Zosyn (piperacillin/tazobactam) reformulation: Expanded compatibility and coadministration with lactated Ringer's solutions and selected aminoglycosides.

Zosyn® , also known as Tazocin® for injection, contains piperacillin and tazobactam and was approved by the US Food and Drug Administration in 1993 for the treatment of indicated serious infections. In 1995, United States Pharmacopoeia and European Pharmacopoeias reduced the particulate limit for injectables by 40%, based on general safety concerns. Wyeth attempted to control sporadic batch failures (associated with increased particulate formation) by shortening product expiration dating from 36 to 24 months and optimizing the stopper siliconization process. These modifications did not correct the problem completely. Wyeth reformulated Zosyn by incorporating two stabilizing functional excipients, ethylene diamine tetraacetic acid disodium salt (EDTA disodium) and sodium citrate, which solved the particulate formation problem. These two functional excipients also allowed for the first time Y-site coadministration of reformulated Zosyn product with amikacin and gentamicin at specific doses and concentrations, and with certain diluents, and the use of Ca++ ion-containing Lactated Ringer’s for admixture preparation. Reformulated Zosyn (approved 2005) may provide useful options of drug administration to healthcare professionals to lessen levels of particulates. Supportive data is provided for the expanded compatibility of reformulated Zosyn with different types of Ringer’s solutions used globally and for the Y-site coadministration of amikacin and gentamicin aminoglycosides.


Zosyn ® background information
Zosyn ® , also known as Tazocin ® piperacillin/tazobactam (Wyeth Pharmaceuticals Inc., Philadelphia, PA, USA), is an intravenously administered antibiotic which is composed at an 8:1 ratio of piperacillin (a semisynthetic β-lactam) and tazobactam (a β-lactamase inhibitor derived from the penicillin nucleus). The Food and Drug Administration (FDA) approved it for use in the United States in 1993. Zosyn is currently used in hospitals worldwide to treat patients with moderate-to-severe infections caused by piperacillinresistant, piperacillin/tazobactam-susceptible β-lactamase-producing strains of certain specifi ed microorganisms. Zosyn is indicated for the treatment of moderate to severe hospital-acquired pneumonia, complicated intra-abdominal infections, complicated skin and soft tissue infections (Wyeth Prescribing Information [Glass vials] 2007; Wyeth Prescribing Information [Galaxy ® bags] 2007), and moderate community-acquired pneumonia. Zosyn and Tazocin represent the same product sold by Wyeth in different countries and are used interchangeably in this communication.

History of reformulation of Zosyn
Zosyn, as an injectable antibiotic, is reconstituted and admixed with a variety of diluents before it is administered to a patient. Post-approval, during the 1990's, Zosyn had sporadic batch failures related to subvisible particulate matter when conducting the usual Particulate Matter Test (United States Pharmacopoeia [USP] Ͻ788Ͼ), at the new drug application (NDA) limits of 10,000 (particles Ն10 µM per small volume dose)/1,000 (particles Ն25 µM per small volume dose).
Two key factors identifi ed as responsible for excessive particulates were: (1) small traces of nontoxic silicone oil used to lubricate the elastomeric closures were dispersed in the reconstituted product as an emulsion. The dispersed silicone oil emulsion particles were interpreted by the HIAC particle size analyzer as solid particulate matter, contributing to the out of specifi cation results for sub-visible particulate matter and, (2) subvisible particulate counts increased with the age of the fi nished product associated with down-shift in the pH of the reconstituted solution of the drug product towards the acidic side.
In response, Wyeth modifi ed its container closure to control silicone lubricant from the rubber stopper insertion process. In addition, the product expiry dating was shortened from 36 to 24 months, as a slight increase occurred in the particulate matter test results in aged product. However, even with these changes, the product still experienced sporadic failures related to particulates. In 1995, USP and European Pharmacopeias adjusted the limits for particulates downwards in part due to ongoing awareness that particulates have the potential to cause morbidity or mortality in patients and because particulate matter had been linked to injection site reactions and thromboembolic events (Longe 1980;Falchuk et al 1985;Lehr et al 2002).
In 2001, partially in response to FDA requests regarding another Wyeth intravenous (IV) product and partially because of FDA concerns about Zosyn, Wyeth undertook to ensure that Zosyn met the revised (1995) USP Ͻ788Ͼ specifi cations. The revised specifi cations lowered the acceptable levels of subvisible particulate matter from 10,000 (particles Ն10 µM per small volume dose)/1,000 (particles Ն25 µM per small volume dose) to 6,000 (particles Ն10 µM per small volume dose)/600 (particles Ն25 µM per small volume dose). This corresponds to a required reduction in the acceptable number of 10 µm and 25 µm particle counts by 40%.
To accomplish this, Wyeth conducted due diligence investigations to establish the root cause and discovered that low pH and trace metal ions can be encountered (Desai et al 2007a(Desai et al , 2007b through the use of various commercial diluents. Both factors may increase the rate of particulate matter formation in admixtures prepared from Zosyn and potentially penicillins and other β-lactams. This can cause the products to fail USP Ͻ788Ͼ. The particulate failures in Zosyn may occur even though the pH of the drug substance is in its approved range of 5.5 to 6.8. Wyeth took corrective action by reformulating Zosyn.

