- Email: [email protected]
Imaging of in vitro parenteral drug precipitation
International Journal of Pharmaceutics 512 (2016) 219–223
Contents lists available at ScienceDirect
International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Imaging of in vitro parenteral drug precipitation Daniel C. Evansa,* , Michael W. Kudenovb,** , Kimberly C. Sassenratha , Eustace L. Dereniakb , Samuel H. Yalkowskya a b
College of Pharmacy, University of Arizona, 1295 N. Martin PO Box 210202 Tucson, AZ 85721, United States College of Optical Sciences, The University of Arizona, 1630 E. University Blvd., P.O. Box 210094, Tucson, AZ 85721-0094, United States
A R T I C L E I N F O
Article history: Received 12 April 2016 Received in revised form 27 July 2016 Accepted 13 August 2016 Available online 16 August 2016
A B S T R A C T
Solid particulate matter introduced into the bloodstream as a result of parenteral drug administration can produce serious pathological conditions. Particulate matter that cannot be eliminated by pre-infusion filtration is often the result of drug precipitation that occurs when certain parenteral formulations are mixed with blood. A new device is designed to model the mixing of drug formulations with flowing blood utilizing a uniquely designed flow cell and a CCD camera to view the formulation as it is mixed with a blood surrogate in real time. The performance of the proposed device is measured using 3 commercially available parenteral formulations previously tested using a validated in vitro model. ã 2016 Elsevier B.V. All rights reserved.
1. Introduction Particulate matter introduced into the bloodstream while administering a parenteral drug can cause inflammation of both venous cell walls and organs in which the particles are embedded (Barber, 2000). The physiological effect of these particles can range from mild irritation at the site of injection to more serious life threatening formation of emboli (Turco and King, 1979). To eliminate particulate matter in parenteral formulations prior to administration, filtration is used. Filtration, however, cannot remove particulates resulting from drug precipitation that occurs when certain parenteral formulations are mixed with blood. This particulate matter from drug precipitation is especially problematic because it can alter the bioavailability of parenteral drugs and exposed venous endothelial cells to high drug concentrations (Powis and Kovach, 1983). In vitro methods used to optimize drug formulations are important tools for saving time and money in the drug development process (Dai, 2010). The first in vitro model used to screen parenteral drugs for their potential to produce particulate matter
* Correspondence to: NuvOx Pharma, 1635 E. 18th Street, Tucson, AZ 85704, United States. ** Correspondence to: Electrical and Computer Engineering, North Carolina State University, 437 Monteith. Campus Box 7914, NC State University, Raleigh, NC 276957914, United States. E-mail addresses: [email protected] (D.C. Evans), [email protected] (M.W. Kudenov), [email protected] (K.C. Sassenrath), [email protected] (E.L. Dereniak), [email protected] (S.H. Yalkowsky). http://dx.doi.org/10.1016/j.ijpharm.2016.08.030 0378-5173/ã 2016 Elsevier B.V. All rights reserved.
upon administration was developed by Schroeder and DeLuca (1973). Shortly after this, a more realistic model was developed by Yalkowsky and Valvani (Yalkowsky and Valvani, 1977), which was later improved upon and validated (Yalkowsky et al., 1983; Johnson and Yalkowsky, 2003). This model simulates blood flow using a peristaltic pump that directs a blood surrogate (Isotonic Sorensen’s Phosphate buffer, pH 7.4(ISPB)) through Tygon1 tubing into a spectrophotometer flow cell. Parenteral formulations are injected at specified rates into the flowing blood surrogate using an IV Y-site and a syringe pump. If drug precipitation occurs, the resultant turbid solution will enter the flow cell and decrease the amount of transmitted light detected by the spectrophotometer. While this model gives a good indication of the amount of precipitate produced by the formulation administered at a specific injection rate, it does not provide any information on where, with respect to the needle tip, crystallization occurs. Additionally, the spectrophotometer is unable to distinguish between drug precipitation and sources of false positives such as air bubbles and Schlieren patterns, which are caused by the mixing of fluids with different indices of refraction. A new in vitro device was designed to obtain more information about drug precipitation and the dynamics of injecting formulation into a flowing blood surrogate. 2. Materials and methods 2.1. Device design The flow cell that was used to image the needle tip during the dynamic precipitation experiments was fabricated using
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D.C. Evans et al. / International Journal of Pharmaceutics 512 (2016) 219–223
Fig. 1. Illustration of the flow cell used to image parenteral drugs injected into ISPB.
