Stability and compatibility of four anthracyclines: doxorubicin, epirubicin, daunorubicin and pirarubicin with PVC infusion bags T. Dine, J.C. Cazin, B. Gressier, M. Luyckx, C. Brunet, M. Cazin, F. Goudaliez, M.L. Mallevais and I. Toraub Introduction The anthracycline antibiotics, doxorubicin (adriamycin), epirubicin, daunorubicin and pirarubicin (tetrahydropyran-doxorubicin)are antiturnout drugs widely used for the t r e a t m e n t of neoplastic diseases [1]. Chemical structures for these a n t i t u m o u r agents are depicted in Figure 1. Conventionally, the anthracyclines have been administered by intravenous bolus injections, but, because of their cardiotoxicity, continuous infusion therapy has recently been studied [2-4]. Hence, the therapeutic advantages of continuous infusion versus intermittent small-volume infusion or intravenous push have been suggested [5-7]. Therefore, with the increasing use of continuous intravenous infusion and intermittent small-volume intravenous infusion, it is imperative t h a t the stability and compatibility of anthracyclines in administration vehicles and polyvinyl chloride (PVC) containers be investigated. Consequently, when drugs are administered by continuous intravenous infusion with PVC material, knowledge of the rate of drug delivery to the patient is essential [8]. Previous studies have reported t h a t loss of certain drugs (diazepam, nitroglycerin) from aqueous solutions stored in plastic infusion bags for various periods of time [9-11]. Generally, these losses have been attributed to interaction (adsorption or absorption) between the drug and the plastic infusion bag, and they may, in some
Keywords Chromatography, high pressure liquid Compatibility Daunorubicin Doxorubicin Drug stability Epirubicin Infusions Pirarubicin Polyvinyl chloride T. Dine (correspondence), J.C. Cazin, B. Gressier, M. Luyckx, C. Brunet and M. Cazin: Laboratoire de Pharmacologie, Pharmacocin~tique et Pharmacie Clinique, Facult~ des Sciences Pharmaceutiques et Biologiques, rue du Professeur Laguesse, B.P. 83, 59006 Lille C~dex, France. F. Goudaliez, M.L. Mallevais and I. Toraub: Laboratoires Macopharma, rue du PontRompue, B.P. 464, 59338 Tourcoing C~dex, France.
14(6) 1992
cases, diminish therapeutic response due to a reduced drug delivery to the patient. Other studies have shown a loss of anthracyclines in glass bottles due to adsorption [12]. Only a minimal amount of information is available concerning the compatibility of anthracyclines administered by PVC infusion bags and intravenous administration sets [13 14]. Such information is important when drug solutions are to be infused over long periods of time. The present study was u n d e r t a k e n with the following objectives: - to survey a range of drugs (anthracyclines), including those presently being administered by intravenous infusion, for possible interactions with PVC infusion bags; - to study the behaviour of these drugs in simulated infusion using PVC containers and administration sets; - to adapt infusions to the conditions routinely used in hospitals (infusion flow rate, dose, volume, temperature, light ...); - to determine the differences in possible interactions between PVC containers and administration sets, as well as the differences in stability of the drugs in 0.9% NaC1 and 5% glucose. A high pressure liquid chromatographic (HPLC) method used in the present study has been developed in our laboratory. This method allowed the rapid determination of doxorubicin, epirubicin, daunorubicin and pirarubicin, a new
Dine T, Cazin JC, Gressier B, Luyckx M, Brunet C, Cazin M, Goudaliez F, Mallevais ML, Toraub I. Stability and compatibility of four anthracyclines: doxorubicin, epirubicin, daunorubicin and pirarubicin with PVC infusion bags. Pharm Weekbl [Sci] 1992;14(6):365-9. Abstract
A rapid isocratic technique was developed for the analysis of four anthracyclines (doxorubicin, epirubicin, dannorubicin and pirarubicin) in parenteral solutions using high pressure liquid chromatography (HPLC) with fluorescence detection and a C18 Hypersil ODS column. The availability and compatibility of these drugs from solutions infused via PVC infusion bags through PVC administration sets have been examined. No significant drug loss was observed during simulated infusions (n = 4) for 24 h using PVC infusion bags and administration sets. No significant difference was found between infusion solutions (5% glucose or 0.9% NaC1), except for pirarubicin. The reconstitution of pirarubicin in 0.9% NaC1 was impossible, because we observed a precipitation of the compound in solution. The stability of the drugs was also studied in solution, in PVC bags after storage at 4~ with protection from light. The results show the stability of doxorubicin, epirubicin and daunorubicin during 7 days of storage to be satisfactory, irrespective of the infusion solution (5% glucose or 0.9% NaC1). In the case of pirm'ubicin, the stability of the drug was satisfactory during 5 days of storage in 5% glucose, but beyond, we observed a degradation of the compound with formation of doxorubicin in the infusion solution. Accepted August 1992.
Pharmaceutisch Weekblad Scientific edition
365
~
0
0 II
OH
0 - CHIOH
Methods
OH
~
COCH20H "'OH
H
o
c%o
OH
OCH~
6
Nit,
Epirubicin
Doxorubicin
CH30
0
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o
0
~
OH
OH
CH 30
6
0
OH COCH.OH "'"OH
0
OH
0
IIo
Pirarubicin
Daunorubicin
Materials The drug substances studied were commercial products suitable for clinical use. Doxorubicin and epirubicin were obtained from F a r m i t a l i a Carlo E r b a Laboratories (Milan, Italy) in vials of 10 m g sterile powder for injection. Daunorubicin and pirarubicin were obtained from Roger Bellon Laboratories (Neuilly sur Seine, France) in vials of 20 and 50 m g sterile powder for injection, respectively. Acetonitrile (HPLC grade), ammonia and formic acid (analytical grade) were obtained from Prolabo (Paris, France). W a t e r for injection (sterile and apyrogenic) obtained from M a c o p h a r m a Laboratories (Tourcoing, France) was used for buffers, dilutions and standard solutions.
