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Stability of tacrolimus solutions in polyolefin containers Jun H. Lee, Pharm.D., Ph.D., Pharmaceutical Development Section, Pharmacy Department, National Institutes of Health Clinical Center, Bethesda, MD. Barry R. Goldspiel, Pharm.D., FASHP, Pharmacy Department, National Institutes of Health Clinical Center, Bethesda, MD. Sujung Ryu, Ph.D., Pharmaceutical Development Section, Pharmacy Department, National Institutes of Health Clinical Center, Bethesda, MD. Gopal K. Potti, Ph.D., Pharmaceutical Development Section, Pharmacy Department, National Institutes of Health Clinical Center, Bethesda, MD.

Purpose. Results of a study to determine the stability of tacrolimus solutions stored in polyolefin containers under various temperature conditions are reported. Methods. Triplicate solutions of tacrolimus (0.001, 0.01, and 0.1 mg/mL) in 0.9% sodium chloride injection or 5% dextrose injection were prepared in polyolefin containers. Some samples were stored at room temperature (20–25 °C); others were refrigerated (2–8 °C) for 20 hours and then stored at room temperature for up to 28 hours. The solutions were analyzed by stability-indicating high-performance liquid chromatography (HPLC) assay at specified time points over 48 hours. Solution pH was measured and containers were visually inspected at each time point. Stability was defined as retention of at least 90% of the initial tacrolimus concentration. Results. All tested solutions retained over 90% of the initial tacrolimus concentration at all time points, with the exception of the 0.001-mg/mL solution prepared in 0.9% sodium chloride injection, which was deemed unstable beyond 24 hours. At all evaluated concentrations, mean solution pH values did not change significantly over 48 hours; no particle formation was detected. Conclusion. During storage in polyolefin bags at room temperature, a 0.001-mg/mL solution of tacrolimus was stable for 24 hours when prepared in 0.9% sodium chloride injection and for at least 48 hours when prepared in 5% dextrose injection. Solutions of 0.01 and 0.1 mg/mL prepared in either diluent were stable for at least 48 hours, and the 0.01-mg/ mL tacrolimus solution was also found to be stable throughout a sequential temperature protocol. Am J Health-Syst Pharm. 2016;73:137-42

T

DOI 10.2146/ajhp150264

acrolimus is a calcineurininhibitor immunosuppressant indicated for the prophylaxis of organ rejection in patients receiving allogeneic liver, kidney, or heart transplants. Tacrolimus is also indicated for immunosuppression in kidney and heart transplant recipients when used concomitantly with adrenal corticosteroids and in conjunction with azathioprine or mycophenolate mofetil.1 Tacrolimus can also be used for prevention and treatment of graftversus-host disease after hematopoietic stem cell transplantation.2,3 The oral route is preferred for tacrolimus administration. When patients cannot tolerate oral administration, which is common in graft-



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Address correspondence to Dr. Goldspiel ([email protected]).

versus-host disease due to gastrointestinal sequelae, the injectable form must be used. The injectable form is given as a continuous infusion. The manufacturer recommends preparation of tacrolimus for infusion in glass or polyethylene containers using 0.9% sodium chloride injection or 5% dextrose injection, with dilution of tacrolimus injection to a concentration of 0.004–0.02 mg/mL and an expiration time of 24 hours after preparation.1 The solution should not be prepared in polyvinylchloride (PVC) containers due to the potential for adsorption leading to decreased stability and possible leaching of plasticizer (via phthalate extraction) into the infusion solution1; the man-