Additional limitations of the original formulation and associated stability problems
In 1993, when Zosyn was introduced, its label instructed that the mixing of Zosyn with an aminoglycoside in vitro can result in substantial inactivation of the aminoglycoside. Chemical degradation of the aminoglycoside (possibly involving microbiologically inactive penicillin-aminoglycoside complexes or metal ion leachables) was observed to result in subpotent doses of the aminoglycoside. Such coadministration was also believed to lead to an unacceptably high particulate load (Desai et al 2007a(Desai et al , 2007b. In addition, the label of Zosyn instructed that lactated Ringer's injection solution (LRS) was not compatible with Zosyn and hence could not be used as a reconstitution or admixture diluent.
Transition metal ions as leachables from exogenous/endogenous sources Contaminants in a drug product solution are defi ned as "leachables." Potentially contaminating components of the drug product's container system are termed "extractables." Potential links between these two types of compounds have formed much of the basis for the increased scrutiny associated with revisions to the USP-NF and its General Chapter Ͻ788Ͼ standards for particulate matter in injectable solutions. Jenke (2005) recently proposed a paradigm for the assessment of interaction between these two classes of compounds based upon the origin of and ability to detect extractables in containers, relative to drug substances they may contain. This report cites the example of a regulatory body imposing a requirement of knowledge of such a linkage upon a drug manufacturer, and its potential impact upon a product's life cycle (Jenke 2005). colleagues (2007a, 2007b) recently reported that there is signifi cant inter-and intra-batch variability in both pH and zinc ion content in commercially available IV diluents and solutions across different US manufacturers. Healthcare practitioners administering drugs in these diluents are generally not aware of this variability. In addition, since zinc Expanded compatibility of reformulated version of Zosyn ® is a common transition metal in drug-container packaging and delivery systems, it can leach into the IV solution from container components as well, and inactivate a drug via catalysis (Desai et al 2007a(Desai et al , 2007b.

