Activity, Reduced Toxicity, and Scale-Up Synthesis of Amphotericin B

Institute of Drug Research, School of Pharmacy, Faculty of Medicine, The Hebrew University ... Publication Date (Web): April 29, 2014 .... Effect of a...
0 downloads 0 Views 2MB Size
Article pubs.acs.org/Biomac

Activity, Reduced Toxicity, and Scale-Up Synthesis of Amphotericin B‑Conjugated Polysaccharide Diana E. Ickowicz,†,§ Shimon Farber,†,§ Edward Sionov,‡ Sarah Kagan,‡ Amnon Hoffman,† Itzhack Polacheck,‡ and Abraham J. Domb*,†,⊥ †

Institute of Drug Research, School of Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem 91120, Israel Department of Clinical Microbiology and Infectious Diseases, Hadassah-Hebrew University Medical Center, Jerusalem 91120, Israel



ABSTRACT: Amphotericin B (AMB) arabinogalactan (AG) conjugate was synthesized by the conjugation of AMB to oxidized AG by reductive amination. The conjugate was evaluated for in vitro antifungal activity and in vivo toxicity. Optimization of the conjugation process was investigated using large batches of 100 g, which are 20 times larger than previously reported for AMB− AG conjugation. The efficacy of AMB−AG conjugates was studied as a function of reaction conditions and time, aldehyde/ reducing agent mole ratio, and purification procedure. The most potent AMB−AG conjugate having low minimal inhibitory concentration (MIC) and high maximal tolerated dose (MTD) was obtained following reduction with NaBH4 at 1:2 mol ratio (AG units/NaBH4) at 25 °C for 24 h. AMB−AG conjugate prepared under these conditions demonstrated MIC of 0.5 mg/L (equiv of AMB) in Candida albicans, and an MTD of 60 mg/kg (equiv of AMB) in mice, while AMB clinical formulation (Fungizone) demonstrated high toxicity (MTD = 3 mg/kg). These findings confirm the simplicity and reproducibility of the conjugation allowing this method to be applied on larger scale production.

1. INTRODUCTION Opportunistic fungal infections have emerged as an important cause of mortality in immune compromised and severely ill patients, such as those who have undergone organ transplantation, cancer patients, and patients with AIDS. The formulation of amphotericin B (AMB) with sodium deoxycholate (Fungizone) remains one of the most effective and widely used drugs for treating systemic fungal infections.1 The main limitation of using Fungizone is its toxicity. In addition to infusion-related adverse effects such as fever, chills, hypotension, thrombophlebitis, dyspnea and nausea, its major limiting factor is cumulative toxicity, particularly nephrotoxicity and renal failure.2 AMB lipid-based delivery systems were developed to overcome this toxicity; these include liposomes,3,4 nanospheres,5 and cochleates.6,7 Lipid-based formulations of AMB such as AmBisome, Amphocil, and Abelcet demonstrate reduced toxicity relative to Fungizone. However, these lipidbased particulate dispersions are of limited stability, and entail manufacturing complications and high cost per treatment. The development of a water-soluble AMB derivative is an alternative to lipid-based particulate formulations. Compared © 2014 American Chemical Society

with AMB physical formulations, water-soluble AMB−polysaccharide conjugate should possess unique biodistribution, reduced toxicity, prolonged plasma circulation and altered physical properties of the drug. Different polymeric drug carriers have been widely studied for this purpose, including polysaccharides, which possess high water-solubility, low toxicity, a high degree of biodegradability, and biocompatibility. Pharmacokinetic of polysaccharides is largely affected by its average molecular weight and distribution, electric charge, chemical modifications and branching. Their applications range from delivery of small drug molecules to preparation of protein conjugates.8−11 This study examines the antifungal activity of AMB grafted onto natural polysaccharide chains (arabinogalactan) via reductive amination prepared on a large scale (Figure 1). Recently, the authors have reported on amphotericin Barabinogalactan conjugate (AMB−AGC), which efficiently Received: February 11, 2014 Revised: April 21, 2014 Published: April 29, 2014 2079

dx.doi.org/10.1021/bm5002125 | Biomacromolecules 2014, 15, 2079−2089

Biomacromolecules

Article

Figure 1. Grafting amphotericin B on arabinogalactan by reductive amination.