NOTES ufacturer also recommends using non-PVC infusion tubing to minimize the possibility of tacrolimus adsorption onto the tubing during administration. Some information on the stability of tacrolimus beyond 24 hours has been reported.4,5 Taormina et al.4 studied tacrolimus concentrations of 0.1 mg/mL diluted in 0.9% sodium chloride injection or 5% dextrose and prepared in glass, PVC, or polyolefin containers. The researchers evaluated solutions prepared from a 10mg/mL tacrolimus injection solution that was investigational at the time of the study. These investigators found that tacrolimus concentrations were greater than 90% at 48 hours after preparation when solutions were stored at room temperature in polyolefin containers. Solutions prepared in glass bottles retained greater than 90% of the original concentration when prepared using 5% dextrose injection but not when prepared using 0.9% sodium chloride injection. The tacrolimus concentration of solutions prepared in PVC bags decreased to less than 90% of the initial concentration within 6 hours, most likely due to adsorption onto the PVC material, leading to the recommendation that tacrolimus not be prepared in PVC containers. Ku et al.5 studied 0.01-mg/mL solutions of tacrolimus in 0.9% sodium chloride injection prepared in glass volumetric flasks using the 5-mg/mL injection formulation. They found that greater than 90% of the original tacrolimus concentration was maintained at 48 hours when the solutions were stored at room temperature. The combination of giving a drug by continuous infusion and a 24-hour expiration time poses a challenge for pharmacies. Either there must be careful coordination between pharmacy and nursing personnel to ensure the availability of medication for infusion or the amount of solution to be infused during a 24-hour period must be divided into several bags. While tacrolimus appears to be stable

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KEY POINTS • This study provides information that simplifies and facilitates the administration of tacrolimus injection as a continuous infusion. • Tacrolimus solutions, when prepared in 5% dextrose injection at a concentration of 0.001 mg/mL, are stable for 20 hours in the refrigerator and then at least 28 hours at room temperature; this allows preparation in advance of the time needed. • Tacrolimus solutions, when prepared at concentrations of 0.01 and 0.1 mg/mL in either 0.9% sodium chloride injection or 5% dextrose injection or at a concentration of 0.001 mg/ mL in 5% dextrose injection, are stable for at least 48 hours at room temperature. • Tacrolimus solutions, when prepared at a concentration of 0.001 mg/mL in 0.9% sodium chloride injection, are stable for 24 hours at room temperature.

in glass containers, glass is no longer commonly used to prepare injectable preparations. In addition, the tacrolimus formulation has changed over the years. To provide additional stability information using the current formulation, we studied tacrolimus solutions of various concentrations stored in polyolefin bags under different temperature conditions.

Methods Sample preparation. Solutions of tacrolimusa at concentrations of 0.001, 0.01, and 0.1 mg/mL were prepared in 150-mL polyolefin bagsb of 0.9% sodium chloride injectionc or 5% dextrose injection.d Each preparation was agitated 10 times to homogenize the contents of the bag.

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Each concentration was prepared in triplicate and stored at room temperature (20–25 °C). Another set of solutions of tacrolimus (0.01 mg/mL) in 0.9% sodium chloride injection or 5% dextrose injection was prepared and stored in a refrigerator (2–8 °C) for 20 hours and then at room temperature for additional periods of up to 28 hours. High-performance liquid chromatography assay. The assay method in the United States Pharmacopeia (USP) monograph for tacrolimus was modified, validated, and used in our study.6 A five-component high-performance liquid chromatography (HPLC) systeme was used. A C18 columnf (4 mm, 3.9 × 150 mm) maintained at 60 °C by the column compartment was used for separation. The elution was run isocratically with a mobile phase consisting of 6 mM phosphoric acid (600 mL), acetonitrile (335 mL), and tert-butyl methyl ether (55 mL) at a flow rate of 1 mL/min. An injection volume of 10 mL was used for the 0.1-mg/ mL preparation, with injection volumes of 50 and 100 mL used to assay the 0.01- and 0.001-mg/mL preparations, respectively. The HPLC limit of detection was set at 205 nm. ChemStation software, version A.02.11 (Agilent Technologies, Santa Clara, CA), was used for instrument operation and data collection and processing. USP guidelines were followed in our analysis; that is, system suitability was established by consecutively injecting a reference standard into the HPLC system six times such that variations of the sum of the tacrolimus 19-epimer and tacrolimus peaks were less than 3%. Two reference standards were prepared for each evaluated tacrolimus preparation and injected before and after sample injections, with observed variations of the reference factor of less than 2%. Assay validation. We performed a limited validation of the assay because it was adapted from a validated assay procedure published in the