commercially available quartz glass tubing. The core of this flow cell (illustrated in Fig. 1) is a round quartz tube (length = 36 cm) with an inner diameter (ID) of 4 mm which is within the observed diameter of the forearm Basilic vein (Baptista-Silva et al., 2003). The round quartz tube was attached to Tygon1 tubing using an IV Y-site connector (front) and a polytetrafluoroethylene (PTFE) fitting (back). This tube was placed inside a larger square quartz tube (length = 30.4 cm ID = 10 mm), and sealed using a thermoplastic polymer. The space between the inner and outer tubes was filled with polyethylene glycol 200 (PEG-200), which has a similar refractive index (1.4585 (Malitson, 1965)) as quartz glass. This outer quartz jacket and PEG-200 cosolvent reduced the amount of reflected and refracted light off the curved surface of the inner quartz tube and increased the sensitivity of the in vitro device. The flow cell is illuminated with two flexible fiber optic light guides, powered by a Vlux 1000 light source. The light guides are positioned above and at a slight angle normal to the top of the flow cell. Scattered light caused by drug precipitation was measured with a monochrome charge-coupled device (CCD) camera (The Imaging Source1 DMK 41BUO2) attached to a Nikon AF Nikkor 28–80 mm SLR lens, using a 21 mm lens tube. The camera was positioned on one side of the flow cell 90 degrees from the fiber optic light guides. Both the camera and the light guides were attached to a linear motion system constructed from T-slot aluminum extrusion. This allowed us to determine the amount of drug precipitation at the needle tip and 12 cm downstream of the needle tip.
the drug reported in literature (Sw) and the predicted solubility of the drug in the formulation solution (St). Amiodarone hydrochloride and phenytoin sodium have been reported to show significant amounts of precipitation when mixed with ISPB while furosemide did not. Triplicate formulations were prepared for each drug according to the information provided in the package inserts or in the Handbook of Injectable Drugs (Trissel, 1994). ISPB was prepared according to instructions listed in the Documenta Geigy Scientific Tables (Geigy, 1970). All formulations were filtered with a 0.45 mm PTFE Acrodisc1 syringe filter and the ISPB was filtered with 0.45 mm Nalgene1 nylon filter system. Images were obtained using IC Capture (The Imaging Source1) at 1 frame per second. Formulations were injected for 60 s at 1 mL/min and 5 mL/min into the flowing (5 mL/ min) ISPB blood surrogate (25 2 C). Additionally, the phenytoin sodium formulations were injected at 1 mL/min into ISPB flowing at 15 mL/min to test the effect of higher blood surrogate flow rates. For each product tested, a placebo formulation, which contained only the inactive ingredients, was also injected onto the device. These images were used to subtract background noise from images taken during the injection of formulations containing the API. The phenytoin placebo was also injected with a blue food dye to illustrate the dynamics associated with the mixing of two fluids. All image processing was done using Matlab’s1 Image Processing Toolbox.
2.2. Parenteral formulations
Fig. 2 shows images of the needle tip (left) and 12 cm downstream (right) during the 1 mL/min injection of amiodarone HCl (top), furosemide (middle) and phenytoin sodium (bottom) formulations. Images taken near the needle tip show that injecting formulations of amiodarone and phenytoin into the flowing ISPB produced a significant amount of drug precipitation immediately
Three commercially available parenteral drug formulations, listed in Table 1, were chosen to test the new in vitro device based on the results of the validation study done by Johnson and Yalkowsky (2003). Table 1 also includes the aqueous solubility of
3. Results
Table 1 Commercially available formulations tested on in vitro device. API
Sw (mg/mL)
Sta (mg/mL)
Dose (mg/ mL)
Formulation Contents
Amiodarone HCl
<1.3e-6 (Bergström et al., 2004) 0.018 (Granero et al., 2010)
3.5 (Alvarez-Núñez and Yalkowsky, 2000; Li et al., 1999) 18 (Granero et al., 2010)
1.5b
0.018 (Schwartz et al., 1977)
88 (Millard et al., 2002; Li et al., 1999)
50
Each mL of solution contains 50 mg amiodarone HCl, 20.2 mg benzyl alcohol, 100 mg of polysorbate 80, and water for injection (pH = 3.5–4.5) Hospira (2016a) Each mL of solution contains 10 mg furosemide HCl, sodium chloride for tonicity adjustment, and enough sodium hydroxide/hydrochloric acid to adjust the pH to 9.0 (Hospira, 2016b) Each mL of solution contains 50 mg phenytoin Na, 0.4 mL propylene glycol, 0.1 mL ethanol, and water for injection with sodium hydroxide for pH adjustment to 10–12.3 (Pharmaceuticals,2016)
Furosemide
Phenytoin Sodium
a b
10
Predicted solubility. 3 mL of 50 mg/mL solution diluted with 100 mL of 5% dextrose solution.
