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Abstract
Objective To evaluate the stability of temocillin solution in two elastomeric infusion devices – Easypump II LT 270–27- S and Dosi-Fusor L25915-250D1 for OPAT administration during 14 days of 5°C±3°C fridge storage followed by 24 hour exposure at an in-use temperature of 32°C, when reconstituted with 0.3% citrate buffer at pH7.
Methods Stability testing was conducted in accordance with standard protocols in the UK National Health Service Yellow Cover Document (YCD). A stability indicating assay method was applied using a high-performance liquid chromatography (HPLC) system with a photodiode array detector. Low (500 mg/240 mL), intermediate (4000 mg/240 mL) and high (6000 mg/240 mL) temocillin concentrations were tested in triplicate devices with duplicate samples taken at 11 time points during fridge storage and subsequent in-use temperature exposure.
Result The percentage of temocillin remaining after 14 days of fridge storage was greater than 97% in both devices and at all concentrations tested. During subsequent in-use temperature exposure, a 95% stability limit was achieved for 12 hours except for the high concentration (25 mg/mL) in the Dosi-Fusor device. It met this criterion for only 10 hours — the percent of temocillin remaining at 12 hours was 94.5%. However, for all devices and the doses tested, the degradation of temocillin was <9% at the end of 24 hours in-use temperature exposure.
Conclusion Temocillin reconstituted with 0.3% citrate buffer at pH7 in elastomeric infusion devices can be stored in a fridge (2°C–8°C) for 14 days meeting the YCD acceptance criteria. Considering <5% degradation, the current data supports twice daily dosing of temocillin within the OPAT setting. In jurisdictions where a <10% degradation limit is acceptable, once daily dosing with 24-hour continuous infusion may be considered. Temocillin is a useful alternative to other broad-spectrum anti-Gram-negative agents currently utilised in the OPAT setting and supports the wider antimicrobial stewardship agenda.
- Administration, Intravenous
- Drug Monitoring
- Drug Compounding
- Quality Assurance, Health Care
- Pharmacology
Data availability statement
Data are available upon reasonable request.
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- Administration, Intravenous
- Drug Monitoring
- Drug Compounding
- Quality Assurance, Health Care
- Pharmacology
What is already known on this topic
Temocillin is used in some European OPAT programmes; however, stability data are not yet available for elastomeric devices compliant with the National Health Service (NHS) Pharmaceutical Quality Assurance Committee Yellow Cover Document (YCD).
What this study adds
Temocillin reconstituted with 0.3% citrate buffer in elastomeric infusion devices is stable at 2°C–8°C fridge storage for 14 days; and at an in-use temperature of 32°C for 12 hours. This complies with the NHS YCD acceptance criteria of <5% degradation.
Degradation was <10% after 24-hour exposure to 32°C in-use temperatures.
How this study might affect research, practice, or policy
The data support the use of temocillin within OPAT as a 24-hour continuous infusion in jurisdictions where <10% degradation is acceptable; and as a twice daily dosing regimen if <5% degradation must be strictly followed.
The use of temocillin within OPAT supports the wider antimicrobial stewardship agenda.
