Article pubs.acs.org/molecularpharmaceutics
Noncovalent Complexation of Amphotericin‑B with Poly(α-glutamic acid) Abeer H. A. Mohamed-Ahmed,†,‡ Karolina A. Les,† Karin Seifert,‡ Simon L. Croft,‡ and Stephen Brocchini*,† †
UCL School of Pharmacy, University College London, 29-39 Brunswick Square, London WC1N 1AX, U.K. Faculty of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine, Keppel Street, WC1E 7HT, U.K.
‡
S Supporting Information *
ABSTRACT: A noncovalent complex of amphotericin B (AmB) and poly(α-glutamic acid) (PGA) was prepared to develop a safe and stable formulation for the treatment of leishmaniasis. The loading of AmB in the complex was in the range of ∼20−50%. AmB was in a highly aggregated state with an aggregation ratio often above 2.0. This complex (AmB−PGA) was shown to be stable and to have reduced toxicity to human red blood cells and KB cells compared to the parent compound; cell viability was not affected at an AmB concentration as high as 50 and 200 μg/mL respectively. This AmB−PGA complex retained AmB activity against intracellular Leishmania major amastigotes in the differentiated THP-1 cells with an EC50 of 0.07 ± 0.03−0.08 ± 0.01 μg/mL, which is similar to Fungizone (EC50 of 0.06 ± 0.01 μg/mL). The in vitro antileishmanial activity of the complex against Leishmania donovani was retained after storage at 37 °C for 7 days in the form of a solution (EC50 of 0.27 ± 0.03 to 0.35 ± 0.04 μg/mL) and for 30 days as a solid (EC50 of 0.41 ± 0.07 to 0.63 ± 0.25 μg/mL). These encouraging results indicate that the AmB−PGA complex has the potential for further development. KEYWORDS: noncovalent polymer complex, enhanced solubility, reduced toxicity, amphotericin B, leishmaniasis
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INTRODUCTION
Toxic and poorly soluble injectable drugs can be either (i) covalently conjugated or (ii) noncovalently associated to a water-soluble polymer.1,2 Drug toxicity can be reduced because the polymer is not systemically distributed throughout the body.3,4 Noncovalent complexes may be preferred when it is necessary to avoid chemical modification of the drug, especially for drugs that are already clinically used. Amphotericin B (AmB) 1 is a toxic, poorly soluble, chemically labile polyene antibiotic that has a propensity to aggregate.5,6 AmB has been used for decades in a micellar form using a deoxycholate formulation (AmB-D) (Fungizone) to treat systemic fungal infections7,8 and is also highly effective against visceral leishmaniasis (VL),9,10 which is fatal if untreated.11 To reduce the known toxicities of AmB, many delivery systems have been described in efforts to improve its efficacy. Three lipid based AmB formulations were introduced to the clinic in 1990s,12−14 and of these, AmBisome (AmB-L) is the most effective to treat VL.15 Unfortunately, its use to treat VL has been limited by high cost and the requirement for a cold chain.11,16 Since lipids are the majority components of the formulation, it is difficult to envisage the sustainable cost of AmB-L ever becoming affordable. Also AmB-L is made by a complex process,17,18 and efforts to make generic forms have not entirely succeeded to produce formulations of comparable efficacy.14,19 While AmB-D is much less expensive, it is toxic and requires treatment regimens © 2012 American Chemical Society
lasting 30 days.20,21 VL is endemic in resource limited regions of the world where it is difficult to ensure a cold chain for labile medicines (i.e., AmB-L) and it is difficult to treat patients over a prolonged period (i.e., AmB-D). To address (i) the cost and stability issues resulting from the use of lipids in AmB-L and (ii) the toxicity of AmB, we hypothesized that lipids could be replaced with a water-soluble polymer to closely associate with AmB in a more simple fabrication process. We have found that poly(α-glutamic acid) (PGA) 2 forms a stable water-soluble, noncovalent complex with AmB (Scheme 1) by a simple mixing process. PGA 2 was Received: Revised: Accepted: Published: 940
June 19, 2012 December 2, 2012 December 12, 2012 December 12, 2012 dx.doi.org/10.1021/mp300339p | Mol. Pharmaceutics 2013, 10, 940−950
Molecular Pharmaceutics
Article
double distilled water was 7.39 ± 0.23 (average mean ± STDV (n = 6)). Determination of AmB Loading in the AmB−PGA Complex 3. The loading of AmB in the AmB−PGA complex 3 was determined by UV using a Lambda 25 (PerkinElmer) UV/visible spectrophotometer. Monomeric AmB has a maximum absorbance wavelength (λmax) at 409 nm. Standard calibration curves of serial concentrations were constructed using serial dilutions of AmB in aqueous methanol (50% v/v) to ensure that AmB remained in the monomeric form. The highest AmB concentration was 10 μg/mL over six concentrations using 2-fold dilutions. The loading of AmB, complexation efficiency, yield, and theoretical AmB loading were calculated according to eqs 1−4. Theoretical AmB loading is AmB loading assuming 100% complexation efficiency.