D.C. Evans et al. / International Journal of Pharmaceutics 512 (2016) 219–223
221
Fig. 2. Images taken near the needle tip (left) and 12 cm downstream of the needle tip (right) during the 1 mL/min injection of amiodarone (top), furosemide (middle) and phenytoin (bottom) after background subtraction.
upon mixing with the ISPB blood surrogate. Downstream of the needle tip, as mixing between these two drug formulations and the ISPB becomes more complete the amount of precipitation increases. Unlike the precipitation of amiodarone, the turbidity in the flow cell caused by injecting the formulation containing phenytoin was less uniform due to the formation of large needleshaped crystals. This effect became more pronounced as the formulation moved downstream due to the increased size of the crystals. The downstream precipitation of amiodarone presented as a suspension of tiny particulates that grew in number but not noticeably in size. Images taken at the needle tip and 12 cm downstream during the injection of formulations containing furosemide show no light scattering caused by drug precipitation at either injection rate. To obtain a quantitative measure of precipitation, the mean grayscale intensity was determined for images taken during the injection of all three formulations at each injection rate and image location. These intensity values, summarized as bar plots in Fig. 3, were obtained after background subtraction and range from 0 (no turbidity) to 1, which represents complete saturation of the camera by the scattered light from a turbid solution.
Injections of amiodarone at 1 mL/min produced a small amount of precipitation near the needle tip that increased in intensity as the formulation moved further down the flow cell. As Fig. 2 shows, the area of the flow cell occupied by this turbid solution remained constant as the formulation moved downstream of the needle tip for the 1 mL/min injection rate. Increasing the injection rate of amiodarone from 1 mL/min to 5 mL/min did not significantly change the intensity of drug precipitation or the precipitation area near the needle tip. Downstream of the needle tip, however, the precipitation of amiodarone became more evenly distributed throughout the flow cell, while the intensity of the turbid solution decreased at the higher flow rate. Even at the higher injection rate the precipitation of amiodarone moved down the length of the flow cell as a turbid suspension without producing larger crystalline particles, such as those produced by the formulation containing phenytoin. At the needle tip, injecting phenytoin sodium into the ISPB produced a significant amount of precipitation that increased in both area and intensity as the injection rate was increased from 1 mL/min to 5 mL/min. At both the 1 mL/min and 5 mL/min injection rates the precipitation intensity for phenytoin
Fig. 3. Plots of the mean grayscale intensity, used as a measure of the amount of precipitation, for Amiodarone, Furosemide, and Phenytoin.
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D.C. Evans et al. / International Journal of Pharmaceutics 512 (2016) 219–223
Fig. 4. Time-lapsed images taken during the injection (1 mL/min) of phenytoin sodium (50 mg/mL) into the phosphate buffered saline flowing at 15 mL/min. Time points of the images are 0 min (top left), 1 min (top right), 3 min (bottom left), and 5 min (bottom right).
increased as the formulation moved downstream of the needle tip. Downstream of the needle tip, the area of the flow cell occupied by precipitation increased for the 1 mL/min injection rate however during the 5 mL/min injection the area decreased as the turbid solution becomes concentrated towards the bottom of the flow cell. As previously mentioned, furosemide did not show any precipitation at either injection rate or image location. The results obtained in this study are in general agreement with those obtained in the validation study published by Johnson and Yalkowsky (2003). In this validation study the turbidity detected by UV spectrophotometry was averaged over three different injection rates (5, 10, and 15 mL/min) and reported as the average opacity (AO). Formulations of phenytoin/Dilantin (AO = 1.196) produced more precipitation than formulations containing amiodarone/Cordarone (AO = 0.315) while formulations containing furosemide did not produce any precipitation (AO = 0.0). Interestingly, Johnson and Yalkowsky observed that injections of
phenytoin at 5 mL/min gave a lower opacity reading than injections at 1 mL/min whereas in our study the opposite was observed. This difference can be reconciled by the differences in the fluid dynamic characteristics of the flow cell used in the two studies. The ISPB flow rate of 5 mL/min, which was used in this study and in the validation study done by Johnson, represents a worst case scenario that may be encountered in patients with compromised blood flow. Forearm blood flow rates recorded in literature for healthy patients however, range from 7 to 268 mL/ min (Longhurst et al., 1974). To determine what effect the ISBP flow rate would have on the results obtained from in vitro parenteral drug testing, phenytoin sodium was injected at/min into ISPB flowing at 15 mL/min. Time-lapsed images taken during this injection (Fig. 4) show the formation of needle-shaped crystals that, over time, cover the surface of the tip of the needle. The effect of the higher ISPB flow rate was simply to move most of the observable precipitation downstream.