Reformulation of Zosyn to overcome limitations
Commonly used IV diluents may contain small amounts of a variety of exogenous inorganic ions and/or organic substances extracted from the containers and closures into the product during manufacturing or storage. Zinc oxide is used as fi ller in rubber elastomeric closures of vials, septa at the necks of infusion bags, or in the plungers of infusion syringes. Zinc-based organic substances are also used as polymerization initiators for the polymers used for the construction of admixture bags or infusion line tubing. Zinc and other transition metal ions are known to catalyze chemical degradation of drug molecules (Bandoh et al 1991;Desai et al 2007aDesai et al , 2007b. Degradation pathways of antibiotics may include acid hydrolysis, ß-lactam ring opening (Bandoh et al 1991), or epimerization. In the case of ionizable drugs generating anionic species, zinc, calcium, and other metal ions are known to form insoluble solid particulates. In the clinical fi eld, risks associated with the injection of intravenous/ intrathecal solutions with particulate contamination are well documented (Nath et al 2004).
The target pH listed on the diluent bags from commercial suppliers can be 3.2 to 6.5 for 5% dextrose injection, and 4.5 to 7.0 for 0.9% sodium chloride injection. The low pH extremes of 3.2 (for 5% dextrose injection) and 4.5 (for 0.9% sodium chloride injection) are considered to be acidic, thus having the potential to accelerate drug degradation, especially in the presence of zinc (Desai et al 2007a(Desai et al , 2007b.
Thus, for the therapeutic performance and safety of drug admixtures infused by the IV route, having data of the pH and levels of zinc in commercial diluents from various parenteral manufacturers is crucial. Since this information is not readily available to the health professionals preparing the solution for parenteral administration, the availability of a drug product resilient to pH or trace metal ion levels commonly found in the clinical setting is preferable.
As part of the root cause analysis, the particulates were isolated and confi rmed to be dimers of piperacillin by spectroscopic characterization. The chemical pathway for the dimer formation is shown in Figure 1.
After extensive research, particulate formation in Zosyn was found to be related to the opening of the β-lactam ring of piperacillin, which is accelerated in acidic pH solutions, and is followed by the dimerization of piperacillin molecules that may be further catalyzed by zinc ions.
In summary, the presence of zinc may contribute to incompatibility by: (1) catalysis of drug degradation and potential underdosing, and (2) generation of insoluble solid particulate matter from the dimer formation and/or chelation with ionic species of the drug. Thus, colleagues (2007a, 2007b)concluded that it is highly desirable that all new drug molecules be screened in the early product development phase for susceptibility to catalysis by zinc, and that the drug product be designed from the outset with suffi cient robustness to withstand exposure to variable amounts of zinc ion. It is also recommended that USP monographs address the potential issues posed by zinc in the drug manufacturing and dosage form infusion process.

Combination therapy with Zosyn and amikacin/gentamicin
Because of the prevalent clinical need to administer piperacillin/tazobactam in combination with the aminoglycosides amikacin or gentamicin to treat nosocomial pneumonia caused by Pseudomonas aeruginosa as well as conditions caused by other pathogens, the clinician often faces operational challenges: either administer Zosyn and amikacin or gentamicin separately, or through the same intravenous line, typically through a Y-site tube. The latter mode, if possible, would expedite the treatment of the patients in critical care. Mixing of the original formulation of Zosyn with an aminoglycoside in vitro resulted in substantial inactivation of the aminoglycoside by piperacillin. It was postulated that penicillin-aminoglycoside complexes are created that are microbiologically inactive and are of unknown toxicity (Benveniste and Davis 1973;Glew and Pavuk 1983).

Evaluation of reformulated Zosyn to expand compatibility
The compatibility of reformulated Zosyn was evaluated by different physicochemical and spectroscopic methods to show that it can be coadministered as admixture with different types of Ringer's solutions which was not feasible with the original Zosyn. Also, the feasibility of Y-site coadministration with amikacin or gentamicin aminoglycosides was evaluated as shown in the following sections.