Scheme 1. Formation of the Noncovalent Complex 3 between AmB 1 and PGA 2
selected because it is a biodegradable and biocompatible polymer that has been evaluated clinically in covalent conjugates developed for oncology.22,23
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EXPERIMENTAL SECTION Materials. Poly(α-glutamic acid) sodium salt (20−40 and 50−70 kDa) was purchased from Sigma Aldrich (catalogue no.: G0546 and G0421). Amphotericin B (injectable grade with purity of 98%, batch no: HAN0604301) was purchased by DNDi from EgChemicals (China). Dimethyl sulfoxide (DMSO) 99.9% (anhydrous grade) was purchased from Sigma Aldrich. Sodium hydroxide (1 M) was purchased from Fischer Scientific. All reactions were carried out at ambient temperature. AmBisome (AmB-L)was a gift from Gilead Sciences (U.K.). Fungizone (AmB-D) was purchased from AAH pharmaceuticals (U.K.). Mouse serum (CD-1) mixed gender pooled was purchased from Sera Laboratories International (West Sussex, U.K.). Methods. Protonation of PGA. Poly(α-glutamic acid) sodium salt (20−40 or 50−70 kDa) was converted into acidic form PGA 2 by dialysis (12−14 kDa visking membrane) of aqueous solution of salt form (10 or 20 mg/mL) against aqueous hydrochloric acid (0.01 M, 1 L) for 24 h and then against double distilled water (5 L) for 24 h to remove sodium chloride.24 The polymer solution was then freeze-dried. The protonation of PGA 2 was checked by IR spectroscopy using the freeze-dried form of PGA 2 (Figure S1 in the Supporting Information). Preparation of AmB−PGA Complex 3. The procedure to prepare the AmB−PGA complex 3 was to dissolve a defined amount of PGA 2 (20−40 or 50−70 kDa) (30 mg) in dry DMSO (0.6 mL) overnight in a glass vial (7 mL). Depending on the desired AmB loading, a specified amount of AmB was dissolved in a separate solution of dry DMSO (0.6 mL) for 1 h. For example to prepare AmB−PGA complex 3 having 50% AmB loading 45 mg of AmB was dissolved in DMSO (0.6 mL) in a glass vial (14 mL). The PGA 2 solution was added dropwise to the AmB solution. The mixture was stirred for 1 h, and then sodium hydroxide (2 equivalents to polymer, 1 M, 464 μL) was divided into two portions. The first portion of 232 μL of sodium hydroxide was added dropwise (1 drop every 5 s), and then the second portion (232 μL) was diluted in water (1 mL). The diluted sodium hydroxide solution was then added dropwise followed by the addition of water (12 mL). The reaction mixture was stirred at room temperature for 1 h and then purified by dialysis (Mw cutoff 12−14 kDa). The dialysis water was changed 6 times every 2 h for the first 8 h then left O/N. The solution was filtered using a microsyringe filter (0.22 μm, PES Millipore) and then was freeze-dried to yield a yellow fluffy product. The reaction mixture was protected from light during the process of preparation and purification. The pH of complex 3 upon reconstitution of the freeze-dried powder in
AmB loading (%) =
concn of AmB determined by UV (μg/mL) initial concn of the complex (μg/mL)
(1)
× 100 complexation efficiency (%) =
achieved AmB loading × 100 theoretical AmB loading
(2)
theoretical loading (%) =
amount of AmB added to complexation reaction (mg) × 100 AmB amount + polymer amount added to reaction (mg)
(3) yield (%) =
wt of the complex after freeze drying (mg) × 100 amount of AmB + amount of polymer (mg)
(4)
HPLC Analysis for Quantification of AmB. High performance liquid chromatography analysis was conducted using C18 column (particle size 5 μm, length 250 mm) following an analytical method described by the literature.25 The mobile phase used was acetonitrile 52%, water 43.7%, and acetic acid 4.3%. The injection volume was 100 μL, and flow rate was 1 mL/min. The concentration of AmB was detected at 407 nm with a run time of 20 min. Under these conditions the retention time of AmB was 8 min. The sensitivity of the system was 0.016 μg/mL, and the detection limit was 0.054 μg/mL AmB. Determination of AmB Aggregation in the AmB−PGA Complex 3. The aggregation state of AmB was determined by scanning the UV absorbance of AmB in the range 300−450 nm using Lambda 25 (PerkinElmer) UV/visible spectrophotometer. AmB gives four absorption peaks. The aggregation ratio was calculated as the ratio of peak I (315−318 nm) to peak IV (409 nm). 