosis, but is rapidly egested, thereby necessitating high doses of a
49
highly toxic INH (Sharma, 2004). For this reason, the development of drug delivery systems
50
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
53
its unique properties, including biocompatibility, biodegradability, mucoadhesive properties,
54
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
56
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
60
and raising INH bioaccessibility, provide controlled drug release as a result of desorption,
61
diffusion of adsorbed INH, and polymer biodegradation. Parameters of the release process
62
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
3 Page 3 of 24
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
71
(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
74
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
84
was 0.95 (determined by 1H NMR spectroscopy); MW was 37,000 (determined by viscometry
85
(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
88
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
92
using a Vario Micro Cube Elemental СНNS analyzer; lyophilization of samples was carried
93
out in a CoolSafe 110-4 freeze dryer; the viscosity of samples was measured using a
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Brookfield LV viscometer.
4 Page 4 of 24
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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
101
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;
103
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
109
dissolved in 0.1 М HCl and dialyzed against distilled water for 2 days using an Orange
110
Scientific 8030T2 membrane (MWCO = 6000–8000). The degree of substitution (DSCE =
111
1.28) was calculated from the 1Н NMR data as previously described (Skorik, Gomes,
112
Vasconcelos, & Yatluk, 2003).
Ac ce p
te
d
M
106
113
2.4. Preparation of isoniazid conjugates with N-(2-carboxyethyl)chitosan
114
The CEC-INH conjugates were obtained according to the general procedure, which
115
includes varying the molar ratios of reactants and the pH of the reaction mixture. The
116
synthesis conditions and some characteristics of the products are given in Table 1.
117
For example, the 6b conjugate was prepared according to the following procedure: 500
118
mg of CEC was dissolved in 100 mL of water, and then 740 mg of INH was added. The
119
mixture was cooled down to 5oC, and then 100 mg of N-(3-dimethylaminopropyl)-N'-
120
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
122
for 1 h and then heated slowly over 1 h until it reached the ambient temperature; stirring was
123
continued for another 20 h. Then 2 mL of methanesulfonic acid and 300 mg of anhydrous
124
P2O5 were added to the solution (Nishi, Nishimura, Ebina, Tsutsumi, & Tokura, 1984). The
125
reaction mixture was stirred at 5oC for 18 h. The resulting product was precipitated with
126
acetone, dissolved in 0.1 M HCl, and dialyzed against distilled water for 2 days. The mass
127
fraction of INH was calculated by elemental analysis and UV-spectroscopy (Table 1); the
128
purity of the product was evaluated by 1Н NMR spectroscopy.
131
cr
us
130
2.5. Preparation of isoniazid conjugates with N-(3-chloro-2hydroxypropyl)chitosan
an
129
ip t
121
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
133
added, and the pH of the solution was brought to 5.5 using 1 M NaOH. The reaction mixture
134
was stirred at 30oC for 8 h, and then (without isolation of CHPC) 100 mg of INH was added,
135
and the pH of the mixture was brought up to 6.0. The solution was stirred for 20 h at 60 оС.
136
The resulting product was precipitated with acetone, dissolved in 0.1 M HCl, and washed
137
repeatedly with acetone. Then 480 g of the obtained conjugate was dissolved in 0.1 М HCl,
138
and 2 mL of methanesulfonic acid and 300 mg of anhydrous P2O5 were added (Nishi et al.,
139
1984). The reaction mixture was stirred at 5oC for 18 h, the product of which was precipitated
140
with acetone, dissolved in 0.1 М HCl, and dialyzed against distilled water for 48 h. The mass
141
fraction of INH was calculated using elemental analysis and UV-spectroscopy data (Table 1);
142
the purity of the product was evaluated by 1Н NMR spectroscopy.
