2013 Volume 61 Issue 5 Pages 567-571
The purpose of the study was to evaluate the compatibility of ozagrel sodium solution and calcium-containing transfusions using solubility product constants. We calculated the solubility product constant of mixtures of ozagrel sodium and calcium chloride and evaluated the compatibility of ozagrel sodium solution (both the original and generic products) with calcium chloride solution using a light obscuration particle counter. Various volumes of ozagrel solution were added to the calcium solutions to make final ozagrel concentrations of 0, 0.8, 1.6, 2.0, 2.4, 3.2 and 4.0 mmol/L. The solutions were gently agitated and stored at 25 and 40°C. The ozagrel concentration, calcium ion concentration and number of microparticles were measured. The solubility product constants obtained were 11.89×10−9 mol3/L3 (at 25°C) and 7.82×10−9 mol3/L3 (40°C). The number of insoluble microparticles was significantly increased when the ionic product was larger than the solubility product constant. In all ozagrel sodium products, the number of insoluble microparticles was within the allowable range according to the Japanese Pharmacopoeia. These results suggest that mixing ozagrel sodium with calcium-containing products is safe and without appreciable risk of incompatibility under clinical conditions.
Information on compatibility is important in the preparation of solutions for parenteral injection. Generally, signs of incompatibility would be observed visually in clinical practice. However, microparticles of less than 50 µm diameter are barely visible to the naked eye, so in this instance it is not possible to confirm incompatibility by visual observation. A light obscuration particle counter, able to detect barely visible small particles, would therefore be useful to confirm the compatibility of mixed injection fluids.
Incompatibilities can mainly be classified as physical or chemical. The most common instigator of incompatibility is a change of pH (physical incompatibility). Therefore, the pH change test is often used to predict incompatibility. One useful predictor of incompatibility is solubility.1) The likelihood/risk of the appearance of an insoluble salt may be evaluated using the solubility product constant (Ksp) and the saturation index (SI), with which the likelihood/risk of incompatibility in injection preparations can be scored. SI is defined as the ratio of the ionic product to Ksp. A score SI<1.0 indicates that the solution is unsaturated, with any insoluble salt invisible to the naked eye, while SI>1.0 indicates that the solution is supersaturated, hence there is a high risk of insoluble salt formation.2,3)
Ozagrel sodium is used in the treatment of cerebral infarction, but severe renal insufficiency has been reported in association with its administration,4) the cause of which is still unknown. Ozagrel is known to interact with calcium ions,4) so one possible reason is incompatibility, although it is stated in the package insert that ozagrel should not be mixed with calcium-containing products. It is possible that ozagrel may be mixed with calcium-containing products after dilution with a calcium-free solution.5) There have been no reports of incompatibility under these circumstances. The Ksp of ozagrel–calcium has not been yet reported.
Ozagrel sodium is not a cheap product, so in clinical practice there may be pressure for generic substitution. Comparisons of quality between original and generic products have frequently been reported,6,7) but in general, these comparisons have not included the compatibility of fluids for injection. We have previously reported that the compatibility of the original manufacturer’s ceftriaxone sodium preparation for injection with calcium chloride is different from that of seven generic versions of ceftriaxone sodium.8) In the present study, the Ksp of ozagrel–calcium was calculated and evaluated for different combinations of ozagrel and calcium. The compatibility of the original ozagrel sodium product with calcium chloride was compared with that of four generic products.
Ozagrel sodium (AK Scientific Inc., CA, U.S.A.), special reagent grade, was used as an standard solution for the determination of the solubility product. Calcium chloride injection 2% (Otsuka Pharmaceutical Factory, Inc., Tokyo, Japan) were also used in this study.
Six different preparations of ozagrel sodium were used in the present study: the original product, Cataclot® 40 mg for Injection and Cataclot® injection 40 mg (Ono Pharmaceutical Co., Ltd., Osaka, Japan) and the following four generic products: Oguzalot® for Injection 40 mg (Sandoz Co., Ltd., Yamagata, Japan), Xaclot inj. 80 mg syringe (i’rom Pharmaceutical Co., Ltd., Tokyo, Japan), Athrombone® Injection 80 mg (Takata Seiyaku Co., Ltd., Tokyo, Japan) and Ozagrel Na intravenous (i.v.) infusion 80 mg (Kobayashi Kako Co., Ltd., Fukui, Japan). The original products were designated A and B, and the four generic products were randomly designated C to F. Products B and C were powders and the other products were solutions.