26 Fungizone was scanned as standard at concentration of 10−5 M, and AmB−PGA complex 3 was scanned at the same concentration (10−5 M). Determination of Size and Charge of AmB−PGA Complex 3. The size of AmB−PGA complex 3 was measured using dynamic light scattering (Zetaziser, nanoseries Nano-ZS (Malvern)). The charge of complex 3 was determined using Zetasizer Nano-Zeta potential measurements. The concentration of AmB−PGA complex 3 used was 10−4 M. Samples were filtered using 0.22 μm syringe filter (PES, Millipore) to remove contamination with dust particles. AmBisome was included for comparison. Size data is expressed as Z average and polydispersity index (PDI). Z average is the intensity weighted mean hydrodynamic size of the collection of particles measured by dynamic light scattering (DLS). The polydisper941
dx.doi.org/10.1021/mp300339p | Mol. Pharmaceutics 2013, 10, 940−950
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incubated at 37 °C for 24 h. Each concentration was tested in triplicate. Triton X (nonionic surfactant) at a concentration of 0.1% was used as a positive control for 100% cell lysis. The result was expressed graphically as percentage of 100% cell lysis and was calculated as shown:
sity index (PDI) describes the width of the particle size distribution. Stability of AmB−PGA Complex 3 in Phosphate Buffered Saline (PBS) and Serum. In vitro release of AmB from AmB− PGA complex 3 in PBS and serum was evaluated by dialysis.27 The quantification was carried out using HPLC as described below. A stock solution of AmB−PGA complex 3 was prepared in water at a concentration of 1 mg/mL AmB equivalent. The aqueous solution of complex 3 (1 mL) was added to either PBS (1 mL, containing DMSO (5%), pH 7.4) or serum (1 mL, containing DMSO (5%)). The final concentration of AmB− PGA complex 3 was 500 μg/mL AmB equivalents (2 mL) (total amount of AmB was 1 mg). The complex 3 solution (2 mL) was placed in a dialysis bag having molecular weight cutoff 12−14 kDa and dialyzed against 50 mL of PBS/DMSO (95% containing DMSO (5%), pH 7.4) at 37 °C. The whole dialysis medium was removed after 0, 1, 4, 8, and 24 h for drug concentration analysis and replaced by fresh medium to prevent drug saturation (maintaining strict sink conditions throughout the experiment). AmB was quantified by HPLC at 407 nm in aqueous methanol (50% v/v). AmB concentration was extrapolated from the calibration curve of AmB in aqueous methanol (50% v/v). The results were expressed graphically as a cumulative percentage release of the total amount of AmB (% w/w) versus time according to the equation
hemolysis (%) =
Abs − Abs0 × 100 Abs100 − Abs0
where Abs, Abs0, and Abs100 are the absorbance for the sample, control blank medium, and control in the presence of hemolytic dose of 0.1% Triton X, respectively.28,29 In Vitro Cytotoxicity Testing. KB cells (HeLa contaminant, cervical adenocarcinoma-derived epithelial cells; ATCC) were plated in 96-well plates at a density of 4 × 104 cells/mL in RPMI 1640 medium +10% hi-FCS and allowed to adhere overnight at 37 °C, 5% CO2. 2-fold serial dilutions over eight concentrations of the formulation were prepared in 24-well plates with starting concentration of 200 μg/mL (AmB equivalents) and the polymer starting concentrations were adjusted to the amount of polymer present in complex 3 according to the loading percent. The cells were treated by adding 100 μL of formulation into the respective well. Podophyllotoxin (Sigma Aldrich) was included as a control at a starting concentration of 0.05 μg/mL in DMSO. For human leukemia THP-1 cells (monocytes derived from peripheral blood of patient with acute monocytic leukemia, ATCC), serial dilutions of the formulation were prepared in 96-well plates and a suspension of THP-1 cells in RPMI 1640 medium +10% hiFCS was added at a density of 5 × 105 cells/mL. Then plates were incubated for 72 h at 37 °C, 5% CO2. For the last 6 h, 20 μL of Alamar Blue (ABD Serotec, U.K.) was added to the plates before they were read on a Gemini plate reader (EX/EM 530/ 580 nm, cutoff 550 nm, 5 readings per well). Data were graphically expressed as percentage cell viability of control calculated as follows:
