Application of in situ potentiometric sensors to study dissolution-precipitation Application of in situ potentiometric sensors to study dissolution-precipitation behavior of electrospray-generated loperamide nanoparticles ktsinman@pion-inc.com behavior of electrospray-generated loperamide nanoparticles ktsinman@pion-inc.com K. Tsinman 1 , O. Tsinman 1 , R. Lingamaneni 1 , J. Patel 1 , C.J. Batty 2 , D. Thao 2 , J.P. Wyman 2 and R.A. Hoerr 2 K. Tsinman , O. Tsinman , R. Lingamaneni , J. Patel , C.J. Batty , D. Thao , J.P. Wyman and R.A. Hoerr 1 Pion Inc, 10 Cook Street, Billerica, MA 01821, USA; 2 Nanocopoeia LLC, 1246 University Ave W, St. Paul, MN 55104, USA Flux of LPD through Double Sink™ PAMPA Membrane Supersaturation Monitoring by FDS INTRODUCTION Flux of LPD through Double Sink™ PAMPA Membrane Supersaturation Monitoring by FDS INTRODUCTION To study LPD formulation effect on flux of the API through artificial membranes, 0.8 mL of the suspension prepared according to Table 1 was added to the corresponding donor chamber of μFLUX system and topped up with 19.2 mL of FaSSIF buffer . Ability of FDS to monitor supersaturating solutions was evaluated by adding excess amount of LPD powder to buffers at different pH values. Figure 7 and 8 show examples of LPD dissolution behavior at pH 8.0 and 6.5. Compound specific potentiometric sensors can be used for real time concentration monitoring of ionizable organic compounds often replacing time consuming HPLC methods 1 . Theoretically, such free drug sensors (FDS) are supposed to detect only to Table 1 was added to the corresponding donor chamber of μFLUX system and topped up with 19.2 mL of FaSSIF blank buffer . different pH values. Figure 7 and 8 show examples of LPD dissolution behavior at pH 8.0 and 6.5. FSD real-time detection showed that during a short supersaturation period the concentration of LPD reached 5 - 10 times often replacing time consuming HPLC methods 1 . Theoretically, such free drug sensors (FDS) are supposed to detect only dissolved molecules since large complexes like nanoparticles cannot penetrate the sensors membrane. The purpose of this FSD real-time detection showed that during a short supersaturation period the concentration of LPD reached 5 - 10 times higher than its equilibrium solubility even without nanoparticle formulation. The supersaturation followed by precipitation after Table 1. Formulations used in the Flux experiment dissolved molecules since large complexes like nanoparticles cannot penetrate the sensors membrane. The purpose of this study was to determine applicability and limitations of this potentiometric technique to measure free drug concentration in the about 20 min (Figure 7). This behavior was confirmed by in situ UV concentration monitoring (Figure 8). Table 1. Formulations used in the Flux experiment presence of nanoparticles. Weight of LPD in Weight of Excipient Volume of Load of LPD in MATERIALS AND METHODS LPD Dissolution-Precipitation, pH 8.0 LPD Dissolution-Precipitation, pH 6.5 LPD in mixture Excipient in mixture Weight of Volume of FaSSIF blank Load of LPD in Suspension MATERIALS AND METHODS 25 LPD Dissolution-Precipitation, pH 8.0 1000 LPD Dissolution-Precipitation, pH 6.5 Formulation (mg) (mg) mixture blank (mL) (mg/mL) LPD HCl PW 25.02 N/A 25.02 2 12.51 Nanoparticle powders of loperamide HCl (LPD, Figure 1) 20 25 800 900 1000 LPD HCl PW 25.02 N/A 25.02 2 12.51 LPD Kollidon Nano 25.02 25.02 50.05 2 12.51 were made using a multi-jet, multi-nozzle ElectroNanospray™ system (ENS, Nanocopoeia, LLC, Loperamide 15 20 mL 600 700 800 mL LPD Kollidon Nano 25.02 25.02 50.05 2 12.51 LPD Soluplus Nano 25.15 25.15 50.30 2 12.58 ElectroNanospray™ system (ENS, Nanocopoeia, LLC, Figure 