Ac ce p
te
d
M
132
6 Page 6 of 24
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
6g
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
Ch0.05Cs0.95HP0.35INH0.231.3H2O
13.3
12.2
–
Ch0.05Cs0.95HP0.35INH0.23(PO3H2)0.631.1H2O 11.1
11.7
–
Ch0.05Cs0.95CE1.28INH0.24(PO3H2)0.521.4H2O 9.3
10.7
–
Ch0.05Cs0.95CE1.28INH0.39(PO3H2)0.671.4H2O 13.9
12.4
us
6a
Solubility in watera
M an
Compound Synthesis conditions
cr
Тable 1 Synthesis conditions and some characteristics of the conjugates.
ed
143
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
7 Page 7 of 24
150
2.6. Enzymatic biodegradation
151
The biodegradation of the chitosan derivatives and INH conjugates was studied using the procedure described earlier (Kast & Bernkop-Schnuerch, 2001). A 300 mg sample
153
(chitosan, CEC, CHPC, or conjugate) was dissolved in 2.4 mL of 1 М HCl, 7.3 mL of distilled
154
water was added, and the pH was adjusted to 5.0 using 1 М NaOH. Then, 1.5 mg of chicken
155
egg white lysozyme per 1 mL of the polymer was added to each solution. At specific times,
156
the viscosities of the reaction mixtures were measured at 37.0 ± 0.1°C using a Brookfield LV
157
viscometer. The viscometer was connected to a PC for the purpose of adjusting analysis
158
parameters, processing, and recording the data with the help of the Rheo Win Pro 2.64
159
program. Polymer solutions containing no lysozyme were used as reference solutions.
an
us
cr
ip t
152
2.7. Determination of minimum inhibitory concentration (MIC)
161
The MIC values were determined using the M. tuberculosis H37Rv laboratory strain
M
160
(which is sensitive to antituberculosis drugs). Tuberculostatic activity was studied using the
163
vertical diffusion method (growth medium: 0.05% KH2PO4, 0.05% sodium citrate, 0.05%
164
MgSO4, 0.2% sodium pyruvate, 3.6% glycerol, 0.036 malachite green, 50% chicken egg
165
yolk). Parameters of mycobacterial growth were evaluated in the presence of the conjugates
166
at various concentrations according to the standard procedure (Kudon & Kudon, 1973). The
167
results were compared to those obtained for the strain growth in the same medium without
168
the conjugates. The appearance of growth inhibition zones indicated tuberculostatic
169
properties of the compounds at the given concentration. All experiments were carried out in
170
triplicate.
Ac ce p
te
d
162
171
2.8. Determination of median lethal dose (LD50)
172
Acute toxicity of the conjugates was tested using white outbred CD-I mice weighing 18–
173
25 g; various doses of INH or conjugates dissolved in physiological saline were introduced
174
intraperitoneally. Observations were made for 2 weeks. Toxicity was evaluated using the
175
median lethal doses (LD50) obtained for test and control groups. The experiments were
Page 8 of 24
176
conducted in agreement with the Rules of Animal Care and Use Committee of the St.
177
Petersburg State Chemical Pharmaceutical Academy, which comply fully with European
178
Community Council directives 86/609/EEC. Naive animals were used for all the experiments. 2.9. Slug mucosal irritation test using Limax flavus
180
Toxicity of INH and the novel conjugates was additionally estimated using the previously
ip t
179
described slug mucosal irritation test (Adriaens et al., 2001). Two days prior to the
182
experiments, Limax flavus slugs (3–4 g in weight) were placed in beakers filled with tissue
183
paper, moistened with a pH 7.4 phosphate buffer solution (PBS, 0.01 M phosphate buffer,
184
0.0027 M KCl and 0.137 M NaCl), and kept at room temperature. Each slug was weighed
185
and then placed in a Petri dish containing a paper filter treated with 1% solution of INH (or a
186
conjugate with a similar INH content) in PBS. The positive control was 1% benzalkonium
187
chloride in PBS, and PBS was used as a negative control. After 1 hour, slugs were removed
188
from the Petri dishes, washed with PBS, dried with filter paper, and weighed again. Slug
189
weight loss was used to estimate the mass fraction of liberated mucus (MP):
192 193
us
an
M
d
te
191
,
where ma and mb are the masses of the slugs before and after the experiment,
Ac ce p
190
cr
181
respectively. Each experiment was carried out in triplicate.