Solubility ProductCalcium solutions were diluted in ultra-pure water to final concentrations of 2.5, 3.75, 5, 7.5 and 10 mmol/L. Ozagrel solutions were also prepared using ultra-pure water. Various amounts of ozagrel solution were added to the calcium solutions to make final ozagrel concentrations of 16.05, 21.4, 32.1, 42.8 and 64.2 mmol/L. The solutions were stored at 25 and 40°C. After 48 h, samples were filtered through a 0.22 µm filter (DISMIC®-25HP, Toyo Roshi Kaisha, Ltd., Tokyo, Japan) and the concentrations of ozagrel, calcium and calcium ion determined.
The ozagrel concentrations were measured using HPLC. The method was essentially the same as the ‘Ozagrel Sodium Assay’ described in the 16th edition of the Japanese Pharmacopoeia.9) For HPLC, 10 µL was injected onto a chromatograph (LC-10AT, Shimadzu, Kyoto, Japan) equipped with a UV detector (SPD-10AV, Shimadzu), an injector (C-R6A, Shimadzu Corporation) and reverse-phase column (Capcell Pak C18UG120 S5: 4.6 mm i.d.×150 mm, Shiseido Co., Ltd., Tokyo, Japan). The column was kept at a constant temperature of 25°C. The mobile phase consisted of ammonium acetate solution (3→1000)–methanol (4 : 1), at a flow rate of 0.6 mL/min. The wavelength was set at 272 nm. The calcium concentration was measured using a methyl-xynol blue assay kit (CalciumE-test Wako, Wako Pure Chemical Industries, Ltd., Osaka, Japan). The absorbance of the resultant solution was measured at 610 nm using a microplate reader (Sunrise™, Tecan Group Ltd., Männedorf, Switzerland). The calcium ion concentration was measured using a calcium electrode (D-53, Horiba Co., Ltd., Kyoto, Japan). The Ksp was calculated from the following equation:
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Calcium solutions were diluted in ultra-pure water to a final concentration of 2.5 mmol/L. Various volumes of ozagrel solution were added to the calcium solutions to make final ozagrel concentrations of 0, 0.8, 1.6, 2.0, 2.4, 3.2 and 4.0 mmol/L. The solutions were gently agitated and stored at 25 and 40°C. Samples stored without adding calcium were prepared as controls. The insoluble microparticles were measured using a light obscuration particle counter (KL-04, Rion Co., Ltd., Tokyo, Japan) 24 h after mixing. The method used was essentially the same as that described in the 16th edition of the Japanese Pharmacopoeia (2011) ‘Insoluble particulate Matter Test for Injection by Method 1. Light Obscuration Test for Injection Count Test.’10)
All procedures were carried out at a clean bench. The thresholds for microparticle size were 1.3, 2.0, 5.0, 10.0, 20.0, 25.0, 40.0, 50.0, and 100.0 µm. The volume of each sample was 1 mL. Measurements were repeated twice and data from the second measurement were used. Before the sample was measured, we confirmed that the number of microparticles with diameter ≥10 µm was very low or not detectable in the ultra-pure water medium. All instruments were washed with ultra-pure water to eliminate insoluble microparticles derived from the devices, and a silicon-free syringe was used to minimize the introduction of insoluble microparticles. Gas bubbles of ≥10 µm diameter in the sample solution were eliminated by letting the solution stand for 2 min.
For injection preparations administered at a volume of ≥100 mL, the tolerated number of insoluble microparticles with a diameter of ≥10 µm is ≤25 per mL, and with a diameter ≥25 µm, ≤3 per mL.
Comparison of the Incompatibility of Ozagrel Sodium ProductsOzagrel sodium solutions were prepared from each of the listed products by dissolving the ozagrel sodium powder in isotonic sodium chloride solution to a final concentration of 20 mg/mL. The purified water solutions containing 80 mg of ozagrel sodium were added to 300 mL of a solution with a calcium ion concentration of 2.5 mmol/L, to make a final ozagrel concentration of 1.07 mmol/L. The number of insoluble microparticles in each solution was determined according to a previously described method10) using a light obscuration particle counter, 0, 3, 6 and 24 h after mixing.
Statistical AnalysisThe relationship between the number of insoluble microparticles and the ozagrel concentration was analyzed using Dunnett’s test; the relationship between the number of insoluble microparticles and the temperature was analyzed using the Tukey test; statistical significance was accepted at the p<0.01 level.
The solubility product constant, Ksp, calculated by analyzing the ozagrel and calcium ion concentrations is shown in Fig. 1. The solubility product constant was 11.89×10−9 mol3/L3 at 25°C and 7.82×10−9 mol3/L3 at 40°C. It was suggested that the difference between the result at these two temperatures is due to the fact that the number of molecular collisions increases in proportion to the rise in temperature; therefore, the Ksp of the high-temperature sample was lower than that measured at the lower temperature. It is suggested that ozagrel–calcium salt precipitated when the ionic product, [Ca2+]×[ozagrel−]2, was higher than the calculated Ksp. For example, when the calcium ion concentration was 2.5 mmol/L, the ozagrel concentration was 2.18 mmol/L (25°C) or 1.78 mmol/L (40°C); thus the ionic product exceeded the Ksp.