2). Solutions of drug with either SoluPlus® or N Cl 10 15 nc µg/m 400 500 600 nc µg/m LPD +Soluplus Mixture 25.30 25.34 50.64 2 12.65 Figure 2). Solutions of drug with either SoluPlus® or Kollidon® K17 (1% w/v each) in isopropanol:CH 2 Cl 2 (10:90 v/v) were fed at 100 μL/min/nozzle and then stored in N N O 40 5 10 Con 200 300 400 Con The slope of concentration-time profiles in the receiver compartment can be used to calculate the flux of LPD v/v) were fed at 100 μL/min/nozzle and then stored in sealed vials until use. Particle size of dry and water- Base: pK a 8.70; logP 3.90 N OH 35 40 LPD PWD 0 5 0 100 200 compartment can be used to calculate the flux of LPD through artificial membrane at particular time point. As Figure 1. Chemical structure and physico-chemical properties of LPD sealed vials until use. Particle size of dry and water- suspended ENS powders was assessed by SEM imaging a Figure 2. Multi-jet nozzle with cone-jet spray plumes. 30 35 LPD-PVP Nanosusp 0 0 5 10 15 20 25 30 35 40 Time, min 0 0 10 20 30 40 50 60 70 80 Time, min through artificial membrane at particular time point. As evident from Figure 11, flux of LPD from physico-chemical properties of LPD and laser diffraction, respectively (Figure 3a,b). cone-jet spray plumes. 25 30 LPD-Soluplus Nanosusp Time, min Time, min nanosuspension with Soluplus was the highest and it was ~ 2 times higher than one from LPD powder . Flux Free Drug Sensors (FDS, Pion Inc., Figure 4) 100 nm (D50) 20 25 LPD+Soluplus Mixture Figure 7. Concentration-time profile of LPD measured in situ by FDS: 4 mg of LPD powder was added to 20 mL of pH 8.0 buffer (a); 20 mg of LPD added to 20 mL of FaSSIF blank buffer at pH 6.5 (b). was ~ 2 times higher than one from LPD powder . Flux from nanosuspension of LPD-Kollidon K17 and from Free Drug Sensors (FDS, Pion Inc., Figure 4) were conditioned in the solution of LPD at the pH 100 nm (D50) 15 20 added to 20 mL of FaSSIF blank buffer at pH 6.5 (b). from nanosuspension of LPD-Kollidon K17 and from the physical mixture of LPD with Soluplus were comparable in value and both were higher than flux value where LPD was fully ionized. After conditioning sensors became specific to the drugs 10.9 μm 81.0 m 10 comparable in value and both were higher than flux from LPD powder . Flux values for the initial period (30 conditioning sensors became specific to the drugs they had been conditioned with and could be used 81.0 μm 5 from LPD powder . Flux values for the initial period (30 – 60 min) and closer to the end of the assay (120 – 180 min) are compiled in the Table 2. they had been conditioned with and could be used to measure concentration of these compounds in situ through monitoring of the change in mV 0 0 50 100 150 200 250 180 min) are compiled in the Table 2. situ through monitoring of the change in mV response of the sensors in different Figure 3. Particle size distribution of loperamide- SoluPlus ENS powders (a) by SEM of dry powder 0 50 100 150 200 250 response of the sensors in different concentrations of LPD. SoluPlus ENS powders (a) by SEM of dry powder showing diameters in fractional μm and (b) by laser diffraction 60 min after suspension in water. Figure 11. Concentration-time profile of LPD in receiver compartment of μFLUX system when introduced as 0.5 mg/mL load from different forms . diffraction 60 min after suspension in water. Table 2. Flux calculated from the slopes of the curves on Figure 11. when introduced as 0.5 mg/mL load from different forms . Flux, µg/(min·cm2) Formulation 30 - 60 min 120 - 180 min LPD PWD 1.18 0.92 LPD PWD 1.18 0.92 LPD-PVP Nano 1.46 1.25 Figure 8. Concentration-time profile of LPD measured in situ by FO UV. Loads correspond to ones described on Figure 