194
3. RESULTS AND DISCUSSION
195
3.1. Synthesis and characterization of chitosan-isoniazid conjugates
196
The INH-chitosan conjugates can be synthesized in two possible ways: (1) by
197
attachment of a INH-spacer block to a polymer or (2) by consecutive attachment of a spacer
198
and INH to a polymer. In the present work, we used the second way; the first step involved
199
modification of chitosan with epichlorohydrin (Scheme 1, 2). Intermolecular crosslinking was
200
prevented by carrying out the reaction at pH 5.5 and at a temperature of 30°С for 8 h; the
201
polymer concentration in the reaction mixture was low (0.25–0.50%) (Berezin, Ishmetova, 9 Page 9 of 24
202
Rusinov, & Skorik, 2014). The degree of chlorohydroxypropylation of 2 (DSHP, Table 1) was
203
determined by elemental analysis of the product isolated from a portion of the reaction
204
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
209
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''
7
O R''
R''
Scheme 1. Synthesis of chitosan-INH conjugates.
d
208
NH
O
O OH HO
N H
te
207
5
O
O HO
R''=
OH
O
, NHS, EDAC
N
O OH
N
;
R'= PO 3H2 , H.
M
NH
O
206
O
O
N
1
OH O
HO
H N
H N
an
H2 N
N H
us
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.
212
Under these conditions, the maximum degree of INH substitution (DSINH) in conjugate 3 was
213
reached in 20 h (Fig. 1). The composition of conjugate 3, calculated from the elemental
214
analysis data, indicates that approximately 60% of chlorohydroxypropyl groups reacted with
215
INH (DSHP = 0.35, DSINH = 0.23). However, no chlorine was detected by the elemental
216
analysis of 3, indicating the complete conversion of chlorohydroxypropyl groups in CHPC.
217
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.
219 220
Ac ce p
210
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
221
(Table 2). This may be caused both by the introduction of bulky substituents into the polymer
222
and by partial intermolecular crosslinking.
224
Тable 2
225
Viscosity of solutions of chitosan and its derivatives (pH 5.0, 25.0oC). 1
2
3
6a
6с
[η] (dL/g)
2.37
4.40
6.15
2.55
2.98
227
6f
3.90
5.43
us
226
6e
cr
Compound
ip t
223
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
231
undesirable polymer degradation. Thus, after sonication of the reaction mixture for 2 h, the
232
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
234
ultrasound sonication has been observed elsewhere (Berezin et al., 2014; Manaenkov,
235
Kamenshchikov, Kislitsa, Stepanenko, & Sulman, 2011).
Ac ce p
te
d
M
an
228
236
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
238
al., 1984) that increased solubility under physiological conditions (Scheme 1, 4).
239
11 Page 11 of 24
ip t cr us an
240
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).
d
M
241
te
243
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.
253
Ac ce p
244
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.
cr
us
an
M
d
te
Ac ce p
273
ip t
256
Although only 10 to 30% of CEC carboxylic groups reacted with INH, and the remaining
274
carboxylic groups were expected to provide water solubility, the CEC-INH conjugates with
275
high DSINH demonstrated low solubility at physiological conditions. Additional phosphorylation
276
was applied to increase the solubility of the conjugates (Scheme 1, 7e, f).
277
The introduction of INH into the CEC structure was confirmed by 1H NMR spectroscopy.
278
The spectra of the conjugates showed slightly shifted signals attributed to the pyridine
279
protons of INH (as compared with INH itself in Fig.3).
280 281
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
283
polymer chains and by intermolecular crosslinking.
an
us
cr
ip t
282
M
284
Fig. 2. The influence of the CEC:INH molar ratio and pH of the reaction mixture on DSINH in
286
CEC-INH conjugates.