The Ksp calculated from the ozagrel and calcium ion concentrations in ozagrel–calcium supersaturated solution was 11.89×10−9 mol3/L3 (25°C) and 7.82×10−9 mol3/L3 (40°C). The dotted line represents mean Ksp.
Figure 2 shows the relation between the ozagrel and calcium concentrations and the formation of the ozagrel–calcium complex. Decreases in calcium and ozagrel concentrations were subtracted from the measured initial concentration. This showed that the concentrations decreased by 1 mol (calcium) and 2 mol (ozagrel). The linear regression analysis was y=0.46x+0.01, r=0.97 and the slope, which defines the ratio of ozagrel and calcium in the ozagrel–calcium salt, was 0.46. This indicates that calcium–ozagrel precipitate consists of a 1 : 2 complex of ozagrel and calcium.4) (Fig. 3)
The concentration of calcium–ozagrel salt was determined by measuring the decrease in concentrations of calcium and ozagrel. Linear regression analysis showed y=0.46x+0.01, r=0.97. Calcium–ozagrel precipitated as a 1 : 2 complex.
The sodium ion from ozagrel sodium interacts with the calcium ion to form insoluble calcium salts.
The number of microparticles measured in the ozagrel and calcium solutions 24 h after mixing is shown in Fig. 4. The number of microparticles increases with increasing ozagrel concentrations and higher temperatures. The number of micoparticles with a diameter <10 µm increased significantly when the ozagrel concentration of the sample solution was ≥2.4 mmol/L (25°C) or ≥2.0 mmol/L (40°C), i.e., when the ionic product was above the Ksp.
The number of microparticles with a diameter <10 µm (A), ≥10 µm (B) and ≥25 µm (C) formed 24 h after ozagrel solution and calcium solution were mixed and stored at 25°C (■) and 40°C (▲). Mean of three observations. Dunnett test, ** p<0.01 versus control; Tukey test, †† p<0.01.
The number of microparticles in the ozagrel–calcium solution prepared according to the method described in the package insert, increased with increasing storage time. The number of microparticles varied between preparations (Fig. 5). However, in all cases, the number of microparticles with diameters ≥10 µm and ≥25 µm, was within the permissible range given in the 16th edition of the Japanese Pharmacopoeia.10) It is suggested that the greater number of microparticles with diameters ≥10 µm in preparation C was due to the gas bubbles generated when the powdery preparation was dissolved.
The number of microparticles with a diameter <10 µm (A), ≥10 µm (B) and ≥25 µm (C) formed when ozagrel and calcium solutions were mixed and stored at 25°C, measured using a light obscuration particle counter. A and B, Original products of ozagrel; C–F, Generic products of ozagrel. Mean of three observations.
The ionic product of ozagrel–calcium is 2.86×10−9 mol3/L3 which is lower than the Ksp. Ceftriaxone also interacts with calcium ions to form a ceftriaxone–calcium salt.11) The solubility product constant of ceftriaxone–calcium reported by Shiffman et al. was 1.62×10−6 mol2/L2.3) These authors also report that precipitates were observed at an ionic product concentration ([Ca2+]×[ceftriaxone3−]) of 1.69×10−5 mol2/L2, which is more than 10 times greater than the Ksp, i.e. the SI was 10.3) In clinical practice, the administration of ceftriaxone and a calcium infusion may also be considered. The calculated ionic product ([Ca2+]×[ceftriaxone3−]) in these circumstances would be 1.13–2.25×10−5 mol2/L2, which exceeds the Ksp; that is, the SI would be 7.0–13.9 when the ceftriaxone concentration is 5–10 mg/mL and the calcium ion concentration 1.25 mmol/L. It is suggested that any precipitate may be difficult to observe with the naked eye when the SI is below 10. The mixture of ceftriaxone and calcium ion is known to have the potential to cause incompatibility, resulting in biliary sludge and pseudocholelithiasis; even death has been reported.12–17)
In the case of ozagrel–calcium, the ionic product was less than 25% of the Ksp. That is, the SI was <1, indicating that the solution was unsaturated, and the presence of ozagrel–calcium salts is unlikely. According to this result, the number of insoluble microparticles would not exceed the permissible range defined in the Japanese Pharmacopoeia. It is therefore suggested that the ozagrel sodium preparation is compatible with calcium-containing products in vitro.12)
The risk of in vivo interactions between ozagrel and calcium can be calculated as follows: The maximum ozagrel concentration was reported to be 1139.82 ng/mL in human plasma at the end of an intravenous administration of 80 mg of ozagrel over a period of 2 h.18) The ionic product ([Ca2+]×[ozagrel−]2) in the plasma was 2.6×10−14 mol3/L3, which is 3.0×105 times lower than the Ksp for ozagrel–calcium salt, when the calcium ion concentration in the plasma is 1.25 mmol/L. The half-life of ozagrel is short.4,18,19) It is therefore unlikely that the co-administration of ozagrel and calcium would give rise to incompatibility in human plasma.