inhibits pathogenic yeast growth.12,13 It is highly water-soluble, relatively nontoxic, biodegradable, and biocompatible. AMB− AGC was found to be highly effective against Candida albicans, Cryptococcus neoformans,1213 and Aspergillus fumigatus,14 and relatively nontoxic compared with the native drug, both in vitro and in vivo.12 The conjugates are about 1000 times less hemolytic than the free drug against erythrocytes, and about 20 times less toxic in a mouse model of acute toxicity. Additionally, the conjugation to AG neutralized the toxic effect of AMB on mammalian cells, including membrane perforation, membrane trafficking and endosomal alkalization,15 induction of high levels of IL-1beta and TNF-alpha in kidneys, and apoptosis in renal tubular cells.16 Previously, the authors demonstrated the powerful antifungal activity of this water-soluble conjugate. The challenge was to determine a set of optimal conditions for the synthesis of AMB−AG conjugate on a pilot scale (100 g), which has yet not been reported. The aim of this study is to determine optimal conditions as defined by the following parameters: reaction time, temperature, reduction conditions, and purification methods. A further goal is to evaluate the effect of these findings on the bioactivity of the AMB−AG conjugate. The scale-up process involved oxidation of arabinogalactan, conjugation of the oxidized polysaccharide with amphotericin B by Schiff base formation, reduction of the imines to obtain amine-based conjugate, purification by cross-flow filtration, and freeze-drying of the product. The antifungal activity of the

conjugates was evaluated in vitro and in vivo. Chemical properties of the conjugates including solubility, molecular weight, pyrogenicity, and osmolarity were also studied.

2. MATERIALS AND METHODS 2.1. Materials. AMB powder was purchased from Alpharma, Copenhagen, Denmark. Water-soluble larch AG of average molecular weight (17 kDa [UF powder Lot 2UF06307]) was obtained from Lonza, Inc., Allendale, NJ, USA. Fungizone was purchased from Bristol Myers Squibb, New York, NY, USA. Potassium periodate (KIO4), sodium borohydride (NaBH4), sodium hydroxide (NaOH), boric acid, dimethyl sulfoxide (DMSO), RPMI 1640 broth medium, and 3-(Nmorpholino)propanesulfonic acid (MOPS) were all purchased from Sigma-Aldrich (Rehovot, Israel). Dowex-1 × 4 acetate form was purchased from Sigma-Aldrich. Water for irrigation (WFI) was purchased from TEVA Medical, Israel. Solutions were filtered prior to HPLC/GPC analysis using Minisart RC15 0.2 μm membrane filters. All solvents and reagents were of analytical grade and were used as indicated. Pall Minisette Casettes was purchased from Pall Corporation, East Hills, NY, USA. Average molecular weights of the conjugates were estimated by GPC-Spectra Physics instrument (Darmstadt, Germany) containing a pump, column (Shodex SB-803HQ) and refractive index (RI) detector.17 This was done according to pullulan standards (PSS, Mainz, Germany) with molecular weights between 5800 and 212 000. Eluent used was 0.05 M NaNO3. HPLC was carried out on a size exclusion column Shodex SB-803-HQ (Phenomenex, Japan) using a UV detector with a mobile phase of acetonitrile (30%): double deionized water (DDW) (70%). The flow rate used 1 mL/min, 2080

