Francine
Edwards
a,
Christina
Tsakmaka
a,
Stephan
Mohr
a,
Peter R.
Fielden
a,
Nick J.
Goddard
a,
Jonathan
Booth
b and
Kin Y.
Tam
*c
aSchool of Chemical Engineering and Analytical Science, The University of Manchester, PO Box 88, Sackville Street, Manchester M60 1QD, UK
bAstraZeneca, Charter Way, Macclesfield, Cheshire, SK10 2NA, UK
cAstraZeneca, Mereside, Alderley Park, Macclesfield, Cheshire SK10 4TG, UK. E-mail: kin_tam@hotmail.com
First published on 6th November 2012
The purpose of this study is to develop a droplet-based microfluidic device capable of monitoring drug precipitation upon a shift from gastric pH (pH 1.5) to intestinal pH (pH 6.5–7.0). The extent of precipitation occurring in droplets over time was measured using a novel on-chip laser scattering technique specifically developed for this study. The precipitation of ketoconazole, a poorly water-soluble basic drug, was investigated under different concentrations and pH values. It has been shown that the drug precipitates rapidly under supersaturation. Two water-soluble aqueous polymers, namely, polyvinylpyrrolidone (PVP) and hydroxypropylmethylcellulose (HPMC) have been evaluated as precipitation inhibitors. HPMC was shown to be the most potent precipitation inhibitor. It is envisaged that the microfluidic pH-shift method developed in this study would form a proof-of-concept study, towards the development of a high throughput method for screening pharmaceutical excipients/precipitation inhibitors.
A number of formulation methods have been reported for overcoming or mitigating low aqueous solubility of oral drugs.2 One proven approach is to maintain an intraluminal concentration that is above the intrinsic solubility of the active pharmaceutical ingredient (API). This strategy is referred to as a supersaturating drug delivery system,3 and can be de-convoluted into two stages: (1) rapid dissolution of the solid/solution formulation in the gastro-intestinal tract, e.g. in the stomach, to achieve a supersaturation, and (2) delay of API precipitation in the gastro-intestinal tract, e.g. in the small intestine to prolong supersaturation and maximize the chance of absorption through the intestinal epithelium cells. A broad range of strategies have been developed to achieve rapid drug dissolution, for example salts, high energy polymorphs, amorphous solid dispersions, nano-sized crystals, solid and liquid lipid based formulations.4 As for the delay of API precipitation, surfactants, such as sodium dodecyl sulphate, and water-soluble polymers, such as polyvinylpyrrolidone (PVP) and hydroxypropylmethylcellulose (HPMC), are commonly employed as precipitation inhibitors.3 It is believed that stabilization of a supersaturated state is accomplished via the kinetic control of API precipitation, typically through hydrophobic interactions and hydrogen bonding between the drug and excipient.5,6 However, the mechanisms of precipitation inhibition are not fully understood,6 and the search for suitable precipitation inhibitors for novel chemical entities rely mostly on trial-and-error experimental evaluation.
In the development of pharmaceutical formulations, screening methods to evaluate the compatibility between the API and precipitation inhibitors are of great interest.3,6,7 Typically, these methods involve monitoring API concentration as a function of time during incubation of the precipitation inhibitor with the API, which is initially in a supersaturated state. However, most of these methods allow only a limited number of excipients to be tested in one experiment, and concentration determination is usually accomplished using either UV-vis spectroscopy or HPLC after separation of solids by centrifugation or filtration.8,9 In this case there is clearly a time delay between sampling and solids separation, therefore the concentration determined will not necessarily reflect the in situ concentration at the time of sampling.