cumulative release (%) amount of AmB released at time t (mg) = × 100 total amount of AmB (1 mg)
where the amount of AmB released at time t is a cumulative amount. For example the amount of AmB released after 1 h is calculated by multiplying AmB concentration obtained from HPLC by total volume of the dialysate (50 mL). Cumulative amount of AmB after 4 h is total amount released at time points 0, 1 and 4 h. Determination of Shape of Complex 3. The shape of complex 3 was determined by transmission electron scan microscopy (TEM). Complex 3 was tested at a concentration of 10 μg/mL AmB equivalents in double distilled water. Hemolysis Assay. Human blood was obtained from healthy donors in London School of Hygiene & Tropical medicine in a falcon tube (50 mL) containing heparin (500 μL). Red blood cells (RBCs) were isolated from whole blood using density gradient centrifugation. Whole blood (7 mL) was slowly added on top of 4 mL of Ficol-Paque Plus solution and then centrifuged at 2000 rpm for 30 min without applying brake. Supernatant was discarded, and red blood cells were collected. RBCs were washed with phosphate buffer saline (pH 7.4) three times by centrifugation at 2000 rpm for 30 min (without brake). A stock solution of RBCs was prepared at 4% v/v in RPMI (Sigma Aldrich) containing L-glutamine (Sigma Aldrich) and no serum. Stock solution of AmB−PGA complex 3 (1 mg/mL AmB equivalent) was prepared in double distilled water and sterilized by filtration (0.22 μm, PES, Millipore). Fungizone (AmB-D) and AmBisome (AmB-L) were included as controls and prepared as mentioned in the manufacturer's protocol in double distilled sterile water (Sigma Aldrich). Serial 2-fold dilution of complexes 3 and controls over six concentrations was prepared in RPMI medium. RBC solution (4%) was added to 96-well plates (100 μL). Serial dilutions (100 μL) of the complexes and controls were added to the plated RBCs and
cell viability (%) =
FI drug treated cells × 100 FI untreated control
where FI is the fluorescence intensity emission In Vitro Antileishmanial Activity against Intracellular Amastigotes. Leishmania major (MHOM/SA/85/JISH118) promastigotes were cultured in Schneider’s media (Sigma Aldrich) supplemented with 10% heat inactivated fetal calf serum. The promastigotes were maintained at 26 °C and cultured for 5 days to ensure their development into the infective stage. Leishmania donovani (MHOM/ET/67/HU3) was maintained in RAG-1 mice (LSHTM colony maintained at Harlan, U.K.) and amastigotes were harvested from the spleen of infected mice. THP-1 cells were maintained in suspension in RPMI media containing 10% FCS and differentiated by incubation in RPMI (10% FSC) containing 20 ng/mL phorbol 12-myristate 13acetate (PMA) (Sigma Aldrich) for 48 h at density of 5 × 104 cells/well. Then cells were rested in PMA-free medium overnight before infection at 37 °C. Differentiated THP-1 cells were infected with L. major promastigotes at parasite/cell ratio of 10:1 or L. donovani amastigotes at ratio of 7:1 and incubated at 34 °C for L. major and 37 °C for L. donovani. Stock solution of complex 3 (1 mg/mL AmB equivalent) was prepared in sterile double distilled water (Sigma Aldrich). Fungizone and AmBisome were reconstituted in sterile double distilled water according to the manufacturer's protocol. 942
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Table 1. Conversion, Yields, and Solubility of AmB−PGA Complexes 3 (n = 3) Using Different Amounts of AmB 1 and PGA 2 sample
AmB (mg)
PGA (mg)
PGA MW (kDa)
theor loading (%)
1 2 3 4 5 6
15.0 20.0 45.0 20.0 30.0 45.0
30.0 30.0 30.0 30.0 30.0 30.0
50−70 50−70 50−70 20−40 20−40 20−40
33.3 40.0 60.0 40.0 50.0 60.0
actual AmB loading (%) 25.2 32.9 53.3 24.6 34.4 50.9
± ± ± ± ± ±
1.1 2.4 4.1 1.9 3.4 1.3
complexation efficiency (%) 76.0 82.3 90.8 61.5 68.9 84.8
± ± ± ± ± ±