Ac ce p
te
d
285
287 288
Fig. 3. 1H NMR spectra of CEC (a), CEC-INH conjugate 7e (b), and INH (с); (400 MHz, D2O,
289
50°C). 14 Page 14 of 24
290
3.2. Enzymatic biodegradation
291
The biodegradation study of chitosan, CEC, CHPC, and the chitosan-INH conjugates under the action of lysozyme revealed considerable differences in the biodegradation of
293
these polymers. As described earlier (Ferrari, Rossi, Bonferoni, & Caramella, 1997; Lee, Ha,
294
& Park, 1995), cleavage of polymer chains causes a decrease in the viscosity of their
295
solutions. The time dependence of a relative decrease in viscosity of a polymer solution upon
296
enzymatic hydrolysis is given in Fig. 4. The viscosity of chitosan in the absence of lysozyme
297
remained constant at a given temperature of 37C; addition of the enzyme led to an almost
298
70% decrease in solution viscosity after 2 h. These results confirm the capability of chitosan
299
to degrade in the human organism, because lysozyme is present in different biological fluids
300
and tissues (e.g., blood serum, mucus, and mucous membranes).
301
Ac ce p
te
d
M
an
us
cr
ip t
292
302
Fig. 4. The dependence of the relative decrease in viscosity of polymer solutions of 1 (1), 5
303
(2), 7f (3), 2 (4), 4 (5) on the duration of enzymatic hydrolysis by lysozyme (pH 5.0,
304
37.0±0.1°C).
305
15 Page 15 of 24
306
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
308
tuberculostatic activity. The second aspect is the decrease in MW of the conjugates caused
309
by the chemical or enzymatic hydrolysis of glycosidic bonds in chitosan chains. This process
310
facilitates removal of the macromolecules from the organism, because the conjugates cannot
311
be filtered by the kidney prior to biodegradation due to their large size. Both types of
312
biodegradation can occur simultaneously (e.g., in lysosomes), but can also take place at
313
different times. In this case, INH is first cleaved from the polymer carrier in the blood vessels,
314
and then the polymer is transported to cells where biodegradation occurs (Labarre, Ponchel,
315
& Vauthier, 2011).
cr
us
an
316
ip t
307
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
318
experiments indicate that CEC is more prone to enzyme-induced biodegradation when
319
compared to CHPC, as viscosity decreases by 47% and 21% after 2 h for the CEC and
320
CHPC solutions, respectively. The addition of lysozyme to the conjugate solutions causes an
321
even slower degradation compared to that of the chitosan, CEC, and CHPC. The
322
biodegradation ability is lower for CHPC-INH than for the CEC-INH conjugate (36% vs. 13%
323
decrease after 2 h, respectively). This decrease in biodegradability may be caused both by
324
steric hindrances (caused by introducing bulky INH) and by the enzyme-inhibiting effect of
325
the conjugates. Chitosan with immobilized thiol functional groups has previously been shown
326
to have the same effect on various enzymes (Kast & Bernkop-Schnuerch, 2001; Varum,
327
Holme, Izume, Stokke, & Smidsrod, 1996).
Ac ce p
te
d
M
317
328
3.3. Antimycobacterial activity of chitosan-INH conjugates
329
The conjugates were studied in vitro by testing their antituberculosis activity against M.
330
tuberculosis H37Rv (Table 3). The conjugates demonstrated similar (for CEC-INH
331
conjugates) or higher (for CHPC-INH conjugates) MIC values in the screening tests when
16 Page 16 of 24
332
compared with INH (taking the INH mass fraction in the conjugates into account). The
333
decrease in the activity of INH-chitosan conjugates containing hydroxypropyl spacers is
334
probably caused by limited availability of the bound INH for the target and/or incomplete
335
cleavage of the strong С–N bond between INH and the polymer.
ip t
336
Таble 3
338
Acute toxicity and tuberculostatic activity of INH and its conjugates against M. tuberculosis
339
H37Rv.