Introduction
Temocillin is a semi-synthetic derivative of penicillin initially designed to achieve high stability to beta-lactamase enzymatic degradation while maintaining activity against a broad range of Gram-negative bacteria.1 2 Temocillin is a potential alternative to carbapenem agents due to its excellent resistance to extended spectrum beta-lactamases (ESBL) and AmpC beta-lactamases.3–6
Temocillin is mainly used in Europe where it is recommended to treat bacteraemia, urinary tract infections, and lower respiratory tract infections caused by susceptible Gram-negative bacilli.6 It is useful in antimicrobial stewardship programmes for several reasons, not least as it can spare carbapenem use when resistance to other commonly indicated antibiotics is observed.3 7 Temocillin’s narrow spectrum of activity is useful in directed therapy, limiting resistance selection for Gram-positives, anaerobes, and most non-fermenting Gram-negatives including Acinetobacter baumanii and Pseudomonas aeruginosa.7 It is also advantageous to minimise the risk of colonisation with Clostridioides difficile or third generation cephalosporin-resistant Enterobacterales. A recent randomised multicentre trial comparing temocillin and cefotaxime in adults with febrile urinary tract infections demonstrated that temocillin exhibited less selective pressure.8
Outpatient parenteral antimicrobial therapy (OPAT) is increasingly used for conditions where suitable oral alternatives are frequently unavailable, including resistant Gram-negative urinary tract and lower respiratory tract infections.9–11 Once daily bolus dosing of antibiotics is one of the ideal administration methods for OPAT agents. Where this is not available and where drug stability allows, elastomeric devices can be a convenient way of delivering treatment for patients and service providers alike. Temocillin has been identified as one of the drugs suitable for OPAT use both due to the increased exposure achieved from continuous infusion of its total daily dose, and its clinical indications. Studies have shown continuous infusion administration of temocillin achieves favourable exposures that cover for the high clinical breakpoint targets of up to 16 mg/L.12–14 Currently, temocillin is used in some European OPAT programmes. It is commonly used to treat urinary tract infections15 where it has enabled high clinical cure rates and demonstrated a favourable safety profile.16
Although it is being used for OPAT, as for most other antibiotics, stability data for temocillin in elastomeric devices that comply with the National Health Service (NHS) Pharmaceutical Quality Assurance Committee Yellow Cover Document (YCD)17 are not yet available.18 Carryn et al 19 assessed temocillin stability in two elastomeric infusion pumps during long-term fridge storage (4 weeks) and 24 hour room temperature storage at a concentration of 10 or 20 g/L in water for injection. They showed that temocillin stability was greater than 90% during both fridge storage and 24 hour exposure at room temperature. However, the study did not comply with the YCD testing protocols; for example, concentrations did not cover for all clinically relevant concentrations, did not test the maximum 32°C in-use temperature condition, and did not analyse subvisible particles.
We therefore conducted our study to evaluate the stability of temocillin in two commonly used elastomeric infusion devices (Easypump II LT 270–27- S and Dosi-Fusor L25915-250D1), in accordance with the YCD testing protocol recommendations to provide data for OPAT services.
Materials and methods
Materials
The solvents used were high performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometry (LC-MS) grade: Acetonitrile HPLC Lichrosolv (Merck, Darmstadt, Germany) and Methanol Optima LC-MS Grade (Fisher Chemicals, Fair Lawn, USA). Disodium hydrogen phosphate was analytical reagent (AR) grade from Merck (Darmstadt, Germany). Ultrapure water was obtained from a Milli-Q Direct water purification system. Pure temocillin disodium was obtained from Alsachim (Illkirch, France). Temocillin powder for injection (Negaban 2 g, Batch: L163407) was supplied by Eumedica SA (Manage, Belgium). Trisodium citrate dihydrate (Sigma-Aldrich, Lot # SLCG13355, Victoria 3029, Australia) and citric acid anhydrous (Sigma-Aldrich, Lot # STBJ8530, Victoria 3029, Australia) were used to make 0.3% citrate buffer pH7. Two elastomeric infusion devices from different manufacturers were used: Easypump II LT 270–27- S (B. Braun Ltd, Sheffield, UK, Lot # 19E29GE221) and Dosi-Fusor L25915-250D1 (Spirit Medical Ltd, Derbyshire, UK, Lot # 211 005 L).
Assay method
A stability indicating assay method was adapted from Kahsay et al, 2014.20
Chromatographic apparatus and conditions
The stability indicating assay method was conducted with a Nexera X2 HPLC system comprising two LC-30AD pumps with degassers, SIL-30AC autosampler, CTO-30AD oven and SPD-M30A photodiode array detector, controlled by LabSolutions software (Shimadzu Corp., Kyoto, Japan).
The stationary phase was a Symmetry C18, 100×2.1 mm (3.5 µm) analytical column (Waters, Milford, USA) preceded by a SecurityGuard Ultra C18 guard column (Phenomenex, Torrance, USA) held at 30°C. Mobile phase A was 30 mM sodium phosphate buffer solution at pH7. Mobile phase B was Acetonitrile/Methanol/Water (50/10/40, v/v/v). Delivery of the mobile phase was by gradient at 0.2 mL/min (online supplemental figure S1).
Supplemental material
Comparison of percentage of temocillin remaining during fridge storage (5°C±3°C) for 14 days by dose and device type.
The autosampler was held at 4°C, and injection volume was 0.5 µL. The photodiode-array detector scanned from 200 to 400 nm, and quantification was at 235 nm.
