Stability Studies of Ternary Mixtures Containing Fosaprepitant, Dexamethasone, Ondansetron and Granisetron Used in Clinical Practice

Chemotherapy-induced nausea and vomiting (CINV) is a frequent and potentially treatment-limiting complication of cancer therapy, which is associated with a significant deterioration in quality of life. The emetogenicity depends on factors related to the drug, the combination of antineoplastic drugs administered pharmacotherapy scheme, as well as factors related to the patient. The temporal pattern of appearance of emesis after chemotherapy can be acute or late. Acute emesis occurs within the first 24 hours after chemotherapy; it is the most intense emesis and is related to the release of serotonin and 5-HT3 receptors. The late emesis happens after the first 24 hours post-chemotherapy, there is evidence that the mechanisms involved begin at 8 hours and related to substance P and the NK1 receptors. With the correct use of antiemetic drugs, CINV can be prevented in almost 70%, and even up to 80% of patients [1].

5-hydroxytryptamine 3 (5-HT3)- receptor antagonists are now the standard therapy for preventing CINV, because emesis is caused by stimulation of 5-HT3 receptors located on vagal afferents by serotonin released from enterochromaffin cells in the small intestine. The first-generation 5-HT3-receptor antagonists, ondansetron (OND), granisetron (GRA), dolasetron, and tropisetron, show considerable efficacy in preventing acute CINV, with acute responses for single agents ranging from 50% to 70%. However, acute responses are further increased when used in combination with the glucocorticoid dexamethasone (DEX) [2]. More recently, understanding the importance of the neurokinin-1 (NK-1) receptor in the emetic pathway in late emesis has led to the development of a new class of effective antiemetics, the NK-1 receptor antagonist (aprepitant, fosaprepitant (FOS)) [3].

According to currently available Multinational Association of Supportive Care in Cancer (MASCC), European Society for Medical Oncology (ESMO) and American Society of Clinical Oncology (ASCO) guidelines, the use of a combination of 5HT₃ receptor antagonist, a NK-1 receptor antagonist and DEX has been classified to be state of the art in patients receiving highly as well as moderately emetogenic chemotherapy [4,5]. In everyday clinical practice, and adhoc admixture of antiemetic drugs in the same IV infusion solution is often highly preferred to accelerate the management of ambulatory procedures, related to reducing administration time and number of administered intravenous preparations. All this would improve patient safety and comfort. So, physico-chemical stability data of ternary mixtures of 5HT₃ receptor antagonist/NK-1 receptor antagonist/ DEX are needed before to avoid unexpected drug loss or even precipitation.

In data base Stabilis [6], information about physico-chemical stability of single solutions of FOS, DEX, GRA or OND and the binary mixtures DEX/OND and DEX/GRA is available. Furthermore, Sun et al. evaluate physical compatibility of ternary mixtures containing FOS, DEX and GRA (FDG) and FOS, DEX and OND (FDO) [3]. However, until now and to our knowledge, there are no published articles that evaluate chemical and physical stability of both ternary mixtures.

So, the development of an appropriately designed stability study, following the Pharmacopoeia guidelines and the recommendations of the International Committee on Harmonization (ICH), including chemical and physical stability, will allow know stability data of ternary mixtures of FDG or FDO at the concentration levels and storage conditions used in clinical practice [7-9].

Physical stability of drugs in mixture is usually evaluated by measurement of pH of mixture, visual inspection of colour changes, cloudiness (turbidity) and/or precipitation and gravimetry to analyze the water loss measurement [10,11]. Evaluation of chemical stability consists in quantifying concentration of each drug in mixture at different time in order to detect degradation of drugs; in this sense, High Performance Liquid Chromatography (HPLC) has been widely employed due to its high-resolution capacity, sensitivity and specificity [9]. In case of mixtures of more than one drug, the development of a chromatographic method that allows simultaneously quantify all drugs in mixtures should be carried out.

Therefore, the aim of this study was to determine the physicochemical stability of the ternary mixtures FDG and FDO in 50, 100 and 250 ml of 0.9 g/dl sodium chloride (NaCl) under different storage conditions of light and temperature.

