http://informahealthcare.com/phd ISSN: 1083-7450 (print), 1097-9867 (electronic) Pharm Dev Technol, Early Online: 1–7 ! 2013 Informa Healthcare USA, Inc. DOI: 10.3109/10837450.2013.852574
RESEARCH ARTICLE
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Formulation approaches to improving the delivery of an antiviral drug with activity against seasonal flu Srinivasa M. Sammeta1, Li Wang1, Shravan K. Mutyam1, Kathleen O’Loughlin1, Carol E. Green1, Milton H. Werner2, Terence Kelly3, and Gita N. Shankar1 1
Pharmaceutical Development, Biosciences Division, SRI International, Menlo Park, CA, USA, 2Inhibikase Therapeutics, Inc., Suite, Atlanta, GA, USA, and 3Kelly Pharma Research Consulting LLC, Ridgefield, CT, USA
Abstract
Keywords
The main objective of the present study was to develop formulations of noscapine hydrochloride hydrate with enhanced solubility and bioavailability using co-solvent- and cyclodextrin-based approaches. Different combinations of co-solvents, which were selected on the basis of high-throughput solubility screening, were subjected to in vitro intestinal drug permeability studies conducted with Ussing chambers. Vitamin E tocopherol polyethylene glycol succinate and propylene glycol based co-solvent formulations provided the maximum permeability coefficient for the drug. Inclusion complexes of the drug were prepared using hydroxypropyl-b-cyclodextrin and sulphobutylether cyclodextrins. Pharmacokinetic studies were carried out in male Sprague–Dawley rats for the selected formulations. The relative bioavailabilities of the drug with the co-solvent- and cyclodextrin-based formulations were found to be similar.
Bioavailability, co-solvents, cyclodextrins, solubility, Ussing chambers
Introduction Noscapine hydrochloride hydrate, a phthalideisoquinoline alkaloid without sedative, euphoric, analgesic or respiratory depressant properties, is used as an antitussive agent worldwide. Recent studies have shown that noscapine is also tubulin-binding and can be used for its anticancer properties1–4. The oral route for drug delivery has been preferred because of its ease of administration and patient compliance. However, relatively high doses of the drug (ED50 300 mg/kg body weight) are essential for induction of anticancer activity. To date, development of oral formulations of the drug has been limited because of the large dose required, poor absorption, low dissolution or aqueous solubility, and extensive first-pass metabolism5,6. To overcome these solubility and bioavailability limitations, different formulation approaches based on co-solvents and cyclodextrins (CDs) can be used. Co-solvents are mixtures of miscible solvents that can be used to solubilize noscapine (hereafter, generally termed ‘‘the drug’’). The excipients used as co-solvents can be water-soluble like polyethylene glycol (PEG) 400, PEG 300, propylene glycol and glycerin; water-insoluble like oleic acid, castor oil and peppermint oil; or surfactants like Tween 80, Tween 20, Cremophor and tocopherol polyethylene glycol succinate (TPGS)7. CDs are cyclic oligosaccharides made of (a-1,4)-linked a-d-glucopyranose units with a hydrophilic outer surface and a lipophilic central cavity. a-CD, b-CD and g-CD, the most common natural CDs, have limited aqueous solubility. CD derivatives of pharmaceutical significance with enhanced solubility include the hydroxypropyl
Address for correspondence: Gita N. Shankar, Pharmaceutical Development, Biosciences Division, SRI International, Menlo Park, CA 94025-3493, USA. Tel: +1-650-859-5197. Fax: +1-650-859-2889. E-mail: [email protected]
History Received 1 August 2013 Accepted 1 October 2013 Published online 8 November 2013
derivatives of b- and g-CD, the randomly methylated b-CD, and sulphobutylether b-CD (SBECD). CDs are used as complexing agents to increase the aqueous solubility of drugs and thus enhance bioavailability and stability8,9. In the present study, oral formulations of noscapine hydrochloride hydrate were developed using co-solvent- and CD-based formulation approaches and evaluated using in vitro permeability and in vivo pharmacokinetic studies.
Materials and methods Chemicals Noscapine hydrochloride hydrate and glycerol were purchased from Sigma-Aldrich (St. Louis, MO). PEG 400, propylene glycol, potassium chloride, Tween 80 and Tween 20 were obtained from Spectrum Chemicals (Gardena, CA). Vitamin E TPGS was purchased from Eastman Chemical Company (Kingsport, TN). Boric acid, sodium hydroxide and sodium phosphate dibasic were obtained from Mallinckrodt Chemicals (Phillipsburg, NJ). b-CD from ISP Technologies Inc (Wayne, NJ), hydroxypropyl-b-CD (HP-b-CD) from Roquette (Keokuk, IA) and SBECD from Cydex Pharmaceuticals Inc (Lenexa, KS) were gift samples. All other chemical were obtained from Fisher Scientific. The pH solubility of the drug The solubility of the drug was determined in different pH buffers2–10. Mcllvaine–Whitting buffer containing various amounts of 0.1 M citric acid and 0.2 M disodium hydrogen phosphate were used to make buffers of pH 2–7. Standard buffer solutions containing different amounts of 0.2 M boric acid and potassium chloride and 0.2 M sodium hydroxide were used to make buffers of pH 8–10. The samples for the pH solubility study
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were prepared by adding an excess of drug to 1 mL of the appropriate pH buffer in a 4-mL vial. The solution was mixed using a vortex genie for approximately 30 s and then placed on an orbital shaker at room temperature. At regular time intervals (24 and 48 h), the vials were removed from the orbital shaker, and the pH of the solutions was measured for each sample. If the solution was turbid and the pH difference between two consecutive days was 50.2 pH units, the solution was considered to have reached equilibrium. Samples were filtered through a 0.45-mm syringe filter and analyzed using high-performance liquid chromatography (HPLC).