Solutions for analysis
The temocillin reference material was prepared in mobile phase A (30 mM sodium phosphate buffer solution at pH 7) to concentrations of 560, 640, 720, 800, 880, 960 µg/mL as calibration standards. Quality controls were prepared by dissolving the test formulation in a test buffer to test concentrations of 25.0, 16.6 and 2.08 mg/mL). Aliquots were stored at −80°C until use.
In an assay in mobile phase A we diluted batch test samples and quality controls to bring the nominal concentration to 800 µg/mL. Diluted test samples and quality controls were injected with a set of calibrators. Samples for injection were held at 4°C and all batches were injected over less than 24 hours.
Validation of the HPLC method
The area of the two epimer peaks for temocillin were summed to give a temocillin area. A straight-line calibration curve was generated from the temocillin peak area (no weighting, not forced through zero). Detector linearity was demonstrated from 560 to 960 µg/mL with r2 values of 0.9990, 0.9970 and 0.9987. Slopes were 2309, 2268 and 2536, respectively. The precision and accuracy of the assay were assessed from duplicate analysis quality controls at each of the test concentrations across three batches. Precision was 0.9%, 1.3% and 1.6%. Accuracy was −0.4%, 0.9% and −0.6% at 2.08, 16.6 and 25.0 mg/mL, respectively.
Forced degradation was undertaken using 0.1 M hydrochloric acid, 0.1 M sodium hydroxide and 3% hydrogen peroxide. Temocillin was tested at 10 mg/mL at room temperature and at 50°C for over 3 hours.
Preparation of antibiotic-filled infuser devices
Temocillin vials were reconstituted with the diluent (0.3% citrate buffer at pH7). The required number of reconstituted vials were then transferred into a sterile measuring cylinder in a Class II biosafety cabinet, and subsequently diluted to volume to make a centralised stock solution at the desired concentration. Three concentrations of temocillin were tested. These concentrations were selected to cover the clinical range of doses (concentrations) corresponding to low dose (500 mg/240 mL=2.17 mg/mL), intermediate dose (4000 mg/240 mL=16.67 mg/mL) and high dose (6000 mg/ 240 mL=25 mg/mL) when the devices are filled to the volume of 240 mL. The nominal reservoir fill volume of 240 mL was transferred from the central stock solution to each device using a 60 mL syringe. Devices were prepared in triplicate at each concentration. Flow restrictors and in-line filters were removed from all devices to enable sampling; the outlet line was clamped.
Temocillin filled devices were stored in a refrigerator (2°C to 8°C) for 14 days without exposure to ultraviolet (UV) light. Each device was wrapped with aluminium foil to completely cover its surfaces and prevent exposure to light during storage and sampling. Following the 14 days of refrigeration, the devices were stored in an incubator at the maximum expected in-use temperature of 32°C for 24 hours. Duplicate samples were collected from each individual device for the two device types that were tested at three concentrations in triplicate devices. Samples were collected at 11 different time points including 0, 24, 48, 96, 168, 240 and 336 hours at refrigeration temperature and 340, 344, 356, and 360 hours at 32°C in-use temperature (running phase). The whole test was run in one batch. A total of 396 samples were collected and an aliquot of 0.5 mL of each sample was immediately stored in a −80°C freezer for concentration measurement.
Samples were collected from each device for visual analysis of colour, clarity, and any precipitation at similar time points of sampling for concentration measurement. The power of hydrogen (pH) of these samples was measured using Orion double junction semi-micro pH electrode with Eutech pH700 pH meter. Subvisible particle assessment was performed using Zetasizer Nano series (Malvern Instruments Ltd, Worcestershire, UK).
Results
Colour, clarity, and precipitation
No visible precipitation was observed for all samples from both devices during fridge storage and in-use temperature. Similarly, all samples appeared clear with no visible turbidity. All samples appeared colourless during fridge storage. However, during in-use temperature, samples taken from devices filled with intermediate and high doses had a faint yellowish appearance. All devices filled with the low dose appeared colourless during both in-use and fridge storage.
pH change
Tables 1 and 2 summarise the change in pH of temocillin solution during fridge storage and in-use temperature exposure for the Easypump and Dosi-Fusor devices, respectively. The 0.3% citrate buffer was able to maintain the pH at seven only for the low concentration of temocillin. At the intermediate and high concentrations, the baseline pH of temocillin solution was lower at 6.84 and 6.81, respectively. However, at all concentrations the respective baseline pH was generally stable during fridge storage until the subsequent exposure to in-use temperature of 32°C, when substantial reduction in pH was observed (tables 1 and 2).