Figure 1 Chemical structures of the anthracyclines studied
semisynthetic anthracycline, in infusion solutions (5% glucose and 0.9% NaC1) using a suitable chromatographic column and mobile phase. We h a v e used this analytical technique to investigate the compatibility of the drugs with PVC containers and PVC infusion sets both during simulated infusions and during storage at 4 ~ in PVC bags used in the hospital p h a r m a c y d e p a r t m e n t where the reconstitution of cytostatics is centralized.
Chromatographic conditions and instrumentation Chromatographic analysis was performed with an H P 1090 high pressure liquid chromatograph (Hewlett Packard, Orsay, France), equipped with a variab]e-volume injector, an automatic sampling system and a H P 1046A fluorescence detector operating at excitation and emission wavelengths 254 n m and 565 nm, respectively. The output from the detector was connected to a Hewlett P a c k a r d 9000 model 300 integrator and the data recorded on an H P Thinkjet t e r m i n a l printer. Analyses were performed on a 5 t~m C18 Hypersil ODS column (100 x 4,6 m m i.d.) (Hewlett Packard, Orsay, France) operating at room t e m p e r a t u r e . The analytical column was protected by a small pre-column (20 m m x 4.6 m m i.d.) packed with C18 Hypersil ODS 5 ~m (Hewlett Packard). Compounds were eluted isocratically with a mobile phase consisting of acetonitrile and formate buffer m i x t u r e (45+55; vo]/vol) at a flow
Table 1 Validation data of the H P L C assay procedure (n = 5)
Sample substance
Internal standard
Concentrations (~g/ml)
Average Coefficient of variation Accuracy Linear regression " concentrations (%) equation found • SD intraassay interassay (y= a x + b ) (~g/ml) (%) (%)
Doxorubicin
daunorubicin
6.25 12.5 25
6.263• 12.506• 25.012•
0.24 0.42 0.92
1.28 0.32 0.56
100.21 100.05 100.04
y = 0.057x - 0.127
0.999
6.25 12.5 25
6.274• 12.506• 25.014•
0.34 0.41 0.32
1.86 1.08 0.76
100.38 100.05 100.06
y = 0.047x + 0.104
0.999
6.25 12.5 25
6.241• 12.508• 25.010•
0.98 0.88 0.69
1.30 0.10 0.41
99.86 100.06 100.04
y = 0.090x - 0.347
0.999
6.25 12.5 25
6.232• 12.487• 24.975•
0.22 0.26 0.30
1.04 0.38 0.67
99.71 99.89 99.90
y = 0.036x
0.999
Epirubicin
Daunorubicin
Pirarubicin
366
daunorubicin
epirubicin
daunorubicin
-
0.131
P h a r m a c e u t i s c h Weekblad Scientific edition
Correlation coefficient (r)
14(6) 1992
r~
,:,-
r a t e of 2 ml/min. The formate buffer was prepared in w a t e r with a m m o n i a adjusted to pH 4 with pure formic acid. For simulated infusions, we used a volumetric infusion p u m p (VIP II) and PVC infusion sets (IS 05) obtained from Becton Dickinson Laboratories, Divison Vial Medical (SaintEtienne de Saint-Geoirs, France). Macoflex ~ PVC infusion bags (50, 250 and 500 ml) were kindly provided by M a c o p h a r m a Laboratories. The bags were filled with 0.9% NaC1 or 5% glucose in w a t e r to study the possible influence of solvent on the interaction of the drug.
c (i.s.)
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(c)
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~ 1 ~ - i ~ 1
(d)
,~
~
r 1
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~
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1
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r
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Figure 2 Examples of chromatographic analysis. (a): doxorubicin with internal standard daunorubicin; (b): epirubicin with internal standard daunorubicin; (c): daunorubicin with internal standard epirubicin; (d): pirarubicin with internal standard daunorubicin
To obtain standard stock solutions, all drugs were reconstituted with distilled w a t e r to give a drug concentration of 50 t~g/m] in all four cases. These standard solutions were protected from light. Working solutions were p r e p a r e d from the standard solutions by suitable dilutions with distilled w a t e r in polypropylene tubes. Calibration curves were constructed in all four cases between 6.25 ~g/ml and 25 ~g/ml. For quantification of drugs, an internal standard was employed to dilute the samples. The use of an internal standard was needed to reduce the errors of dilution, because the drug concentrations were high in the PVC bags during simulated infusions and storage. The p e a k ratio (drug p e a k area/internal standard p e a k area) was calculated and the a m o u n t of drug determined by reference to the calibration curve. Daunorubicin was used as the internal standard to quantify doxorubicin, epirubicin and pirarubicin, while epirubicin was employed in the case of daunorubicin.
Simulated infusions
lO,O
10,0
9,5
9,5
A
9,0
9,o
OONC (mg/500rlll)
C
a.5
8,5 B,0 7,5 (mg/5OOm0 7,0
7,5 7,o
6,5 6,0 5,5
--[ .
.
.
................. 1
.
from infusion sets -
~
-
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5,0 5
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6
8
10
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S
8
10
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12,0 -
12,0.
11,5 11,0 10,5
---[ -
................ l
-
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....
-
~1,o -
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(mg/500ml) 9,5
9,0
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8,5
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15
30
1
2
4
TIME (rain. h.)
6
B
10
24
5
15
30
1
2
4
5
8
TIME (rain. h.)