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Solubility of the drug in different solvents Initially, the solubility of drug in different co-solvent combinations was determined using SRI International’s (SRI’s) proprietary high-throughput solubility screening (HTSS) system. The system uses a high-throughput liquid handling system (TECAN Freedom EVO) to prepare the solvent combinations. Freedom EVOware, the software associated with the liquid handling system, is used to aspirate and dispense a predetermined volume of selected solvents into each well in a microtiter plate. Before the addition of the drug, the baseline turbidities of solvents are read using a Microplate reader. A stock solution of drug prepared in dimethylsulfoxide (DMSO) is added to the plates, which are prefilled with premixed cosolvent combinations. The plates are shaken after addition of the drug, and the turbidity is measured again. The solubility of the drug is assessed by the determining the change in turbidity before and after addition of the drug in the solvent system. Forty-five solvent combinations were tested using HTSS. From the HTSS results, nine solvent combinations were selected and their solubility confirmed manually. Manual solubility assessment testing omits the DMSO necessary in the HTSS process. In this approach, a weighed amount of drug is added directly to the solvent system and mixed by vortexing or sonication. In vitro permeability studies The in vitro permeability for the various drug formulations was determined in Ussing chambers. Isolated membrane taken from the jejunum segment, devoid of Peyer’s patches, of male Sprague– Dawley rat intestine was mounted between the tissue slider and placed between mucosal and serosal Ussing chambers. The effective exposure area of tissue in the Ussing chambers was 0.5 cm2. Initially, 5 mL of Kreb’s ringer bicarbonate (KRB) buffer, pH 4.0, was added to each of the two chambers and allowed to equilibrate for 20 min at 37 C with continuous bubbling of 100% oxygen. After the equilibration of the tissue, the mucosal KRB buffer was replaced either with drug dissolved in KRB buffer or a drug formulation to achieve a final concentration of 5 mg/mL in the mucosal chamber. About 2 mL of solution was sampled from the serosal side at 0.5, 1, 1.5, 2, and 2.5 h and replaced with an equal volume of fresh KRB buffer. The samples collected were analyzed by HPLC to determine the percentage of drug transported across the intestinal tissue. The apparent permeability coefficient (Papp) was calculated using Equation (1)10. Papp ¼
dQ 1 ðcm=sÞ dt AC0
ð1Þ
Where dQ/dt is the steady-state appearance rate on the serosal side of the membrane, A is the exposed tissue surface area and C0 is the initial concentration of the drug on the mucosal side of the chamber. All the results are represented as the mean standard deviation (SD) of 3–5 trials.
Effect of CDs on the solubility of the drug The effect of CD in enhancing the solubility of drug was studied using three CD types: b-CD, HP-b-CD and SBECD. A phase-solubility assay was performed to determine the stoichiometry of a drug-CD binary system11. Various concentrations of b-CD (2–15 mM), HP-b-CD (35–357 mM) and SBECD (11–185 mM) were prepared in phosphate buffered saline (PBS) solution, pH 7.4. The concentration range for all the CDs was set on the basis of their maximum solubility in PBS. About 2 mL of varying concentrations of the CDs was added to glass vials, and an excess amount of drug was added, with the contents stirred for 48 h at 37 1 C using a shaker bath. Excess drug was also added to PBS, pH 7.4, which served as the control. After equilibrium, the samples were filtered using a 0.22-mm membrane filter and analyzed for drug content using HPLC. The inclusion complexes of drug-HP-b-CD and drug-SBECD were prepared using freeze drying8. Drug and CD were dissolved in water in 1:1 mM, and the resulting solution was stirred for 24 h in an orbit shaker at room temperature. Subsequently, the solution was lyophilized, and the sample obtained was collected and passed through a # 100-mm sieve. The thermal behavior of the drug, HPb-CD, SBECD, the drug-HP-b-CD inclusion complex and the drug-SBECD inclusion complex were studied using a differential scanning calorimeter (DSC) Q200 V24.4 Build 116 (Universal V4.5A TA Instruments, New Castle, DE) thermal analyzer. In vivo pharmacokinetic studies Pharmacokinetic studies were carried out in male Sprague– Dawley rats (300–350 g) procured from Charles River Laboratories. The rats were maintained on a 12 h light/12 h dark cycle with access to food and water ad libitum. All animal experiments complied with the standards set out in the guidelines of SRI’s Institutional Animal Care and Use Committee. Three drug formulations were administered as a single oral gavage (po) dose in three groups (n ¼ 3) of animals. The dose of drug in the three formulations was 200 mg/kg. Group 1 rats received the first formulation with the drug dissolved in 10% propylene glycol/90% sterile water and group 2 rats received the drug dissolved in 20% vitamin E TPGS/80% sterile water. The third formulation of drug– SBECD inclusion complex dissolved in sterile water was administered to group 3 rats. About 300 mL of blood was collected at 5, 15 and 30 min, and 1, 2, 4, 8, 12 and 24 h postdose from each rat via the jugular vein catheter and placed in tubes containing K3 ethylenediaminetetraacetic acid (K3EDTA) for plasma evaluation. Plasma was prepared by centrifuging the samples at 4 C and storing them at approximately 70 C until analysis took place. Analytical method HPLC analysis of the drug from in vitro studies was performed using an Agilent 1100 system containing a Phenomenex Luna (C8) column (150 4.6 mm, 5 mm) and ultraviolet (UV) detector. The drug was analyzed at 235 nm using a mobile phase of a mixture of 20 mM ammonium acetate, adjusted to pH 4.5 and of acetonitrile (65:35 v/v) with a flow rate of 1.0 mL/min at 25 C. The sample solutions were diluted to the linear range of the calibration standards using a 50:50 (v/v) mixture of 20 mM ammonium acetate and of acetonitrile. The amount of drug in the plasma samples was analyzed using a liquid chromatography/tandem mass spectrometry (LC/MS/MS) method with narcotine-N-oxide as the internal standard. To 50 mL of plasma, 50 mL of internal standard was added, and plasma proteins were precipitated by the addition of 200 mL of acetonitrile and methanol (8:2, v/v), followed by vortex and
Formulation approaches to improving the delivery of an antiviral drug
DOI: 10.3109/10837450.2013.852574
centrifugation at 4000 rpm for 5 min at 5 C. The supernatant was then collected and injected to LC/MS/MS. Data analysis
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The plasma drug level data were analyzed using WinNonlinÕ , version 5.2 professional, by noncompartmental modeling. The maximal plasma concentration (Cmax), the time to maximum plasma concentration (Tmax), the area under the plasma concentration–time curve up to the last time point (AUClast), the area under the plasma concentration–time curve extrapolated to infinity (AUCinf) and the terminal elimination half-life (t1/2) were determined.
