Plasma Protein Binding and PK/PD of Total and Unbound Temocillin Non-ICU Patients

Multidrug resistance towards Gram-negative pathogens makes essential the re-examination of older compounds. Temocillin is a penicillin originally marketed in the 1980s but then largely abandoned. It, however, shows a marked ß-lactamase stability (including most classical and extended-spectrum TEM, SHV, CTX-M enzymes and AmpC ß-lactamase). Temocillin is approved for the treatment of bacterial infections of the chest, the lungs, the kidney, the bladder, as well as bacterial infections of the bloodstream and wound infections.

Temocillin efficacy depends primarily from the time interval during which the unbound plasma concentration remains above the minimal inhibitory concentration (MIC) of the antibiotic against the target organism(s). Unfortunately, no comprehensive pharmacokinetic data are available in non-critically-ill patients.

The primary objective of the study is characterize the pharmacokinetics of total and unbound temocillin in non-ICU patients, and, on this basis, to propose optimized dosage regimens in this population. The secondary objectives are (i) to look for possible correlations between the plasma protein profile and the unbound temocillin concentrations; (ii) to investigate the impact of the level and nature of circulating plasma proteins on the unbound temocillin concentration.

The study will be non-randomized, uncontrolled, prospective, open label, interventional, and monocentric. It will include a population pharmacokinetic-pharmacodynamic analysis of the data obtained. The study will enroll patients ≥ 18 years in need of a treatment with temocillin for (i) complicated urinary tract infection and pyelonephritis (associated or not with bacteremia), or (ii) lower respiratory tract infection, or (iii) abdominal infection, and requiring ≥ 4 days of hospitalization. Blood samples will be obtained at day 0 (control) and after 2 and 4 days of drug treatment (full pharmacokinetic evaluation over 8 to 12 h post-administration). Total and unbound temocillin concentrations in plasma will be quantified by a validated analytical method.

A population pharmacokinetic/pharmacodynamics model of plasma total and unbound concentrations of temocillin will be obtained by Bayesian algorithms using Pmetrics software, driven by the predicted plasma total and unbound concentration. The model will be used to assess the probability of target attainment of temocillin.

1.1. Introduction.

Multidrug resistance towards gram-negative pathogens has been increasing dramatically over the last decades. Due to insufficient discovery of drugs acting on novel targets, re-examination of older compounds, such as temocillin, for which resistance is still low, has proven to be of great therapeutic interest.

As all the β-lactams, temocillin efficacy depends primarily on the time interval during which the plasma concentration remains above the minimal inhibitory concentration (MIC) of the antibiotic against the target organism(s) (Craig, 1998). It is generally accepted that the unbound concentration of the antibiotic must remain above the MIC for at least 40 to 70% of the interval between two successive administrations, and should even reach 100% for severe infections in patients hospitalized in Intensive Care Units (MacGowan, 2011).

Temocillin (Negaban®), a ß-lactamase-resistant penicillin antibiotic, was first marketed in the 1980s but then abandoned due to a lack of activity against anaerobes, gram-positive bacteria and Pseudomonas aeruginosa (Livermore et al., 2006; Zykov et al., 2016). However, temocillin stability to ß-lactamases (including classical and extended-spectrum TEM, SHV, CTX-M enzymes and AmpC ß-lactamase) has found great appreciation amongst a large number of professionals in the field (Balakrishnan et al., 2011; Livermore and Tulkens, 2009). It is approved for intravenous and intramuscular administration at a dose ranging between 1-2 g twice - thrice - a day for the treatment of bacterial infections of the chest, the lungs, the kidney, the bladder, as well as bacterial infections of the bloodstream, abdominal infection, and wound infections (RCP Temocillin, 2014).

Studies performed in critical ill patients show important variations in the level of plasma proteins and rapid and unpredictable fluctuations in renal function (Beumier et al., 2015; Goncalves-Pereira and Povoa, 2011; Roberts and Lipman, 2009), both of which are known to modulate the pharmacokinetics of β-lactams (Goncalves-Pereira and Povoa, 2011; Hayashi et al., 2013; Sime et al., 2012; Udy et al., 2012; Wong et al., 2013). As a consequence, the unbound concentration of β-lactams with high protein binding (Schleibinger et al., 2015; Ulldemolins et al., 2011; Van Dalen et al., 1987; Wong et al., 2013), and renal elimination (Carlier et al., 2013; Simon et al., 2006; Vandecasteele et al., 2015) such as temocillin will be markedly modified in these patients.

