The use of liquid chromatography-tandem mass spectrometry for therapeutic drug monitoring of antibiotics in cancer patients

Introduction

According to the World Health Organization (WHO) the number of cancer related deaths reached 8.2 million in 2012. By 2025 this ailment is expected to affect 22 million individuals worldwide. A large body of evidence shows that increased risk of mortality and morbidity of critically ill/immunocompromised cancer patients is associated with their high susceptibility for infection such as severe pneumonia [1], [2], urinary tract infection [2], and blood stream infection [3] to name a few. Even though inadequacy of the antimicrobial therapy recommended for critically ill patients in intensive care units (ICU) is well known, the dramatic failure reported for cancer patients is alarming. The high percentage of infection-induced deaths in cancer patients is extremely high [4] and can increase even to 100%, depending on the strength of infection [5]. Poulikakos et al. show that during the period of 2013–2014 none of the critically ill cancer patients admitted to ICU due to H1N1 infection survived to discharge. Even after treatment with oseltamivir, all patients died from respiratory tract infection and multiple organs failure [5]. Recent studies show that the dosing regimen of two β-lactam antibiotics (cefepime and piperacillin), prescribed for febrile neutropenic patients with hematological malignancies, requires adjustment [6], [7] especially in the presence of organ dysfunction [8]. The need for dose adjustment is mainly due to the fact that the pathophysiological changes encountered in ICU patients substantially affect the pharmacokinetic profile of a drug predisposing the patients to toxic or sub-therapeutic concentrations, when standard doses are applied. In the latter condition an increase in the risk of acquiring bacterial resistance is also of concern [3], [9], [10]. This is alarming especially in cancer patients with neutropenia who are at higher risk for severe sepsis and, therefore, for poor clinical outcome. In addition to failure in the antibiotic therapy, another factor such as the prevalence of drug-drug interaction among antibiotics and antineoplastic agents could also exacerbate the failure/toxicity of the antimicrobial therapy in critically ill cancer patients [11]. Collectively, as the pathophysiological changes observed in critically ill patients differ widely inter- and intra-individually and due to the potential occurrence of drug-drug interaction, the establishment of specific guidelines to be used by the clinicians is unlikely to be successful [12], [13], [14]. Consequently, by tailoring the antimicrobial therapy and monitoring antibiotic-antineoplastic interaction, therapeutic drug monitoring (TDM) would serve as an invaluable tool to increase the efficacy and safe therapeutic use of both classes of drugs in critically ill/immunocompromised patients [15]. To be applied in routine analysis the method(s) used for TDM has to be fast, accurate and cost effective. Nowadays, liquid chromatography tandem mass spectrometry (LC-MS/MS) is the method of choice due to its accuracy and specificity along with the short turnover of analysis it offers. To date several reviews have been published focusing on the importance of TDM in improving antimicrobial treatment in critically ill patients, yet, to our knowledge none has tackled its relevance with respect to cancer patients and drug-drug interaction. In addition, no other reviews summarized the current LC-MS/MS methods developed for the simultaneous determination of different classes of antibiotics used as a first and second line of treatments in ICU. Therefore, this review will discuss briefly the drawbacks of the current antimicrobial therapy with specific emphasis on the importance of TDM in dose adjustment and in the study of antibiotic-antineoplastic drug interaction. As the focus of this review is to optimize the antimicrobial therapy of critically ill cancer patients, we will delineate only the analytical methods used for antibiotics determination. We will also shed the light on the main points that should be taken into account while developing a new method for the simultaneous analysis of the different classes of antibiotics used in ICU.

