In vitro protein binding of teicoplanin to neonatal serum

J Infect Chemother (2004) 10:280–283 DOI 10.1007/s10156-004-0339-x

© Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases 2004

ORIGINAL ARTICLE Mitsuru Sando · Yoshitake Sato · Satoshi Iwata Hironobu Akita · Keisuke Sunakawa

In vitro protein binding of teicoplanin to neonatal serum

Received: March 3, 2004 / Accepted: August 3, 2004

Abstract The protein-binding ability of teicoplanin (TEIC) to neonatal serum was investigated and the following results were obtained. (1) The protein binding ability of TEIC at concentrations ranging from 10 to 100 µg/ml, to neonatal serum was 80.5%–71.9% and was generally low as compared with that for adult serum. (2) The protein binding ability of TEIC (20 µg/ml) to albumin in neonatal serum was not concentration-dependent, and the correlation between the protein-binding ability and albumin concentration was low. Namely, it was suggested that the protein-binding ability of TEIC to neonatal serum was not dependent merely on the albumin concentration, and there was a possibility that the protein-binding ability also depended on qualitative changes in albumin. (3) The protein-binding ability of TEIC (20 µg/ml) to total bilirubin and free bilirubin was not concentration-dependent, and the correlation was extremely low, indicating that there is a low possibility that the protein-binding ability of TEIC induces an increase in free bilirubin. Key words Neonates · Protein-binding · Teicoplanin · Albumin · Total bilirubin · Free bilirubin

M. Sando (*) Department of Pharmacy, Ota General Hospital, Fuji Heavy Industries Ltd. Health Insurance Society, 29-5 Hachimancho, Ota, Gunma 373-8585, Japan Tel. ⫹81-276-22-6631; Fax ⫹81-276-25-7498 Y. Sato Department of Pediatrics, Ota General Hospital, Fuji Heavy Industries Ltd. Health Insurance Society, Gunma, Japan S. Iwata Department of Pediatrics, National Tokyo Medical Center, Tokyo, Japan H. Akita Department of Pediatrics, St. Marianna University School of Medicine, Yokohama City Seibu Hospital, Yokohama, Japan K. Sunakawa Department of Infectious Diseases, Kitasato University School of Medicine, Kanagawa, Japan

Introduction There have been many reports of in vitro basic studies of the serum protein-binding ability of antibiotics.1–4 However, adult serum or albumin has been used as a test material in most of these studies, and there have been few reports of the protein-binding ability of antibiotics to neonatal serum. The binding ability of antibiotics to serum protein is considered to be related to the persistence of blood concentrations, body distribution, excretion, and metabolism of antibiotics. Blood concentrations of antibiotics persist especially when the antibiotic is bound to serum protein, but because the free antibiotic exerts the antibacterial activity, the binding to serum protein is an important problem. However, it is pointed out that, in neonates there is a danger of promoting the liberation of bilirubin from albumin by preventing albumin-bilirubin conjugation, when various kinds of drugs are used.5–7 Therefore, we investigated the in vitro protein-binding of teicoplanin (TEIC), a glycopeptide antibiotic, to neonatal serum and the effects of this test drug on free bilirubin values. In this article we report the results obtained.

Subjects and methods Test samples and study period One hundred and ninety-four serum samples were obtained from healthy neonates who were delivered at Ota General Hospital (Fuji Heavy Industries Ltd. Health Insurance Society) during the 10 months from May 2000 to March 2001. Of the residual serum samples of blood that was collected from these healthy newborns for tests for bilirubin, congenital metabolic disorders, and vitamin K deficiency, only the samples for which informed consent from the parents was obtained were used as the test samples. Because the residual samples used for the study were small amounts, sera from 3 to 4 neonates were pooled randomly and used as one test sample. A total of 37 serum samples were used.

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For the measurement of protein-binding ability, serum samples from the neonates and blank serum samples obtained from healthy adults were used. TEIC was added to the samples to obtain concentrations of 10, 20, 50, and 100 µg/ml (each concentration, six samples). Preparation of serum and method of determination of protein binding TEIC (supplied by Aventis Pharma, Tokyo, Japan) powder (23 mg, corresponding to 20 mg TEIC) was dissolved in 1/ 15 mol/l phosphate buffer (pH, 7.4) to adjust the volume to 10 ml, and the obtained 2.00 mg/ml solution was diluted with phosphate buffer to adjust the concentration to 1000, 500, 200, 100, 50, and 20 µg/ml. The TEIC concentration was determined by bioassay. TEIC was added to the serum samples obtained from the neonates and the serum samples (controls) obtained from the healthy adults, so that concentrations of 10, 20, 50, and 100 µg/ml were obtained (six samples for each of these concentrations), and the serum protein-binding ability of TEIC was determined by the equilibrium dialysis method.8 Dialyser cells (equilibrium type, sample-holding capacity: 0.5 ml, 10 holes, EC-0; Samplatec, Osaka Japan) and cellulose dialysis membrane (visking sheet, VC-10, Wako Pure Chemical Industries, Tokyo, Japan) were used. Phosphate buffer powder (pH 7.4, Wako Pure Chemical Industries) was dissolved in 50 mmol/l NaCl solution to make 1/15 mol/l and the solution was used as a buffer solution. After penetration at 37°C for 44 h, the concentrations of the test drug in the serum and buffer solution were determined and the protein-binding ability was calculated. The proteinbinding ability was determined by the following formula:

Albumin solution (concentration at dialysis: 1/5 of the prepared concentration) Human albumin (0.75, 1.0, and 1.25 g) was dissolved in phosphate buffer to adjust the volume to 5 ml, and 15, 20, and 25 g/dl albumin solutions (AL-L, AL-S, and AL-H, respectively) were obtained. Bilirubin solution (concentration at dialysis: 1/5 of the prepared concentration) Bilirubin (10 mg) was dissolved in 2 ml of 0.1 mol/l sodium hydroxide and adjusted with phosphate buffer to a volume of 20 ml, and 50 mg/dl bilirubin solution (BL-EH) was obtained. This solution (1 and 5 ml) was adjusted with phosphate buffer to a volume of 10 ml, and 5 and 25 mg/dl bilirubin solutions (BL-S and BL-H, respectively) were obtained. Free fatty acid solution (concentration at dialysis: 1/50 of the prepared concentration)

Protein binding ability (%)⫽(serum concentration ⫺ buffer solution concentration) / serum concentration ⫻ 100

Palmitic acid (MW, 256.43; 310 mg), palmitoleic acid (MW, 254.41; 56 mg), stearic acid (MW, 284.49; 130 mg), oleic acid (MW, 282.47; 340 mg), and linoleic acid (MW, 290.45; 310 mg) were mixed, dissolved in 50 ml ethanol, and adjusted with 0.1 mol/l sodium hydroxide to a volume of 100 ml, and 41.8 mmol/l free fatty acid solutions (FFA-H), containing the above acids at concentrations of 12, 2.2, 4.6, 12, and 11 mmol/l, respectively, were obtained. These solutions (2.5 and 5 ml) were mixed with ethanol and 0.1 mol/l sodium hydroxide (1 : 1, v/v) to adjust the volume to 10 ml, and 10.5 and 20.9 mmol/l free fatty acid solutions (FFA-L and FFA-S, respectively) were obtained. The artificial serum was prepared by mixing the albumin solution, bilirubin solution, and free fatty acid solution at each concentration, and adding TEIC standard solution and phosphate buffer.

Effects of albumin, total bilirubin, and free bilirubin on protein-binding of TEIC

Results

TEIC was added to the 37 neonatal serum samples and to artificial serum samples (containing human albumin, bilirubin, and free fatty acids in the range of serum concentrations present in healthy humans) so that a concentration of 20 µg/ml of the test drug was obtained, and the correlation between protein–binding ability and albumin, total bilirubin, or free bilirubin in each serum sample was investigated. Albumin in each serum sample was measured by the bromcresol green (BCG) method. For bilirubin, the total bilirubin concentration and free bilirubin concentration were determined, using an UV analyzer, by the peroxidase oxidation method, based on changes in absorption due to enzymatic oxidation of bilirubin. The artificial serum was prepared by the following methods.

Figure 1 shows TEIC protein-binding ability in the neonatal and adult sera at TEIC concentrations of 10, 20, 50, and 100 µg/ml (each concentration, six samples) in serum samples from the neonates and blank serum samples from the healthy adults. The protein-binding ability of TEIC in adult sera was 86.17 ⫾ 0.63% for 10 µg/ml of TEIC; 83.85 ⫾ 0.63% for 20 µg/ml; 77.13 ⫾ 1.17% for 50 µg/ml; and 76.53 ⫾ 1.04% for 100 µg/ml. The protein-binding ability tended to decrease with an increase in the serum concentration of TEIC. Similarly, the protein-binding ability in neonatal sera was 80.58 ⫾ 0.73% for 10 µg/ml of TEIC; 76.33 ⫾ 0.65% for 20 µg/ml; 74.55 ⫾ 1.44% for 50 µg/ml; and 71.90 ⫾ 1.28% for 100 µg/ml, and the variations in protein-binding ability were concentration-dependent, as was the case in the adult sera. The serum protein-binding ability in neonatal sera was lower at all the drug concentrations as compared with that in adults. In particular, the serum protein-binding ability of

