Drugs R D (2015) 15:253-259
`DOI 10.1007/s40268-015-0098-4
`
`ORIGINAL RESEARCH ARTICLE
`
`CrossMark
`
`Stability of Melphalan in 0.9 % Sodium Chloride Solutions
`Prepared in Polyvinyl Chloride Bags for Intravenous Injection
`
`Romain-Pacome Desmaris1
`
`• Lionel Mercier1
`
`• Angelo Paci1
`
`Published online: 16 July 2015
`©The Author(s) 2015. This article is published with open access at Springerlink.com
`
`Abstract Melphalan is an alkylating agent frequently
`used in an intravenous formulation to treat hematologic
`malignancies and solid tumors in both adults and children.
`According to the manufacturer, melphalan is stable in
`sterile 0.9 % sodium chloride for 90 min at room temper(cid:173)
`ature (RT). Several authors have studied the stability of
`different concentrations of melphalan; however, most were
`not adapted to the current manufacturing process applied in
`pharmaceutical centralized units. This study was conducted
`to determine the stability of melphalan in 0.9 % sodium
`chloride solutions at concentrations used for intravenous
`injection in practice. Melphalan is commonly prepared in
`diluted solutions ranging from 2 to 4 mg/ml for the treat(cid:173)
`ment of adult patients and at lower concentrations (down to
`0.5 mg/ml) for pediatric use. Accordingly, these were the
`three concentrations chosen for this study. Melphalan
`concentrations were measured with high-performance thin(cid:173)
`layer chromatography (HPTLC). At RT, admixtures pre(cid:173)
`pared at 4 mg/ml were stable for up to 8 h without pro(cid:173)
`tection from light; however, at lower concentrations, such
`as 0.5 and 2 mg/ml, stability did not exceed 2 h. When
`refrigerated, melphalan was stable for 24 h at 2 mg/ml;
`however, at 0.5 and 4 mg/ml, the drug was not stable.
`
`181 Romain-Pacome Desmaris
`romain.desmaris@gustaveroussy.fr
`
`Lionel Mercier
`lionel.mercier@gustaveroussy.fr
`
`Angelo Paci
`angelo.paci@gustaveroussy.fr
`
`Department of Clinical Pharmacy, Gustave Roussy Hospital,
`114 Rue Edouard V aillant, 94 805 Villejuif Cedex, France
`
`Melphalan solutions present with limited stability at 0.5, 2,
`and 4 mg/ml and are not adapted for delayed administra(cid:173)
`tion in pharmaceutical centralized units. However, at 4 mg/
`ml and at RT, a stability of 8 h is very interesting in
`practice and allows sufficient time for preparation, phar(cid:173)
`maceutical control, transport, and administration.
`
`1 Introduction
`
`Melphalan is an alkylating agent available as either an oral
`or an intravenous formulation and used to treat malignancy
`in both adult and pediatric patients. Melphalan is a major
`drug against hematological malignancies (multiple mye(cid:173)
`loma, Hodgkin or non-Hodgkin lymphoma, lymphoid
`leukemia, or myeloid acute leukemia), solid tumors such as
`ovarian or breast cancers, and children's neuroblastoma
`with posologies comprising between 100 and 200 mg/m2
`according to
`the summary of product characteristics
`(SmPC) [1]. Melphalan is mainly used as intensive treat(cid:173)
`ment supported by autologous stem-cell transplantation
`and administered alone
`in multiple myeloma at
`140--200 mg/m2
`, and at 100--140 mg/m2 in combination
`with other cytotoxic drugs (such as busulfan) in pediatric
`solid tumors (neuroblastoma, Ewing's sarcoma, peripheral
`neuroectodermal tumor, rhabdomyosarcoma) [2-4]. In our
`institution, an off-label use of melphalan includes isolated
`limb or pelvic perfusion at 1.5 mg/kg [5]. According to the
`manufacturer, melphalan is stable in 0.9 % sodium chlo(cid:173)
`ride for no longer than 90 min at room temperature (RT)
`[l ]. Melphalan is commonly used in diluted solutions
`ranging from 2 to 4 mg/ml for adults; for pediatric use,
`lower concentrations (down to 0.5 mg/ml) are frequently
`used in pediatric patients.
`
`L\Adis
`
`Hospira, Exh. 2027, p. 1
`
`

`
`254
`
`2 Aim of the Study
`
`Several works have studied the stability of melphalan in
`polyvinyl chloride (PVC) bags. Beitz and colleagues [6, 7]
`reported a stability of 24 h when 0.9 % sodium chloride
`solutions at 0.06 mg/ml were stored at 4 °C, while this
`duration was reduced to 90 min when those solutions were
`stored at 23 °C without protection from light exposure.