Instrumentation and chromatographic conditions

An Agilent Technologies 1100 liquid chromatograph with a quaternary pump, a diode array detector (DAD), a thermostated column compartment, an autosampler and a HP Compaq computer equipped with Agilent-Chemstation software was used. 10 μL of each solution was injected, by duplicate, into the chromatograph through a Rheodyne valve (Cotati, CA), with a 20 μ loop. Kromasil^® C18 column of 5 μm particle size (250 × 4.6 mm inner diameter, Análisis Vínicos, Spain) was used. Mobile phase was ortophosphoric acid (0.1%)-acetonitrile (50:50, v/v); the flow rate was set to 0.8 ml/min, temperature to 20°C and detection to 254 nm. The column was equilibrated with mobile phase for 30 min prior to injection of the drug solution. Ortophosphoric acid and acetonitrile solutions were previously vacuum-filtered through 0.45 μm nylon membranes (Micron Separations, Westboro, MA) and sonicated prior to HPLC analysis.

A pH meter (model 3510, Jenway, UK) connected to a glass pHelectrode and an analytical balance (GF-200, A&D Instruments Ltd, UK) were used to measure the pH and weight, respectively.

Chemicals

For the preparation of mixtures, FOS (Ivemend^® 150 mg; Merck Sharp & Dohme, Spain), DEX (Fortecortin^® 40 mg/5 ml; Merck, Spain), OND (8 mg/4 ml; Normon, Spain) and GRA (3 mg/3 ml; Genéricos Españoles Laboratorios, Spain) were used. 0.9 g/dl NaCl intravenous infusion BP Viaflo^® 50, 100 and 250 ml were purchased from Baxter (Spain), too.

Acetonitrile (Scharlab SL, Spain), ortophosphoric acid (Fluka Analytical, Sweden) and sterile water for injection (Grifols, Spain) were used to prepare the mobile phase used in chromatographic analysis.

Solutions preparation for calibration curves

For each drug, calibration curves were done with six standards prepared by making serial dilutions with 0.9 g/dl NaCl from commercial formulations from DEX, OND, GRA and FOS solution obtained after reconstitution of drug powder content in commercially vial (Ivemend^®) with 5 ml of 0.9 g/dl NaCl. The concentration range assayed for each drug was: FOS, 0.15-4.00 mg/ml; DEX and OND, 0.016-0.300 mg/ml; GRA, 0.005-0.100 mg/ml.

Mixtures preparation and storage conditions

24 mixtures were prepared in the same way as those prepared for hospital clinical practice following the guidelines of “Pharmaceutical Compounding: Sterile Preparations” of the United States Pharmacopeia (USP) [12-14]. 12 mixtures contained 150 mg of FOS, 8 mg of DEX and 8 mg of OND in 50 ml (mixtures 1-4), 100 ml (mixtures 5-8) and 250 ml (mixtures 9-12) of 0.9 g/dl NaCl. 12 mixtures contained 150 mg of FOS, 8 mg of DEX and 3 mg of GRA in 50 ml (mixtures 13-16), 100 ml (mixtures 17-20) and 250 ml (mixtures 21-24) of 0.9 g/dl NaCl.

For mixtures with the same volume of NaCl, two mixtures were introduced in protective bags for ambient light (PL); one of them was stored at room temperature (27.6°C, IC95% 26.6 to 28.7°C; RT) and the other at 5.8°C (IC95% 3.4 to 8.1°C; F). The other two mixtures were exposed to light (L) and one was stored at RT and the other at F.

Time of study was 15 days, and the assays were performed every 24 hours, and in the first 24 hours were performed at 3 hours, 6 hours and 12 hours, too Tables 1 and 2 summarizes the storage conditions of each mixture assayed.

Chromatographic method validation

Table 1: Concentration and conditions of storage of mixtures of FDO.

Table 2: Concentration and conditions of storage of mixtures of FDG.

The developed chromatographic method was validated with each drug for linearity, specificity, accuracy, precision, limit of detection and limit of quantification, in accordance with ICH guidelines [15]. The chromatograms were evaluated on the basis of the peak area of each drug. So, for each drug were evaluated the following parameters:

Linearity: The graph mean absorbance (y-axis) versus concentration (x-axis) was plotted and correlation coefficient (r), y-intercept and slope of regression line were estimated.

Specificity: The specificity of the method was ascertained by evaluating the presence of interferences at the retention time of drug.

Accuracy (% Recovery): The accuracy of the method was determined by calculating recoveries by method of standard additions; known amount of drug (0%, 50%, 100%, 150%) were added to a prequantified sample solution and was determined.

Method precision (Repeatability): Three standards of drug were analyzed six times and relative standard deviation (%RSD) was calculated for each concentration level.

Intermediate precision (Reproducibility): Intra-day precision was determined by analyzing three standards for three times in the same day and inter-day precision, by analyzing three standards daily for five days.