Figure 3 Concentration kinetics of doxorubicin (A), epirubicin (B), daunorubicin (C) and pirarubicin (D) during simulated infusions (n = 4) using plastic infusion bags and sets
14(6) 1992
P h a r m a c e u t i s c h Weekblad Scientific edition
Infusions were simulated out under laboratory conditions i m i t a t i n g infusions to patients routinely used in hospitals. For this purpose, we used an infusion p u m p and PVC a d m i n i s t r a t i o n sets. The respective drug concentrations in solution were 8 rag/500 ml (16 ~g/ml) for doxorubicin and daunorubicin, and 10 mg/500 ml (20 #m/ml) for epirubicin and pirarubicin. The simulated infusions were carried out over a period of 24 h at a flow r a t e of 21 ml/min. Infusion solutions of drugs were prepared in PVC infusion bags containing 500 ml 5% glucose or 0.9% NaC1 i m m e d i a t e l y before the infusion. The bag containing drug was t h e n attached to an administration set connected to the infusion p u m p t h a t allowed the solution to flow t h r o u g h at a constant rate. At specified times of infusion, 1 ml samples were w i t h d r a w n at r e g u l a r intervals from the PVC bags, and at the same t i m e an aliquot of effluent (1 ml) was collected from the administration set. Samples were kept frozen in polypropylene tubes at - 2 0 ~ until analysis by HPLC. All simulated infusions were carried out at least in quadruplicate (n = 4: two infusions in 0.9% NaC1 and two infusions in 5% glucose) at a m b i e n t t e m p e r a t u r e (20-24~ without protection from light.
367
Storage in infusion bags Insofar as it was possible, we employed conditions in conformity with the drug concentrations normally used in hospital pharmacy departments for the storage of drugs in infusion bags. To infusion bags containing 50, 250 or 500 ml of 0.9% NaC1 or 5% glucose solution, a known amount of drugs was added to achieve the following concentrations which are most often used in hospitals: doxorubicin, 40 ~g/ml; epirubicin, 40 ~g/ml; daunorubicin, 16 t~g/ml; pirarubicin, 800 ~g/ml. After mixing the drug in the bag by rapid shaking, i ml samples were withdrawn at regular intervals and stored in polypropylene tubes at - 2 0 ~ until HPLC analysis. Infusion bags containing the drug were stored at 4~ for a period of 7 days with protection from light. Drug storage in these bags was carried out in 0.9% NaC1 and 5% glucose. Sample preparation Before HPLC analysis, each defrozen sample was suitably diluted with an internal standard solution in order to obtain a drug concentration within the calibration range. Results and discussion
High pressure liquid chromatography The chromatograms of the four drugs with the respective internal standards in solution obtained immediately after mixing, are illustrated in Figure 2. The drugs were simultaneously and rapidly separated, identified and quantified. The components were satisfactorily resolved by this HPLC method and had retention times of 1.65 min (doxorubicin), 1.80 min (epirubicin), 2.65 min (daunorubicin) and 3.75 min (pirarubicin). Table 1 summarizes the validation data of the assay procedure for each drug. We observed good linearity between peak area ratios and concentrations. The calibration curves were fitted by the least-square method for the peak area ratio of the sample substance and the internal standard (y) versus the concentration of the analysed product (x). The correlation coefficients were all above 0.999 and no significant differences were observed between the equation parameters. To assess reproducibility, the same concentration was analysed five times for each point of the calibration curves. The results demonstrate that this analytical method has acceptable accuracy and precision in every case.
Figure 4 HPL chromatogram obtained after injection of a sample ofpirarubicin (D) obtained after 5 days of storage in a PVC bag at 4 ~ A: degradation product identified as doxorubicin; C: daunorubicin (internal standard)
(Ls.)
o
Time (rnln,)
Stability of anthracyclines during simulated infusions using P V C infusion bags and sets The analysis of each sample was performed by HPLC after a suitable dilution in the mobile phase in order to fit the calibration curves. Figure 3 depicts the concentration kinetics of all four drugs during simulated infusions (n = 4), using plastic infusion bags and sets. When solutions of doxorubicin, epirubicin and daunorubicin were infused through PVC infusion sets from PVC infusion bags over a period of 24 h, the variation in drug concentration in both the PVC bags and effluent in no case exceeded 10%. This demonstrates that the three drugs were not sorbed by the PVC infusion bags and sets during infusion at ambient temperature. No significant difference was observed with respect to drug stability during simulated infusions using 5% glucose or 0.9% NaC1. In contrast, in the case of pirarubicin the study had been only effected in 5% glucose, because the drug was not sufficiently soluble in 0.9% NaC1 and a precipitation of compound was rapidly observed in the solution. In addition, the simulated infusion showed variations more important than in the case of the other drugs, as well as more irregular concentration kinetics. However, the variation in drug concentration in both the PVC bags and effluent did not exceed 10% during simulated infusions over a period of 24 h. No degradation product was detected by HPLC in infusion solution.
Table 2 Concentrations (zg/ml) present in solution after storage in plastic bags at 4 oC
Drug
Doxorubicin
Infusion solution
0.9% NaC1
5% glucose
0.9% NaC1
5% glucose
0.9% NaC1
5% glucose
5% glucose
Initial 1 day 3 days 5 days 7 days
40.00 40.20+_0.12 39.68_+0.16 39.40_+0.10 37.56-+0.08
40.00 39.00+0.14 37.96+_0.12 37.08_+0.11 36.08-+0.07
40.00 40.20_+0.24 38.60+0.32 37.76_+0.14 36.32+0.18
40.00 39.68+_0.27 39.84+0.34 39.56_+0.22 40.56+0.19
16.00 16.10_+0.06 16.28+0.12 15.92_+0.17 15.94_+0.12
16.00 16.90• 16.46_+0.18 15.88_+0.22 16.24_+0.13
800.00 769.80+_24.16 816.40_+12.34 791.20_+16.32 719.20_+13.48
Pharmaceutisch Weekblad Scientific edition
14(6) 1992
368
Epirubicin
Daunorubicin
Pirarubicin
Stability of anthracyclines in infusion bags during storage at 4~ with protection from light The analysis of each sample was performed by HPLC after a suitable dilution in the mobile phase in order to fit the calibration curves. The concentrations of all four drugs in solutions after various periods of storage in PVC infusion bags at 4~ with protection from light are listed in Table 2. No significant disappearance of drug (exceeding 10%) was observed in PVC infusion bags for doxorubicin, epirubicin and daunorubicin over a period of 7 days of storage, irrespective of the infusion solution (5% glucose or 0.9% NaC1). In contrast, in the case of pirarubicin, the study had only been effected in 5% glucose. We did note a significant disappearance of drug during a period of 7 days of storage. Table 2 shows that the loss of drug exceeded 10% after 5 days of storage. This loss could be explained by the formation of a degradation product of pirarubicin, which was identified as doxorubicin. Figure 4 shows an HPL chromatogram obtained after injection of 10 ~1 of sample obtained after 5 days of storage at 4~ The detection of doxorubicin in the infusion solution could lead to a modified therapeutic response to t r e a t m e n t using. In this and an other study [15], the results show, respectively, the in vitro and in vivo formation of doxorubicin from pirarubicin.