From previous reports,18–21) we have estimated ozagrel concentrations in the kidney at the end of an intravenous administration of 80 mg ozagrel over a period of 2 h in patients with chronic renal failure. Incompatibility was estimated using the SI. Firstly, the plasma concentration of ozagrel was compared in healthy individuals and patients with chronic renal failure. In patients with chronic renal failure, the maximum ozagrel concentration after the continuous administration of 80 mg ozagrel over a period of 24 h was 230 ng/mL (Cp1).20) The administration rate was estimated as 1.1 µg/kg/min and the average bodyweight assumed to be 50 kg. The maximum ozagrel concentration in healthy individuals was 97 ng/mL in plasma 2 h after intravenous administration at a rate of 1 µg/kg/min.19) If the administration rate were increased to 1.1 µg/kg/min, the plasma concentration would be predicted to increase to 106.8 ng/mL (Cp2). The plasma concentration of ozagrel in patients with chronic renal failure was estimated to be 2.2 times that of healthy individuals.
The concentration of ozagrel in the kidney was estimated by extrapolation from rat data as there are no data on ozagrel concentrations in the human kidney. In a previous study in rats, high concentrations of ozagrel were found in the liver, kidney and intestinal tract. The maximum ozagrel concentration in plasma 5 min after intravenous administration of 10 mg/kg ozagrel in a single injection was 19.68 µg/mL (Cp3), and the maximum ozagrel concentration in the kidney was 81.26 µg/mL (Ck1).21) The concentration in the kidney was therefore approximately four times that in the plasma.
The maximum ozagrel concentration in the kidney at the end of an intravenous administration of 80 mg ozagrel over a period of 2 h in patients with chronic renal failure was calculated on the basis of the rat data and data on the maximum plasma ozagrel concentrations after intravenous administration of 80 mg ozagrel over a period of 2 h in healthy individuals, which was 1139.82 ng/mL (Cp4).18) This led to an estimation of the maximum ozagrel concentration in kidney after intravenous administration of 80 mg ozagrel over a period of 2 h in patients with chronic renal failure, of 10.3 µg/mL (Ck2=Cp4×Cp1/Cp2×Ck1/Cp3), which is nine times higher than the plasma ozagrel concentration in healthy individuals. When the ozagrel and calcium ion concentrations were assumed to be 10.3 µg/mL and 1.25 mmol/L, respectively, the ionic product was calculated as 2.2×10−12 mol3/L3, which is 3.6×103 times lower than the Ksp for a ozagrel–calcium salt. This suggests that ozagrel–calcium salts would be unlikely to appear in the kidney if ozagrel were to be administered to patients with chronic renal failure.
It is pertinent to note the interaction of ceftriaxone and calcium ions reported in a previous article.22) Ceftriaxone was found at a high concentration in the gall bladder; the average concentration of ceftriaxone determined 1–3 h after intravenous administration of 1 g ceftriaxone, was 989 µg/mL in the bile duct.23) When the calcium ion concentration in plasma was assumed to be 1.25 mmol/L, the ionic product ([Ca2+]×[ceftriaxone3−]) in the bile duct was 2.01×10−6 mol2/L2, which is 1.2 times greater than the Ksp for the ceftriaxone–calcium salt. Ceftriaxone may therefore be incompatible in vivo with solutions containing calcium ions. In the case of ozagrel, however, the ionic product was lower than the Ksp both in vitro and in vivo. This supports our contention that ozagrel preparations can be used safely in combination with calcium-containing transfusions in clinical practice.
Each calcium salt has different dissociation characteristics. The ratio between the degree of dissociation in calcium chloride and calcium gluconate was 3 : 1. Nakai et al. reported that, with ceftriaxone, when the calcium concentrations were the same, precipitates were formed more easily with calcium chloride than with calcium gluconate.22) It is therefore suggested that calcium chloride may more easily form precipitates with ozagrel than calcium gluconate.
This study showed that the ionic product of ozagrel and calcium was below the Ksp. This result suggests that ozagrel sodium preparations will be compatible with calcium chloride-containing transfusion fluids, although direct mixture of ozagrel sodium preparations with calcium preparations should be avoided in case the ionic product exceeds the Ksp, and the mixture causes incompatibility. The SI is a useful index in the evaluation of incompatibility.
We thank Ms. Y. Umeki, Ms. R. Tanaka, and Ms. K. Matsuda of Mukogawa Women’s University for their assistance with this experiment.