dx.doi.org/10.1021/bm5002125 | Biomacromolecules 2014, 15, 2079−2089

Biomacromolecules

Article

periodate was added at 1:1 and 1:2 mol ratios (IO4−/AMB). Samples were incubated with KIO4 for 0, 1, 2, 4, 24, and 48 h at room temperature. AMB concentration was determined by UV at 390 nm using a calibration curve prepared with AMB. In addition, samples were evaluated for their in vitro activity. Determination of the Reducing Agent (NaBH4) Effect on Amphotericin B and Its Conjugate. Fifty milligrams of AMB dissolved in 20 mL of borate buffer (0.1 N, pH 11) was prepared. Sodium borohydride at 1:1, 1:2, and 1:4 mol ratios (NaBH4/AG unit) was added to these solutions. Samples were incubated for 0, 1, 2, 4, 24, and 48 h at room temperature. AMB concentration was analyzed by UV at 390 nm using a calibration curve prepared with AMB. In addition, samples were evaluated for their in vitro activity against C. albicans. Solutions of AMB−AG conjugate were also evaluated in the same manner. Ultrafiltration Procedure. The purification was performed on a cross-flow filtration device (Filtron, Pall, USA), using two manometers placed at the module inlet and outlet, a valve placed at the module outlet, and a 603S pump (Watson Marlow, UK). Membrane pressure (Poly(ether sulfone)) at the module inlet was 2 bar, while pressure at the module outlet was 0 bar, and pressure on the filtrate side was 0 bar. Prior to the filtration, membranes were conditioned with water to eliminate residues of NaOH. The purification procedure was carried out with 5 L solution (feed volume) at 22 °C. Permeate flow returned to the feed container. The filtrate flow rate was 6 L/hour. Three liters of DDW or WFI was added to the container every 30 min. The pH of the medium was maintained at 6.5. At the end of each experiment, the ultrafiltration membranes were regenerated by flushing with pure water for 30 min, followed by NaOH solution (1 N) for 30 min, and finally NaOH (0.1 N) for 30 min. The membranes were stored in the final solution (0.1 N NaOH). Quantitative Determination of Periodate (IO4−) and Iodate (IO3−) Traces in Oxidized Arabinogalactan by Iodometric Method. A solution of oxidized AG (20 mg, 0.125 mmol) in 5 mL of DDW was shaken for 2 h at 37 °C until full dissolution. Fifty microliters of H2SO4 0.01 N was added to reach pH 1−3, followed by addition of 80 mg of KI. The solution was stirred for 15 min at room temperature. No color change (yellow-brown) was observed. Two milliliters of indicator starch solution (0.1 g per 20 mL boiled water) was added to the acidic AG solution to determinate traces of periodate (IO4−) and iodate (IO3−). The solution was stirred for an additional 15 min at room temperature. No pale blue color was produced, indicating undetectable levels of periodate and iodate ions in the oxidized AG. Potassium periodate (KIO4), applied as standard, resulted in yellowbrown colored solution. Test for Sterility of the AMB−AG Conjugates. The test was applied for pilot-scale samples only, and was performed by Aminolab Ltd., Nes Ziona, Israel, using Steritest (Membrane filtration method), according to USP specifications.20 Test for Pyrogenicity of the AMB−AG Conjugates. The presence of pyrogens was determined by the Limulus amebocyte lysate (LAL) testing-kinetic turbidometric technique. The test was applied for pilotscale samples only and was performed by Aminolab. Briefly, tested solution was prepared in a concentration of 1 mg/mL. One hundred microliters of the solution was inserted into each of four wells (two wells for testing the sample endotoxin, and two for spiking determination of a positive product control, PPC). One hundred microliters of lysate was added to each well, and to the spiking wells. Ten microliters of 5 EU/mL Control Standard endotoxin (CSE) were added. Pyrogen-free water (100 μL) was used as a negative control. The test was performed in duplicate for each sample in a 96-well plate. The endotoxin units were less than 0.5 EU/ml or 20 EU/device, indicating a pyrogen-free sample. 2.2.2. In Vitro Studies. Broth Dilution Method: Antifungal Activity. C. albicans strain ATCC 90028 (The American Type Culture Collection, ATCC, Manassas, VA) was used for susceptibility testing. Minimal inhibitory concentration (MIC) values were determined according to Clinical Laboratory Standards Institute (CLSI) recommendations.21 A stock solution of 5 mg/mL was prepared in dimethyl sulfoxide for free AMB; AMB−AGC was prepared in water.