Conclusion In conclusion, the HPLC procedure described in this paper, rapidly and reproducibly determines anthracyclines in parenteral solutions. With the increasing use of continuous intravenous infusion of anthracyclines [16-18], the present study has examined the kinetics of doxorubicin, epirubicin, daunorubicin and pirarubicin concentration during simulated infusion using PVC infusion bags and administration sets. The results demonstrate a satisfactory compatibility of anthracyc]ines with PVC infusion material over a 24-h infusion 9 It is likely t h a t other drugs interact with PVC infusion bags and administration sets, leading to a reduction in the clinical effectiveness of the drug [19]. This type of study is important with regard to the packaging of pharmaceuticals in plastic containers in general [20], and might be carried out for all drugs administered in PVC infusion bags.
Acknowledgement This work was supported by grants of the Comit~ du Nord de la Ligue Nationale Franr Contre le Cancer and the Conseil R~gional du Nord-Pas de Calais. The authors also wish to t h a n k Macopharma, F a r m i t a l i a Carlo Erba and Roger Bellon Laboratories for co-operation in this study.
14(6) 1992
Pharmaceutisch Weekblad Scientific edition
References 1 Lavelle F. Structure et activit6s des anthracyclines. Pathol Biol (Paris) 1987;35:11-9. 2 Greidanus J, Willemse PH, Uges DR, Oremus ET, De Langen ZJ, De Vries EG. Continuous infusion of low-dose doxorubicin, epirubicin and mitoxantrone in cancer chemotherapy: a review. Pharm Weekbl [Sci] 1988;10:237-45. 3 De Vries EG, Nanninga AG, Greidanus J, Oremus ET, Verschueren RC, Mulder NH, et al. A phase II study of a 21 days continuous infusion schedule with epirubicin in advanced gastric cancer. Eur J Cancer Clin Oncol 1989;25:1509-10. 4 Speth PA, Linssen PC, Boezeman JB, Wesssels HM, Haanen C. Leukemic cell and plasma daunomycin concentrations after bolus injection and 72 h infusion. Cancer Chemother Pharmacol 1987;20:311-5. 5 Carlson RW, Sikic BI. Continuous infusion or bolus injection in cancer chemotherapy. Ann Intern Med 1983; 99:823-3. 6 Paul C, Tidefelt U, Liliemark J, Peterson C. Increasing the accumulation of daunorubicin in human cells by prolonging the infusion time. Leuk Res 1989;13:191-6. 7 Greidanus J, Willemse PH, Sleijfer DT, Mulder NH, Verschueren RC, Nieweg R, et al. Phase II study of a 21-day continuous infusion schedule with epirubicin in metastatic colorectal cancer. Eur J Cancer Clin Oncol 1988;24:801-2. 8 D'Arcy PF. Drug interactions and reactions update. Drugs Intell Clin Pharm 1983;17:726-31. 9 Moorhatch P, Chiou WL. Interactions between drugs and plastic intravenous fluid bags. Part I: sorption studies on 17 drugs. Am J Hosp Pharm 1974;31:72-8. 10 Benvenuto JA, Anderson RW, Kerkof K, Smith RG, Loo TL. Stability and compatibility of antitumor agents in glass and plastic containers. Am J Hosp Pharm 1981;38:1914-8. 11 Blum L. Bundgaard H. Sorption of drugs by plastic infusion bags. Int J Pharm 1982;10:339-51. 12 Bots AM, Van Oort WJ, Noordhoek J, Van Dijk A, Klein SW, Van Haesel QG. Analysis of adriamycin and adriamycinol in micro volumes of rat plasma. J Chromatogr 1983;272:421-7. 13 Poochikian GK, Cradock JC, Flora KP. Stability of anthracycline antitumor agents in four infusion fluids. Am J Hosp Pharm 1981;38:483-6. 14 Bosanquet AG. Stability of solutions of antineoplastic agents during preparation and storage for in vitro assays. II. Assay methods, adriamycin and the other antitumor antibiotics. Cancer Chem Pharm 1986;17: L10. 15 Robert J, David M, Huet S, Chauvergne J, Monnier A. Pharmacocin~tique de la pirarubicine (THP-doxorubicine) chez des malades canc~reux. Bull Cancer 1989;76:889-92. 16 Speth PA, Linssen PC, Boezeman JB, Wessels HM, Haanen C. Cellular and plasma adriamycin concentrations in long-term infusion therapy of leukemia 9patients. Cancer Chemother Pharm 1987;20:305-10. 17 Ackland SP, Ratain MJ, Vogelzang NJ, Choi KE, Ruane M, Sinkule JA. Pharmacokinetics and pharmacodynamics of long-term continuous-infusion doxorubicin. C]in Pharmacol Ther 1989;45:340-7. 18 Liso V, Specchia G, Pavone V, Capalbo S, Dione R. Continuous infusion chemotherapy with epirubicin and vincristine in relapsed and refractory acute leukemia. Acta Haematol 1990;83:116-9. 19 Kowaluk EA, Roberts MS, Blackburn HP, Polack AE. Interactions between drugs and polyvinyl chloride im fusion bags. Am J Hosp Pharm 1981;38:1308-14. 20 Magnam J, Martin JP. Les anticanc6reux sont-ils compatibles avec les mati~res plastiques? Lett QS Cancer 1988;72:1-6.