Change in pH of temocillin solution in Easypump II LT 270–27- S elastomeric devices filled at low, intermediate, and high doses during fridge and in-use temperature storage
Change in pH of temocillin solution in Dosi-Fusor L25915-250D1S elastomeric devices filled at low, intermediate, and high dose during fridge and in-use temperature storage
Temocillin concentration
Figure 1 presents a comparison of the percentage of temocillin remaining during the 14 days of fridge storage (5°C±3°C) for the Easypump and Dosi-Fusor devices at the different doses tested. Temocillin concentration declined only gradually during fridge storage with the percentage remaining within the devices maintained well above 95% for 14 days and at all concentrations. However, there was a relatively rapid decline in temocillin concentration during the subsequent 24 hour in-use temperature exposure (figures 2 and 3). The mean±SD of percentages of temocillin remaining at the end of 24 hour in-use temperature exposure at the low, intermediate and high doses tested were 92.9±0.3, 92±1.4 and 91.9±1.8, respectively for the Easypump device; and 93.5±0.9, 92.4±0.9, and 91.1±1.4, respectively for the Dosi-Fusor device (online supplemental tables S1-S3).
Percentage of temocillin remaining during 24 hour in-use temperature exposure (32°C) following 14 days of fridge storage at high (6000 mg/240 mL), intermediate (4000 mg/240 mL) and low (500 mg/240 mL) dose in Easypump II LT 270–27- S elastomeric infusion device. h, hour.
Percentage of temocillin remaining during 24 hour in-use temperature exposure (32°C) following 14 days of fridge storage at high (6000 mg/240 mL), intermediate (4000 mg/240 mL) and low (500 mg/240 mL) dose in Dosi-Fusor L25915-250D1 elastomeric infusion device.
Degradation products
Degradation peaks from acidic degradation (n=3), basic degradation (n=4) and oxidation (n=6) were eluted separately from temocillin. For the analysis of test samples only five degradation peaks were present: designated A, B, C, D and E (figure 4). The cumulative amount of the five degradation products relative to the corresponding temocillin amount (% mean±SD) at the end of 24 hour in-use temperature exposure was 3.6±0.3, 3.3±0.1, and 4.3±0.6 for the Easypump device at low, intermediate and high concentrations respectively, and 4.2±0.8, 3.4±0.3 and 4.6±0.6 respectively for the Dosi-Fusor device (online supplemental tables S4-S6).
Temocillin chromatogram showing five major degradation peaks A, B, C, D and E.
Subvisible particles
The maximum particle size measured was 5.5 microns. There was no difference in the measured particle sizes either by concentration, device type, or storage temperature. No trend of increasing particle size was noted during either fridge storage or in-use temperature exposure.
Discussion
Our results show that temocillin (with 0.3% citrate buffer) was stable during refrigeration for 14 days in the concentration range of 2.17 mg/ml to 25 mg/mL in two commercially available elastomeric infusion devices. Subsequent warming to 32°C, however, led to increased degradation and a loss of >5% of active drug beyond the 12 hour time point but in all cases, less than 10% after 24 hours.
Long-term stability of temocillin solution during fridge storage is consistent with previous studies19 21 and supports use in the outpatient setting. At the end of 24 hour in-use temperature exposure however, the percentage of temocillin remaining was below the 95% YCD standard acceptance criteria. This could limit its use via 24 hour continuous infusion in jurisdictions such as the UK. Extrapolating from the best fit regression lines, the 95% stability limit was maintained for 12 hours for all doses and devices tested except for the high dose of the Dosi-Fusor device which was at 94.5% remaining at 12 hours. Greater than 91% of active drug remained at all concentrations in both devices at the end of the 24 hour period which may allow for continuous infusion in jurisdictions that accept up to 10% degradation. We were unable to find another published study in elastomeric devices with a 32°C in-use temperature. Other studies have reported >95% stability following fridge storage of 48 hours22 and 2 weeks19 followed by 24 hours of exposure to room temperature. However, these studies did not meet the YCD criteria for a body worn device. Importantly, in some hot tropical climates such as in Australia, in-use temperatures are often in excess of usual room temperature.23 24 De Jongh et al 14 evaluated the stability of temocillin solution in Milli-Q water at a concentration of 83.4 g/L when exposed to temperatures up to 37°C. The authors reported <2% degradation for up to 24 hours. However, again these studies were not conducted with YCD testing requirements. Furthermore, their data shows no loss when stored at a temperature as high as 30°C for up to 24 hours. We performed pre-scoping work using unbuffered water for injection (results not included here), however, we were unable to reproduce these findings. The percentage of temocillin remaining was <95% but greater than 91% when reconstituted in water for injection and stored at 32°C for 24 hours, consistent with other results in the current study.