detecting the eluent at 406 nm. All samples were dissolved in DDW (100 mg/mL of AMB−AGC); dissolution of the samples in the mobile phase resulted in immediate precipitation. 1H NMR spectra (D2O and DMSO) were obtained on a Varian 300-MHz spectrometer in 5 mm o.d. tubes. D2O and DMSO containing tetramethylsilane served as the solvent and shift reference. Fourier transform infrared (FT-IR) spectra were recorded on a PerkinElmer 2000 FTIR. The degree of conjugation was estimated by UV absorbance of AMB−AGC at 390 nm based on the equation obtained from the AMB calibration curve. UV measurements were done on an Uvikon 930 spectrophotometer, and samples were dissolved in a mixture of DDW and DMSO (1:200 v/v). The osmolarity of the samples including AMB, Fungizone, and AMB−AGC compounds was estimated using a 5500 vapor pressure osmometer, Wescor. The standard solutions used were optimole Wescor standards. 2.2. Methods. 2.2.1. Polymer Synthesis. Oxidation of Arabinogalactan at Lab Scale. Arabinogalactan (AG) (10 g, 62.5 mmol of AG units) was dissolved in 100 mL of DDW, and potassium periodate was gradually added at a 1:1 mol ratio (IO4−/AG units) to avoid overheating of the reaction flask. The reaction mixture was allowed to mix in the dark at 25 ± 2 °C for 2 h, and the polyaldehyde was then purified by anion exchange chromatography (Dowex-1 in acetate form), followed by extensive dialysis against DDW (5L X 4) (12 000 cutoff cellulose tubing) at 4 °C for 3 days. After purification, the polyaldehyde was freeze-dried to obtain white powder in 75% ±5% (w/w) average yield. FT-IR (KBr); 1724 cm−1 (CO). The aldehyde content was determined by the hydroxylamine hydrochloride method.18 Oxidation of Arabinogalactan at Pilot Scale. AG (120 g, 0.75 mol of sugar units) was dissolved in 1.2 L of DDW. Potassium periodate 1:1 mol ratio (IO4−/saccharide) was added, and the mixture was stirred in the dark at room temperature for 2 h. The resultant oxidized AG was purified from iodate (IO3−) and unreacted periodate (IO4−) by passing the reaction solution through a column (9 cm diameter, 120 cm height) filled with 2 kg Dowex 1 × 4 acetate form, at a flow rate of 25 mL per minute. The clear solution of oxidized AG was purified from acetic acid and low molecular weight residues of oxidized arabinogalactan by ultrafiltration using “Minisette Casettes” with a 5 kDa MWCO filter and freeze-dried. Average yield: 75% (w/w). FT-IR (KBr) = 1724 cm−1 (CO). Synthesis of Amphotericin B−Arabinogalactan at Laboratory Scale. AMB−AG was synthesized as reported previously.19 In brief, oxidized AG (500 mg, 3.87 mmol/g aldehyde groups) was dissolved in 50 mL of 0.1 M borate buffer (pH 11 ± 0.1) at room temperature. To the clear solution, 125 mg of AMB (950 U/mg) was added, and the pH of the mixture was maintained at 11 ± 0.1. The mixture was gently stirred in a light-protected container at room temperature for 48 h and dialyzed against DDW (4 × 5 L) at 4 °C, applying 12 000 cutoff cellulose tubing followed by lyophilization to obtain a deep yelloworange imine-based conjugate in 85% overall yield. The amine-based conjugate was obtained after reducing the imines groups with excess sodium borohydride (189 mg, 5 mmol) at room temperature for 20 h. The resultant light-yellow solution was dialyzed as described above followed by freeze-drying to obtain a yellowish amine-based conjugate in 80% w/w overall yield and stored at 4 °C. AMB content in the product was 20 ± 2% w/w as determined by spectrophotometric measurement of AMB (UV, λ = 390 nm). 1 H NMR (D2O): 0.5−2.5 ppm (bm, AMB), 3.73 ppm (bm, AG hydrogens), 5.22 ppm (m, 1H, anomeric hydrogen of AG), 6.47 ppm (m, 7H, polyene hydrogens of AMB). FT-IR (KBr): 1070 (C−O−C), 1417 (CHR1CHR2 cis), 1572 (polyene CC), 1645 (conj. CC), 1792 (CO), 2116 (intramolecular bonded OH), 3390 (OH), 2933 (anomeric C−H) cm−1 Pilot Scale Synthesis of AMB−AG. AMB−AG conjugate at a 50 g AG scale was obtained using the lab scale method described above using 33 L of water for the ultrafiltration step followed by freeze-drying at 85% w/w overall yield. Determination of the Oxidizing Agent (KIO 4 ) Effect on Amphotericin B and Its Conjugate. Fifty milligrams of AMB was dissolved in 20 mL of borate buffer (0.1N, pH 11) and potassium 2081