Results and discussion The pH solubility of the drug Solubility of the drug in different pH buffers as estimated by HPLC analysis is shown in Figure 1. The drug was found to be more soluble at an acidic pH and less soluble at neutral and basic pH conditions. After 48 h, the drug solutions in pH 2 and 3 buffers changed color to pale yellow and hence were not considered for further solubility analysis. The drug had a highest solubility of 93 mg/mL in the pH 4 buffer. Solubility of the drug in different co-solvent systems The solubility of the drug in different combinations of solvents was determined using HTSS. Ten solvents (100% ethanol, 50% w/w glycerol in water, 100% PEG 400, 100% propylene glycol, 20% w/w Plasdone K29/32 in water, 40% w/w Solutol HS 15 in water, 100% Transcutol, 20% w/w Tween 20 in water, 15% w/w Tween 80 in water and 20% w/w Vitamin E TPGS in water) were used to create 45 combinations, with the solubility of drug in
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each combination assessed (data not shown). Using the HTSS results, nine optimal combinations of solvents were selected, and the solubility of the drug was manually confirmed at 100 and 200 mg/mL; data are shown in Table 1. Except for two combinations, all combinations of solvents achieved a drug solubility of 200 mg/mL. In vitro permeability studies On the basis of the solubility data, five co-solvent formulations were developed (Table 2). The amounts of excipients were within the maximum levels used in preclinical formulations12. In the in vitro permeability studies, KRB buffer, pH 4.0, was used in place of HN-2-hydroxyethylpiperazine-N0 -2-ethanesulfonic acid (HEPES) buffer, pH 7.4, because of the low solubility of the drug in HEPES (1.4 mg/mL). The Papp from the control formulation across the jejunum segment of the intestine was found to be 3.21 106 cm/s. The Papps of the drug in different co-solvent formulations are shown in Table 2 and Figure 2. All the co-solvent formulations led to an increase in Papp (2–5-fold) compared with the control. The formulation made with TPGS (column F1 in Figure 2) as co-solvent showed the highest permeability among the co-solvent-based formulations, followed by propylene glycol as co-solvent (column F3 in Figure 2). The increase in permeability with co-solvents could be due to an increase in drug solubility and/or the action of co-solvents on the intestinal membrane. Effect of CDs on the solubility of the drug Figure 3 shows the solubility of drug at different concentrations of b-CD. Drug solubility in PBS alone was found to be 1.206 mg/mL. The addition of b-CD resulted in enhanced drug solubility (5-fold) compared with the control, but no linear increase in drug solubility occurred with increasing b-CD concentration. That finding indicates that the drug did not form a 1:1 inclusion complex with b-CD. Figure 4 indicates drug solubility at different concentrations of HP-b-CD. The addition of HP-b-CD resulted in enhanced drug solubility compared with the control, and a linear increase in drug Table 2. Co-solvent-based drug formulations with Papp values.
Formulation # Control F1 F2 F3 F4 F5 Figure 1. pH solubility curve of the drug in different pH buffers estimated by the HPLC method.
Co-solvents pH 4.0 KRB buffer TPGS/water TPGS/transcutol/water Propylene glycol/water PEG 400/propylene glycol/water PEG 400/propylene glycol/Tween 80/water
Ratio of solvents (% v/v)
Papp (average SD) 106 cm/s
100 20/80 20/20/60 10/90 20/10/70
3.21 0.86 15.21 6.28 5.55 0.49 6.20 1.35 5.68 2.83
20/10/1/69
4.94 0.99
Table 1. Manual assessment of drug solubility in different co-solvent combinations. Combination 1 2 3 4 5 6 7 8 9
Solvent 1
Solvent 2
PEG 400 Propylene glycol PEG 400 PEG 400 Propylene glycol PEG 400 Propylene glycol TPGS TPGS
Propylene glycol Glycerol Glycerol Tween 80 Tween 80 Propylene glycol Water Water Transcutol
Solvent 3 – Water Water Water Water Water – – Water
Ratio of solvents (% v/v)
Solubility (mg/mL)
50/50 50/25/25 50/25/25 50/10/40 50/10/40 20/10/70 10/90 20/80 20/20/60
100 200 200 200 100 200 200 200 200
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Figure 2. Papp of the drug for different formulations. Figure 5. Phase solubility diagram of the drug in PBS, pH 7.4 and SBECD solution.
Figure 3. Phase solubility diagram of the drug in PBS, pH 7.4 and b-CD solution.
Figure 5 indicates drug solubility at different concentrations of SBECD. The addition of SBECD resulted in enhanced drug solubility compared with the control, and a linear increase in drug solubility took place with increasing SBECD concentration. Drug solubility in PBS was enhanced 35-fold (43.17 mg/mL). A linear (AL) type of curve was obtained from the phase solubility study, indicating the formation of a 1:1 drug and SBECD complex. The stability constant (Kc) of the drug and SBECD complex (1:1) was calculated as 0.370 mM1 from the linear plot of the phase solubility diagram using Equation (2). The formation of a 1:1 inclusion complex in the solid state was determined using DSC (Figure 6a–e). The DSC curve of neat drug showed a peak near 225.24 C that is indicative of its melting point (221–223 C). A thermogram of the inclusion complex of drug-HP-b-CD and drug-SBECD prepared by freeze drying (1:1) indicated complete disappearance of the melting point peak characteristic of drug. In vivo pharmacokinetic studies
Figure 4. Phase solubility diagram of the drug in PBS, pH 7.4 and HPb-CD solution.
solubility occurred with increasing HP-b-CD concentration. Drug solubility in PBS was enhanced 18-fold (22.49 mg/mL). An AL type of curve was obtained from the phase solubility study, indicating the formation of a 1:1 drug and HP-b-CD complex11. The apparent stability constant (Kc) of the drug and HP-b-CD complex (1:1) was calculated as 0.052 mM1 from the linear plot of the phase solubility diagram using Equation (2). Kc ¼
slope Soð1 slopeÞ
where, So is the solubility of the drug in the absence of CD.