`Pinguet et al. [8] reported a stability of 6 h when 0.9 %
`sodium chloride solutions of melphalan prepared at
`0.2 mg/ml were stored at 4 °C and protected from light.
`Chang et al. [9] reported that melphalan hydrolysis
`decreased when sodium concentration increased [9]. These
`data were confirmed by Pinguet et al. [8], who showed that
`melphalan was stabile for longer when it was diluted in a
`3 % sodium chloride solution. In our institution, to avoid
`error with the isotonic form, the use of hypertonic sodium
`chloride solution is not recommended. More recently, B.
`BRAUN AG, Melsungen, Germany, medical devices
`manufacturer, studied the stability of melphalan in 0.9 %
`sodium chloride solutions in Ecoflac® bags (flexible bags
`made
`from polyethylene). They demonstrated
`that
`0.06 mg/ml melphalan solution was stable for 24 h when
`stored at 4 °C and for 2 h at 22 °C under the same con(cid:173)
`ditions [10]. These few published data provide information
`about the manufacturing process for very low concentra(cid:173)
`tions of melphalan administered to patients. Hospital
`pharmacists need more detailed information about the
`stability of this diluted drug at higher concentrations,
`according to current clinical practice. Further data regard(cid:173)
`ing the stability of melphalan in solutions prepared at
`2-4 mg/ml are currently lacking. In order to acquire novel
`experience on the stability of melphalan at these higher
`concentrations commonly used in daily clinical practice,
`we conducted a physico-chemical stability study of mel(cid:173)
`phalan solutions prepared at 0.5, 2, and 4 mg/ml in 0.9 %
`sodium chloride stored at both refrigerated temperatures
`(5 ± 3 °C) and RT (25 ± 2 °C). By conducting this study,
`we intended to optimize the manufacturing production
`schedule in a centralized unit to improve patient care
`management with the possibility of preparing these infu(cid:173)
`sion bags with a prolonged duration of storage.
`
`3 Materials and Methods
`
`3.1 Materials
`
`Alkeran® (melphalan 5 mg/ml) for intravenous use from
`GlaxoSmithKline (batch no. 7004) was used. Melphalan
`(Fig. 1) was reconstituted with its specific solvent (10 ml
`
`L\Adis
`
`R. Desmaris et al.
`
`Fig. 1 Chemical structure of melphalan 2-amino-3-[4-(2-(dichlor(cid:173)
`oethyl)amino)phenyl] propanolc acid
`
`of sterile water with a mixture of sodium citrate, propylene
`glycol, and alcohol in injectable water) to obtain a final
`concentration of 5 mg/ml in each glass vial as recom(cid:173)
`mended in the SmPC by the manufacturer [1 ]. Emptied
`PVC bags by Maco Pharma with a 100 ml volume of
`capacity (batch 07B15H) were used. For the degradation
`study, 0.1 M hydrochloric acid (0.1 M HCl) and 0.1 M
`sodium hydroxide (0.1 M NaOH) were provided by Pro(cid:173)
`labo-VWR International SA, and 10 % hydrogen peroxide
`(10 % H102) by Cooper.
`
`3.2 Preparation, Storage, and Sampling
`
`Melphalan admixtures (diluted solutions) were prepared
`by filling emptied PVC bags with melphalan reconsti(cid:173)
`tuted solution at 5 mg/ml and 0.9 % sodium chloride
`solution to achieve the three drug concentration levels
`(4, 2, and 0.5 mg/ml). PVC bags were filled to final
`volume of 50 ml. All admixtures ( 18 total, six for each
`concentration and three for each concentration and each
`temperature) were prepared in a vertical laminar airflow
`hood to ensure aseptic conditions and to protect opera(cid:173)
`tors from toxic effects. Concentration ranges of mel(cid:173)
`phalan were defined according to clinical practice in our
`hospital (0.5 mg/ml for pediatric use, 2 and 4 mg/ml for
`adults).
`The effect of temperature during storage was investi(cid:173)
`gated. Bags were stored at refrigerated
`temperature
`(5 ± 3 °C) or at RT (25 ± 2 °C). Bags stored at RT were
`not protected from light, while bags at 5 °C were protected
`from light. However, almost no data on the influence of
`light on melphalan stability are available.
`At the end of the preparation, the admixture samples
`were removed at defined intervals ranging from 0 to 24 h.