369
Keywords Chromatography, high pressure liquid Compatibility Daunorubicin Doxorubicin Drug stability Epirubicin Infusions Pirarubicin Polyvinyl chloride T. Dine (correspondence), J.C. Cazin, B. Gressier, M. Luyckx, C. Brunet and M. Cazin: Laboratoire de Pharmacologie, Pharmacocin~tique et Pharmacie Clinique, Facult~ des Sciences Pharmaceutiques et Biologiques, rue du Professeur Laguesse, B.P. 83, 59006 Lille C~dex, France. F. Goudaliez, M.L. Mallevais and I. Toraub: Laboratoires Macopharma, rue du PontRompue, B.P. 464, 59338 Tourcoing C~dex, France.
14(6) 1992
cases, diminish therapeutic response due to a reduced drug delivery to the patient. Other studies have shown a loss of anthracyclines in glass bottles due to adsorption [12]. Only a minimal amount of information is available concerning the compatibility of anthracyclines administered by PVC infusion bags and intravenous administration sets [13 14]. Such information is important when drug solutions are to be infused over long periods of time. The present study was u n d e r t a k e n with the following objectives: - to survey a range of drugs (anthracyclines), including those presently being administered by intravenous infusion, for possible interactions with PVC infusion bags; - to study the behaviour of these drugs in simulated infusion using PVC containers and administration sets; - to adapt infusions to the conditions routinely used in hospitals (infusion flow rate, dose, volume, temperature, light ...); - to determine the differences in possible interactions between PVC containers and administration sets, as well as the differences in stability of the drugs in 0.9% NaC1 and 5% glucose. A high pressure liquid chromatographic (HPLC) method used in the present study has been developed in our laboratory. This method allowed the rapid determination of doxorubicin, epirubicin, daunorubicin and pirarubicin, a new
Dine T, Cazin JC, Gressier B, Luyckx M, Brunet C, Cazin M, Goudaliez F, Mallevais ML, Toraub I. Stability and compatibility of four anthracyclines: doxorubicin, epirubicin, daunorubicin and pirarubicin with PVC infusion bags. Pharm Weekbl [Sci] 1992;14(6):365-9. Abstract
A rapid isocratic technique was developed for the analysis of four anthracyclines (doxorubicin, epirubicin, dannorubicin and pirarubicin) in parenteral solutions using high pressure liquid chromatography (HPLC) with fluorescence detection and a C18 Hypersil ODS column. The availability and compatibility of these drugs from solutions infused via PVC infusion bags through PVC administration sets have been examined. No significant drug loss was observed during simulated infusions (n = 4) for 24 h using PVC infusion bags and administration sets. No significant difference was found between infusion solutions (5% glucose or 0.9% NaC1), except for pirarubicin. The reconstitution of pirarubicin in 0.9% NaC1 was impossible, because we observed a precipitation of the compound in solution. The stability of the drugs was also studied in solution, in PVC bags after storage at 4~ with protection from light. The results show the stability of doxorubicin, epirubicin and daunorubicin during 7 days of storage to be satisfactory, irrespective of the infusion solution (5% glucose or 0.9% NaC1). In the case of pirm'ubicin, the stability of the drug was satisfactory during 5 days of storage in 5% glucose, but beyond, we observed a degradation of the compound with formation of doxorubicin in the infusion solution. Accepted August 1992.
Pharmaceutisch Weekblad Scientific edition
365
~
0
0 II
OH
0 - CHIOH
Methods
OH
~
COCH20H "'OH
H
o
c%o
OH
OCH~
6
Nit,
Epirubicin
Doxorubicin
CH30
0
OH 0
HO NH2
o
0
~
OH
OH
CH 30
6
0
OH COCH.OH "'"OH
0
OH
0
IIo
Pirarubicin
Daunorubicin
Materials The drug substances studied were commercial products suitable for clinical use. Doxorubicin and epirubicin were obtained from F a r m i t a l i a Carlo E r b a Laboratories (Milan, Italy) in vials of 10 m g sterile powder for injection. Daunorubicin and pirarubicin were obtained from Roger Bellon Laboratories (Neuilly sur Seine, France) in vials of 20 and 50 m g sterile powder for injection, respectively. Acetonitrile (HPLC grade), ammonia and formic acid (analytical grade) were obtained from Prolabo (Paris, France). W a t e r for injection (sterile and apyrogenic) obtained from M a c o p h a r m a Laboratories (Tourcoing, France) was used for buffers, dilutions and standard solutions.
Figure 1 Chemical structures of the anthracyclines studied
semisynthetic anthracycline, in infusion solutions (5% glucose and 0.9% NaC1) using a suitable chromatographic column and mobile phase. We h a v e used this analytical technique to investigate the compatibility of the drugs with PVC containers and PVC infusion sets both during simulated infusions and during storage at 4 ~ in PVC bags used in the hospital p h a r m a c y d e p a r t m e n t where the reconstitution of cytostatics is centralized.