dx.doi.org/10.1021/bm5002125 | Biomacromolecules 2014, 15, 2079−2089

Biomacromolecules

Article

The growth in each well was then estimated visually. The MIC was defined as the lowest drug concentration that resulted in complete inhibition of visible growth. In Vitro Toxicity Study on Red Blood Cells (Hemolysis). Sheep red blood cells (SRBCs) were suspended (5%, v/v) in phosphate buffer saline (PBS; (Oxoid Ltd., Basingstoke, UK) and were washed twice in the same buffer by centrifugation (1000g, 10 min,). The hemolysis reaction was conducted in glass tubes containing 0.1 mL of the serially diluted drug and 0.9 mL of SRBCs. The tubes were centrifuged after 1 h of incubation at 37 °C in a water bath, and the results were assessed visually. In Vitro Toxicity in VERO Kidney Cells. The cell viability of VERO kidney cell line was tested in various concentrations of both AMB and the AMB−AGC as described by Kagan et al.15). Briefly, the cells were inoculated in a 96-well plate at a concentration of 1.6 × 105 cells per well (DMEM medium containing 10% FCS, Biological Industries, Beit Haemek, Israel) and incubated at 37 °C, 5% CO2 for 24 h. The drug (stock solution 20 mg AMB/ml in DMSO) was added to the cells following a 1:10 dilution in the medium at various concentrations (1, 5, 10, 20, 60, 180, 400 μg/mL). The cells with the drug were incubated for another 2 h. DMSO was used as a control. The cells were then rinsed and tested for viability by the XTT kit (Biological Industries, Beit Haemek, Israel). The results were read by a Microwell System Reader (Organon Technica, Oss, The Netherlands) at 490 nm. 2.2.3. In Vivo Toxicity Study. Evaluation of Acute Toxicity in Mice. Male albino ICR mice weighing ∼30 g were injected through the tail vein with various doses of each AMB formulation. Each dosage was administered intravenously as consecutive bolus injections of 0.1−0.2 mL every 10 min to a group of 3 mice, until death was observed. If mice survived the highest dose injected, the survival was monitored for 7−8 days to determine the maximal tolerated dose (MTD) (mg/kg). AMB−AGC formulations were prepared in 5% dextrose and were filter sterilized prior to injection through a sterile 0.2-μm pore-size cellulose acetate sterile filter (Schleicher & Schuell, Dassel, Germany). The formulations were protected from light and kept at 4 °C until use within 24 h after preparation. Multiple Dose Toxicity (Subacute Toxicity) in Rats. Four-week-old male albino LEW/SsNHsd rats weighing ∼150 g were injected through the tail vein with various doses of each AMB or AMB−AGC formulation (10, 20, 30 mg/kg/day for AMB−AGC and 1 mg/kg/day for Fungizone). Each dosage was administered for 28 consecutive days by daily single bolus injections to a group of 5 rats. Formulations of AMB were prepared as described above. The gross toxicity of AMB− AGC was evaluated in rats through daily observation of physical condition and changes in body weight. The animals were kept under specific-pathogen-free environment at the animal facilities in the Faculty of Medicine at the Hebrew University and given free access to irradiated food and acidified water throughout the experiment. Ethics Statement. Procedures, care, and treatment of the mice were carried out according to the principles of humane treatment outlined by the Guide for the Care and Use of Laboratory Animals of the Hebrew University, approved by the Committee for Ethical Conduct in the Care and Use of Laboratory Animals (approval number OPRRA01-5011) and in full compliance with the Israel animal welfare act (law 5754-1994 and 5761-2001). The study was approved by the joint ethics committee for animal welfare (IACUC) of the Hebrew University and the Hadassah Medical Centre (Jerusalem). The facilities of the Hebrew University are accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (accreditation number 1285), experiments were performed in full compliance with national and international guidelines. Therapeutic Efficacy Study. C. albicans ATCC 90028 was used to induce systemic murine candidosis for the efficacy studies. Yeast inoculum was injected into the tail veins of male ICR mice (weight, 30 ± 3 g) by administration of a single bolus of a 0.2-mL suspension in PBS. The inocula was 5 × 104 yeast cells per mouse from a 24 h culture for C. albicans on Sabouraud dextrose agar (SDA) at 30 °C. Systemic infections following these inocula are regularly produced in

