Evaluation of in Vitro Precipitation Methods

PING LI, RAO VISHNUVAJJALA, S. ESMAIL TABIBI,

AND

SAMUEL H. YALKOWSKY*,

Contribution from Department of Pharmaceutical Sciences, College of Pharmacy, University of Arizona, Tucson, Arizona 85721, and The National Cancer Institute, Bethesda, Maryland 20892. Received July 10, 1997. Accepted for publication November 10, 1997.
formulation includes hydroxypropyl -cyclodextrin (HPCD) which is added to retard precipitation of the drug when administered to patients. We are exploring possible new formulations of flavopiridol that do not require HPCD. Flavopiridol is a weakly basic compound with an intrinsic solubility of 0.02 mg/mL and a pKa of 5.68.7 The water solubility of this drug is high at acidic pH (10 mg/mL) where the drug is formulated, but relatively low (0.02 mg/ mL) at the physiologic pH of 7.4. The preclinical formulation composed of 2 mg/mL of flavopiridol in citric acid (pH 3.0) gives rise to the possibility of intravascular precipitation.7 Diazepam is chosen as a reference because it is known to produce phlebitis if injected too rapidly and to be safe if injected properly.3,6 Thus it represents the limit of pharmaceutical acceptability.

Abstract 0 Four in vitro precipitation methods were tested and evaluated using flavopiridol and diazepam formulations. The methods include static serial dilution, dynamic injection, and dropwise addition with and without stirring. The results generated from these methods are comparable and complementary. The static serial dilution method is most effective in quantifying the amount of precipitation and more descriptive of the formation and redissolution of the precipitate than the others. The dynamic injection method, however, has its merit in more realistically simulating the physiological environment of drugblood interaction near the injection sites.

Introduction
Pharmaceuticals that have poor water solubility are often formulated with the aid of cosolvents and/or by pH adjustment. Some of these products tend to precipitate when they are diluted in blood or other aqueous fluids.1 For example, diazepam injection is known for precipitating in the blood stream, thereby forming crystals which can produce severe pain and phlebitis, as well as alter bioavailability.1-4 In contrast to the inherent difficulty of performing in vivo studies, in vitro precipitation can be easily and accurately measured, and the results can be related to in vivo phlebitis.4 A few in vitro methods have been proposed for studying precipitation: static serial dilution, dynamic injection, and dropwise dilution with or without stirring. In the static serial dilution method, the formulation is sequentially diluted in a one-to-one ratio with solutions such as normal saline, 5% dextrose in water (D5W), human plasma, Tris buffer, and isotonic Sorensens phosphate buffer (ISPB). The precipitate formed can be visually observed and quantified.1,5,6,7 In the dynamic injection method, the formulation is added into an infusion fluid through an injector. The formulation and fluid flow rates can be adjusted to mimic those of the in vivo injection site. When precipitation occurs, the resulting opacity is measured in a flow cell by a spectrophotometer.5,6,8 In the method of dropwise addition, small aliquots of formulation are added serially to ISPB. The precipitate which would not redissolve in 30 s without stirring is determined visually. Alternatively, the latter procedure can be performed with stirring. In this paper, these four in vitro precipitation methods are evaluated using flavopiridol and diazepam formulations. At present, flavopiridol is undergoing clinical trails for the treatment of breast cancer tumors at the National Cancer Institute (NCI). Phase I studies of this agent have been completed and it will be evaluated extensively in broad phase II clinical trails. The current (intravenous)
* To whom correspondence should be addressed. University of Arizona. The National Cancer Institute.