Chromatographic conditions and instrumentation Chromatographic analysis was performed with an H P 1090 high pressure liquid chromatograph (Hewlett Packard, Orsay, France), equipped with a variab]e-volume injector, an automatic sampling system and a H P 1046A fluorescence detector operating at excitation and emission wavelengths 254 n m and 565 nm, respectively. The output from the detector was connected to a Hewlett P a c k a r d 9000 model 300 integrator and the data recorded on an H P Thinkjet t e r m i n a l printer. Analyses were performed on a 5 t~m C18 Hypersil ODS column (100 x 4,6 m m i.d.) (Hewlett Packard, Orsay, France) operating at room t e m p e r a t u r e . The analytical column was protected by a small pre-column (20 m m x 4.6 m m i.d.) packed with C18 Hypersil ODS 5 ~m (Hewlett Packard). Compounds were eluted isocratically with a mobile phase consisting of acetonitrile and formate buffer m i x t u r e (45+55; vo]/vol) at a flow
Table 1 Validation data of the H P L C assay procedure (n = 5)
Sample substance
Internal standard
Concentrations (~g/ml)
Average Coefficient of variation Accuracy Linear regression " concentrations (%) equation found • SD intraassay interassay (y= a x + b ) (~g/ml) (%) (%)
Doxorubicin
daunorubicin
6.25 12.5 25
6.263• 12.506• 25.012•
0.24 0.42 0.92
1.28 0.32 0.56
100.21 100.05 100.04
y = 0.057x - 0.127
0.999
6.25 12.5 25
6.274• 12.506• 25.014•
0.34 0.41 0.32
1.86 1.08 0.76
100.38 100.05 100.06
y = 0.047x + 0.104
0.999
6.25 12.5 25
6.241• 12.508• 25.010•
0.98 0.88 0.69
1.30 0.10 0.41
99.86 100.06 100.04
y = 0.090x - 0.347
0.999
6.25 12.5 25
6.232• 12.487• 24.975•
0.22 0.26 0.30
1.04 0.38 0.67
99.71 99.89 99.90
y = 0.036x
0.999
Epirubicin
Daunorubicin
Pirarubicin
366
daunorubicin
epirubicin
daunorubicin
-
0.131
P h a r m a c e u t i s c h Weekblad Scientific edition
Correlation coefficient (r)
14(6) 1992
r~
,:,-
r a t e of 2 ml/min. The formate buffer was prepared in w a t e r with a m m o n i a adjusted to pH 4 with pure formic acid. For simulated infusions, we used a volumetric infusion p u m p (VIP II) and PVC infusion sets (IS 05) obtained from Becton Dickinson Laboratories, Divison Vial Medical (SaintEtienne de Saint-Geoirs, France). Macoflex ~ PVC infusion bags (50, 250 and 500 ml) were kindly provided by M a c o p h a r m a Laboratories. The bags were filled with 0.9% NaC1 or 5% glucose in w a t e r to study the possible influence of solvent on the interaction of the drug.
c (i.s.)
F,-
c 0.s.)
A
B(~.)
c (Ls,)
a_
Preparation of standard solutions
D
x
L
(a) --
(c)
(b)
~ 1 ~ - i ~ 1
(d)
,~
~
r 1
2
~
[
1
I
I
I
r
I
"
T i m e (rain.)
Figure 2 Examples of chromatographic analysis. (a): doxorubicin with internal standard daunorubicin; (b): epirubicin with internal standard daunorubicin; (c): daunorubicin with internal standard epirubicin; (d): pirarubicin with internal standard daunorubicin
To obtain standard stock solutions, all drugs were reconstituted with distilled w a t e r to give a drug concentration of 50 t~g/m] in all four cases. These standard solutions were protected from light. Working solutions were p r e p a r e d from the standard solutions by suitable dilutions with distilled w a t e r in polypropylene tubes. Calibration curves were constructed in all four cases between 6.25 ~g/ml and 25 ~g/ml. For quantification of drugs, an internal standard was employed to dilute the samples. The use of an internal standard was needed to reduce the errors of dilution, because the drug concentrations were high in the PVC bags during simulated infusions and storage. The p e a k ratio (drug p e a k area/internal standard p e a k area) was calculated and the a m o u n t of drug determined by reference to the calibration curve. Daunorubicin was used as the internal standard to quantify doxorubicin, epirubicin and pirarubicin, while epirubicin was employed in the case of daunorubicin.
Simulated infusions
lO,O
10,0
9,5
9,5
A
9,0
9,o
OONC (mg/500rlll)
C
a.5
8,5 B,0 7,5 (mg/5OOm0 7,0
7,5 7,o
6,5 6,0 5,5
--[ .
.
.
................. 1
.
from infusion sets -
~
-
6,0 5,5 5,0
5,0 5
15
30
1 2 4 l I M E (rain h )
6
8
10
24
5
15
3O
2
-4
S
8
10
24
10
24
TIME (rain h)
12,0 -
12,0.
11,5 11,0 10,5
---[ -
................ l
-
~- from infusion sets -
....
-
~1,o -
10,5
CONC 100 (mg/500m) 9,5 :
(mg/500ml) 9,5
9,0
B
9,0.
8,5
8,5 aJ 5
15
30
1
2
4
TIME (rain. h.)
6
B
10
24
5
15
30
1
2
4
5
8
TIME (rain. h.)
Figure 3 Concentration kinetics of doxorubicin (A), epirubicin (B), daunorubicin (C) and pirarubicin (D) during simulated infusions (n = 4) using plastic infusion bags and sets
14(6) 1992
P h a r m a c e u t i s c h Weekblad Scientific edition
Infusions were simulated out under laboratory conditions i m i t a t i n g infusions to patients routinely used in hospitals. For this purpose, we used an infusion p u m p and PVC a d m i n i s t r a t i o n sets. The respective drug concentrations in solution were 8 rag/500 ml (16 ~g/ml) for doxorubicin and daunorubicin, and 10 mg/500 ml (20 #m/ml) for epirubicin and pirarubicin. The simulated infusions were carried out over a period of 24 h at a flow r a t e of 21 ml/min. Infusion solutions of drugs were prepared in PVC infusion bags containing 500 ml 5% glucose or 0.9% NaC1 i m m e d i a t e l y before the infusion. The bag containing drug was t h e n attached to an administration set connected to the infusion p u m p t h a t allowed the solution to flow t h r o u g h at a constant rate. At specified times of infusion, 1 ml samples were w i t h d r a w n at r e g u l a r intervals from the PVC bags, and at the same t i m e an aliquot of effluent (1 ml) was collected from the administration set. Samples were kept frozen in polypropylene tubes at - 2 0 ~ until analysis by HPLC. All simulated infusions were carried out at least in quadruplicate (n = 4: two infusions in 0.9% NaC1 and two infusions in 5% glucose) at a m b i e n t t e m p e r a t u r e (20-24~ without protection from light.