ð2Þ
Pharmacokinetic studies were carried out in rats to determine the best two co-solvent formulations, based on in vitro permeability studies; and the drug–SBECD inclusion complex, based on phase solubility and complexation efficiency. TPGS- and propyleneglycol-based co-solvent formulations were selected. All the formulations were tested at 200 mg/kg. Figure 7 shows the mean plasma drug concentrations in the three formulation groups. Plasma drug levels increased rapidly after administration and reached the observed peak concentration at about 0.5–1 h. Drug levels decreased thereafter and were below the lower limit of quantitation of 100 ng/mL by 24 h in nearly all samples. The plasma drug concentrations for group 1 (the drug in 10% propylene glycol/90% sterile water) and for group 2 (the drug in 20% vitamin E TPGS/80% sterile water) were similar, but for group 3 (the drug–SBECD in sterile water) the drug levels were lower. Otherwise, the plasma drug profile was the same in the three groups. The results of the pharmacokinetics analysis are presented in Table 3. The Tmax was 0.5–1 for all rats. The mean t1/2, which did not vary significantly among the three groups, was about 4–5 h. The Cmax was highest in group 1 (12 788 2316 ng/mL), followed by group 2 (11 434 4918 ng/mL), and lowest in group 3 (10 471 1610 ng/mL), although the differences were not statistically significant. AUClast and AUCinf showed a similar pattern, although the exposure based on AUC in group 3 animals was lower than in groups 1 and 2.
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DOI: 10.3109/10837450.2013.852574
Figure 6. DSC thermogram of (a) the drug, (b) HP-b-CD, (c) the inclusion complex of the drug and HP-b-CD, (d) SBECD, and (e) the inclusion complex of the drug and SBECD.
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Figure 6. Continued.
Table 3. Pharmacokinetic parameters of the drug in Sprague–Dawley rats.
Rat 1 2 3 4 5 6 7 8 9 Figure 7. Plasma concentrations of Noscapine hydrochloride hydrate in male Sprague–Dawley rats (oral; 200 mg/kg).
Group
t1/2 (h)
Tmax (h)
Cmax (ng/mL)
AUClast (h ng/mL)
AUCinf (h ng/mL)
1
4.5 8.7 2.6 5.3 3.1 6.2 3.9 2.4 4.1 1.9 2.8 3.5 7.6 4.6 2.6
0.5 1 0.5 0.7 0.3 0.5 1 1 0.8 0.3 0.5 0.5 0.5 0.5 0.0
12 539 10 606 15 218 12 788 0.2316 13 349 15 106 5847 11 434 4918 9228 9894 12 290 10 471 1610
66 668 79 177 132 402 92 749 34 905 97 363 139 740 41 654 92 919 49 194 59 616 50 497 64 399 58 171 7063
74 639 131 870 132 902 113 137 33 344 125 748 141 400 43 181 103 443 52 772 60 915 56 650 102 230 73 265 25 175
Mean SD 2 Mean SD 3 Mean SD
DOI: 10.3109/10837450.2013.852574
Formulation approaches to improving the delivery of an antiviral drug
Conclusion The study results indicate that co-solvent formulations of noscapine can be developed that have enhanced solubility and permeability levels. The drug formed an inclusion complex with the more soluble forms of CDs – HP-b-CD and SBECD – which enhanced solubility. Pharmacokinetic studies carried out in rats show that the relative bioavailability of the drug from the two cosolvent formulations and the CD formulation were close to each other, indicating the feasibility of including both co-solvent and CD based formulations in further study.
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Acknowledgements The authors would like to thank Dr Joan-Huey Dow for HPLC analysis of formulation samples, Dr Robert Swezey for bioanalysis of the plasma samples and Mr. Paul Penwell for DSC determinations.
Declaration of interest This project has been funded in whole or part with federal funds from NIAID, NIH, DHHS under Contract No. HHSN266200600011C/N01-AI60011. The authors report no declarations of interest.
References 1. Mahmoudian M, Rahimi-Moghaddam P. The anti-cancer activity of Noscapine: a review. Recent Pat Anticancer Drug Discov 2009;4: 92–97.
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2. Martindale WA. The extra pharmacopoeia. London: The Pharmaceutical Press; 1997. 3. Aneja A, Dhiman N, Idnani J, et al. Preclinical pharmacokinetics and bioavailability of Noscapine, a tubulin-binding anticancer agent. Cancer Chemother Pharmacol 2007;60:831–839. 4. Landen JW, Lang R, McMohan SJ, et al. Noscapine alters microtubule dynamics in living cells and inhibits the progression of melanoma. Clin Cancer Res 2002;62:4109–4114. 5. Landen JW, Hau V, Wang M, et al. Noscapine crosses the bloodbrain barrier and inhibits glioblastoma growth. Clin Cancer Res 2004;10:5187–5201. 6. Madan J, Dhiman N, Parmar VK, et al. Inclusion complexes of Noscapine in b-cyclodextrin offer better solubility and improved pharmacokinetics. Cancer Chemother Pharmacol 2010; 65:537–548. 7. Strickley RG. Solubilizing excipients in oral and injectable formulations. Pharm Res 2004;21:201–230. 8. Carrier RL, Miller LA, Ahmed I. The utility of cyclodextrins for enhancing oral bioavailability. J Control Release 2007;123: 78–99. 9. Loftsson T, Jarho P, Masson M, Jarvinen T. Cyclodextrins in drug delivery. Expert Opin Drug Deliv 2005;2:335–351. 10. Ungell AL, Nylander S, Bergstrand S, et al. Membrane transport of drugs in different regions of the intestinal tract of the rat. J Pharm Sci 1998;87:360–366. 11. Higuchi T, Connors AK. Phase solubility techniques. Adv Anal Chem Instrum 1965;4:117–212. 12. Neervannan S. Preclinical formulations for discovery and toxicology: physicochemical challenges. Expert Opin Drug Metab Toxicol 2006;2:715–731.