Current antibiotic therapy in ICU patients

The antimicrobial therapy of infection in ICU patients often relies on the use of the β-lactam class of antibiotics (carbapenems, penicillins, cephalosporins and monobactams) as well as other classes/antibiotics such as fluoroquinolones, sulfamide, oxalizidione and tigecyclin. Every treatment regimen aims to attain the pharmacokinetic/pharmacodynamics (PK/PD) target: maintain a proper concentration to induce the desired effects. Depending on the class of the antibiotics the PD kill characteristic can be (1) time-dependent (i.e.: β-lactams, carbapenems, linezolid), (2) concentration-dependent (i.e.: fluoroquinolones, daptomycin), and (3) concentration-dependent with time-dependence (i.e.: tygecyclin, linezolid, fluoroquinolones, aminoglycosides) [16], [17]. For time dependent antibiotics such as β-lactams the antibacterial activity is related to the time for which their unbound plasma concentration is maintained above the minimum inhibitory concentration (MIC) during a dosing interval (fT_>MIC) [18], [19]. For several β-lactam antibiotics the PK/PD targets ranges from 40% to 70% of the dosing interval (40% fT_>MIC to 70% fT_>MIC) [18], [19]. On the other hand, for fluoroquinolones which exhibit concentration-dependent killing behavior [20], the best predictor of bacterial killing is the 24h AUC_{0−24 h}/MIC (area under the concentration–time curve/MIC) while the peak plasma concentration C_max/MIC is important to prevent the emergence of bacterial resistance [10]. Data based on clinical trials suggests that AUC_{0−24 h}/MIC of >100 and <250 yield bacterial killing at slow rate (7 days), whereas AUC_{0−24 h}/MIC of >250 ensure rapid bactericidal action (24 h) of fluoroquinolones for both Gram-negative and Gram-positive bacteria [20]. To avoid resistance of Gram-negative pathogens to fluoroquinolones the C_max/MIC ratio should be >10–12 [20], [21].

Impact of pathophysiological changes on the antimicrobial therapy

The current antibiotic dosing regimen is based on studies performed with healthy volunteers or non-critically ill patients. Yet, the dramatic pathophysiological changes such as alterations in drug’s clearance and protein binding, volume of distribution (Vd) and acid–base balance, which change drug’s distribution confronted in ICU patients affect significantly the pharmacokinetic (PK) profile of the antibiotics used [22]. Furthermore, patients, presenting dysfunction in vital organs such as kidney and liver that represent the major routes for drug elimination, are at increased risk of the prompt onset of toxic drug concentration [12], [23]. On the other hand, patients with augmented renal clearance [24] or prescribed with continuous renal therapy suffer from decreased levels of antibiotics necessary to achieve a positive clinical outcome [25]. It is evident that different antibiotics are differently affected by the pathophysiological changes encountered in ICU patients. While hydrophilic antibiotics such as β-lactams are mostly affected by the pathophysiological changes in Vd and creatinine clearance [16], lipophilic antibiotics such as fluoroquinolones and tigecyclins have, nonetheless, lesser Vd alterations but may develop altered drug clearance [16], [17]. Several studies published so far support the idea that using the conventional dosage regimens recommended for patients with minor infections is not adequate for critically ill patients with severe infections. Compelling evidence demonstrates that the dosing recommendation of β-lactam antibiotics is inadequate in critically ill patients [19] and that the poor clinical outcome observed to date is related to poor antibiotics exposure [13], [18], [19], [26], [27], [28]. In an empirical study published in 2014 by De Waele et al. [19] data analysis from 68 ICU across 10 countries shows that around 20% of critically ill patients did not reach the most conservative β-lactams PK/PD target (50% fT_>MIC), and 40% of them did not reach the 100% fT_>MIC, which was independently associated with increased creatinine clearance. Furthermore, other studies indicate the large variations in the levels of the different classes of antibiotics used in ICU: cefepime [29], meropenem [30], piperacillin [14], linezolid [31], [32], [33], sulfamethoxazole/trimethoprim [34], fluconazole [13], [35], flucloxacillin [36], and ciprofloxacin [9], [10], [37]. In addition to the extensive inter-individual variation, intra-individual variation was observed for piperacillin [14] and linezolid [33].

Besides the documented poor exposure of antibiotics in ICU, emerging evidence supports the correlation of increased levels of β-lactams with increased toxicities such as increased occurrence of neurological deterioration (tazobactam and meropemen) [38], and seizure (cefepime) in critically ill patients [38], [39]. Collectively, the current knowledge proves the need for dose adjustment (amount and duration) in order to achieve a positive clinical outcome in critically ill patients. Even though all of the aforementioned studies were performed in critically ill non-cancer patients, similar results are anticipated due to the similar pathophysiological changes encountered in cancer patients as with other critically ill patients. Furthermore, convincing evidence shows that increased morbidity and mortality of cancer patients infected with resistant bacteria are more likely to occur following receipt of inadequate initial empirical antibiotic treatment [40].