`The sampling was as follows: at the end of the preparation
`(HO), after 1 h (Hl), 2 h (H2), 4 h (H4), 6 h (H6), 12 h
`(H12), and 24 h (H24). We expected the stability of mel(cid:173)
`phalan solutions at RT to be reduced compared with those
`stored at 5 °C; therefore, additional samples after 8 h (H8)
`and 16 h (H16) were obtained for bags stored at RT.
`Samples were not treated immediately but stored in ade(cid:173)
`quate conditions protected from light and at -20 °C.
`
`Hospira, Exh. 2027, p. 2
`
`

`
`Melphalan in Polyvinyl Chloride Bags for IV Use
`
`255
`
`3.3 Chromatographic Analysis and Analytical
`Validation
`
`Table 2 Validation of the repeatability and intermediate precision
`study
`
`Melphalan concentrations were measured via high-perfor(cid:173)
`mance thin-layer chromatography (HPTLC CAMAG®)
`combined to an ultraviolet (UV) detection with fluores(cid:173)
`cence extinction quantitation. This analytical method has
`been previously developed and published and has been
`routinely used in our quality control (QC) laboratory for
`several years for 39 compounds, including melphalan. The
`assessment of the temperature storage conditions has been
`validated during the analytical validation carried out for
`each developed analytical method. Samples of melphalan
`can be frozen without degradation [11- 13]. Validation was
`conducted by evaluating common parameters defined by
`the International Conference of Harmonization (ICH) [14]
`such as specificity, linearity, accuracy, precision (repeata(cid:173)
`bility and intermediate precision), and limits of detection
`(LOD) and quantification (LOQ). The parameters were
`determined by the statistical analysis of six calibration
`plots. The stationary phases (Lichrospher® Si60F254 nm),
`manufactured by Merck, were made of uniform 0.2 nm
`thin silica layers that were placed on a glass surface of
`20 x 10 cm. Sample application was standardized: auto(cid:173)
`mated TLC sampler m® devices take into account defined
`parameters such as the volume, size of the sprayed band,
`and accurate positioning on the chromatography plate.
`These parameters were computerized by ATS m® soft(cid:173)
`ware. After migration, the plate was analysed on a TLC
`scanner 3® densitometer driven by CATS 4® software as
`previously reported [11]. The HPTLC analysis station by
`CAMAG® is composed of a HPTLC Vario-<:hamber for
`mobile phase optimization, automated sample applicators
`(TLC Sampler III), which manage the calibrated applica(cid:173)
`tions of sampled solutions (volume required is 500 nL),
`solid Teflon® (PFTE) migration chambers, and a TLC
`Scanner 3® densitometer driven by a winCATS® software.
`Accuracy results, providing information about the recovery
`of the analyte from the sample, are summarized in Table 1.
`Means and relative standard deviation (RSD) values were
`calculated from the six determinations of each QC with
`three different known concentrations of melphalan, which
`were classified as low, medium, and high (QCL, QCM, and
`
`Table 1 Results of the accuracy study
`
`CV,(%)•
`CV1(%t
`
`QCL
`
`0.7
`3.9
`
`QCM
`
`1.0
`3.9
`
`QCH
`
`1.4
`3.9
`
`CV; coefficient of variation of intermediate precision, CV, coefficient
`of variation of repeatability, QCH high quality control, QCL low
`quality control, QCM medium quality control
`• The mean and CV values were calculated on six different
`measurements
`
`QCH, respectively). Precision contains three components
`according to the ICH guidelines: repeatability, intermediate
`precision, and reproducibility (not studied for this latter).
`Repeatability and intermediate precision are reported in
`Table 2. Repeatability, expressed as RSD, or coefficient of
`variation of repeatability (CVr), consisted of the analysis of
`each QC six times according to the same analytical pro(cid:173)
`cedure with the same equipment in the same laboratory.
`Calibration curves were generated by non-linear Michae(cid:173)
`lis-Menten-like regression and were validated by quality
`controls. Intermediate precision evaluates the reliability of
`the method in an environment other than that used during
`development of the method. Determination, expressed as
`RSD or coefficient of variation of intermediate precision
`(CV J, consisted of the analysis of each QC six times under
`the same analytical conditions but on multiple days, by
`different analysts and using different equipment, except the
`HPTLC workstation. For the forced degradation study,
`melphalan concentrations were also measured via HPTLC
`CAMAG®, which is a relevant separating method accord(cid:173)
`ing to the most recent guidelines for practical stability
`studies of anti-cancer drugs [15].
`
`3.4 Physico-Chemical Investigations
`
`We investigated all admixtures for particulate matter: color
`change, clarity, and pH values, which were measured with
`a digital pH meter.