RESEARCH ARTICLE
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Formulation approaches to improving the delivery of an antiviral drug with activity against seasonal flu Srinivasa M. Sammeta1, Li Wang1, Shravan K. Mutyam1, Kathleen O’Loughlin1, Carol E. Green1, Milton H. Werner2, Terence Kelly3, and Gita N. Shankar1 1
Pharmaceutical Development, Biosciences Division, SRI International, Menlo Park, CA, USA, 2Inhibikase Therapeutics, Inc., Suite, Atlanta, GA, USA, and 3Kelly Pharma Research Consulting LLC, Ridgefield, CT, USA
Abstract
Keywords
The main objective of the present study was to develop formulations of noscapine hydrochloride hydrate with enhanced solubility and bioavailability using co-solvent- and cyclodextrin-based approaches. Different combinations of co-solvents, which were selected on the basis of high-throughput solubility screening, were subjected to in vitro intestinal drug permeability studies conducted with Ussing chambers. Vitamin E tocopherol polyethylene glycol succinate and propylene glycol based co-solvent formulations provided the maximum permeability coefficient for the drug. Inclusion complexes of the drug were prepared using hydroxypropyl-b-cyclodextrin and sulphobutylether cyclodextrins. Pharmacokinetic studies were carried out in male Sprague–Dawley rats for the selected formulations. The relative bioavailabilities of the drug with the co-solvent- and cyclodextrin-based formulations were found to be similar.
Bioavailability, co-solvents, cyclodextrins, solubility, Ussing chambers
Introduction Noscapine hydrochloride hydrate, a phthalideisoquinoline alkaloid without sedative, euphoric, analgesic or respiratory depressant properties, is used as an antitussive agent worldwide. Recent studies have shown that noscapine is also tubulin-binding and can be used for its anticancer properties1–4. The oral route for drug delivery has been preferred because of its ease of administration and patient compliance. However, relatively high doses of the drug (ED50 300 mg/kg body weight) are essential for induction of anticancer activity. To date, development of oral formulations of the drug has been limited because of the large dose required, poor absorption, low dissolution or aqueous solubility, and extensive first-pass metabolism5,6. To overcome these solubility and bioavailability limitations, different formulation approaches based on co-solvents and cyclodextrins (CDs) can be used. Co-solvents are mixtures of miscible solvents that can be used to solubilize noscapine (hereafter, generally termed ‘‘the drug’’). The excipients used as co-solvents can be water-soluble like polyethylene glycol (PEG) 400, PEG 300, propylene glycol and glycerin; water-insoluble like oleic acid, castor oil and peppermint oil; or surfactants like Tween 80, Tween 20, Cremophor and tocopherol polyethylene glycol succinate (TPGS)7. CDs are cyclic oligosaccharides made of (a-1,4)-linked a-d-glucopyranose units with a hydrophilic outer surface and a lipophilic central cavity. a-CD, b-CD and g-CD, the most common natural CDs, have limited aqueous solubility. CD derivatives of pharmaceutical significance with enhanced solubility include the hydroxypropyl
Address for correspondence: Gita N. Shankar, Pharmaceutical Development, Biosciences Division, SRI International, Menlo Park, CA 94025-3493, USA. Tel: +1-650-859-5197. Fax: +1-650-859-2889. E-mail: [email protected]
History Received 1 August 2013 Accepted 1 October 2013 Published online 8 November 2013
derivatives of b- and g-CD, the randomly methylated b-CD, and sulphobutylether b-CD (SBECD). CDs are used as complexing agents to increase the aqueous solubility of drugs and thus enhance bioavailability and stability8,9. In the present study, oral formulations of noscapine hydrochloride hydrate were developed using co-solvent- and CD-based formulation approaches and evaluated using in vitro permeability and in vivo pharmacokinetic studies.
Materials and methods Chemicals Noscapine hydrochloride hydrate and glycerol were purchased from Sigma-Aldrich (St. Louis, MO). PEG 400, propylene glycol, potassium chloride, Tween 80 and Tween 20 were obtained from Spectrum Chemicals (Gardena, CA). Vitamin E TPGS was purchased from Eastman Chemical Company (Kingsport, TN). Boric acid, sodium hydroxide and sodium phosphate dibasic were obtained from Mallinckrodt Chemicals (Phillipsburg, NJ). b-CD from ISP Technologies Inc (Wayne, NJ), hydroxypropyl-b-CD (HP-b-CD) from Roquette (Keokuk, IA) and SBECD from Cydex Pharmaceuticals Inc (Lenexa, KS) were gift samples. All other chemical were obtained from Fisher Scientific. The pH solubility of the drug The solubility of the drug was determined in different pH buffers2–10. Mcllvaine–Whitting buffer containing various amounts of 0.1 M citric acid and 0.2 M disodium hydrogen phosphate were used to make buffers of pH 2–7. Standard buffer solutions containing different amounts of 0.2 M boric acid and potassium chloride and 0.2 M sodium hydroxide were used to make buffers of pH 8–10. The samples for the pH solubility study
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were prepared by adding an excess of drug to 1 mL of the appropriate pH buffer in a 4-mL vial. The solution was mixed using a vortex genie for approximately 30 s and then placed on an orbital shaker at room temperature. At regular time intervals (24 and 48 h), the vials were removed from the orbital shaker, and the pH of the solutions was measured for each sample. If the solution was turbid and the pH difference between two consecutive days was 50.2 pH units, the solution was considered to have reached equilibrium. Samples were filtered through a 0.45-mm syringe filter and analyzed using high-performance liquid chromatography (HPLC).