mice, and they cause total killing within 10 to 20 days. The appropriate inoculum for the experiments was experimentally determined. The yeast concentration was determined by counting with a hemacytometer. The viable count was measured as the number of colony forming units (CFUs) on SDA plates after 24−48 h of incubation at 30 °C. Infected mice were treated with AMB−DOC (Fungizone) and AMB−AGC at various doses. Ten mice were used for each treatment and maintained in separate cages. Treatment was started 24 h after the initiation of the infection by intravenous injection of a daily single bolus (0.2 mL) of the drug for 5 consecutive days. Each dose comprised an AMB equivalent in a dosage range of 1 to 8 mg/kg/day, depending on the specific treatment. In addition, a control group of 10 infected mice was treated with PBS instead of the conjugate. The number of surviving animals in each group was recorded daily over a period of 30 days.

3. RESULTS 3.1. Synthesis and Characterization of Amphotericin B−Arabinogalactan at Gram and 100 g Quantities. Oxidized arabinogalactan (AG) was obtained by reacting polysaccharide in aqueous medium with potassium periodate at 1:1 mol ratio (IO4−/AG unit) for 2 h in a light-protected container (Table 1).). Oxidized AG was purified by DOWEX-1 Table 1. Equipment Characteristics for Lab and Pilot Scale: Oxidation of Arabinogalactan characteristics

laboratory scale (L)

pilot scale (L)

reactor capacity volume of the reaction ion-exchange column volume ion-exchange dry resin volume