`Stability was defined as the presence of at least 90 % of
`melphalan concentration according to reported recommen(cid:173)
`dations [16]. Each sample was analyzed in triplicate. A five(cid:173)
`level calibration range was set and validated by three levels
`of QC to conduct the sample analysis of melphalan solutions.
`
`QCL
`
`QCM
`
`QCH
`
`150.5 (150)
`5.89
`
`250.0 (250)
`9.83
`
`458.2 (450)
`17.99
`
`4 Results
`
`QCH high quality control, QCL low quality control, QCM medium
`quality control, RSD relative standard deviation
`a Values in brackets correspond to the target values; the mean and
`RSD values were calculated on six different measurements
`
`4.1 Organoleptic Characteristics
`
`All the admixtures stored at RT and prepared at 0.5, 2, and
`4 mg/ml remained clear for 24 h. When stored at 5 °C, the
`
`b.Adis
`
`Hospira, Exh. 2027, p. 3
`
`

`
`256
`
`R. Desmaris et al.
`
`Chromatogram A
`
`Chromatogram B I
`
`I Chromatogram C
`
`t-~-~ .. ~
`
`0-0- 1
`
`...
`
`I-+_.,.";.,.."··~ .. ~~~· -
`
`--!- - - - -
`
`""
`
`Chromatogram D
`
`Chromatogram E
`
`Fig. 2 Chromatograms of 2 mg/ml melphalan solution submitted to various stress testing of forced degradation study
`
`Table 3 Concentration evolution of melphalan at 4 °C
`
`Hours
`
`HO
`
`H1
`
`H2
`
`H4
`
`H6
`
`H12
`
`H24
`
`0.5
`Mean
`SD
`A(%Ho)
`2
`Mean
`SD
`A{%Ho)
`4
`Mean
`SD
`A(%Ho)
`
`0.485
`1.41 x 10-3
`
`0.362
`4.24 x 10-3
`25.3
`
`0.373
`16.97 x 10-3
`23.1
`
`0.390
`12.73 x 10-3
`19.6
`
`0.380
`15.56 x 10-3
`21.6
`
`0.355
`4.95 x 10-3
`26.8
`
`0.351
`7.78 x 10-3
`27.2
`
`2.063
`4.04 x 10-2
`
`2.002
`7.64 x 10-3
`3.0
`
`2.055
`6.24 x 10-2
`0.4
`
`4.230
`1.73 x 10-2
`
`4.053
`4.93 x 10-2
`4.2
`
`4.167
`5.13 x 10-2
`1.5
`
`1.985
`5.07 x 10-2
`3.7
`
`4.120
`1.70 x 10-1
`2.6
`
`1.960
`2.78 x 10-2
`5.0
`
`4.160
`1.17 x 10-1
`1.7
`
`1.900
`5.50 x 10-2
`7.9
`
`1.930
`8.66 x 10-3
`6.4
`
`4.100
`1.70 x 10-1
`3.1
`
`3.917
`1.20 x 10-1
`7.4
`
`SD standard deviation
`
`admixtures remained clear at 0.5 and 2 mg/ml for 24 h. For
`those prepared at 4 mg/ml, the solution was turbid and
`colored from H4.
`
`4.2 Forced Degradation Study
`
`Figure 2 shows the results of the forced degradation study
`for melphalan solutions at 2 mg/ml in various stressed
`conditions and after 6 h of exposition according to the ICH
`
`[14]. Chromatogram A shows the result of a solution of
`melphalan diluted in 0.9 % sodium chloride solution.
`Chromatogram B shows the result of a solution of mel(cid:173)
`phalan diluted in 0.1 M HCL Chromatogram C shows the
`result of a solution of melphalan diluted in 0.1 M NaOH.
`Chromatogram D shows the results of a solution of mel(cid:173)
`phalan diluted in H20 2 10 %. Curve E shows the results of
`a solution of melphalan diluted in 0.1 M HCI with zinc
`powder. The mean of the concentration variation values for
`
`b.Adis
`
`Hospira, Exh. 2027, p. 4
`
`

`
`Melphalan in Polyvinyl Chloride Bags for IV Use
`
`257
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`the melphalan solutions after 6 h of exposure were calcu-
`lated on six different measurements. The loss of melphalan
`was calculated at 22.5 % for melphalan diluted in 0.9 %
`sodium chloride solution, 13.6 % for melphalan diluted in
`0.1 M HCl, 70.0 % for melphalan diluted in 0.1 M NaOH,
`39.l % for melphalan diluted in H20 2 10 %, and 18.4 %
`for melphalan diluted in 0.1 M HCl with zinc powder.