Experimental Section
MaterialssFlavopiridol was obtained from the National Cancer Institute and used as received. All other chemicals were reagent grade and purchased from Sigma or Aldrich. Diazepam was injectable Valium (5 mg/mL, 10 mL vial, Roche). The ISPB was prepared according to Scientific Tables9 and filtered through a 0.45 m filter prior to use. Flavopiridol was formulated to a concentration of 2 mg/mL in 0.1 M citric acid (pH 3.0) and filtered. A Master Flex pump (model 7520-00, Cole-Parmer Instrument Co.), a syringe infusion pump (model 22, Harvard Apparatus), a spectrophotometer (model DU-7, Beckman), and a flow cell with a volume of 0.5 mL were used for the dynamic precipitation studies. MethodssAssaysThe solution concentrations of both flavopiridol and diazepam were measured by ultraviolet spectrophotometry at 350 nm. (1) In Vitro Static Serial DilutionsOne milliliter of the formulation was added to 1 mL of ISPB and agitated. One milliliter of the resulting solution was then mixed with another 1 mL of ISPB and agitated. This step was repeated until 10 dilutions were obtained. This gives the last one a concentration of less than 1/1000 of the original formulation concentration. Visual observations were used to determine the presence or absence of a precipitate upon mixing. Following these initial observations, the formulation-buffer mixtures were placed on a test tube rotator for 24 h and then filtered by a 0.45 m filter. The equilibrium concentrations in each diluted solutions were determined spectrophotometrically. The difference between the total theoretical concentration and the measured concentration in each dilution is equal to the amount of drug that precipitated from 1 mL of original formulation. The formulation-diluent ratio is defined as the ratio of the volume of formulation to the total volume (volume of formulation + volume of ISPB). (2) In Vitro Dynamic InjectionsTo make different formulationdiluent ratios, 1 mL of formulation was injected at several constant rates (0.2, 0.5, 1.0, 2.0, 5.0, 10.0, and 20.0 mL/min) into ISPB which was pumped at 20 mL/min. Precipitation was determined as turbidity, which was measured by the decrease in the transmittance of 470 nm light through the flow cell in the spectrophotometer. Since neither drug nor the formulation components absorb at this wavelength, the peak height or turbidity is a measure of the amount of the precipitate in the flow cell. The formulationdiluent ratio is defined in the same way as in the static serial dilution method with the injection rates taking the place of the volumes.

196 / Journal of Pharmaceutical Sciences Vol. 87, No. 2, February 1998

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Figure 1sFlavopiridol concentration versus formulationdiluent ratio in the static serial dilutions (opened circles, no precipitation; closed circles, precipitation).
(3) Dropwise Addition with StirringsSmall aliquots of formulation were added sequentially to 10 mL of ISPB. After each addition, the samples were gently stirred for 30 s. If no precipitate was found in the solution, another aliquot of formulation was added. This procedure was repeated until a precipitate was formed that would not redissolve in 30 s or until the amount of formulation exceeded the amount of ISPB. (4) Dropwise Dilution without StirringsThe above-mentioned dropwise addition procedure3 was performed without agitation until a precipitate formed that did not redissolve in 30 s.

Figure 2sThe amount of the flavopiridol and diazepam precipitate formed at each formulationdiluent ratio in the static serial dilutions (opened symbols, no precipitation; closed symbols, precipitation).

Results and Discussion

The solution concentrations of flavopiridol versus formulation-diluent ratios in static serial dilution are given in Figure 1. The dashed line represents the total theoretical concentration of flavopiridol in the serial dilutions, i.e., the concentration that would be found if no precipitation occurred. The solid line represents the equilibrium concentration of flavopiridol (which was determined after removal of the precipitate). When pure formulation is first diluted, the equilibrium concentration points (represented by open circles) coincide with the theoretical concentration curve; i.e., no precipitation is observed. As dilution continues and the ratio reaches 0.25, the equilibrium concentration points fall below the total theoretical concentration curve and a precipitate is observed visually (represented by closed circles). This phenomenon persists over all formulation-diluent ratios ranging from 0.25 to 0.03. Below the ratio of 0.03, the equilibrium concentration points rejoin the total theoretical curve with the observation that all precipitate is redissolved. The vertical difference between the two curves represents the amount of flavopiridol precipitate. The amounts of flavopiridol and diazepam precipitate formed at each formulation-diluent ratio are shown in Figure 2. In comparison with the diazepam formulation, flavopiridol displays only minimal precipitation. At a formulation-diluent ratio of 0.5, diazepam reaches its maximum precipitation, yielding approximately 1490 g (corresponding to 30% of the formulation), while flavopiridol has its maximum precipitation of 145 g (corresponding to only 7% of the formulation) at the ratio of 0.125. It is also observed that little or no precipitate is found for both flavopiridol or diazepam when the formulations are further diluted below a ratio of 0.015. This is expected since the drug concentrations are lower than their solubilities.