Fig. 5. Time-lapsed images taken during the injection of a phenytoin placebo formulation containing bromophenol blue at 0 (top left), 8 (top right), 13 (bottom left), and 24 (bottom right) seconds after injection to illustrate the effect of eddies (vortices) on the fluid dynamics of a simulated in vitro injection.
D.C. Evans et al. / International Journal of Pharmaceutics 512 (2016) 219–223
One unexpected observation during the injection of phenytoin was the presence of crystals on the back of the needle tip and upstream of the needle opening. These crystals are the result of vortices, or eddies, that occur as a result of the ISPB flow past the needle. To better illustrate these fluid dynamics, Fig. 5 shows the injection of a blue dye mixed with the phenytoin placebo formulation. The vortices and “areas of recirculation” observed in our experiments, as well as in fluid dynamic experiments using catheters (Rotman et al., 2013), are the result of fluid moving past an obstruction (needle tip). While the fluid dynamics in the in vitro device presented in this paper do not precisely match the fluid dyamics in the cephalic or basic vein, a needle or catheter placed in the flowing blood can be expected to cause the formation of eddies, or vortices. This fact may explain why formulations associated with precipitation during administration such as phenytoin and amiodarone are associated with a higher incidence of phlebitis or skin irritation at the site of injection. 4. Conclusion The new in vitro device to predict drug precipitation upon administration yielded valuable qualitative and quantitative information useful for optimizing parenteral formulations. The flow cell designed for this study allows detection of drug precipitation at the site of injection where clinical symptoms of infusion related injuries are often observed. Having a photo record of the experiment allows the user to observe the performance of the formulation after the experiment has concluded. This feature, along with image processing techniques, also allows the user to minimize sources of false positive readings such as air bubbles and Schlieren patterns caused by the mixing of fluids with different indices of refraction. Finally the observed fluid dynamics of injection may explain how particulates present at the needle tip may cause local injury of venous cell walls clinically observed as phlebitis. References Alvarez-Núñez, F.A., Yalkowsky, S.H., 2000. Relationship between Polysorbate 80 solubilization descriptors and octanol–water partition coefficients of drugs. Int. J. Pharma. 200 (2), 217–222.
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Baptista-Silva, J., Dias, A., Cricenti, S., Burihan, E., 2003. Anatomy of the Basilic Vein in the Arm and its Importance for Surgery. Braz. J. Morphol. Sci. 20 (3), 171–175. Barber, T.A., 2000. Control of Particulate Matter Contamination in Healthcare Manufacturing, 1st ed. Interpharm Press, Englewood. Bergström, C.A.S., Luthman, K., Artursson, P., 2004. Accuracy of calculated pHdependent aqueous drug solubility. Eur. J. Pharm. Sci. 22 (5), 387–398. Dai, W.-G., 2010. In vitro methods to assess drug precipitation. Int. J. Pharm. 393 (1–2), 1–16. Geigy, D., 1970. In: Geigy, J.R., Ardsley, S.A. (Eds.), Scientific Tables, 7th ed. . Granero, G.E., Longhi, M.R., Mora, M.J., Junginger, H.E., Midha, K.K., Shah, V.P., Stavchansky, S., Dressman, J.B., Barends, D.M., 2010. Biowaiver monographs for immediate release solid oral dosage forms: furosemide. J. Pharm. Sci. 99 (6), 2544–2556. Hospira. Products and Services > Drugs > Amiodarone Hydrochloride Injection. https://www.hospira.com/en/products_and_services/drugs/ AMIODARONE_HYDROCHLORIDE (accessed 23.07.16). Hospira. Products and Services > Drugs > Furosemide Injection, USP. https://www. hospira.com/en/products_and_services/drugs/FUROSEMIDE_INJECTION (accessed 23.07.16). Johnson, J.L., Yalkowsky, S.H., 2003. Prediction of precipitation-induced phlebitis: a statistical validation of an in-vitro model. J. Pharm. Sci. 92, 1574–1581. Li, P., Tabibi, S.E., Yalkowsky, S.H., 1999. Solubilization of flavopiridol by pH control combined with cosolvents, surfactants, or complexants. J. Pharm. Sci. 88 (9), 945–947. Longhurst, J., Capone, R.J., Mason, D.T., Zelis, R., 1974. Comparison of blood flow measured by plethysmograph and flowmeter during steady state forearm exercise. Circulation 49, 535–540. Malitson, I.H., 1965. Interspecimen comparision of the refractive index of fused silica. J. Opt. Soc. Am. 55 (10), 1205–1208. Millard, J.W., Alvarez-Núñez, F.A., Yalkowsky, S.H., 2002. Solubilization by cosolvents: establishing useful constants for the log-linear model. Int. J. Pharm. 