Microfluidic technology offers the advantage of studying the precipitation process with minimal sample consumption and precise control of temperature and concentrations. Moreover, the generation of monodisperse droplets in microfluidic channels allows numerous independent crystallization experiments to be performed without concern for cross-contamination. To date, research in this area has predominately focused on protein crystallisation. Examples include the optimisation of crystallisation conditions to obtain diffraction quality crystals using a concentration gradient,10 decoupling protein nucleation and growth kinetics through droplet-merging,11 and droplet concentration control through a permeable PDMS membrane.12 However, research examining the use of droplet-based microfluidics to study precipitation of smaller molecules is much more limited. Salmon et al. reported a microfluidic device on which hundreds of droplets of varying concentration could be stored under the application of a temperature gradient to investigate the solubility diagram of adipic acid.13 Droplet-based microfluidics have also been used to examine the crystal morphology of potassium nitrate.14 Ali et al. have applied a continuous phase microfluidic reactor to study the precipitation of prednisolone in the presence of anti-solvent to identify the variables affecting the precipitation process.15 Yalkowsky et al. have developed a dynamic in vitro assay to simulate the injection of a parenteral formulation into a vein, with the aim to assess drug precipitation upon dilution in a continuous phase.16,17 As far as we are aware, no work has been reported on the use of droplet-based microfluidic technology to investigate the precipitation of pharmaceuticals and the evaluation of precipitation inhibitors. We believe that this technology could offer a potentially more elegant, efficient and automatable approach to the analysis of precipitation phenomena.
In this work, we have developed a droplet-based microfluidic device to study the precipitation of a poorly water-soluble, weakly basic drug, ketoconazole. As a weak base, ketoconazole exhibits greater solubility in acidic media where ionization occurs, for example under gastric conditions (pH 1.5–2.0). At higher pHs (5.0–8.0), corresponding to gastric emptying into the small intestine, the solubility of the drug decreases and precipitation can occur. Rapid precipitation of the drug in the intestine results in low and variable oral bioavailability.18 In our microfluidic implementation, the drug is pre-dissolved as supersaturated solutions in gastric pH, to simulate rapid dissolution in a gastric environment. These solutions are then mixed with a basic buffer solution of appropriate concentration to simulate the change of pH upon gastric emptying into the small intestine. We refer to this as the pH-shift method.3 Following the pH-shift, precipitation occurring in the droplets was monitored as a function of time using an on-chip light scattering technique specifically developed for this study. With the same microfluidic device, we report the effects of precipitation inhibitors on the extent of precipitation of ketoconazole using the same pH-shift method.
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Fig. 1 The microfluidic chip (a), the experimental configuration (b), and a schematic of droplet formation and composition (c) used to study ketoconazole precipitation. |
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Fig. 2 Measured solubility (C) of ketoconazole as a function of pH (symbols). The line represents the calculated solubility profile: C = C0(1 + 10(pKa−pH)) where C and C0 represent the solubility and intrinsic solubility (2.4 μg mL−1) respectively. Solubility data at pH 5.0, 6.0 and 7.8 were quoted from ref. 22 and 23. |
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Fig. 3 Scattered light intensity (arbitrary units) by latex bead solutions as a function of concentration. (♦ 460 nm, ▲ 600 nm, ● 800 nm bead diameter.) |
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Fig. 4 Scattered light intensity as a function of time for the precipitation of ketoconazole after a pH-shift to pH 7.0. (▲ S = 161, ■ S = 97, ♦ S = 48, – S = 32, ● blank.) |
As shown in Fig. 4, the scattered light intensity approaches its maximum after five minutes. This enabled us to measure the average maximum intensity for a particular experiment, which we define as the average maximum intensity values from 5 to 7 minutes. As revealed from ex situ DLS measurements carried out at a supersaturation of 48 (see Section 3.4, Fig. 7), the effective particle diameter shows little growth beyond 8 minutes, which is consistent with the on-chip light scattering data as shown in Fig. 4. Additional ex situ DLS measurements at different supersaturations (data not shown) suggested that particle sizes appear to reach steady levels beyond 8 minutes. Interestingly, the particle growth rates were found to be slower at lower supersaturations, resulting in smaller particle diameters. It is envisaged that both the particle size and particle concentrations contribute to the scattering signal. However an unambiguous delineation of these two components from the scattering signal is beyond the scope of this preliminary study, and would merit further investigation.