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Solubility of the drug in different solvents Initially, the solubility of drug in different co-solvent combinations was determined using SRI International’s (SRI’s) proprietary high-throughput solubility screening (HTSS) system. The system uses a high-throughput liquid handling system (TECAN Freedom EVO) to prepare the solvent combinations. Freedom EVOware, the software associated with the liquid handling system, is used to aspirate and dispense a predetermined volume of selected solvents into each well in a microtiter plate. Before the addition of the drug, the baseline turbidities of solvents are read using a Microplate reader. A stock solution of drug prepared in dimethylsulfoxide (DMSO) is added to the plates, which are prefilled with premixed cosolvent combinations. The plates are shaken after addition of the drug, and the turbidity is measured again. The solubility of the drug is assessed by the determining the change in turbidity before and after addition of the drug in the solvent system. Forty-five solvent combinations were tested using HTSS. From the HTSS results, nine solvent combinations were selected and their solubility confirmed manually. Manual solubility assessment testing omits the DMSO necessary in the HTSS process. In this approach, a weighed amount of drug is added directly to the solvent system and mixed by vortexing or sonication. In vitro permeability studies The in vitro permeability for the various drug formulations was determined in Ussing chambers. Isolated membrane taken from the jejunum segment, devoid of Peyer’s patches, of male Sprague– Dawley rat intestine was mounted between the tissue slider and placed between mucosal and serosal Ussing chambers. The effective exposure area of tissue in the Ussing chambers was 0.5 cm2. Initially, 5 mL of Kreb’s ringer bicarbonate (KRB) buffer, pH 4.0, was added to each of the two chambers and allowed to equilibrate for 20 min at 37 C with continuous bubbling of 100% oxygen. After the equilibration of the tissue, the mucosal KRB buffer was replaced either with drug dissolved in KRB buffer or a drug formulation to achieve a final concentration of 5 mg/mL in the mucosal chamber. About 2 mL of solution was sampled from the serosal side at 0.5, 1, 1.5, 2, and 2.5 h and replaced with an equal volume of fresh KRB buffer. The samples collected were analyzed by HPLC to determine the percentage of drug transported across the intestinal tissue. The apparent permeability coefficient (Papp) was calculated using Equation (1)10. Papp ¼
dQ 1 ðcm=sÞ dt AC0
ð1Þ
Where dQ/dt is the steady-state appearance rate on the serosal side of the membrane, A is the exposed tissue surface area and C0 is the initial concentration of the drug on the mucosal side of the chamber. All the results are represented as the mean standard deviation (SD) of 3–5 trials.
Effect of CDs on the solubility of the drug The effect of CD in enhancing the solubility of drug was studied using three CD types: b-CD, HP-b-CD and SBECD. A phase-solubility assay was performed to determine the stoichiometry of a drug-CD binary system11. Various concentrations of b-CD (2–15 mM), HP-b-CD (35–357 mM) and SBECD (11–185 mM) were prepared in phosphate buffered saline (PBS) solution, pH 7.4. The concentration range for all the CDs was set on the basis of their maximum solubility in PBS. About 2 mL of varying concentrations of the CDs was added to glass vials, and an excess amount of drug was added, with the contents stirred for 48 h at 37 1 C using a shaker bath. Excess drug was also added to PBS, pH 7.4, which served as the control. After equilibrium, the samples were filtered using a 0.22-mm membrane filter and analyzed for drug content using HPLC. The inclusion complexes of drug-HP-b-CD and drug-SBECD were prepared using freeze drying8. Drug and CD were dissolved in water in 1:1 mM, and the resulting solution was stirred for 24 h in an orbit shaker at room temperature. Subsequently, the solution was lyophilized, and the sample obtained was collected and passed through a # 100-mm sieve. The thermal behavior of the drug, HPb-CD, SBECD, the drug-HP-b-CD inclusion complex and the drug-SBECD inclusion complex were studied using a differential scanning calorimeter (DSC) Q200 V24.4 Build 116 (Universal V4.5A TA Instruments, New Castle, DE) thermal analyzer. In vivo pharmacokinetic studies Pharmacokinetic studies were carried out in male Sprague– Dawley rats (300–350 g) procured from Charles River Laboratories. The rats were maintained on a 12 h light/12 h dark cycle with access to food and water ad libitum. All animal experiments complied with the standards set out in the guidelines of SRI’s Institutional Animal Care and Use Committee. Three drug formulations were administered as a single oral gavage (po) dose in three groups (n ¼ 3) of animals. The dose of drug in the three formulations was 200 mg/kg. Group 1 rats received the first formulation with the drug dissolved in 10% propylene glycol/90% sterile water and group 2 rats received the drug dissolved in 20% vitamin E TPGS/80% sterile water. The third formulation of drug– SBECD inclusion complex dissolved in sterile water was administered to group 3 rats. About 300 mL of blood was collected at 5, 15 and 30 min, and 1, 2, 4, 8, 12 and 24 h postdose from each rat via the jugular vein catheter and placed in tubes containing K3 ethylenediaminetetraacetic acid (K3EDTA) for plasma evaluation. Plasma was prepared by centrifuging the samples at 4 C and storing them at approximately 70 C until analysis took place. Analytical method HPLC analysis of the drug from in vitro studies was performed using an Agilent 1100 system containing a Phenomenex Luna (C8) column (150 4.6 mm, 5 mm) and ultraviolet (UV) detector. The drug was analyzed at 235 nm using a mobile phase of a mixture of 20 mM ammonium acetate, adjusted to pH 4.5 and of acetonitrile (65:35 v/v) with a flow rate of 1.0 mL/min at 25 C. The sample solutions were diluted to the linear range of the calibration standards using a 50:50 (v/v) mixture of 20 mM ammonium acetate and of acetonitrile. The amount of drug in the plasma samples was analyzed using a liquid chromatography/tandem mass spectrometry (LC/MS/MS) method with narcotine-N-oxide as the internal standard. To 50 mL of plasma, 50 mL of internal standard was added, and plasma proteins were precipitated by the addition of 200 mL of acetonitrile and methanol (8:2, v/v), followed by vortex and
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centrifugation at 4000 rpm for 5 min at 5 C. The supernatant was then collected and injected to LC/MS/MS. Data analysis
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The plasma drug level data were analyzed using WinNonlinÕ , version 5.2 professional, by noncompartmental modeling. The maximal plasma concentration (Cmax), the time to maximum plasma concentration (Tmax), the area under the plasma concentration–time curve up to the last time point (AUClast), the area under the plasma concentration–time curve extrapolated to infinity (AUCinf) and the terminal elimination half-life (t1/2) were determined.