Figure 3sThe relative amount of precipitation produced by flavopiridol and diazepam formulations using the dynamic injection (opened symbols, no precipitation; closed symbols, precipitation).

In Figure 3, we see the results from the dynamic injection studies. The turbidities (represented by peak height) produced by the precipitation of flavopiridol and diazepam are plotted against the formulation-diluent ratios. As shown in the figure, diazepam (triangles) again produces much more precipitate than does flavopiridol (circles). It is observed that very slight flavopiridol precipitation (closed circles) occurs at a medium range of injection rates between 10.0 and 2.0 mL/min, with little or no precipitation at either higher or lower injection rates; while for diazepam, precipitation (closed triangles) starts at a formulation-diluent ratio of 0.5 and extends down to a ratio of 0.05. Comparison of Figures 2 and 3 indicates that the precipitation follows similar trends in both the static serial dilution and the dynamic injection methods. It is seen that the precipitation is associated with a broad medium range of formulation-diluent ratios, approximately 0.50 to 0.03 for flavopiridol and 0.5 to 0.05 for diazepam; and the precipitation is reduced to undetectable levels for both drugs at lower or higher ratios. However, there is a slight difference in the value of the formulation-diluent ratio at which maximum precipitation is formed. In the static serial dilution method, the ratio is Journal of Pharmaceutical Sciences / 197 Vol. 87, No. 2, February 1998

Figure 4sA longitudinal section of tubing illustrating postinjection precipitation: (A) complete mixing, (B) partial mixing (plug flow).

0.125, while in the dynamic injection method, the ratio moves up to 0.25 for flavopiridol. Such a difference could be a result of differences in the type of mixing that occurs under different methods. In the static serial dilution method, the equilibrium is reached for 24 h (see Figure 4A) as all of the formulation mixes with all of the diluent. While in the dynamic dilution method the mixing of formulation with the buffer is relatively slow, and the formulation forms a plume in the flowing stream. This leads to incomplete or delayed mixing and may well result in maximum precipitation at a slightly higher formulation-diluent ratio. If the formulation is injected very rapidly, the plume will become a plug of undiluted formulation. In this case, mixing with the ISPB will only occur at the leading and the tailing edges of the plug, as illustrated in Figure 4B. The results from a 45 s injection of flavopiridol and diazepam are consistent with the above explanation. As shown in Figure 5A, an injection rate of 2.0 mL/min of flavopiridol formulation produces a single peak which corresponds to the passage of the immediately diluted formulation containing precipitated drug; an injection rate of 10.0 mL/min of the same formulation (Figure 5B) causes two shorter peaks separated by a region of low opacity. The first peak is produced by precipitation at the leading edge of the plug as this region is mixed with diluent. As the initial precipitate travels out of the flow cell it is followed by the relatively unmixed (i.e., concentrated) formulation plug, as recorded by a low opacity value. When the central portion of the plug has passed through the flow cell, the trailing edge, diluted to the point of precipitation, enters and produces the second peak. Similarly, diazepam shows evidence of plug flow when tested in the system. At a low injection rate of 2 mL/min, the thorough mixing of formulation and infusion fluid results in a single peak of diazepam precipitate (Figure 5C), while at high injection rate of 20 mL/min, the formulation plug leads to two distinct peaks separated by a low opacity region (Figure 5D). For both drugs, the precipitate amount per 1 mL of original formulation is much less under plug flow than under conditions which produce complete mixing. Under plug flow as under complete mixing, diazepam precipitates to a greater extent than flavopiridol. Note that while the peak heights appear comparable, the scales differ by a factor of 150, indicating the far greater magnitude of diazepam precipitation. Table 1 shows the formulation-diluent ratios at which precipitation occurs in the experiments of static serial dilution, dynamic injection, and the two dropwise addition methods. When the formulation is added dropwise into 198 / Journal of Pharmaceutical Sciences Vol. 87, No. 2, February 1998