Fig. 5 shows the average maximum intensity against the supersaturations of ketoconazole. It can be seen that the average maximum intensity increases with supersaturation in a linear fashion (up to a supersaturation of 110), as expected if all the drug in a supersaturated state has precipitated, suggesting the average maximum intensity can be used to study drug precipitation using our microfluidic system. At higher supersaturations, the average maximum intensity begins to show a negative deviation from linearity. The linear supersaturation of 110 in our microfluidic system is well above the supersaturations of ketoconazole at FaSSIF media, and pH 6.5 buffer, corresponding to the expected minimum intraluminal concentrations for a 200 mg dose of ketoconazole (see Section 3.1). It is noted that the maximum intraluminal concentrations (correspond to supersaturations of 67 and 170, in FaSSIF and pH 6.5 aqueous buffer, respectively) represent the worst cases as the estimates were derived by neglecting any convective dilution in the lumen. Nevertheless, the linear supersaturation of 110 is still above the maximum intraluminal concentration at FaSSIF, which is a more physiologically relevant matrix. The slight negative deviation from linear beyond a supersaturation of 110 does not preclude the use of average maximum intensity for detecting drug precipitation. This is illustrated in Fig. 4, where the data obtained at a supersaturation of 160 still permits a clear identification of precipitation.
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Fig. 5 Average maximum light scattering intensity as a function of supersaturation (S): ♦ pH 6.5, ● pH 7.0. The line represents the linear regression line (R2 = 0.96) generated using data up to a supersaturation of 110. |
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Fig. 6 Average maximum light scattering intensity as a function of HPMC (top) and PVP (bottom) concentration, after a shift to pH 7.0 using a ketoconazole supersaturation of 48. |
As shown in Fig. 6, the average maximum intensities decrease in the presence of HPMC and PVP, suggesting both polymers are able to inhibit precipitation. However, the potency of precipitation inhibition is considerably better for HPMC than that of PVP. For example, with 0.05 mM HPMC, the drop in the average maximum intensity and hence precipitation inhibition was shown to outperform that of 1.7 mM PVP. Ketoconazole has eight hydrogen bond acceptors but no hydrogen bond donors. It is expected that greater interaction with the drug would be observed for a polymer capable of forming hydrogen bonds such as HPMC than a polymer which itself contains no proton bond donor, like PVP. The ex situ DLS data in Fig. 7 shows a marked decrease in effective diameter of the precipitated particles in the presence of both 0.8 mM PVP and 0.1 mM HPMC. The effect of HPMC was more pronounced resulting in a lower rate of particle growth (approximately 400 nm versus 600 nm after 10 minutes for HPMC and PVP, respectively). No scattering signal was measured from 5 mM polymer solutions (greater than the highest concentration used in the precipitation experiments), indicating the intensities observed in Fig. 7 result from precipitated drug. It is evident that HPMC is more effective at inhibiting particle growth than that of PVP in the case of ketoconazole. This finding is consistent with data reported by Taylor et al. showing that HPMC is more effective at maintaining supersaturation than PVP for the two model drugs, felodipine and indomethacin.25
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Fig. 7 DLS measurements to illustrate the effect of HPMC and PVP on the effective diameter of the precipitated ketoconazole particles. Data obtained at pH 7.0 with a ketoconazole supersaturation of 48. (♦ no polymer, ■ 0.8 mM PVP, ▲ 0.1 mM HPMC.) |
This work has demonstrated the potential of the microfluidic pH-shift method for screening candidate precipitation inhibitors in the development of pharmaceutical formulations. Further work to develop this concept for the simultaneous screening of multiple precipitation inhibitors is being carried out in our laboratories, and will be reported in a future publication.
The microfluidic pH-shift experiment developed in this study offers a novel way for precisely controlled in situ monitoring of drug precipitation. This is a step forward compared to traditional approaches that require solid separation and off-line sample quantification. We envisage that our methodology is amenable to further develop into a high throughput screen for selecting optimal pharmaceutical excipients/precipitation inhibitors, which would improve the speed and quality for the development of pharmaceutical formulations with the potential to become supersaturated during gastro-intestinal transit.
This journal is © The Royal Society of Chemistry 2013 |