Materials and methods
The samples of reformulated Zosyn from Wyeth, used in the admixture diluent compatibility and Y-site aminoglycoside studies, were manufactured and released as per approved product specifi cations. Commercial intravenous diluents, amikacin and gentamicin antibiotic products were purchased from US/German or other European countries and were used within expiry dating.
For the simulated Y-site administration study, reformulated Zosyn was admixed in a (high dose, low dose) crossover with two different gentamicin drug products in a (high dose, low dose) matrix design. The crossover pairings of reformulated Zosyn and gentamicin were based on the dose extremes expected in clinical use and were derived from the label dosing instructions listed on the package insert for each drug product.
The subvisible particulate (10 and 25 µm) counts were measured by the HIAC light obscuration method described in USPϽ788Ͼ and equivalent EU pharmacopeia 5.0 (2004) 2.9.19 sections.
Typical HPLC assay methods were used for potency determinations of amikacin, piperacillin, and tazobactam in the presence of reformulated Zosyn components.
For gentamicin potency determinations in the presence of reformulated Zosyn components, it was necessary to design a customized nuclear magnetic resonance (NMR) method. For the NMR study, it was necessary to remove the water from the commercial gentamicin product by lyophilization. The simulated Y-site samples were prepared by dissolving lyophilized gentamicin and reformulated Zosyn (lyophilized powder) in D 2 O in accordance with the concentration matrix described in Table 1.
Initially, the NMR method by Professor Holzgrabe (Winters 2005) was evaluated to quantify gentamicin potency in the presence of reformulated Zosyn as a function of time. The NMR method appears to be applicable only to Notes: a The concentrations listed represent the calculated theoretical high and low concentrations that a hospital pharmacist would prepare in separate intravenous infusion bags for reformulated Tazocin and gentamicin; b When infused through a Y-site tube, each drug solution will be infused at the same rate. Thus, the resulting admixture solution infused intravenously to the patient will have a fi nal concentration that is half of the concentrations listed above in each individual bag. Therefore, to simulate this practice, the fi nal admixtures will be prepared at concentrations that are half what is listed in the table (eg, For "High Tazocin" -"High Gentamicin" Y-site infusion simulation the sample will be prepared at approximately 20 mg/ml of piperacillin, 2.5 mg/ml of tazobactam, and 1.66 mg/ml of gentamicin in D 2 O.).
Expanded compatibility of reformulated version of Zosyn ® gentamicin drug product as-is, with a low ambient pH of about 4 to 4.6. Upon the addition of reformulated Zosyn, the fi nal pH of the simulated Y-site solution with gentamicin is raised to about 6, so the ionic environment of the anomeric protons and the -N-methyl protons is changed, and hence the NMR spectrum is totally different than that of gentamicin drug product alone as observed by the Holzgrabe group. For the customized NMR method, two groups of proton resonances of gentamicin were isolated from other proton resonances and were explored for the quantifi cation of gentamicin. One is from the anomeric protons at δ H 5.84 (doublet) and δ H 5.80 (multiplet). The other is from the N-methyl group at δ H 2.85 (singlet). Although the anomeric proton resonances were well separated, due to the relatively low concentration of gentamicin used in the clinical admixtures, the observed signal-to-noise ratio for the anomeric protons was very low. The N-methyl proton resonance peaks (with very good separation from the other peaks, and a peak intensity signifi cantly higher than the anomeric proton peaks) were considered more appropriate to provide quantifi cation of clinically relevant parenteral admixtures with low levels of gentamicin in the presence of very high levels of reformulated Zosyn.
NMR experiments were performed at room temperature on a Bruker DRX-500 NMR spectrometer, operating at 500.13 MHz ( 1 H), equipped with TOPSPIN software (Version 1.3). 112 scans were collected into 64 k data points giving a digital resolution of 0.16 Hz per point. The spectral width was 10330 Hz, the transmitter offset at 6.17 ppm, and the fl ip angle was 90°. Using an acquisition time of 3.17 s and an additional delay of 1 s, the pulse repetition period was about 4.17 s. Samples were measured in D 2 O at 298 K. Each 1 H NMR data point for gentamicin represents cumulative scans collected over ten minutes.
Hydroquinone monoethyl ether was used as an internal standard due to the absence of chemical interactions with gentamicin and all components of reformulated Zosyn. Also, the associated peaks of the hydroquinone monoethyl ether protons do not overlap or cause interference with the N-methyl protons of gentamicin that were used for quantifi cation. This customized NMR method provides a kinetic snapshot at 10-minute intervals over a period of 1 hour for gentamicin strength in the presence of reformulated Zosyn.