245 (1–2), 153–166. Pharmaceuticals, W.-W. West-Ward Pharmaceuticals > Products > Phenytoin Sodium Injection, USP. http://www.west-ward.com/en/Products/ProductsRoot/ Phenytoin%20Sodium%20Injection%20USP (accessed 23.07.16). Powis, G., Kovach, J.S., 1983. Disposition of bisantrene in humans. Cancer Res. 43, 925–929. Rotman, O.M., Shav, D., Raz, S., Zaretsky, U., Einav, S., 2013. Biomechanical aspects of catheter-related thrombophlebitis. J. Biomed. Sci. Eng. 6, 6–13. Schroeder, H.G., DeLuca, P.B., 1973. A study on the in vitro precipitation of poorly soluble drugs from nonaqueous vehicles in human plasma. Bull. Parenter. Drug Assoc. 28, 1–14. Schwartz, P.A., Rhodes, C.T., Cooper, J.W., 1977. Solubility and ionization characteristics of phenytoin. J. Pharm. Sci. 66 (7), 994–997. Trissel, L., 1994. Handbook of Injectable Drugs, 8th ed. American Society of HealthSystem Pharmacists, Bethesda. Turco, S.J., King, R.E., 1979. Sterile Dosage Forms: Their Preparation and Clinical Application, 2th ed. Lea & Febiger, Philidelphia. Yalkowsky, S.H., Valvani, S.C., 1977. Precipitation of solubilized drugs due to injection or dilution. Drug Intell. Clin. Pharm. 11, 417–419. Yalkowsky, S.H., Valvani, S.C., Johnson, B.J., 1983. In vitro method for detecting precipitation of parenteral formulations after injection. J. Pharm. Sci. 72, 1014– 1017.
Contents lists available at ScienceDirect
International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Imaging of in vitro parenteral drug precipitation Daniel C. Evansa,* , Michael W. Kudenovb,** , Kimberly C. Sassenratha , Eustace L. Dereniakb , Samuel H. Yalkowskya a b
College of Pharmacy, University of Arizona, 1295 N. Martin PO Box 210202 Tucson, AZ 85721, United States College of Optical Sciences, The University of Arizona, 1630 E. University Blvd., P.O. Box 210094, Tucson, AZ 85721-0094, United States
A R T I C L E I N F O
Article history: Received 12 April 2016 Received in revised form 27 July 2016 Accepted 13 August 2016 Available online 16 August 2016
A B S T R A C T
Solid particulate matter introduced into the bloodstream as a result of parenteral drug administration can produce serious pathological conditions. Particulate matter that cannot be eliminated by pre-infusion filtration is often the result of drug precipitation that occurs when certain parenteral formulations are mixed with blood. A new device is designed to model the mixing of drug formulations with flowing blood utilizing a uniquely designed flow cell and a CCD camera to view the formulation as it is mixed with a blood surrogate in real time. The performance of the proposed device is measured using 3 commercially available parenteral formulations previously tested using a validated in vitro model. ã 2016 Elsevier B.V. All rights reserved.
1. Introduction Particulate matter introduced into the bloodstream while administering a parenteral drug can cause inflammation of both venous cell walls and organs in which the particles are embedded (Barber, 2000). The physiological effect of these particles can range from mild irritation at the site of injection to more serious life threatening formation of emboli (Turco and King, 1979). To eliminate particulate matter in parenteral formulations prior to administration, filtration is used. Filtration, however, cannot remove particulates resulting from drug precipitation that occurs when certain parenteral formulations are mixed with blood. This particulate matter from drug precipitation is especially problematic because it can alter the bioavailability of parenteral drugs and exposed venous endothelial cells to high drug concentrations (Powis and Kovach, 1983). In vitro methods used to optimize drug formulations are important tools for saving time and money in the drug development process (Dai, 2010). The first in vitro model used to screen parenteral drugs for their potential to produce particulate matter
* Correspondence to: NuvOx Pharma, 1635 E. 18th Street, Tucson, AZ 85704, United States. ** Correspondence to: Electrical and Computer Engineering, North Carolina State University, 437 Monteith. Campus Box 7914, NC State University, Raleigh, NC 276957914, United States. E-mail addresses: [email protected] (D.C. Evans), [email protected] (M.W. Kudenov), [email protected] (K.C. Sassenrath), [email protected] (E.L. Dereniak), [email protected] (S.H. Yalkowsky). http://dx.doi.org/10.1016/j.ijpharm.2016.08.030 0378-5173/ã 2016 Elsevier B.V. All rights reserved.