`Evolution of melphalan content in different stress testing
`conditions shows that melphalan is greatly decreased in
`alkaline and oxidative conditions, while it remains more
`stable in acidic conditions. Exposure to strong reductive
`conditions also shows a decrease of melphalan concentra-
`tion. On the chromatograms, five peaks of degradation
`products appears with retention factors (Rf) lower than the
`Rf obtained for the peak of melphalan. Therefore, the
`chromatographic method is able to separate its main
`degradation products and is based on loss of parent com-
`pound. No attempt was made to resolve the degradation
`products completely .
`
`4.3 Melphalan Concentration Evolution Study
`
`Table 3 shows that the solutions of melphalan at 0.5 mg/
`ml stored at 5 °C presented the largest changes as com-
`pared with HO. After only 2 h, the calculated change was
`23.1 %, while at 2 and 4 mg/ml the variation was limited,
`with respective degradation percentages calculated at 0.4
`and 1.5 %. This decrease in melphalan concentrations was
`also observed at RT as shown in Table 4. When stored at
`RT, melphalan concentrations decreased between 8 and
`12 hat 4 mg/ml (0.7 % of melphalan degraded at 8 h vs.
`22.8 % at 12 h). For the solutions stored at RT and pre-
`pared at 0.5 and 2 mg/ml, melphalan concentration
`decreased more rapidly. After 2 h, the respective degra-
`dations percentages were 4.2 and 2.7 %, and after 4 h they
`were 12.4 and 17.4 %. Admixtures of melphalan at 4 mg/
`ml were more stable than 0.5 and 2 mg/ml solutions. The
`different concentrations seemed to have an effect on
`melphalan stability, as more concentrated solutions
`(above 2 mg/ml) were found to be more stable than
`diluted solutions (0.5 mg/ml). Bosanquet [17] explained
`that this difference is probably due to the faster decrease
`in pH due to ion chloride liberation concomitantly to the
`degradation of melphalan. This phenomenon was found
`both at low temperatures and at RT. However, concen-
`trations of melphalan decreased more rapidly at RT than
`at low temperature. For the solutions prepared at 2 and
`4 mg/ml and stored at 4 °C, physical stability was
`demonstrated for 24 h (respective degradations of 6.5 and
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`<l
`
`fjj '-'
`<l
`
`E3
`
`not stable. At room temperature, stability did not exceed
`8 h for concentrated solutions (4 mg/ml), and was shorter
`for diluted solutions.
`
`b.Adis
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`Hospira, Exh. 2027, p. 5
`
`

`
`258
`
`R. Desmaris et al.
`
`Table 5 Summarized results of melphalan stabilities at the two studied storage conditions at 0.5, 2, and 4 mg/ml
`
`Concentration (mglml)
`
`Temperature: 5 °C
`
`Temperature: 25 °C
`
`Organoleptic characteristics
`
`Stability (h)
`
`Organoleptic characteristics
`
`Stability (h)
`
`No precipitation
`No precipitation
`Precipitation
`
`<l
`24
`24
`
`No precipitation
`No precipitation
`No precipitation
`
`2
`2
`8
`
`conditions. This work also reports that admixtures prepared
`at 2 mg/ml can be stored for 24 h at refrigerated temper(cid:173)
`atures (5 ± 3 °C). Admixtures prepared at 0.5 and 4 mg/
`ml are not stable in these conditions, and the advance
`preparation of melphalan solutions at these concentrations
`for delayed administration is not recommended.
`
`Acknowledgments This study did not receive any external funding
`and the authors report no relevant financial conflicts of interest.
`
`Open Access This article is distributed under the terms of the
`Creative Commons Attribution-NonCommercial 4.0 International
`License (http://creati.vecommons.org/licenses/by-nc/4.0/), which per(cid:173)
`mits any noncommercial use, distribution, and reproduction in any
`medium, provided you give appropriate credit to the original
`author(s) and the source, provide a link to the Creative Commons
`license, and indicate if changes were made.
`
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`5 Discussion
`
`Solutions of melphalan diluted in PVC bags show limited
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`the
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`high concentrations such as 2 and 4 mg/ml than at 0.5 mg/
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`Temperature has a counter-intuitive effect on melphalan
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`than at RT after preparation. However, concentration levels
`were also important for the stability of these solutions, as
`diluted solutions showed higher instability. We conclude
`that melphalan in 0.9 % sodium chloride at 0.5 mg/ml is
`not stable at both temperatures.
`
`6 Conclusion
`
`It is a very important challenge for pharmacists working in
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`can be stored for 8 h without protection from light. For
`lower concentrations such as 0.5 and 2 mg/ml, no more
`than 2 h of stability can be expected in the same
`
`b.Adis
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`Hospira, Exh. 2027, p. 6
`
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