Results and discussion The pH solubility of the drug Solubility of the drug in different pH buffers as estimated by HPLC analysis is shown in Figure 1. The drug was found to be more soluble at an acidic pH and less soluble at neutral and basic pH conditions. After 48 h, the drug solutions in pH 2 and 3 buffers changed color to pale yellow and hence were not considered for further solubility analysis. The drug had a highest solubility of 93 mg/mL in the pH 4 buffer. Solubility of the drug in different co-solvent systems The solubility of the drug in different combinations of solvents was determined using HTSS. Ten solvents (100% ethanol, 50% w/w glycerol in water, 100% PEG 400, 100% propylene glycol, 20% w/w Plasdone K29/32 in water, 40% w/w Solutol HS 15 in water, 100% Transcutol, 20% w/w Tween 20 in water, 15% w/w Tween 80 in water and 20% w/w Vitamin E TPGS in water) were used to create 45 combinations, with the solubility of drug in
3
each combination assessed (data not shown). Using the HTSS results, nine optimal combinations of solvents were selected, and the solubility of the drug was manually confirmed at 100 and 200 mg/mL; data are shown in Table 1. Except for two combinations, all combinations of solvents achieved a drug solubility of 200 mg/mL. In vitro permeability studies On the basis of the solubility data, five co-solvent formulations were developed (Table 2). The amounts of excipients were within the maximum levels used in preclinical formulations12. In the in vitro permeability studies, KRB buffer, pH 4.0, was used in place of HN-2-hydroxyethylpiperazine-N0 -2-ethanesulfonic acid (HEPES) buffer, pH 7.4, because of the low solubility of the drug in HEPES (1.4 mg/mL). The Papp from the control formulation across the jejunum segment of the intestine was found to be 3.21 106 cm/s. The Papps of the drug in different co-solvent formulations are shown in Table 2 and Figure 2. All the co-solvent formulations led to an increase in Papp (2–5-fold) compared with the control. The formulation made with TPGS (column F1 in Figure 2) as co-solvent showed the highest permeability among the co-solvent-based formulations, followed by propylene glycol as co-solvent (column F3 in Figure 2). The increase in permeability with co-solvents could be due to an increase in drug solubility and/or the action of co-solvents on the intestinal membrane. Effect of CDs on the solubility of the drug Figure 3 shows the solubility of drug at different concentrations of b-CD. Drug solubility in PBS alone was found to be 1.206 mg/mL. The addition of b-CD resulted in enhanced drug solubility (5-fold) compared with the control, but no linear increase in drug solubility occurred with increasing b-CD concentration. That finding indicates that the drug did not form a 1:1 inclusion complex with b-CD. Figure 4 indicates drug solubility at different concentrations of HP-b-CD. The addition of HP-b-CD resulted in enhanced drug solubility compared with the control, and a linear increase in drug Table 2. Co-solvent-based drug formulations with Papp values.
Formulation # Control F1 F2 F3 F4 F5 Figure 1. pH solubility curve of the drug in different pH buffers estimated by the HPLC method.
Co-solvents pH 4.0 KRB buffer TPGS/water TPGS/transcutol/water Propylene glycol/water PEG 400/propylene glycol/water PEG 400/propylene glycol/Tween 80/water
Ratio of solvents (% v/v)
Papp (average SD) 106 cm/s
100 20/80 20/20/60 10/90 20/10/70
3.21 0.86 15.21 6.28 5.55 0.49 6.20 1.35 5.68 2.83
20/10/1/69
4.94 0.99
Table 1. Manual assessment of drug solubility in different co-solvent combinations. Combination 1 2 3 4 5 6 7 8 9
Solvent 1
Solvent 2
PEG 400 Propylene glycol PEG 400 PEG 400 Propylene glycol PEG 400 Propylene glycol TPGS TPGS
Propylene glycol Glycerol Glycerol Tween 80 Tween 80 Propylene glycol Water Water Transcutol
Solvent 3 – Water Water Water Water Water – – Water
Ratio of solvents (% v/v)
Solubility (mg/mL)
50/50 50/25/25 50/25/25 50/10/40 50/10/40 20/10/70 10/90 20/80 20/20/60
100 200 200 200 100 200 200 200 200
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Pharm Dev Technol, Early Online: 1–7
Figure 2. Papp of the drug for different formulations. Figure 5. Phase solubility diagram of the drug in PBS, pH 7.4 and SBECD solution.
Figure 3. Phase solubility diagram of the drug in PBS, pH 7.4 and b-CD solution.
Figure 5 indicates drug solubility at different concentrations of SBECD. The addition of SBECD resulted in enhanced drug solubility compared with the control, and a linear increase in drug solubility took place with increasing SBECD concentration. Drug solubility in PBS was enhanced 35-fold (43.17 mg/mL). A linear (AL) type of curve was obtained from the phase solubility study, indicating the formation of a 1:1 drug and SBECD complex. The stability constant (Kc) of the drug and SBECD complex (1:1) was calculated as 0.370 mM1 from the linear plot of the phase solubility diagram using Equation (2). The formation of a 1:1 inclusion complex in the solid state was determined using DSC (Figure 6a–e). The DSC curve of neat drug showed a peak near 225.24 C that is indicative of its melting point (221–223 C). A thermogram of the inclusion complex of drug-HP-b-CD and drug-SBECD prepared by freeze drying (1:1) indicated complete disappearance of the melting point peak characteristic of drug. In vivo pharmacokinetic studies
Figure 4. Phase solubility diagram of the drug in PBS, pH 7.4 and HPb-CD solution.
solubility occurred with increasing HP-b-CD concentration. Drug solubility in PBS was enhanced 18-fold (22.49 mg/mL). An AL type of curve was obtained from the phase solubility study, indicating the formation of a 1:1 drug and HP-b-CD complex11. The apparent stability constant (Kc) of the drug and HP-b-CD complex (1:1) was calculated as 0.052 mM1 from the linear plot of the phase solubility diagram using Equation (2). Kc ¼
slope Soð1 slopeÞ
where, So is the solubility of the drug in the absence of CD.