0.25 0.1 0.75 0.125

3 1.2 7.5 1

chromatography followed by extensive dialysis against DDW (5 kDa cutoff cellulose tubing) for 48 h at 4 °C and lyophilization to dryness resulting in 75% w/w overall yield. Purification of the oxidized AG in the 100 g scale was done by the ultrafiltration process using a 5 kDa MWCO “Minisette membrane” against DDW followed by concentration of the solution to a volume of 2 L every hour with the addition of 1 L of DDW every 30 min, for 11 cycles. The ultrafiltration process was continued for at least 5 h. The purified polyaldehyde solution was concentrated to 2 L and lyophilized to dryness. Average molecular weights of oxidized AGs were estimated by gel permeation chromatography (GPC) using a pullulan standards table. Oxidation of AG for both scales resulted in a moderate decrease in molecular weight, i.e., 11−12 kDa related to starting AG of 17 kDa. The oxidation took place at the branched side chains, resulting in a moderate chain scission. The resultant oxidized AG demonstrated reduced solubility in water (80 mg/mL), while its precursor, natural AG, was found to be highly soluble in water (2.3 g/mL). The degree of oxidation was determined by the hydroxylamine hydrochloride method.18 Oxidation of AG with periodate at the laboratory scale yielded a 62 ± 3% oxidation of saccharide units (Table 2). Similar aldehyde content was obtained with oxidized AG prepared under similar conditions with an equal amount of periodate at pilot scale, i.e., 60 ± 3%. In addition, no iodate (IO3−) and no reacted periodate (IO4−) impurities were found as estimated by iodometric method. Since no distinct difference was observed in the oxidation degree of the AGs prepared by both techniques, no optimization of the oxidation conditions was needed to produce large amount of the product. On the 2082

dx.doi.org/10.1021/bm5002125 | Biomacromolecules 2014, 15, 2079−2089

Biomacromolecules

Article

WFI. The purification process was monitored by pH measurements of the medium. The final solution (pH 7−8) was concentrated and freeze-dried. Aldehyde content of the AMB−AG conjugate was evaluated prior to and following the addition of sodium borohydride to ensure sufficient reduction. Table 2 represents aldehyde content values obtained with the conjugates at various steps of the AMB−AGC preparation process. According to the hydroxylamine hydrochloride method, negligible aldehyde content was detected, indicating efficient reduction of aldehyde groups with sodium borohydride. All synthetic polymers were characterized for their structure (1H NMR), average molecular weight (GPC), solubility and AMB content (UV spectroscopy) (Table 3). The resultant polymers were highly soluble in water (80 mg/mL for the imine-based form and 170 mg/mL for the amine-based conjugate), while free drug AMB showed negligible solubility in water (i.e., 0.1 mg/mL). AMB−AGC demonstrated reduced solubility in organic solvents such as DMSO in comparison with the pure drug (i.e., 50 mg/mL). In addition, poor solubility of the AMB−AGC was evident in N-methyl pyrrolidone, methanol, ethanol, diethyl ether, dimethylacetamide and acetonitrile. The degree of substitution of AMB− AGC with AMB was estimated by typical UV absorbance of the drug at 300−420 nm. The concentration of the AMB in each sample was calculated at 390 nm, using the equation obtained from the AMB calibration curve. Based on the UV spectroscopy results, unreduced AMB−AGC contains 18% ± 2 (w/w) of

Table 2. Degree of Oxidation of Arabinogalactan and Its Conjugates sample

% oxidationa

arabinogalactan oxidized arabinogalactan (lab-scale) oxidized arabinogalactan (pilot-scale) AMB−AG unreduced AMB−AG reduced (lab-scale) AMB−AG reduced (pilot-scale)

undetectable 62 ± 3 60 ± 3 15−20 undetectable undetectable

a

% Oxidation was determined by hydroxylamine hydrochloride method.18 Values were recorded as percentages of dialdehyde groups per 100 arabinogalactan units.

basis of these results, 1:1 mol ratio (AG unit/periodate) was chosen for further synthesis of oxidized AG at pilot scale. In general, in both scales, AMB was added to a 0.1 N borate buffer solution (pH 11), while at large scale, water for irrigation was used, containing 4 weight equivalents of oxidized AG. Conjugation reaction was carried out in a light-protected container at room temperature for 48 h. The pH of the reaction solution was maintained at 11 using NaOH solution. After the conjugation was completed, part of the conjugates was reduced by the addition of the excess borohydride at room temperature for 24 h at gram scale and 20 h at large scale. Unreduced and reduced conjugates prepared under similar conditions were purified by extensive dialysis against DDW and lyophilized to dryness. The purification of the 100 g conjugate was done by ultrafiltration in a 5 kDa MWCO “Minisette membrane” against