Requirement of TDM to study antibiotic-antineoplastic interaction

Concomitant administration of a wide range of medications along with antineoplastic agents, characterized by their narrow therapeutic index and high level of inter-individual variability, to cancer patients leads to drug-drug interactions [11], [41], which account for about 4% of the reported cancer death [42]. The interaction can be manifested at (1) the pharmacokinetic level, by affecting the absorption, distribution, metabolism, elimination (ADME) of the drug itself or a combination of drugs, and (2) the pharmacodynamics level where the combination result in synergistic, antagonistic or additive effects [11]. Even though the use of antineoplastic agents is generally discontinued if the patients show local signs of infection and develops fever, some antineoplastic agents are not stopped and are co-administered with the prescribed antibiotics. Consequently, the knowledge of possible interaction between antibiotic and antineoplastic agents may be valuable [43]. The drawbacks of combination treatments between antibiotic and anticancer compounds have been documented back to as early as 1967 whereby an antagonism between benzylpenicillin (penicillin G) and the antineoplastic agent actinomycin D was confirmed [44]. Recently, toxicities resulting from a combination therapy of antibiotic and antineoplastic agents have been also documented [45]. Most of the reports are on the effect of the co-administration of antibiotics along with methotrexate (MTX), an anti-folate agent effective against different types of tumors such as lymphoma, osteosarcoma and bladder [45], [46], [47]. The observed toxicity was not limited to the combination of MTX with one class of antibiotics but covered different classes such as fluoroquinolones, β-lactams (i.e.: penicillin and cephalosporin), and sulfonamide. For instance, a life threatening toxicity was observed in a patient receiving ciprofloxacin, a fluoroquinolone, and a high dose of MTX to treat non-Hodgkin lymphomas [45]. The toxicity was due to a prolonged persistence of MTX in the plasma of the patient, probably due to a decrease in its renal elimination [45]. Delay in MTX elimination has been also documented in children concomitantly receiving ciprofloxacin [46]. Competition in renal clearance between MTX and penicillin antibiotics has been also reported three decades ago whereby the simultaneous presence of MTX and phenoxymethylpenicillin (penicillin V) resulted in transient toxicity in a case study of a patient with prostate cancer [47]. Other antibiotics such as amoxicillin, piperacillin, oxacillin and pristinamycin have similarly resulted in delayed clearance of MTX [48], [49]. Blockage of the proximal renal organic anion transporters OAT1 and OAT3 by antimicrobial agents was suggested as the reason for the decreased renal excretion and increased toxicity of MTX [45], [50]. However, along with a decrease in its clearance an additional mechanism has been observed when MTX was combined with trimethoprim (TMP)/sulfamethoxazole (SMZ) whereby an increase in MTX free unbound levels, through displacement from plasma proteins, was observed in children treated for leukemia [51]. As TMP is excreted by the organic cation system and not the organic anion system, the observed decrease in MTX clearance can be due to the inhibition of the latter one by the presence of SMZ [51], [52]. Interestingly, in opposition to the aforementioned antibiotics that enhanced MTX induced toxicities, TMP enhanced MTX anti-folate effect [48]. This is possibly due to the fact that TMP itself is an anti-folate; hence, it can act synergistically with MTX [53]. Drug-drug interaction has been also documented when nilotinib, used to treat chronic myeloid leukemia, was combined with antiretroviral drugs (ritonavir and efavirenz) [54]. The authors recommended TDM to achieve the desired dose and avoid toxic effects when the drugs are co-administered [54]. To date all the studies comments on the anticancer related toxicities associated with the concomitant presence of antibiotics but none evaluated the presence of anticancer drugs on the PK/PD of antibiotics which remains an interesting field of research that merits further investigation. Finally, TDM is not only important to adjust the dose of antimicrobial therapy and the anticancer therapy [15] but such monitoring will also help in identifying and avoiding drug potential interactions.