upon administration was developed by Schroeder and DeLuca (1973). Shortly after this, a more realistic model was developed by Yalkowsky and Valvani (Yalkowsky and Valvani, 1977), which was later improved upon and validated (Yalkowsky et al., 1983; Johnson and Yalkowsky, 2003). This model simulates blood flow using a peristaltic pump that directs a blood surrogate (Isotonic Sorensen’s Phosphate buffer, pH 7.4(ISPB)) through Tygon1 tubing into a spectrophotometer flow cell. Parenteral formulations are injected at specified rates into the flowing blood surrogate using an IV Y-site and a syringe pump. If drug precipitation occurs, the resultant turbid solution will enter the flow cell and decrease the amount of transmitted light detected by the spectrophotometer. While this model gives a good indication of the amount of precipitate produced by the formulation administered at a specific injection rate, it does not provide any information on where, with respect to the needle tip, crystallization occurs. Additionally, the spectrophotometer is unable to distinguish between drug precipitation and sources of false positives such as air bubbles and Schlieren patterns, which are caused by the mixing of fluids with different indices of refraction. A new in vitro device was designed to obtain more information about drug precipitation and the dynamics of injecting formulation into a flowing blood surrogate. 2. Materials and methods 2.1. Device design The flow cell that was used to image the needle tip during the dynamic precipitation experiments was fabricated using
220
D.C. Evans et al. / International Journal of Pharmaceutics 512 (2016) 219–223
Fig. 1. Illustration of the flow cell used to image parenteral drugs injected into ISPB.
commercially available quartz glass tubing. The core of this flow cell (illustrated in Fig. 1) is a round quartz tube (length = 36 cm) with an inner diameter (ID) of 4 mm which is within the observed diameter of the forearm Basilic vein (Baptista-Silva et al., 2003). The round quartz tube was attached to Tygon1 tubing using an IV Y-site connector (front) and a polytetrafluoroethylene (PTFE) fitting (back). This tube was placed inside a larger square quartz tube (length = 30.4 cm ID = 10 mm), and sealed using a thermoplastic polymer. The space between the inner and outer tubes was filled with polyethylene glycol 200 (PEG-200), which has a similar refractive index (1.4585 (Malitson, 1965)) as quartz glass. This outer quartz jacket and PEG-200 cosolvent reduced the amount of reflected and refracted light off the curved surface of the inner quartz tube and increased the sensitivity of the in vitro device. The flow cell is illuminated with two flexible fiber optic light guides, powered by a Vlux 1000 light source. The light guides are positioned above and at a slight angle normal to the top of the flow cell. Scattered light caused by drug precipitation was measured with a monochrome charge-coupled device (CCD) camera (The Imaging Source1 DMK 41BUO2) attached to a Nikon AF Nikkor 28–80 mm SLR lens, using a 21 mm lens tube. The camera was positioned on one side of the flow cell 90 degrees from the fiber optic light guides. Both the camera and the light guides were attached to a linear motion system constructed from T-slot aluminum extrusion. This allowed us to determine the amount of drug precipitation at the needle tip and 12 cm downstream of the needle tip.
the drug reported in literature (Sw) and the predicted solubility of the drug in the formulation solution (St). Amiodarone hydrochloride and phenytoin sodium have been reported to show significant amounts of precipitation when mixed with ISPB while furosemide did not. Triplicate formulations were prepared for each drug according to the information provided in the package inserts or in the Handbook of Injectable Drugs (Trissel, 1994). ISPB was prepared according to instructions listed in the Documenta Geigy Scientific Tables (Geigy, 1970). All formulations were filtered with a 0.45 mm PTFE Acrodisc1 syringe filter and the ISPB was filtered with 0.45 mm Nalgene1 nylon filter system. Images were obtained using IC Capture (The Imaging Source1) at 1 frame per second. Formulations were injected for 60 s at 1 mL/min and 5 mL/min into the flowing (5 mL/ min) ISPB blood surrogate (25 2 C). Additionally, the phenytoin sodium formulations were injected at 1 mL/min into ISPB flowing at 15 mL/min to test the effect of higher blood surrogate flow rates. For each product tested, a placebo formulation, which contained only the inactive ingredients, was also injected onto the device. These images were used to subtract background noise from images taken during the injection of formulations containing the API. The phenytoin placebo was also injected with a blue food dye to illustrate the dynamics associated with the mixing of two fluids. All image processing was done using Matlab’s1 Image Processing Toolbox.