Figure 5sPrecipitation of flavopiridol and diazepam formulations from 45 s injection in dynamic studies; (A) flavopiridol, 2 mL/min; (B) flavopiridol, 10 mL/min; (C) diazepam, 2 mL/min; (D) diazepam, 20 mL/min. Table 1sFormulationDiluent Ratios Which Cause Precipitation in Four in Vitro Precipitation Methods formulations flavopiridol diazepam static serial dilution 0.03 0.03 dynamic injection 0.10 0.10 dropwise with stirring 0.10 0.07 dropwise without stirring 0.30 0.19

ISPB, there appears to be a difference in the precipitation behavior between experiments with and without stirring. In the experiment with stirring, a formulation-diluent ratio of 0.10 leads to flavopiridol precipitation, while in the experiment without stirring, a slightly higher ratio of 0.30 is needed to produce the same phenomenon. When diazepam is used, a formulation-diluent ratio of 0.07 is needed for the experiment with stirring and a ratio of 0.19 for the experiment without stirring. It is speculated that an intact region of formulation exists within the droplet for both flavopiridol and diazepam in the experiments without stirring. Thus the experiment without stirring is analogous to the dynamic injection, whereas dropwise addition with stirring is analogous to the static serial dilution in determining the point at which precipitation occurs. While the dropwise addition methods are rapid and simple, only the static serial dilution and dynamic injection experiments provide information about the amount of precipitation and about the reduction in precipitation at low dilutions.

Conclusion
Four in vitro precipitation methods have been studied. The results generated from those methods are comparable and complementary. The static serial dilution method is most effective in quantifying the amount of precipitation and more descriptive of the formation and dissolution of the precipitate than others. The dynamic injection method, however, has its merit in simulating the physiological

environment of drug-blood interaction, whereas the dropwise addition methods are somewhat simpler to perform.

References and Notes

1. Schroeder, H. G.; DeLuca, P. P. A Study on the In Vitro Precipitation of Poorly Soluble Drug From Nonaqueous Vehicles in Human Plasma. Bull. Parenter. Drug Assoc. 1974, 28, 1-15. 2. Turco, S. J. Infusion Phlebitis: A Review of the Literature. Parenterals 1987, 5, 1-8. 3. Morris, M. E. Compatibility and Stability of Diazepam Injection Following Dilution with Intravenous Fluids. Am. J. Hosp. Pharm. 1978, 35, 669-672. 4. Lewis, G. B. H.; Hecker, J. F. Infusion Thrombophlebitis. Br. J. Anaesth. 1985, 57, 220-233.

5. Ward, G. H.; Yalkowsky, S. H. Studies in Phlebitis VI: Dilution-Induced Precipitation of Amiodarone HCL. J. Parenter. Sci. Technol. 1993, 47, 161-165. 6. Yalkowsky, S. H.; Valvani, S. C.; Johnson, B. W. In vitro method for detecting Precipitation of Parenteral Formulations After Injection. J. Pharm. Sci. 1983, 72, 1014-1017. 7. Dannenfelser, R-M.; Surakitbanharn, Y.; Tabibi, S. E.; Yalkowsky, S. H. Parenteral Formulation of Flavopiridol (NSC-649890). PDA J. Pharm. Sci. Technol. 1996, 50, 356359. 8. Irwin, W. J.; Iqbal, M. Bropirimine formulation: The dynamic testing of injections. Int. J. Pharm. 1992, 83, 241-249. 9. Diem, K.; Lentner, C. Scientific Tables, 7th ed.; Geigy Pharmaceuticals: New York, 1974, pp 280-282.

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