340
us
MIC (μg INH/mL) 0.20 0.25 1.05
LD50 (mg INH/kg) 210 850 650
an
Compound INH CEC-INH (7f) CHPC-INH (4)
cr
337
LD50/MIC 1050 3400 620
3.4. Toxicity of chitosan-INH conjugates
342
The studies of acute toxicity in laboratory animals demonstrated a considerable
M
341
increase (by a factor of 3–4) in the LD50 value for the conjugates when compared to INH
344
(Table 3). Additionally, we applied a slug mucosal irritation test using Limax flavus terrestrial
345
slugs (Adriaens et al., 2001; Adriaens & Remon, 1999; Adriaens & Remon, 2002). Chemical
346
irritation causes the slugs to release mucus, which facilitates their departure from an
347
aggressive medium. This test proved to be a reliable and promising method for the evaluation
348
of chemical toxicity.
te
Ac ce p
349
d
343
Experiments with the positive control showed that the slugs experienced acute irritation
350
and released about 33.1% yellow mucus, while tests with the negative control showed that
351
the slugs released 3.6% colorless mucus. The amount of mucus produced by the slugs in the
352
experiments with the positive and negative controls differed slightly from the data given in a
353
previous experiment (Adriaens et al., 2001) (26.6% and 2.2%, respectively). These variations
354
may be caused by differences in size (mass) of the slugs used by different research groups.
17 Page 17 of 24
355
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
357
the negative control (3.3 and 3.8%, respectively) within the error of measurement. This
358
indicates good biocompatibility and low toxicity of these polymers.
359
Ac ce p
te
d
M
an
us
cr
ip t
356
360
Fig. 5. The toxicity of INH and its conjugates performed in Limax flavus slugs; MP is the
361
mucus production estimated as a slug body weight loss; error bars represent one standard
362
deviation from the mean.
363 364
In the experiments with the chitosan-INH conjugates, slugs produced colorless mucus,
365
thereby indicating a relatively high biocompatibility and relatively low toxicity. The amount of
366
mucus produced by slugs after contact with the conjugates was lower by a factor of 2.8 for
367
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
368
Although we have not found any statistically significant difference in toxicity between CEC-
369
INH (7f) and CHPC-INH (4) conjugates, the toxicity of the latter was somewhat higher in all
370
cases.
371
4. CONCLUSION
373
The present work describes the preparation of novel water-soluble chitosan-INH
ip t
372
conjugates via two different spacers (–CH2–CH(OH)–CH2– and –CH2–CH2–C(O)–). We have
375
found reaction conditions that do not cause destruction or crosslinking of the chitosan matrix
376
and provide a relatively high INH content ((INH) is up to 16%). Ultrasound treatment
377
activated the conjugation processes, but caused partial degradation of the polymer chains.
378
The chitosan-INH conjugates were prone to enzymatic biodegradation, but consecutive
379
modification reduced biodegradability in the following order: chitosan > CEC and CHPC >
380
chitosan-INH conjugate.
us
an
M
Evaluation of the tuberculostatic activity of the chitosan-INH conjugates against M.
d
381
cr
374
tuberculosis H37Rv demonstrated that the MIC values of the conjugates were close to (for
383
CEC-INH conjugates) or higher (for CHPC-INH conjugates) than that of INH itself. Toxicity
384
tests with Limax flavus slugs and with mice showed that immobilization of chitosan
385
decreased INH toxicity by a factor of 3–4.
Ac ce p
386
te
382
CEC is preferred as a carrier over CHPC because the CEC-INH conjugate contained a
387
higher amount of the active substance, showed lower toxicity, and demonstrated a higher
388
therapeutic index (LD50/MIC) than CHPC-INH. The CEC-INH conjugate is therefore a
389
promising substance for further study, with possible applications in antituberculosis therapy.
390 391
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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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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Buys, G. M., du Plessis, L. H., Marais, A. F., Kotze, A. F., Hamman, J. H. (2013). Direct
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Ferrari, F., Rossi, S., Bonferoni, M. C., Caramella, C. (1997). Characterization of rheological
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Kudon, S., Kudon, T. (1973). Study on the isolation culture technique of tubercle bacilli
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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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microspheres as anti-tubercular drug carriers. Journal of Antimicrobial Chemotherapy,
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nanoparticles for pulmonary delivery of isoniazid. Journal of Microencapsulation, 28,
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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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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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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
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between CHPC and INH (CHPC:INH 1:2, pH 6.0, 70оС).
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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).
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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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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.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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