Results and discussion
Improved clinical utility of the reformulated version of Zosyn In 2005 (USP Ͻ788Ͼ 2007), the FDA approved the new formulation of Zosyn that complies with USPϽ788Ͼ particulate specifi cations and has an expanded compatibility profi le with the aminoglycosides, amikacin and gentamicin. The modifi cations to the formulation consisted of the addition of the disodium salt of ethylene diamine tetraacetic acid (disodium EDTA; edetate disodium dihydrate), which acts as a metal-chelating agent, and sodium citrate, which acts as a buffer. These modifi cations lessen the possibility of particulate matter accumulation during storage of the solution form of Zosyn or the development of particulate matter upon reconstitution of Zosyn lyophilized powder with commonly used diluents. In addition, reformulated Zosyn can be administered simultaneously, either with amikacin or gentamicin, via Y-site infusion at specifi c doses and concentrations, and with certain diluents (

Allowance of Y-site coadministration of reformulated Zosyn with aminoglycosides
The inactivation of aminoglycosides in the presence of penicillin-class drugs containing β-lactam rings has been recognized (Benveniste and Davis 1973;Glew and Pavuk 1983). However, amikacin and gentamicin have been shown to be compatible in vitro with reformulated Zosyn containing disodium EDTA supplied in vials or bulk pharmacy containers in certain diluents at specifi c doses and concentrations for a simultaneous Y-site infusion.
Simulated Y-site administration studies by Wyeth were conducted for reformulated Zosyn with amikacin or gentamicin in the concentration ranges and clinical doses described in the labels of Zosyn and each of the aminoglycosides. The simulated studies mimicked Y-site coadministration for Zosyn-amikacin systems and evaluated potency and degradation products by HPLC analyses (Tables 3  and 4). Reformulated Zosyn was shown to be compatible for simultaneous administration via a Y-site intravenous tube with amikacin in the concentration ranges of 2.25 g reformulated Zosyn/150 mL to 4.5 g/50 mL for Zosyn and 1.75 mg/mL to 7.5 mg/mL for amikacin in sterile water for injection, USP and 0.9% sodium chloride injection, USP, 5% dextrose in water for injection, USP and lactated Ringer's injection, USP (Table 5). Degradation products and related compounds in the solution were quantifi ed in a further study, which confi rmed the fi rst study's conclusions, and   showed that reformulated Zosyn (piperacillin/tazobactam) is compatible for Y-site coadministration with amikacin or gentamicin. US-sourced amikacin and gentamicin, evaluated here, represent the composition ranges of the products prescribed globally. The study designs used for Y-site compatibility and admixture stability are based on simulated Y-site injection testing procedures reported by Choi and colleagues (1994) and by Trissel and Martinez (1994a).
A follow-up simulated Y-site compatibility study, conducted by using an innovative NMR method, is supplementary to the previous study using an LC-MS method. The current study differs from the previous study in that it tested the simulated Y-site mixtures of reformulated Tazocin and gentamicin at shorter time intervals of 0, 10, 20, 30, 40, 50, and 60 minutes, while the previous study tested these mixtures at 0, 1, 2, and 4 hours. This change is to collect data at clinically realistic shorter time intervals, because the 4-hour testing time point represents an exaggerated contact time for the Y-site drug compatibility testing. In actual clinical practice, when two drugs are infused through a Y-site the maximum estimated contact time (prior to entering the blood stream) is short, often in the range of 15 minutes and not extending more than 60 minutes (Trissel 1994b;Leissing 1989). In this experiment to simulate Y-site administration, reformulated Tazocin was admixed in a (high dose, low dose) crossover with two gentamicin drug products sourced from Germany in a (high dose, low dose) matrix design, as shown in Table  1. The crossover pairings of reformulated Tazocin and gentamicin were based on the dose extremes expected in clinical use and were derived from the label dosing instructions listed on the package insert for each drug product.