3.5 6.0 8.4 4.7 6.8 2.1
isolated yield (%)
aqueous solubility (mg AmB/mL)
± ± ± ± ± ±
1.5 3.0 3.0 1.5 3.0 3.0
75.1 78.8 72.0 71.1 73.1 66.5
2.4 1.7 5.7 8.8 7.6 8.2
Complex 3 was tested at a starting concentration of 1 μg/mL AmB equivalent for L. major and 5 μg/mL for L. donovani with 3-fold serial dilutions over six concentrations. The highest concentration of the polymer tested was calculated according to the highest amount present in complex 3 at the highest concentration tested. Each concentration was tested in quadruplicate at a volume of 200 μL. Infected cultures were incubated for 72 h at 34 °C. At the end of the incubation period, the slides were fixed with 100% methanol and stained with Giemsa (VWR, U.K.) 10% in water. The level of infection per well was evaluated by counting number of infected macrophages per 100 macrophages under the microscope. Percentage of infection was compared to untreated control wells (100% infection). The result was expressed as percentage reduction in infected macrophages compared to untreated control wells. Data was analyzed with Microsoft xl/fit using the nonlinear sigmoidal curve-fitting Levenberg−Marquardt logarithm, and EC50/EC90 (with 95% confidence limits) values were estimated.
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RESULTS AND DISCUSSION PGA has been used to complex to some drugs during formulation by precipitation or metal complexation,30,31 to positively charge polymers32−34 and surfactants,35 and as an excipient for therapeutic proteins.36 Copolymers with PGA have also been examined37,38 in efforts to encapsulate drugs. However in an effort to maintain costs and to develop a scalable process with the simplest polymer possible, we have sought only to use a PGA homopolymer. It is also known that polyvinylpyrrolidone can complex small amounts of AmB.39 The main drawbacks of the AmB− polyvinylpyrrolidone complex are (1) the AmB loading in this complex was very low (0.249% w/w) and (2) the complexation of polyvinylpyrrolidone with AmB required the use of a large amount of polyvinylpyrrolidone (2 g) and methanol (100 mL).39 This would presumably make the manufacture at large scale of this AmB complex difficult, costly, and in the end not feasible primarily due to the low loading of AmB. Efforts to encapsulate and conjugate AmB have also been described using different water-soluble polymers.40−54 In some of these systems the polymers used synergized the toxicity of AmB55 or the AmB did not display physicochemical properties consistent with the form of AmB in AmB-L.56 Gamma cyclodextrin (CD) can form an inclusion complex with AmB.55,57 Although AmB−CD complexes showed improved water solubility, they were toxic against human RBCs (33.1 ± 2.04% hemolysis after 1 h incubation at an AmB concentration of 100 μg/mL).58 The potential drawbacks of cyclodextrin AmB complexation are (1) the need to use a large amount of cyclodextrin to achieve effective solubilization of AmB and (2) synergistic toxicity caused by gamma cyclodextrin alone.59,60
Figure 1. Superposition of the UV spectra of AmB−PGA complexes 3 showing the hypsochromic shift of the aggregation peak. AmB-D (purple line) and AmB-L (dark green line). (A) Complex 3 (PGA 50− 70 kDa) with AmB loadings of 25% (blue), 31% (light green), and 51% (red); (B) complex 3 (PGA 20−40 kDa) with AmB loadings of 23% (blue), 37% (red), and 52% (green).
Polymeric micelle systems are relatively easy to fabricate in the laboratory, but unfortunately some of these systems can suffer from instability in serum which can reduce in vivo stability.61−63 In some systems where AmB is monomeric, it is not clear if AmB will remain monomeric after a prolonged period of storage (shelf life).40,48,64 Also in some systems the AmB loading is low.64 Our aims are to determine if a stable, simple-to-make nontoxic complex of AmB and PGA 2 could be made that had similar physicochemical characteristics to those displayed by AmB-L. It was necessary to ensure that AmB loading was quite high (>20%) to ensure that it could be administered in a practical way. The low water solubility of AmB 1 (