2.2. Parenteral formulations
Fig. 2 shows images of the needle tip (left) and 12 cm downstream (right) during the 1 mL/min injection of amiodarone HCl (top), furosemide (middle) and phenytoin sodium (bottom) formulations. Images taken near the needle tip show that injecting formulations of amiodarone and phenytoin into the flowing ISPB produced a significant amount of drug precipitation immediately
Three commercially available parenteral drug formulations, listed in Table 1, were chosen to test the new in vitro device based on the results of the validation study done by Johnson and Yalkowsky (2003). Table 1 also includes the aqueous solubility of
3. Results
Table 1 Commercially available formulations tested on in vitro device. API
Sw (mg/mL)
Sta (mg/mL)
Dose (mg/ mL)
Formulation Contents
Amiodarone HCl
<1.3e-6 (Bergström et al., 2004) 0.018 (Granero et al., 2010)
3.5 (Alvarez-Núñez and Yalkowsky, 2000; Li et al., 1999) 18 (Granero et al., 2010)
1.5b
0.018 (Schwartz et al., 1977)
88 (Millard et al., 2002; Li et al., 1999)
50
Each mL of solution contains 50 mg amiodarone HCl, 20.2 mg benzyl alcohol, 100 mg of polysorbate 80, and water for injection (pH = 3.5–4.5) Hospira (2016a) Each mL of solution contains 10 mg furosemide HCl, sodium chloride for tonicity adjustment, and enough sodium hydroxide/hydrochloric acid to adjust the pH to 9.0 (Hospira, 2016b) Each mL of solution contains 50 mg phenytoin Na, 0.4 mL propylene glycol, 0.1 mL ethanol, and water for injection with sodium hydroxide for pH adjustment to 10–12.3 (Pharmaceuticals,2016)
Furosemide
Phenytoin Sodium
a b
10
Predicted solubility. 3 mL of 50 mg/mL solution diluted with 100 mL of 5% dextrose solution.
D.C. Evans et al. / International Journal of Pharmaceutics 512 (2016) 219–223
221
Fig. 2. Images taken near the needle tip (left) and 12 cm downstream of the needle tip (right) during the 1 mL/min injection of amiodarone (top), furosemide (middle) and phenytoin (bottom) after background subtraction.
upon mixing with the ISPB blood surrogate. Downstream of the needle tip, as mixing between these two drug formulations and the ISPB becomes more complete the amount of precipitation increases. Unlike the precipitation of amiodarone, the turbidity in the flow cell caused by injecting the formulation containing phenytoin was less uniform due to the formation of large needleshaped crystals. This effect became more pronounced as the formulation moved downstream due to the increased size of the crystals. The downstream precipitation of amiodarone presented as a suspension of tiny particulates that grew in number but not noticeably in size. Images taken at the needle tip and 12 cm downstream during the injection of formulations containing furosemide show no light scattering caused by drug precipitation at either injection rate. To obtain a quantitative measure of precipitation, the mean grayscale intensity was determined for images taken during the injection of all three formulations at each injection rate and image location. These intensity values, summarized as bar plots in Fig. 3, were obtained after background subtraction and range from 0 (no turbidity) to 1, which represents complete saturation of the camera by the scattered light from a turbid solution.
Injections of amiodarone at 1 mL/min produced a small amount of precipitation near the needle tip that increased in intensity as the formulation moved further down the flow cell. As Fig. 2 shows, the area of the flow cell occupied by this turbid solution remained constant as the formulation moved downstream of the needle tip for the 1 mL/min injection rate. Increasing the injection rate of amiodarone from 1 mL/min to 5 mL/min did not significantly change the intensity of drug precipitation or the precipitation area near the needle tip. Downstream of the needle tip, however, the precipitation of amiodarone became more evenly distributed throughout the flow cell, while the intensity of the turbid solution decreased at the higher flow rate. Even at the higher injection rate the precipitation of amiodarone moved down the length of the flow cell as a turbid suspension without producing larger crystalline particles, such as those produced by the formulation containing phenytoin. At the needle tip, injecting phenytoin sodium into the ISPB produced a significant amount of precipitation that increased in both area and intensity as the injection rate was increased from 1 mL/min to 5 mL/min. At both the 1 mL/min and 5 mL/min injection rates the precipitation intensity for phenytoin
Fig. 3. Plots of the mean grayscale intensity, used as a measure of the amount of precipitation, for Amiodarone, Furosemide, and Phenytoin.