7. LPD-PVP Nano 1.46 1.25 LPD-Soluplus Nano 2.17 2.03 Figure 8. Concentration-time profile of LPD measured in situ by FO UV. Loads correspond to ones described on Figure 7. LPD-Soluplus Nano 2.17 2.03 LPD+Soluplus Mixture 1.39 1.61 CONCLUSIONS Solubility of LPD in the presence of Soluplus® It was shown that FDS could be used in measuring in situ concentration of ionizable compounds in the presence of Solubility of LPD in the presence of Soluplus® Figure 5. A schematic of the μFLUX apparatus showing a pair of It was shown that FDS could be used in measuring in situ concentration of ionizable compounds in the presence of nanoparticles avoiding complicated solid separation methods. LPD nanoparticles seemed to dissolve completely in the water-based nanosuspension at concentration up to 5 mg/mL. Figure 5. A schematic of the μFLUX apparatus showing a pair of the donor and receiver chambers. FO probes attached to the μDISS Profiler monitor concentrations in the donor (left) and Figure 4. Free Drug Sensor and nanoparticles avoiding complicated solid separation methods. This technique can be a powerful addition to in situ UV concentration monitoring in cases when particulate light scattering When suspension (or solution) was added to the buffer LPD was released almost immediately, with a kinetic solubility that reached more than 80 μg/mL (at pH 8.0) before LPD began precipitating from the solution. μDISS Profiler monitor concentrations in the donor (left) and receiver (right) compartments. The chambers can be separated by artificial, cell-based size exclusion, or other types of membranes Figure 4. Free Drug Sensor and Reference Electrode in the solution. This technique can be a powerful addition to in situ UV concentration monitoring in cases when particulate light scattering and additional absorption make spectroscopic measurements difficult. reached more than 80 μg/mL (at pH 8.0) before LPD began precipitating from the solution. One of the outcome of the study was the fact that in the presence of 50% wt/wt of Soluplus the solubility of LPD increased artificial, cell-based size exclusion, or other types of membranes mounted in the Membrane Holder. The limitation of drug selective electrode technology as any potentiometric based technique is that compound must be charged in the media of the interest. One of the outcome of the study was the fact that in the presence of 50% wt/wt of Soluplus the solubility of LPD increased up to 5 mg/mL in the aqueous medium regardless whether it was nanosized or not. Both techniques confirmed that LPD powder got dissolved up to 5 mg/mL level when 5 mg/mL of Soluplus was in the background (Figures 9 and 10) μFLUX Profiler™ fitted with μFLUX apparatus (Pion Inc., Figure 5) was used to measure simultaneously dissolution characteristics (donor chamber) and concentration of LPD permeating through artificial gastro-intestinal tract mimicking charged in the media of the interest. Nano-formulation Soluplus® provided superior solubilization for LPD that also manifested itself in the increased flux. powder got dissolved up to 5 mg/mL level when 5 mg/mL of Soluplus was in the background (Figures 9 and 10) characteristics (donor chamber) and concentration of LPD permeating through artificial gastro-intestinal tract mimicking membranes to the receiver chamber . Nano-formulation Soluplus® provided superior solubilization for LPD that also manifested itself in the increased flux. Additional study should be performed to investigate interference of lipophilic and charged media or formulation components membranes to the receiver chamber . Each pair (Figure 5) of a donor and an receiver compartment were separated by a filter-supported artificial membrane (Double- Additional