Table 3. Effect of the Reduction Conditions on Antifungal Activity of AMB−AGC batch

AMBa (%, w/w)

time (h)

temp. (°C)

AG/NaBH4 (mole ratio)

purificationb

MICc,b (mg/L)

MTDd (mg/kg)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

26.6 20.8 20.5 21.2 20.8 17.7 19.9 18.9 20.6 19.3 21.6 21.1 22 19.9 17.9 21 20 20 24.2 26 22

24 1 1 1 2 4 1 3 1 1 3 1 4 13 4 24 12 18 6 12 24

25 4 4 4 4 4 4 4 4 4 4 25 4 4 25 4 4 4 25 25 25

1:1 1:1 1:1 1:1 1:1 1:1 1:2 1:1 1:2 1:1 1:1 1:2 1:2 1:1 1:2 1:1 1:2 1:2 1:2 1:2 1:2

Dial.10 kDa Dial.10 kDa UF-5 kDa UF-5 kDa UF-5 kDa UF-5 kDa UF-5 kDa UF-5 kDa UF-5 kDa UF-5 kDa UF-5 kDa UF-5 kDa UF-5 kDa UF-5 kDa UF-5 kDa UF-5 kDa UF-5 kDa UF-5 kDa UF-5 kDa UF-5 kDa UF-5 kDa

1 0.25 0.10 0.25 0.25 0.13 0.13 0.13 0.13 0.25 0.13 0.13 0.25 0.13 0.25 0.13 0.25 0.25 0.25 0.25 0.5

108 20 5 20 20 20 20 20 20 10 10 10 10 10 30 10 20 20 20 20 60

All imine conjugates were similarly prepared starting with oxidized AG and AMB. Reduction step was done at changing conditions: 4° and 25 °C; 1, 2, 3, 4, 6, 12, 20, and 24 h; amounts of reducing agent ranging from 1:1 and 1:2 mol ratios (AG unit/NaBH4, respectively) and various purification techniques. aAMB content in corresponding conjugate was determined by spectrophotometric measurement of AMB−AG (UV, λ = 391 nm) using the calibration curve of AMB. bThe purification of the conjugates from buffers, nonconjugated AG, AMB, and small conjugate chains was conducted either by (1) Dialysis (Dial.) done through a 12 kDa cutoff tube against DDW (5L X 4) for 48 h at 4 °C, or (2) Ultrafiltration (UF) done by concentration of the batch combined with a diafiltration steps. The permeate flow returned to the feed container. The filtrate flow rate was 6 L/h. The pH was monitored by litmus pH paper indicator. cMIC of AMB−AGC required for complete inhibition of C. albicans after exposure for 24 h. d MTD was determined in ICR mice by repetitive injections of 10 mg AMB/kg into the tail vein every 10 min of one of the AMB formulations to a group of mice (three animals in each group), and observing their survival for 1 week. 2083

dx.doi.org/10.1021/bm5002125 | Biomacromolecules 2014, 15, 2079−2089

Biomacromolecules

Article

Table 4. Characterization of the Scale-up Conjugates sample

MWa (Da)

P

AMBb (%, w/ w)

free AMBc (%, w/ w)

solubilityd (mg/mL)

water contente (%)

pyrogenes (EU/ vial)

MICf (mg/L)

MTDg (mg/ kg)

BH 102 BH 103 BH 104 Fungizone

24 900 26 531 22 136 --

1.52 1.65 1.48 --

19 ± 2 19 ± 2 19 ± 2 --

0.25% 0.212% 1.39% 100%

185 178 211 --

7.32 7.18 12.6 --