Limit of Detection (LOD) and Limit of Quantification (LOQ): LOD and LOQ were calculated using following equation: LOD=3·σ/S, LOQ=10·σ/S; where σ is the standard deviation of y-intercepts of regression lines and S is the slope of the calibration curve.

Physical and chemical stability assessment

Physical compatibility was evaluated daily by: (1) visual inspection of the mixtures for colour changes, cloudiness (turbidity) and/or precipitation. Incompatibility: appearance of some parameter; (2) loss of volume due to evaporation by gravimetry, weighting each mixture before and after extracting aliquot to HPLC analysis. Incompatibility: loss of weight ≥ 5%; (3) pH of mixtures, measured each two days in an aliquot of 2.5 ml removed from each mixture by inserting into the bag injection port, previous homogenization by double inversion. Incompatibility: variation of pH>20%.

Chemical stability was evaluated daily determining the concentration of each drug in the mixture by HPLC. For this purpose, each day, an aliquot of 150 μl was removed from each mixture, in the same way that for the analysis of pH. For each drug, the pair data concentration and time were adjusted, if it was possible, to a zero- (equation 1) or first-order kinetic equation (equation 2):

C = C₀ – k₀·t (equation 1)

ln C = ln C₀ - k₁·t (equation 2)

being C, drug concentration at a specific time; C₀, drug concentration at t=0; k₀, the zero-order degradation rate constant and k₁, the first-order degradation rate constant.

For each drug in mixture, data were reported as a percentage compared with 100% (concentration determined just after preparation); the parameter T₉₀ (time at witch remaining drug concentration is of 90%) was calculated by: (1) using equation 3 and 4, depending on the order of reaction or (2) if adjust to kinetic equation was not possible, it was considered the maximum time at which remaining drug concentration was ≥ 90%. Caducity of mixture was established by considering the lowest T₉₀ value of the three drugs in the mixture, maximum study time (15 days).

T₉₀ = 0.1·C₀/ k₀ (equation 3)

T₉₀ = 0.105/ k₁ (equation 4)

The present study evaluated the effects of concomitant dilution and storage of FOS with DEX as corticosteroid and OND or GRA as 5-HT₃ antagonists. The reason for evaluating physical and chemical stability of the ternary mixtures FDO and FDG was based on the fact that: (1) ad hoc admixtures of these antiemetic drugs in the same IV infusion solution could alleviate the everyday clinical practice particularly in ambulatory settings; (2) both ternary mixtures may represent two potent antiemetic regimens for first-line treatment in case of highly emetogenic chemotherapy as well as salvage regimens in moderately emetogenic chemotherapy; (3) only compatibility information of both mixtures was publicated.

Optimization and validation of the chromatographic method

To optimize the chromatographic conditions a C18 HPLC column, ortophosphoric acid solution (0.1%) and acetonitrile mixture were found to be the best stationary phase and mobile phase combination to have symmetrical and well-resolved peaks of FOS, DEX and OND or GRA, simultaneously, in 0.9 g/dl NaCl mixtures. The same method was used to analyze both ternary mixtures. The total runtime for the analysis was 5 min and the retention times of drugs were: FOS, 2.4 min; OND, 2.9 min; GRA, 2.9 min and DEX, 4.3 min.

Chromatographic method validation

In the experimental conditions indicated, the analytical performance parameters suggested by ICH guidelines were evaluated: linearity, specificity, accuracy, method precision, intermediate precision, LOD and LOQ. Because of the simplicity of the procedure, no internal standard was needed. Table 3 shows the values of some of the parameters obtained for each drug. Furthermore, specificity was adequate since no interferences were observed at retention time of drugs. So, since all the criteria were acceptable according to ICH guidelines, the proposed chromatographic method was adequate to determine simultaneously FOS, DEX and OND or GRA in mixtures. Figures 1 and 2 show the chromatograms of mixtures of FDO and FDG in 50 ml of 0.9 g/dl NaCl, just after preparation.

Table 3: Validation parameters of chromatographic method.

Figure 1: Chromatogram of mixture containing 150 mg of FOS, 8 mg of DEX and 8 mg of OND in 50 ml 0.9 g/dl NaCl, just after preparation, at the chromatographic conditions. Peaks: FOS, 2.4 min; OND, 2.9 min; DEX, 4.3 min.

Figure 2: Chromatogram of mixture containing 150 mg of FOS, 8 mg of DEX and 3 mg of GRA in 50 ml 0.9 g/dl NaCl, just after preparation, at the chromatographic conditions. Peaks: FOS, 2.4 min; GRA, 2.9 min; DEX, 4.3 min.