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Fig. 1. Protein binding of teicoplanin to neonatal serum. Symbols and vertical bars indicate means ⫾ SD (n ⫽ 6). *P ⬍ 0.05; **P ⬍ 0.01, Student’s t-test

Fig. 2. Relationship between albumin concentration and protein binding of teicoplanin to neonatal serum

10 and 20 µg/ml of TEIC (which are the serum concentrations mainly encountered in actual clinical treatment) in neonatal sera was significantly low as compared with that in adults (P ⬍ 0.01). The relationship between the serum protein-binding ability of TEIC in neonates and serum albumin concentration is shown in Fig. 2. The protein-binding ability of TEIC to artificial serum (control) tended to increase with the albumin concentration. However, the albumin content in neonatal serum ranged from 3.13 to 4.59 g/dl (mean, 3.87 ⫾ 0.31 g/dl), and the correlation coefficient in neonatal serum was 0.0746. There was no significant correlation between the concentration of neonatal albumin and the proteinbinding ability of TEIC (72.3%–81.0%; mean, 76.3 ⫾ 0.65%; P ⫽ 0.66). The relationship between the protein-binding ability of TEIC and the total bilirubin concentration (6.83–16.17 mg/ dl; mean, 12.22 ⫾ 2.27 mg/dl) is shown in Fig. 3. The protein-

Fig. 3. Relationship between total bilirubin concentration and protein binding of teicoplanin to neonatal serum

Fig. 4. Relationship between free bilirubin concentration and protein binding of teicoplanin to neonatal serum (n ⫽ 37)

binding ability to artificial serum tended to decrease with an increase in bilirubin, but the correlation coefficient for neonatal bilirubin content was 0.00346, and there was no significant correlation (P ⫽ 0.98). The relationship between the concentration of free bilirubin and the protein-binding ability of TEIC is shown in Fig. 4. The correlation coefficient was 0.1308, and there was no significant correlation between the concentration of free bilirubin (0.16–0.64 mg/dl; mean, 0.39 ⫾ 0.11 mg/dl) and the protein-binding ability of TEIC (P ⫽ 0.44).

Discussion It is generally known that the serum-protein binding ability of antibiotics in neonates is low as compared with that in older children and adults. The reasons for the low proteinbinding ability are low albumin levels in premature infants,

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the low drug-binding capacity of albumin, and the blocking of the protein-binding of drugs by bilirubin-albumin binding.9–11 In addition, the permeability function of the blood-brain barrier (BBB) in neonates is weak, and physiologically high levels of bilirubin (which has extremely high affinity for albumin) may cross the BBB. However, it is pointed out that there is a danger of development of neonatal jaundice when free bilirubin is increased by the competitive proteinbinding of antibiotics.12,13 The liberation of bilirubin from albumin is considered to be related to the types of drugs used, their blood concentrations, and their protein binding, and it is generally known that there is a danger of the in vitro liberation of bilirubin from albumin by several kinds of representative antibiotics, such as sulfonamides and novobiocin.14 The in vitro protein binding of TEIC in the present study did not vary even at high bilirubin concentrations. Because TEIC is considered not to increase free bilirubin, the risk of development of neonatal jaundice was presumed to be low. On the other hand, as noted above serum concentrations of TEIC used in actual clinical treatment are 10 and 20 µg/ml or more,15–17 and, we found here that the serum protein-binding of TEIC at these concentrations in neonatal sera was significantly lower than that in adult sera (P ⬍ 0.01). We found that the protein-binding ability of TEIC in neonatal sera was as low as 71.9%– 80.5%; judging from the changes in serum concentrations, and free TEIC (active type) in blood is presumed to be present in comparatively high concentrations in neonates. Indeed, the free TEIC concentration in neonatal sera showed about a 40% increase at 10 µg/ml TEIC and about a 46% increase at 20 µg/ml compared with adult sera. This suggests that more marked clinical effects in neonates can be obtained with the same blood concentration as that used in adults; however, the risk of adverse effects may also increase. Further in vivo studies with considerations of clinical effectiveness and safety are necessary. It is generally believed that the serum protein-binding ability of antibiotics accords well with their serum albuminbinding ability, but no correlation of serum protein-binding ability with albumin concentration was found in the present study. This suggests that qualitative changes in albumin may affect drug protein-binding capacity, or alternatively, differences in binding sites may affect this capacity.18 Further investigation is considered to be necessary in the future.

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