ð2Þ
Pharmacokinetic studies were carried out in rats to determine the best two co-solvent formulations, based on in vitro permeability studies; and the drug–SBECD inclusion complex, based on phase solubility and complexation efficiency. TPGS- and propyleneglycol-based co-solvent formulations were selected. All the formulations were tested at 200 mg/kg. Figure 7 shows the mean plasma drug concentrations in the three formulation groups. Plasma drug levels increased rapidly after administration and reached the observed peak concentration at about 0.5–1 h. Drug levels decreased thereafter and were below the lower limit of quantitation of 100 ng/mL by 24 h in nearly all samples. The plasma drug concentrations for group 1 (the drug in 10% propylene glycol/90% sterile water) and for group 2 (the drug in 20% vitamin E TPGS/80% sterile water) were similar, but for group 3 (the drug–SBECD in sterile water) the drug levels were lower. Otherwise, the plasma drug profile was the same in the three groups. The results of the pharmacokinetics analysis are presented in Table 3. The Tmax was 0.5–1 for all rats. The mean t1/2, which did not vary significantly among the three groups, was about 4–5 h. The Cmax was highest in group 1 (12 788 2316 ng/mL), followed by group 2 (11 434 4918 ng/mL), and lowest in group 3 (10 471 1610 ng/mL), although the differences were not statistically significant. AUClast and AUCinf showed a similar pattern, although the exposure based on AUC in group 3 animals was lower than in groups 1 and 2.
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DOI: 10.3109/10837450.2013.852574
Figure 6. DSC thermogram of (a) the drug, (b) HP-b-CD, (c) the inclusion complex of the drug and HP-b-CD, (d) SBECD, and (e) the inclusion complex of the drug and SBECD.
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Figure 6. Continued.
Table 3. Pharmacokinetic parameters of the drug in Sprague–Dawley rats.
Rat 1 2 3 4 5 6 7 8 9 Figure 7. Plasma concentrations of Noscapine hydrochloride hydrate in male Sprague–Dawley rats (oral; 200 mg/kg).
Group
t1/2 (h)
Tmax (h)
Cmax (ng/mL)
AUClast (h ng/mL)
AUCinf (h ng/mL)
1
4.5 8.7 2.6 5.3 3.1 6.2 3.9 2.4 4.1 1.9 2.8 3.5 7.6 4.6 2.6
0.5 1 0.5 0.7 0.3 0.5 1 1 0.8 0.3 0.5 0.5 0.5 0.5 0.0
12 539 10 606 15 218 12 788 0.2316 13 349 15 106 5847 11 434 4918 9228 9894 12 290 10 471 1610
66 668 79 177 132 402 92 749 34 905 97 363 139 740 41 654 92 919 49 194 59 616 50 497 64 399 58 171 7063
74 639 131 870 132 902 113 137 33 344 125 748 141 400 43 181 103 443 52 772 60 915 56 650 102 230 73 265 25 175
Mean SD 2 Mean SD 3 Mean SD
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Formulation approaches to improving the delivery of an antiviral drug
Conclusion The study results indicate that co-solvent formulations of noscapine can be developed that have enhanced solubility and permeability levels. The drug formed an inclusion complex with the more soluble forms of CDs – HP-b-CD and SBECD – which enhanced solubility. Pharmacokinetic studies carried out in rats show that the relative bioavailability of the drug from the two cosolvent formulations and the CD formulation were close to each other, indicating the feasibility of including both co-solvent and CD based formulations in further study.
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Acknowledgements The authors would like to thank Dr Joan-Huey Dow for HPLC analysis of formulation samples, Dr Robert Swezey for bioanalysis of the plasma samples and Mr. Paul Penwell for DSC determinations.
Declaration of interest This project has been funded in whole or part with federal funds from NIAID, NIH, DHHS under Contract No. HHSN266200600011C/N01-AI60011. The authors report no declarations of interest.
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2. Martindale WA. The extra pharmacopoeia. London: The Pharmaceutical Press; 1997. 3. Aneja A, Dhiman N, Idnani J, et al. Preclinical pharmacokinetics and bioavailability of Noscapine, a tubulin-binding anticancer agent. Cancer Chemother Pharmacol 2007;60:831–839. 4. Landen JW, Lang R, McMohan SJ, et al. Noscapine alters microtubule dynamics in living cells and inhibits the progression of melanoma. Clin Cancer Res 2002;62:4109–4114. 5. Landen JW, Hau V, Wang M, et al. Noscapine crosses the bloodbrain barrier and inhibits glioblastoma growth. Clin Cancer Res 2004;10:5187–5201. 6. Madan J, Dhiman N, Parmar VK, et al. Inclusion complexes of Noscapine in b-cyclodextrin offer better solubility and improved pharmacokinetics. Cancer Chemother Pharmacol 2010; 65:537–548. 7. Strickley RG. Solubilizing excipients in oral and injectable formulations. Pharm Res 2004;21:201–230. 8. Carrier RL, Miller LA, Ahmed I. The utility of cyclodextrins for enhancing oral bioavailability. J Control Release 2007;123: 78–99. 9. Loftsson T, Jarho P, Masson M, Jarvinen T. Cyclodextrins in drug delivery. Expert Opin Drug Deliv 2005;2:335–351. 10. Ungell AL, Nylander S, Bergstrand S, et al. Membrane transport of drugs in different regions of the intestinal tract of the rat. J Pharm Sci 1998;87:360–366. 11. Higuchi T, Connors AK. Phase solubility techniques. Adv Anal Chem Instrum 1965;4:117–212. 12. Neervannan S. Preclinical formulations for discovery and toxicology: physicochemical challenges. Expert Opin Drug Metab Toxicol 2006;2:715–731.