NOTES

TACROLIMUS

USP.6 Reference-grade tacrolimusg (purity of 97.6%) was used to prepare the standard solutions. A standard 5-point calibration curve was constructed by linear regression of the peak-area values for tacrolimus at concentrations of 0.001, 0.0025, 0.005, 0.01, and 0.1 mg/mL. An injection volume of 100 mL was used. Stability-indicating assay validation. The assay was shown to be stability indicating through forced degradation of the samples using 0.1N sodium hydroxide, 0.1N hydrochloric acid, and 3% hydrogen peroxide, which demonstrated that the peaks associated with the degradation products did not interfere

with the peak from the intact tacrolimus. Each solution was prepared at a final concentration of 0.1 mg/ mL, including the positive control of intact tacrolimus in sterile water for injection. Two vials were prepared in each condition; one vial was stored at room temperature and the other in an ovenh (at 60 °C) for 24 hours. Sample analysis. Stability assessments were performed after preparation and at 20 (refrigerator samples only), 24, 28, and 48 hours. At each time point, 1 mL was taken from each bag and analyzed in duplicate by HPLC, and 2 mL was taken for pH measurements. The pH meteri was calibrated with standard buffer solu-

tions of pH 4, 7, and 10. The samples were also visually inspected against black and white backgrounds using a high-intensity lamp at each time point to evaluate the physical compatibility of the solutions. Data analysis. All experiments were performed in triplicate. Data are presented as the mean ± S.D. percentage of the initial concentration remaining. Stability was defined as retention of greater than 90% of the initial tacrolimus concentration.

Results and discussion The HPLC assay adapted from the USP assay showed that the curve was linear over the range of evalu-

Figure 1. Representative chromatographic signal lines of intact tacrolimus (A) and samples assayed after storage in polyolefin bags at room temperature (20–25 °C) for 24 hours: tacrolimus diluted with sterile water (B) and tacrolimus subjected to forced degradation by 0.1N hydrochloric acid (C), 0.1N sodium hydroxide (D), and 3% hydrogen peroxide (E).

A

B

C

tacrolimus tacrolimus 19-epimer

D

E 0

2

4

6

8

10

12

14

16

18

Retention Time (min)



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NOTES

TACROLIMUS

ated tacrolimus concentrations (r2 = 0.9999). The limit of detection was 0.00054 mg/mL, and the limit of quantification was 0.00095 mg/mL. The average retention time was 7.3 minutes. The tailing factors of the chromatographic peaks for tacrolimus (a measure of peak asymmetry) were no less than 2. Variations of the sum of the tacrolimus 19-epimer and tacrolimus peaks were less than 3%. Variations of the reference factors for two reference standards were less than 2%. Also, variations of reference factors measured on different days were less than 2%, which showed the interday validity of the assay. Figures 1 and 2 show representative chromatographic signals for tacrolimus

subjected to forced degradation at room temperature and at 60 °C, respectively. The peaks associated with the degradation products did not interfere with the peak from the intact tacrolimus. As seen in Figure 1, with storage at room temperature, tacrolimus was stable in water for injection or 0.1N hydrochloric acid for 24 hours, while interference was 51% with dilution in 3% hydrogen peroxide. However, tacrolimus solutions were highly unstable when stored in basic conditions and under heat. With storage at 60 °C for 24 hours, the tacrolimus peak disappeared in samples degraded by water for injection, 0.1N sodium hydroxide, and 3% hydrogen peroxide; the peak was

decreased by approximately 28% in samples degraded by 0.1N hydrochloric acid. The purity of the USP reference standard was 97.6%; that purity value was applied to calculate the assay results. Different injection volumes were used to improve HPLC signal strength and reduce signal variability during analysis of samples containing lower concentrations of tacrolimus. All samples, with the exception of 0.001-mg/mL solution prepared in 0.9% sodium chloride injection, retained greater than 90% of the initial concentration at all time points, suggesting minimal to no loss of product due to degradation or adsorption (Table 1). The tacrolimus content of

Figure 2. Representative chromatographic signal lines of intact tacrolimus (A) and samples assayed after storage in heat (60 °C) in polyolefin bags for 24 hours: tacrolimus in water for injection (B) and tacrolimus subjected to forced degradation by 0.1N hydrochloric acid (C), 0.1N sodium hydroxide (D), and 3% hydrogen peroxide (E).