The pharmacokinetics of total and unbound temocillin together with its plasma protein binding have been recently investigated in clinical studies performed in healthy volunteers and in critical-ill patients. The first results show that plasma protein binding of temaocillin was concentration-dependent in both populations but much more important in healthy volunteers compared to critically-ill patients), resulting in a lower unbound concentration of temocillin in healthy volunteers as compared to critically ill patients.

These studies were conducted in two extreme populations, but temocillin is more widely used in patients who do not require hospitalization in an intensive care unit. It would therefore be of high interest to study the pharmacokinetics of temocillin in non-critically-ill patients, including its protein binding, in order to estimate whether the current dosing regimen is optimal to reach PK/PD targets predictive of efficacy.

1.2. Study objectives.

1.2.1. Primary objectives.

* To characterize the pharmacokinetics of total and unbound temocillin using population pharmacokinetic and pharmacodynamics modeling and to calculate and assess the values of key pharmacokinetic parameters (total clearance, volume of distribution, constants of elimination, plasma total and unbound maximal and minimal concentrations) in patients hospitalized in standard care wards.
* To propose optimized dosage regimens in this population, as compared to what could be proposed for patients hospitalized in intensive care units.

Secondary objectives:

1.2.2. Secondary objectives.

* To look for possible correlations between the plasma protein profile and the unbound temocillin concentrations;
* To investigate the impact of the level and nature of circulating plasma proteins on the unbound temocillin concentration.

1.3. Study design.

Non-randomized control trial, prospective, open label, interventional, monocentric, population pharmacokinetic and pharmacodynamics.

1.4. Study population.

1.4.1. Sample size calculation.

As a descriptive pharmacokinetic study without formal predefined hypothesis, no calculation of the size of the population has been made. Based on literature data and our own experience, a maximum of 60 patients should be sufficient to draw meaningful conclusions.

1.4.2. Study groups and number of patients

The population studied is divided according to their pathology into 3 groups

* Group 1: 15 to 30 patients with complicated urinary tract infection and pyelonephritis associated or not with bacteraemia.
* Group 2: 15 to 30 patients with lower respiratory tract infection.
* Group 3: 15 to 30 patients with abdominal infection.

In a first step, each group will undergo a thorough pharmacokinetic study. In a second step we will study the pharmacokinetics of all pooled data and the results obtained will make it possible to compare the parameters.

1.4.3. Inclusion criteria (see elsewhere)

1.4.4. Exclusion criteria (see elsewhere)

1.4.5. Reason for withdrawal of study: at the patient's own request; in case of lack of cooperation, change of medication, occurrence of adverse event(s), or other objections to participate in the study in the opinion of the investigator

1.5. Study drug.

Temocillin (NEGABAN®) 2 g will be administered as intravenous infusion over 30 minutes 2 or 3 times a day. The patient's physician will decide on the duration of temocillin use.

1.5.1. Indications.

Temocillin is indicated for treatment of the infections mentioned in the protocol.

1.5.2. Contraindications.

The use of Temocillin (NEGABAN®) is contraindicated in patients with a history of allergic reactions to any of the penicillins or any other type of beta-lactam drug.

1.5.3. Undesirable effects: diarrhoea, pain at site of injection, occasionally rash (urticarial or erythematous), fever, arthralgia or myalgia, sometimes developing more than 48 hours after treatment initiation, angioedema and anaphylaxis. In patients suffering from renal failure, neurological disorders with convulsions have been reported following i.v. injection of high dose of penicillins.

1.6. Study protocol.

Upon signature of the informed consent form and provided that the inclusion/exclusion criteria are fulfilled, subjects will be enrolled in the study. Only the procedure of management of the patients of the hospital where the study will be conducted will be taken into account in this study. Only patients verifying inclusion criteria will be accepted.

1.6.1. Study day.

1.6.1.1. Study day 1.

Control (Blood Sampling before dosing)

1.6.1.2. Study day 2.

Determination of total and unbound temocillin concentrations in plasma, will be performed after intravenous administration. Plasma samples will be collected at defined time points before the dose administration and during up to 8 hours (if the dose is 2g/8h) or up to 12 hours (if the dose is 2g/12h).

1.6.1.3. Study day 4.

Determination of total and unbound temocillin concentrations in plasma, will be performed after intravenous administration of 2g of temocillin. Plasma samples will be collected at defined time points before the dose administration and during up to 8 hours (if the dose is 2 g/8 h) or up to 12 hours (if the dose is 2 g/12 h).