study should be performed to investigate interference of lipophilic and charged media or formulation components on the measurements. Sink™ PAMPA 2 ) with 1.5 cm 2 surface area. The donor compartment was filled with 20 mL of the media of interest while the receiver compartment contained the same volume of Acceptor Sink Buffer at pH 7.4 (ASB-7.4, Pion Inc). The integrated fiber- on the measurements. receiver compartment contained the same volume of Acceptor Sink Buffer at pH 7.4 (ASB-7.4, Pion Inc). The integrated fiber- optic UV probes were positioned in the donor and receiver compartments allowing real time concentration monitoring in all y = 54.211x + 303.99 500 optic UV probes were positioned in the donor and receiver compartments allowing real time concentration monitoring in all chambers. Zero Intercept Method (ZIM) 3 analysis was performed using Au PRO™ software version 5.1 (Pion). REFERENCES After Centrifugation R² = 0.9992 y = 51.455x + 309.43 480 RESULTS AND DISCUSSION REFERENCES After Centrifugation y = 51.455x + 309.43 R² = 0.9987 460 V RESULTS AND DISCUSSION 1. Bohets H. Development of in situ ion selective sensors for dissolution. Analytica Chimica Acta 581 (2007) 181–191. 440 mV In Situ Concentration Measurements using Free Drug Sensors (FDS™) 1. Bohets H. Development of in situ ion selective sensors for dissolution. Analytica Chimica Acta 581 (2007) 181–191. 2. A. Avdeef, O. Tsinman. PAMPA—A drug absorption in vitro model. 13. Chemical selectivity due to membrane hydrogen bonding: In combo 440 Std curve in DI+Soluplus_uncorrected In Situ Concentration Measurements using Free Drug Sensors (FDS™) comparisons of HDM-, DOPC-, and DS-PAMPA models. Eur. J. Pharm Sci. 2006, 28 (1), 43-59. 3. K. Tsinman, O. Tsinman, B. Riebesehl, and M. Juhnke. Comparison of Naproxen Release from Nano- and Micro-Suspensions with Its 420 Std curve in DI+Soluplus_uncorrected Std curve in DI_Soluplus_corrected on 2 mg/mL std Figure 6 shows standard curve for LPD that was used to translate mV response of FDS into concentration information. It has to be noted that unlike FDS Standard Curve 3. K. Tsinman, O. Tsinman, B. Riebesehl, and M. Juhnke. Comparison of Naproxen Release from Nano- and Micro-Suspensions with Its Dissolution from Untreated and Micronized Powder. Poster presented at AAPS Annual Meeting and Exposition. October 14 -18, 2012, Chicago, IL. 400 2.3 2.8 3.3 3.8 response of FDS into concentration information. It has to be noted that unlike linear proportionality of UV absorbance and concentration, potentiometric y = 62.763x + 299.15 370 380 mV IL. 2.3 2.8 3.3 3.8 logC (µg/mL) linear proportionality of UV absorbance and concentration, potentiometric response is linearly proportional to the logarithm of concentration. The R² = 0.99 350 360 onse, m Figure 9. Serial addition of LPD stock solution to the water-based media containing 5 mg/mL of Soluplus. Determined concentration by UV was in Figure 10. Standard curve collected using FDS in water-based media containing 5 mg/mL of Soluplus in the background showed no evidence of standard curve was then used to convert dynamic in situ millivolt response of FDS into concentration allowing real time monitoring of LPD in the presence 330 340 Respo line with amount of LPD added. containing 5 mg/mL of Soluplus in the background showed no evidence of LPD precipitation FDS into concentration allowing real time monitoring of LPD in the presence of nanoparticles. 310 320 FDS of nanoparticles. 0.2 0.4 0.6 0.8 1 1.2 log Conc (µg/mL) log Conc (µg/mL) Figure 6. Standard curve (FDS mV response versus logarithm of concentration) from adding of pre-dissolved logarithm of concentration) from adding of pre-dissolved LPD into pH 8.0 buffer.