Five major degradation products were identified from the temocillin sample chromatograms with distinct degradation peaks emerging before those of the temocillin diastereoisomers (figure 4). The gradual increase in the amount of these degradation products mirrored the progressive decline in temocillin concentration (online supplemental tables S4–S6, figures S2–S4). Arithmetically, the summation of the total amount of degradation products measured by peak areas accounts for temocillin lost at the corresponding time during both the fridge storage and in-use temperature exposure. Acid, alkaline and enzymatic degradation pathways of temocillin have been previously described by Burton et al; 22 major products are penillic acids, penicilloic acids and their derivatives. There is no data to suggest these products are toxic or accumulate in vivo, although this is unlikely given similar compounds are also observed from other penicillins and are likely to undergo renal excretion due to their high polarity. Temocillin itself is excreted via glomerular filtration mostly unmetabolized (up to ~80 %), with a small amount disposed in the bile and by degradation.1 Whether the in vivo and in vitro degradation patterns are similar remains to be studied; however, in vivo formation of penicilloic acids for example, has been previously reported for other penicillins.25 We suggest that evaluation of degradation products should remain an important aspect of antimicrobial stability study protocols, along with a debate on the clinical relevance of limiting degradation to 5% of the starting concentration versus allowing a 10% loss. Future studies evaluating the clinical exposures achieved from once and twice daily temocillin regimens when administered using elastomeric infusion devices would add useful data to the discussion.
Conclusion
This study’s findings suggest temocillin reconstituted with 0.3% citrate buffer in elastomeric infusion devices is stable at 2°C–8°C fridge storage meeting the YCD acceptance criteria of <5% degradation for up to 14 days. At the subsequent 32°C in-use temperature, the YCD acceptance criteria was only met for 12 hours. In jurisdictions where <5% degradation must be strictly followed, twice daily dosing of temocillin within OPAT practice is appropriate. However, given the YCD stresses that a 10% loss may be acceptable for some molecules and the results showing <9% degradation observed at the end of 24 hour in-use temperature exposure, the data may support the use of temocillin for 24 hour continuous infusion in elastomeric infusion devices where this can be justified and the clinical significance is fully understood. For certain Gram-negative infections in the OPAT setting, temocillin should be considered a useful narrow spectrum alternative to other more commonly used parenteral anti-Gram-negative agents including carbapenems and cephalosporins. Therefore, we conclude that using temocillin within OPAT supports the wider antimicrobial stewardship agenda.
Data availability statement
Data are available upon reasonable request.
Ethics statements
Patient consent for publication
Ethics approval
Not applicable.
References
Supplementary materials
Supplementary Data
This web only file has been produced by the BMJ Publishing Group from an electronic file supplied by the author(s) and has not been edited for content.
Supplementary Data
This web only file has been produced by the BMJ Publishing Group from an electronic file supplied by the author(s) and has not been edited for content.
Footnotes
EAHP Statement 3: Production and Compounding.
Correction notice This paper has been updated since it was first published. The funding statement has been changed.
Contributors All authors designed the study. FBS conducted the experiment and drafted the manuscript. SW assayed samples. All authors critically reviewed the draft manuscript and approved the final paper. FBS is responsible for the overall content as a guarantor
Funding The authors would like to thank: EUMEDICA SA Belgium for funding and in-kind provision of temocillin; B. Braun Medical Ltd, for funding and in-kind provision of elastomeric devices; Sean Unwin, Metro South Health, Queensland Government, for in-kind provision of Dosi-Fusor devices; and Dr. James Falconer, School of Pharmacy, the University of Queensland, for access to laboratory facilities. Fekade Sime acknowledges support from the Australian National Health and Medical Research Council (NHMRC), Investigator Grant (APP1197866). J.A. Roberts would like to acknowledge funding from the Australian National Health and Medical Research Council for a Centre of Research Excellence (APP2007007) and an Investigator Grant (APP2009736) as well as an Advancing Queensland Clinical Fellowship.
Competing interests None declared.
Provenance and peer review Not commissioned; externally peer reviewed.
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