1.7. Study duration.

The expected total duration of the trial (recruitment) is 24 months (or more if patient enrollment is slow).

2. Methods.

2.1. Sampling period's instructions.

Blood sampling will be performed over 8 or 12 hours. Venous blood will be drawn from a suitable antecubital vein at defined time points before and after study drug administration.

Time points will be as follows: If dose administration is 2g/8h

* Day 1: Control (before dosing),
* Day 2 and 4: T0 + 30 min, 1, 2, 4, 6, and 8 hours after study drug administration.
* T0: time after end of infusion.

Time points will be as follows: If dose administration is 2g/12h

* Day 1: Control (before dosing)
* Day 2 and 4: T0 + 30 min, 1, 2, 4, 6, 8, and 12 hours after study drug administration.
* T0: time after end of infusion.

2.2. Blood collection tube and volume.

* Blood collection tube: EDTA tube (plasma) without ball or gel
* Blood volume per tube: 5mL
* Total blood volume by kinetics: 45mL The blood samples will be placed on ice immediately after collection and subsequently centrifuged (2000g, 10 minutes, 4°C). Resulting plasma will be aliquoted and frozen at -80°C until analysis.

Collection of bacterial strains from the patient All bacterial strains identified in the biological samples of each patient will be conserved

2.3 and 2.4: Flow Charts (available on request).

2.5. Total and unbound temocillin quantification.

Total and unbound temocillin concentrations in plasma samples will be quantified using a validated liquid chromatography - tandem mass spectrometry (LC-MS/MS) method (Ngougni Pokem et al., 2015).

For determination of the total temocillin,- concentration, assay will be performed after plasma protein precipitation with methanol. Unbound concentrations will be determined after ultracentrifugation of samples using Amicon filter Ultra-15 device; NMWL 30K; Merck Millipore Ltd) TMO.

2.6. Demographic and Laboratory data.

The following laboratory investigations will be collected

* Age,
* Body weight and body mass index (BMI),
* Calculated Glomerular filtration rate (GFR)
* Total protein and albumin level

2.7. Labeling.

2.7.1. Patient identification.

Each enrolled patient will be identified by the first letter of the first name and the first letter of the surname. Patient numbers 01 - 60 will be used in a consecutive order.

2.7.2. Sample identification.

Each sample will be clearly and unequivocally identified with a label resistant to the storage temperature and containing the following information:

* Type of infection
* patient number
* Scheduled time of sampling (hh:min)

2.8. Statistical and pharmacokinetic data analysis.

Statistical analysis will be performed using commercially available softwares (JMP Pro and Graph Pad Prism). Primary pharmacokinetic outcome variables (area under the curve \[AUC\], volume of distribution \[Vd\], total drug clearance \[Cl\]), and maximal and minimal concentration \[Cmax, Cmin\] will be displayed as means ± standard deviation (SD) given normal distribution of the data, or as medians and interquartile range if data are not normally distributed. Normal distribution of the data will be assessed by means of boxplots, Q-Q plots. Graphs showing the concentration-time profiles of total and unbound temocillin will be presented.

2.9. Population pharmacokinetic and pharmacodynamics analysis.

2.9.1. Model building.

The Population pharmacokinetic and pharmacodynamics (Pop-PK/PD) model of plasma total and unbound concentrations of temocillin will be analysed and fitted by Bayesian algorithms using non-commercially available software programs (Pmetrics software version1.4.1 ; LAPKB, Los Angeles, CA, USA.), driven by the predicted plasma total and unbound concentration.

One-, two and three-compartmental pharmacokinetic models will be fitted to the plasma temocillin pharmacokinetic data set. The model which best describes the data will be selected to drive the unbound temocillin concentration pharmacokinetic model in plasma.

Eligibility Criteria:
Inclusion Criteria:

* ≥ 18 years old
* prescribed temocillin for a complicated urinary tract infection and pyelonephritis associated or not with bacteraemia; or a l ower respiratory tract infection; or an abdominal infection
* requiring ≥ 4 days hospitalization
* having signed and informed consent (or signed by the legal representative)

Exclusion Criteria:

* Patients \< 18 years old
* Patients allergic to β-lactams
* Patients Ig-E mediated allergy to penicillin
* Patients with acute or chronic renal failure (GFR \< 30ml/min)
* Patients having participated in another study \< 30 days before inclusion in the present study