A tacrolimus tacrolimus 19-epimer

B C D E

0

2

4

6

8

10

12

14

Retention Time (min)

140 

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16

18

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Table 1. Stability of Tacrolimus Solutions Stored in Polyolefin Infusion Bags at Room Temperature (20–25 °C)a Diluent and Theoretical Concentration (mg/mL)

Mean ± S.D. % Initial Concentration Remaining

Mean ± S.D. Initial Concentration (%)

24 hr

28 hr

48 hr

0.9% sodium chloride injection  0.001



97.8 ± 6.7

94.8 ± 1.6

94.6 ± 6.2

100.2 ± 3.1

 0.01



96.5 ± 1.0

100.4 ± 2.0

101.5 ± 5.0

104.0 ± 6.9

 0.1



99.4 ± 1.1

103.0 ± 0.8

102.6 ± 0.8

103.7 ± 1.2

 0.001



100.7 ± 3.4

101.7 ± 0.4

100.8 ± 0.7

99.3 ± 0.6

 0.01



100.4 ± 0.9

101.3 ± 1.1

102.3 ± 0.8

101.6 ± 1.1

 0.1



101.7 ± 0.4

103.1 ± 0.1

103.4 ± 0.4

103.7 ± 0.7

5% dextrose injection

a

Test results are for all analyzed samples (n = 6) of each preparation.

Table 2. pH of Tacrolimus Solutions Stored in Polyolefin Infusion Bags at Room Temperature (20–25 °C) Diluent and Theoretical Concentration (mg/mL)

Mean ± S.D. pHa 0 hr

24 hr

28 hr

48 hr

 0.001

5.11 ± 0.06

5.08 ± 0.05

5.09 ± 0.07

5.11 ± 0.03

 0.01

5.21 ± 0.08

5.11 ± 0.01

5.05 ± 0.01

5.08 ± 0.01

 0.1

5.19 ± 0.14

5.17 ± 0.08

5.13 ± 0.03

5.18 ± 0.15

 0.001

4.69 ± 0.10

4.68 ± 0.10

4.56 ± 0.05

4.68 ± 0.10

 0.01

4.53 ± 0.07

4.47 ± 0.06

4.46 ± 0.06

4.48 ± 0.06

 0.1

4.68 ± 0.31

4.80 ± 0.08

4.76 ± 0.11

4.77 ± 0.11

0.9% sodium chloride injection

5% dextrose injection

a

Test results are for all analyzed samples (n = 3) of each preparation.

Table 3. Stability and pH of Tacrolimus Solution (0.01 mg/mL) in Polyolefin Infusion Bags During Sequential Temperature Protocola Assay and Diluent

0 hr

20 hr

24 hr

28 hr

48 hr

Stability assayb   0.9% sodium chloride injection

96.1 ± 5.4

99.2 ± 0.7

98.5 ± 4.0

104.0 ± 3.6

101.9 ± 5.6

  5% dextrose injection

101.7 ± 3.0

101.5 ± 1.1

101.2 ± 1.8

102.2 ± 0.6

100.5 ± 1.3

  0.9% sodium chloride injection

5.24 ± 0.07

5.10 ± 0.02

5.10 ± 0.01

5.11 ± 0.06

5.12 ± 0.03

  5% dextrose injection

4.45 ± 0.01

4.45 ± 0.06

4.43 ± 0.06

4.41 ± 0.02

4.40 ± 0.01

pH assayc

Protocol entailed refrigerated storage (2–8 °C) for 20 hr and then storage at room temperature (20–25 °C) for an additional 28 hr. Values are mean ± S.D. percentage of initial concentration remaining. c Values are mean ± S.D. pH. a