Physico-chemical stability assessment of mixtures containing FDO

At the end of the study, none of the mixtures (mixtures 1-12) showed changes in colour, precipitation or measurable losses of volume at the different storage conditions. So, all mixtures were physically stable and so, compatible, during the time of storage.

Table 4 shows initial medium pH values (pH0) of mixtures and variations of pH after 7 and 15 days of storage. In mixtures PL (mixtures 3-4, 7-8, 11-12) mean of pH variations were 0.28% (IC95%, -1.02% to 1.59%) at 7th day and 0.017% (IC95%, -1.404% to 1.438%) at 15^th day; however, in mixtures exposed to L (mixtures 1-2, 5-6, 9-10), mean of pH variations were -6.22% (IC95%, -6.87% to -5.56%) at 7th day and -6.07% (IC95%, -6.74% to -5.40%) at 15th day, being the lowest pH achieved 7.1 what means a reduction of 3.4% respect to blood pH (7.35-7.45). In a previous study carried out by us about physico-chemical stability of FOS in mixtures [11], a maximum decrease in pH after 15 days of storage of 13.6% was observed, being the lowest pH achieved 7.0, too; it was explained by considering the flow of CO₂ through polyolefin bag and the consequent acidification of solutions and no FOS degradation.

Table 4: pH₀ and variation of pH of mixtures after 7 and 15 days of storage.

Table 5 shows the percentage of remaining drugs concentration (%RC) at the different conditions of storage at day 7 and 15. As can be observed, in all mixtures, %RC of FDO were ≥ 90% after 15 days of storage; so, it seems to be that temperature, light and drug concentration do not affect FDO chemical stability. Table 6 shows the T90 values obtained from kinetic adjust and experimental value, being the last one the time at which %RC of all three drugs in the mixture were ≥ 90%; experimental value was in all cases of 15 days.

Table 5: Percentage of remaining concentrations of FDO in mixtures at day 7 and 15 of storage.

Table 6: T₉₀ values for mixtures containing FDO assayed.

To sum up, mixtures containing 150 mg of FOS, 8 mg of DEX and 8 mg of OND in 50, 100 and 250 ml of 0.9 g/dl NaCl were physicochemical stable for 15 days at different conditions of light (PL and L) and temperature (RT and F).

Physico-chemical stability assessment of mixtures containing FDG

At the end of the study, none of the mixtures (mixtures 13-24) showed changes in colour or measurable losses of volume at the different storage conditions. Only in mixtures 13 and 16, precipitate was observed by visual inspection after 7 days of storage. So, mixture 13 and 16 were physically compatible for 6 days while the rest of mixtures were compatible for 15 days.

As can be observed in Table 4, pH0 mean values were higher than pH of mixtures containing FDO. Furthermore, variation of pH with time was highest and increased with increasing 0.9 g/dl NaCl volume (Table 7), being the lowest pH achieved 7.0. As has been commented for mixture FDO, acidification could be a consequence of the flow of CO₂ through polyolefin bag and in mixtures with highest drugs concentrations (mixtures 13-16) it could have provoked precipitation.

Table 7: T₉₀ values for mixtures containing FDG assayed.

Regards chemical stability, after 15 days of storage, %RC of all drugs were ≥ 93.5% for all mixtures (Table 8). Kinetic adjust was not possible in mixtures and experimental T₉₀ values was in all cases of 15 days (Table 9).

Table 8: Mean variation of pH for mixtures containing FDG after 7 and 15 days of storage.

Table 9: Percentage of remaining concentrations of FDG in mixtures at day 7 and 15 of storage.

So, mixtures containing 150 mg of FOS, 8 mg of DEX and 3 mg of GRA in 100 and 250 ml of 0.9 g/dl NaCl were physico-chemical stable for 15 days at different conditions of light (PL and L) and temperature (RT and F). Mixtures 13 and 16, in 50 ml of 0.9 g/dl NaCl, were physicochemical stable for 6 days due to appearance of precipitate after day 7.

The caducity of all-in-one admixtures containing fosaprepitant 150 mg, dexamethasone 8 mg and ondansetron 8 mg or granisetron 3 mg in 50, 100 and 250 ml of 0.9 g/dl NaCl was established at different conditions of light and temperature. The results from this paper represent the first evidence of the physico-chemical stability of both ternary mixtures FDO and FDG used in clinical practice at different drugs concentrations and conditions of storage. These results indicate that advance preparation of these ternary mixtures is possible, reducing waiting times for patients and that their administration simplify the management of these treatments in terms of reducing number of preparations and improving patient safety and comfort.

The authors acknowledge FISABIO for the financial support.