367
Storage in infusion bags Insofar as it was possible, we employed conditions in conformity with the drug concentrations normally used in hospital pharmacy departments for the storage of drugs in infusion bags. To infusion bags containing 50, 250 or 500 ml of 0.9% NaC1 or 5% glucose solution, a known amount of drugs was added to achieve the following concentrations which are most often used in hospitals: doxorubicin, 40 ~g/ml; epirubicin, 40 ~g/ml; daunorubicin, 16 t~g/ml; pirarubicin, 800 ~g/ml. After mixing the drug in the bag by rapid shaking, i ml samples were withdrawn at regular intervals and stored in polypropylene tubes at - 2 0 ~ until HPLC analysis. Infusion bags containing the drug were stored at 4~ for a period of 7 days with protection from light. Drug storage in these bags was carried out in 0.9% NaC1 and 5% glucose. Sample preparation Before HPLC analysis, each defrozen sample was suitably diluted with an internal standard solution in order to obtain a drug concentration within the calibration range. Results and discussion
High pressure liquid chromatography The chromatograms of the four drugs with the respective internal standards in solution obtained immediately after mixing, are illustrated in Figure 2. The drugs were simultaneously and rapidly separated, identified and quantified. The components were satisfactorily resolved by this HPLC method and had retention times of 1.65 min (doxorubicin), 1.80 min (epirubicin), 2.65 min (daunorubicin) and 3.75 min (pirarubicin). Table 1 summarizes the validation data of the assay procedure for each drug. We observed good linearity between peak area ratios and concentrations. The calibration curves were fitted by the least-square method for the peak area ratio of the sample substance and the internal standard (y) versus the concentration of the analysed product (x). The correlation coefficients were all above 0.999 and no significant differences were observed between the equation parameters. To assess reproducibility, the same concentration was analysed five times for each point of the calibration curves. The results demonstrate that this analytical method has acceptable accuracy and precision in every case.
Figure 4 HPL chromatogram obtained after injection of a sample ofpirarubicin (D) obtained after 5 days of storage in a PVC bag at 4 ~ A: degradation product identified as doxorubicin; C: daunorubicin (internal standard)
(Ls.)
o
Time (rnln,)
Stability of anthracyclines during simulated infusions using P V C infusion bags and sets The analysis of each sample was performed by HPLC after a suitable dilution in the mobile phase in order to fit the calibration curves. Figure 3 depicts the concentration kinetics of all four drugs during simulated infusions (n = 4), using plastic infusion bags and sets. When solutions of doxorubicin, epirubicin and daunorubicin were infused through PVC infusion sets from PVC infusion bags over a period of 24 h, the variation in drug concentration in both the PVC bags and effluent in no case exceeded 10%. This demonstrates that the three drugs were not sorbed by the PVC infusion bags and sets during infusion at ambient temperature. No significant difference was observed with respect to drug stability during simulated infusions using 5% glucose or 0.9% NaC1. In contrast, in the case of pirarubicin the study had been only effected in 5% glucose, because the drug was not sufficiently soluble in 0.9% NaC1 and a precipitation of compound was rapidly observed in the solution. In addition, the simulated infusion showed variations more important than in the case of the other drugs, as well as more irregular concentration kinetics. However, the variation in drug concentration in both the PVC bags and effluent did not exceed 10% during simulated infusions over a period of 24 h. No degradation product was detected by HPLC in infusion solution.
Table 2 Concentrations (zg/ml) present in solution after storage in plastic bags at 4 oC
Drug
Doxorubicin
Infusion solution
0.9% NaC1
5% glucose
0.9% NaC1
5% glucose
0.9% NaC1
5% glucose
5% glucose
Initial 1 day 3 days 5 days 7 days
40.00 40.20+_0.12 39.68_+0.16 39.40_+0.10 37.56-+0.08
40.00 39.00+0.14 37.96+_0.12 37.08_+0.11 36.08-+0.07
40.00 40.20_+0.24 38.60+0.32 37.76_+0.14 36.32+0.18
40.00 39.68+_0.27 39.84+0.34 39.56_+0.22 40.56+0.19
16.00 16.10_+0.06 16.28+0.12 15.92_+0.17 15.94_+0.12
16.00 16.90• 16.46_+0.18 15.88_+0.22 16.24_+0.13
800.00 769.80+_24.16 816.40_+12.34 791.20_+16.32 719.20_+13.48
Pharmaceutisch Weekblad Scientific edition
14(6) 1992
368
Epirubicin
Daunorubicin
Pirarubicin
Stability of anthracyclines in infusion bags during storage at 4~ with protection from light The analysis of each sample was performed by HPLC after a suitable dilution in the mobile phase in order to fit the calibration curves. The concentrations of all four drugs in solutions after various periods of storage in PVC infusion bags at 4~ with protection from light are listed in Table 2. No significant disappearance of drug (exceeding 10%) was observed in PVC infusion bags for doxorubicin, epirubicin and daunorubicin over a period of 7 days of storage, irrespective of the infusion solution (5% glucose or 0.9% NaC1). In contrast, in the case of pirarubicin, the study had only been effected in 5% glucose. We did note a significant disappearance of drug during a period of 7 days of storage. Table 2 shows that the loss of drug exceeded 10% after 5 days of storage. This loss could be explained by the formation of a degradation product of pirarubicin, which was identified as doxorubicin. Figure 4 shows an HPL chromatogram obtained after injection of 10 ~1 of sample obtained after 5 days of storage at 4~ The detection of doxorubicin in the infusion solution could lead to a modified therapeutic response to t r e a t m e n t using. In this and an other study [15], the results show, respectively, the in vitro and in vivo formation of doxorubicin from pirarubicin.