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Fig. 4. Time-lapsed images taken during the injection (1 mL/min) of phenytoin sodium (50 mg/mL) into the phosphate buffered saline flowing at 15 mL/min. Time points of the images are 0 min (top left), 1 min (top right), 3 min (bottom left), and 5 min (bottom right).
increased as the formulation moved downstream of the needle tip. Downstream of the needle tip, the area of the flow cell occupied by precipitation increased for the 1 mL/min injection rate however during the 5 mL/min injection the area decreased as the turbid solution becomes concentrated towards the bottom of the flow cell. As previously mentioned, furosemide did not show any precipitation at either injection rate or image location. The results obtained in this study are in general agreement with those obtained in the validation study published by Johnson and Yalkowsky (2003). In this validation study the turbidity detected by UV spectrophotometry was averaged over three different injection rates (5, 10, and 15 mL/min) and reported as the average opacity (AO). Formulations of phenytoin/Dilantin (AO = 1.196) produced more precipitation than formulations containing amiodarone/Cordarone (AO = 0.315) while formulations containing furosemide did not produce any precipitation (AO = 0.0). Interestingly, Johnson and Yalkowsky observed that injections of
phenytoin at 5 mL/min gave a lower opacity reading than injections at 1 mL/min whereas in our study the opposite was observed. This difference can be reconciled by the differences in the fluid dynamic characteristics of the flow cell used in the two studies. The ISPB flow rate of 5 mL/min, which was used in this study and in the validation study done by Johnson, represents a worst case scenario that may be encountered in patients with compromised blood flow. Forearm blood flow rates recorded in literature for healthy patients however, range from 7 to 268 mL/ min (Longhurst et al., 1974). To determine what effect the ISBP flow rate would have on the results obtained from in vitro parenteral drug testing, phenytoin sodium was injected at/min into ISPB flowing at 15 mL/min. Time-lapsed images taken during this injection (Fig. 4) show the formation of needle-shaped crystals that, over time, cover the surface of the tip of the needle. The effect of the higher ISPB flow rate was simply to move most of the observable precipitation downstream.
Fig. 5. Time-lapsed images taken during the injection of a phenytoin placebo formulation containing bromophenol blue at 0 (top left), 8 (top right), 13 (bottom left), and 24 (bottom right) seconds after injection to illustrate the effect of eddies (vortices) on the fluid dynamics of a simulated in vitro injection.
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One unexpected observation during the injection of phenytoin was the presence of crystals on the back of the needle tip and upstream of the needle opening. These crystals are the result of vortices, or eddies, that occur as a result of the ISPB flow past the needle. To better illustrate these fluid dynamics, Fig. 5 shows the injection of a blue dye mixed with the phenytoin placebo formulation. The vortices and “areas of recirculation” observed in our experiments, as well as in fluid dynamic experiments using catheters (Rotman et al., 2013), are the result of fluid moving past an obstruction (needle tip). While the fluid dynamics in the in vitro device presented in this paper do not precisely match the fluid dyamics in the cephalic or basic vein, a needle or catheter placed in the flowing blood can be expected to cause the formation of eddies, or vortices. This fact may explain why formulations associated with precipitation during administration such as phenytoin and amiodarone are associated with a higher incidence of phlebitis or skin irritation at the site of injection. 4. Conclusion The new in vitro device to predict drug precipitation upon administration yielded valuable qualitative and quantitative information useful for optimizing parenteral formulations. The flow cell designed for this study allows detection of drug precipitation at the site of injection where clinical symptoms of infusion related injuries are often observed. Having a photo record of the experiment allows the user to observe the performance of the formulation after the experiment has concluded. This feature, along with image processing techniques, also allows the user to minimize sources of false positive readings such as air bubbles and Schlieren patterns caused by the mixing of fluids with different indices of refraction. Finally the observed fluid dynamics of injection may explain how particulates present at the needle tip may cause local injury of venous cell walls clinically observed as phlebitis. References Alvarez-Núñez, F.A., Yalkowsky, S.H., 2000. Relationship between Polysorbate 80 solubilization descriptors and octanol–water partition coefficients of drugs. Int. J. Pharma. 200 (2), 217–222.
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