Simulated Y-site stability results
The kinetic snapshot at every ten minutes over a period of 0 to 60 minutes of the mixed solutions analyzed by evaluating the -N-methyl proton resonance of the gentamicin molecule in a noninvasive manner at room temperature shows the following: (1) For both gentamicin products in all four combinations with reformulated Tazocin, the potency values of gentamicin at ten minutes are maintained at greater than 98% of the initial. As described earlier, the typical time for gentamicin and reformulated Tazocin to remain in contact during the actual Y-site co-administration in a clinical setting is about 15 minutes. (2) At 30 minutes and 60 minutes, better than 97% and 96% of the initial potency of gentamicin is maintained, respectively, even for the worst-case scenario of a solution mixture of "high" reformulated Tazocin and "low" gentamicin during a simulated Y-site administration. Based on the aminoglycoside and β-lactam interaction chemistry, the highest degradation of gentamicin was expected for this combination.
(3) As expected, for the "high" gentamicin combination with "low" or "high" reformulated Tazocin, the gentamicin strength at 60 minutes was found to be greater than 98%. Representative data for Y-site compatibility at clinically relevant concentration ranges of reformulated Tazocin combined with another German gentamicin commercial drug product (Ratiopharm ® ) are provided in Table 6. Data    for the Rebofacin gentamicin product (not shown here) were found to be similar.
Particulate matter evaluation for reformulated Zosyn -amikacin or gentamicin (simulated Y-site study) The subvisible particle counts for 10 µm and 25 µm size were determined using the HIAC method. The particulate counts at 0 and 4 hours for simulated Y-site mixtures of reformulated Zosyn and gentamicin or amikacin in admixtures of common commercial diluents are provided in Tables 7 and 8. The concentrations of reformulated Zosyn and gentamicin or amikacin were chosen to simulate clinically relevant dilutions in the admixture infused. The particulate counts up to the 4 hour test period for the reformulated Zosyn -amikacin system and reformulated Zosyn -gentamicin system were well within the current USP and EU Pharmacopeia specifi cations. Reformulated Zosyn was shown to be compatible for coadministration via a Y-site intravenous tube with gentamicin under the concentration ranges of 2.25 g reformulated Zosyn/150 mL to 4.5 g/100 mL for Zosyn and 0.7 mg/mL to 3.32 mg/mL for gentamicin in the 0.9% sodium chloride injection USP coadministration of Zosyn with 0.9% sodium chloride.

Conclusions
In summary, the product quality enhancement provides expanded fl exibility for the administration of reformulated Zosyn under variable clinical use conditions: (1) Reformulated Zosyn complies with USP-NF 30 Ͻ788Ͼ, European and Asia Pacifi c Pharmacopoeia specifi cations for particulate matter in injections under all clinical use conditions because it is tolerant to variability in pH and metal ion concentrations of commercial solutions used in the clinical setting. The variabilities of actual pH and metal ion concentrations in commercial IV solutions and diluents are unknown to the pharmacist and nurse end-user. (2) The product maintains chemical and physical stability under the conditions encountered in the clinical fi eld of use where commercial diluents, such as 5% dextrose solution with potential variables of pH and leachable metal ions, are used for admixture preparation for parenteral administration. Regardless of the pH or zinc content of an admixture diluent or IV solution, reformulated Zosyn will maintain potency and lessen the level of particulates infused into patients. (3) The product is compatible with calcium-containing Ringer's solutions (as lactate or acetate) or Hartmann's solution. (4) The reformulated product provides the capability of simultaneous Y-site coadministration of amikacin (with 0.9% NaCl or 5% dextrose) and gentamicin (with 0.9% NaCl), without compromising either drug's potency, and provides useful options for the administration of Zosyn, especially for the treatment of nosocomially-acquired pneumonia.
(5) Based on the recent stability data, the expiration dating of reformulated Zosyn has been extended to 36 months from its previously reduced dating of 24 months.