b



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NOTES the 0.001-mg/mL solution fell to less than 90% of the initial concentration at the 28-hour time point and can therefore be considered stable for only 24 hours (Table 1). The pH of the solutions varied by concentration; however, there were only slight changes in pH over the 48-hour study period for each of the concentrations tested (Table 2). Visual inspection did not demonstrate any particles in the solution containers at any time during the study period. To reflect clinical usage conditions, a sequential temperature protocol was adopted. A solution with a tacrolimus concentration of 0.01 mg/ mL was stored in the refrigerator for 20 hours and then at room temperature for an additional 28 hours (total time, 48 hours). Greater than 90% of tacrolimus concentrations remained for all time points, with slight changes in pH over the study period (Table 3).7

Conclusion During storage in polyolefin bags at room temperature, a 0.001-mg/ mL solution of tacrolimus was stable for 24 hours when prepared in 0.9% sodium chloride injection and for at least 48 hours when prepared in 5% dextrose injection, and solutions of

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0.01 and 0.1 mg/mL prepared in either diluent were stable for at least 48 hours; the 0.01-mg/mL tacrolimus solution was also found to be stable throughout a sequential temperature protocol.

Disclosures The authors have declared no potential conflicts of interest. The views presented here are those of the authors and do not necessarily represent the policy of the National Institutes of Health or the Department of Health and Human Services. Prograf (tacrolimus) injection (5 mg/ mL), Astellas Pharma US, Inc., Northbrook, IL, lot 5A3276C. b 150-mL PAB Mixing container, B. Braun Medical Inc., Irvine, CA, lot Y94002-856. c 0.9% Sodium Chloride Injection, USP, 500 mL, B. Braun Medical, lot J4H634. d 5% Dextrose Injection, USP, 1000 mL, B. Braun Medical, lot J4E697. e Agilent Series 1100 (G1322A degasser, G1311A quaternary pump, G1313A autosampler, G1316A column compartment, and G1314A variable wavelength detector), Agilent Technologies, Santa Clara, CA. f Nova-Pak, Waters Corporation, Milford, MA. g Tacrolimus, USP reference standard, United States Pharmacopeial Convention, Rockville, MD, lot H0M009. a

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Isotemp 500 Series, Fisher Scientific, Pittsburgh, PA. i Beckman 350 pH/Temp/mV meter, Beckman Coulter, Inc., Danvers, MA. h

References 1. Prograf (tacrolimus) injection prescribing information. Northbrook, IL: Astellas Pharma US; 2012 Aug. 2. Choi SW, Reddy P. Current and emerging strategies for the prevention of graft-versus-host disease. Nat Rev Clin Oncol. 2014; 11:536-47. 3. Fortune K, Couriel D. Tacrolimus in hematopoietic stem cell transplantation. Expert Opin Drug Metab Toxicol. 2009; 5:835-41. 4. Taormina D, Abdallah HY, Venkataramanan R et al. Stability and sorption of FK 506 in 5% dextrose injection and 0.9% sodium chloride injection in glass, polyvinyl chloride, and polyolefin containers. Am J Hosp Pharm. 1992; 49:119-22. 5. Ku YM, Min DI, Kumar V, Noormohamed SE. Compatibility of tacrolimus injection with cimetidine hydrochloride injection in 0.9% sodium chloride injection. Am J Health-Syst Pharm. 1995; 52:2024-5. 6. The United States pharmacopeia, 37th rev., and The national formulary, 32nd ed. Rockville, MD: United States Pharmacopeial Convention; 2014:4813-6. 7. Kaester S, Sewell G. A sequential temperature cycling study for the investigation of carboplatin infusion stability to facilitate ‘dosebanding’. J Oncol Pharm Pract. 2007; 12:119-26.

Stability of tacrolimus solutions in polyolefin containers.

Results of a study to determine the stability of tacrolimus solutions stored in polyolefin containers under various temperature conditions are reporte...
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