Conclusion In conclusion, the HPLC procedure described in this paper, rapidly and reproducibly determines anthracyclines in parenteral solutions. With the increasing use of continuous intravenous infusion of anthracyclines [16-18], the present study has examined the kinetics of doxorubicin, epirubicin, daunorubicin and pirarubicin concentration during simulated infusion using PVC infusion bags and administration sets. The results demonstrate a satisfactory compatibility of anthracyc]ines with PVC infusion material over a 24-h infusion 9 It is likely t h a t other drugs interact with PVC infusion bags and administration sets, leading to a reduction in the clinical effectiveness of the drug [19]. This type of study is important with regard to the packaging of pharmaceuticals in plastic containers in general [20], and might be carried out for all drugs administered in PVC infusion bags.
Acknowledgement This work was supported by grants of the Comit~ du Nord de la Ligue Nationale Franr Contre le Cancer and the Conseil R~gional du Nord-Pas de Calais. The authors also wish to t h a n k Macopharma, F a r m i t a l i a Carlo Erba and Roger Bellon Laboratories for co-operation in this study.
14(6) 1992
Pharmaceutisch Weekblad Scientific edition
References 1 Lavelle F. Structure et activit6s des anthracyclines. Pathol Biol (Paris) 1987;35:11-9. 2 Greidanus J, Willemse PH, Uges DR, Oremus ET, De Langen ZJ, De Vries EG. Continuous infusion of low-dose doxorubicin, epirubicin and mitoxantrone in cancer chemotherapy: a review. Pharm Weekbl [Sci] 1988;10:237-45. 3 De Vries EG, Nanninga AG, Greidanus J, Oremus ET, Verschueren RC, Mulder NH, et al. A phase II study of a 21 days continuous infusion schedule with epirubicin in advanced gastric cancer. Eur J Cancer Clin Oncol 1989;25:1509-10. 4 Speth PA, Linssen PC, Boezeman JB, Wesssels HM, Haanen C. Leukemic cell and plasma daunomycin concentrations after bolus injection and 72 h infusion. Cancer Chemother Pharmacol 1987;20:311-5. 5 Carlson RW, Sikic BI. Continuous infusion or bolus injection in cancer chemotherapy. Ann Intern Med 1983; 99:823-3. 6 Paul C, Tidefelt U, Liliemark J, Peterson C. Increasing the accumulation of daunorubicin in human cells by prolonging the infusion time. Leuk Res 1989;13:191-6. 7 Greidanus J, Willemse PH, Sleijfer DT, Mulder NH, Verschueren RC, Nieweg R, et al. Phase II study of a 21-day continuous infusion schedule with epirubicin in metastatic colorectal cancer. Eur J Cancer Clin Oncol 1988;24:801-2. 8 D'Arcy PF. Drug interactions and reactions update. Drugs Intell Clin Pharm 1983;17:726-31. 9 Moorhatch P, Chiou WL. Interactions between drugs and plastic intravenous fluid bags. Part I: sorption studies on 17 drugs. Am J Hosp Pharm 1974;31:72-8. 10 Benvenuto JA, Anderson RW, Kerkof K, Smith RG, Loo TL. Stability and compatibility of antitumor agents in glass and plastic containers. Am J Hosp Pharm 1981;38:1914-8. 11 Blum L. Bundgaard H. Sorption of drugs by plastic infusion bags. Int J Pharm 1982;10:339-51. 12 Bots AM, Van Oort WJ, Noordhoek J, Van Dijk A, Klein SW, Van Haesel QG. Analysis of adriamycin and adriamycinol in micro volumes of rat plasma. J Chromatogr 1983;272:421-7. 13 Poochikian GK, Cradock JC, Flora KP. Stability of anthracycline antitumor agents in four infusion fluids. Am J Hosp Pharm 1981;38:483-6. 14 Bosanquet AG. Stability of solutions of antineoplastic agents during preparation and storage for in vitro assays. II. Assay methods, adriamycin and the other antitumor antibiotics. Cancer Chem Pharm 1986;17: L10. 15 Robert J, David M, Huet S, Chauvergne J, Monnier A. Pharmacocin~tique de la pirarubicine (THP-doxorubicine) chez des malades canc~reux. Bull Cancer 1989;76:889-92. 16 Speth PA, Linssen PC, Boezeman JB, Wessels HM, Haanen C. Cellular and plasma adriamycin concentrations in long-term infusion therapy of leukemia 9patients. Cancer Chemother Pharm 1987;20:305-10. 17 Ackland SP, Ratain MJ, Vogelzang NJ, Choi KE, Ruane M, Sinkule JA. Pharmacokinetics and pharmacodynamics of long-term continuous-infusion doxorubicin. C]in Pharmacol Ther 1989;45:340-7. 18 Liso V, Specchia G, Pavone V, Capalbo S, Dione R. Continuous infusion chemotherapy with epirubicin and vincristine in relapsed and refractory acute leukemia. Acta Haematol 1990;83:116-9. 19 Kowaluk EA, Roberts MS, Blackburn HP, Polack AE. Interactions between drugs and polyvinyl chloride im fusion bags. Am J Hosp Pharm 1981;38:1308-14. 20 Magnam J, Martin JP. Les anticanc6reux sont-ils compatibles avec les mati~res plastiques? Lett QS Cancer 1988;72:1-6.
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