Overview of published analytical methods optimized for therapeutic drug monitoring

Current knowledge highlights the large variation in the levels of different classes of antibiotics prescribed to ICU patients. As these patients are usually prescribed with a cocktail of β-lactams and other classes of antibiotics, therefore, there is an increasing interest in providing a convenient method that enables the simultaneous analysis of the different antibiotics to assist quick decision in dose adjustment. A suitable method should be straightforward, with short turnover, and cost effective. Immunoassays are usually an attractive choice as they do not require special expertise, equipment or significant resources. Yet, immunoassays which have provided such advantages for antibiotics as aminoglycosides, do not exist for β-lactams and attempts for the establishment of such techniques have been difficult [6], [55]. Therefore, with the current limitations the use of immunoassays for antibiotics’ TDM is not considered as a priority for further development and efforts have been directed to explore other alternatives such as the use of chromatographic techniques. Consequently, liquid-chromatography (LC) coupled to UV [28], [56], [57], [58], [59], chemiluminescence (CL) [60], electrochemical (EC) [61], and mass spectrometry (MS) [62], [63], have been widely/successfully used for the detection of β-lactams. Although as compared to mass spectrometry less sophisticated analysis is performed with the use of the other conventional detectors, the speed and sensitivity of the former one surpass those offered by the latter ones (UV, CL, EC). The current interest focuses on LC-MS/MS [60], [64], [65], [66], [67], [68], [69], [70], [71], [72], [73], [74]. This is not surprising as it is well acknowledged that LC-MS/MS has the power of combining the sensitivity of tandem MS and the selectivity of chromatography thus improving the accuracy of quantitating low drug concentrations from different matrices. In many cases the use of LC-MS/MS offers an increase in the sensitivity by several orders of magnitude. For instance, reported lower limit of detection of some β-lactams achieved with LC-UV or LC-EC detectors is 5 mg/L [28], [61], while it reached 10–50 times lower levels (0.1–0.5mg/L) when LC-MS/MS is used [64], [74]. As in the case of critically ill patients very low levels can be anticipated methods that offer higher sensitivity are preferred. Furthermore, the analysis time with conventional detectors is usually lengthier from 10 to 22 min [28], [57] which makes them, in terms of the fast delivery of the results, less attractive for routine analysis when compared to LC-MS/MS which can offer analysis time of less than 5 min. There is no doubt that conventional methods are less expensive and can be also suitable for TDM; nevertheless, careful consideration should be made when availability, costs, speed and accuracy come into the play. Even though it is assumed that the implementation of LC-MS requires extremely sophisticated infrastructure, i.e. highly qualified trained personnel and expensive equipment; yet, in comparison to earlier eras the costs for mass spectrometry machines dropped significantly, used machines are readily available on the market and training of personnel is doable. Nowadays, the number of hospitals incorporating LC-MS in routine analysis is increasing. This is for the reason that the promising reward obtained in terms of fast delivery of results, faster success in patients treatment along with a decrease in the costs of medications and stay of patients in the hospital pay off for what a hospital would invest in the implementation of LC-MS in routine analysis.

LC-MS/MS for antibiotics therapeutic monitoring what shall we know?

Although different analytical methods have been developed for the quantification of individual antibiotics, in the last 5 years several methods have been published for the simultaneous analysis of subsets of antibiotics usually given to critically ill patients [64], [65], [66], [67], [68], [69], [70], [71], [72], [74], and continuous attempts for improved method development are ongoing. This stems from the need for rapid and accurate methods for the detection in one run of as many as possible of the antibiotics used as first and second lines of treatment in critically ill patients. Nonetheless, care must be taken during method development due to the difference in the physicochemical properties of the different antibiotics used in ICU. In comparison to stable antibiotics such as linezolid, β-lactams antibiotics are unstable and highly susceptible for degradation under different analytical settings. Reviewing the chemical characteristics of β-lactams antibiotics is beyond the scope of this review. However, as the current attempt is to optimize methods for the simultaneous analysis of different antibiotics with different reported stabilities it is necessary to consider the different parameters that might affect the stability of the antibiotics ahead of time. Consequently, this part will shed light on the key steps that have been recognized to be essential to improve the LC-MS/MS analysis of antibiotics used in critically ill patients with specific emphasis on β-lactams due to their instability.

Popular antibiotics measured by LC-MS/MS

A thorough evaluation/comparison of the developed methods and the types of the studied antibiotics shows that in the 11 described methods 37 antibiotics belonging to different classes have been analyzed, yet at different rates. A ranking of the different antibiotics according to the frequency of their inclusion in the developed methods is highlighted in Table 4. Eight of the 11 described methods share, although to a different extent, the analysis of 13 out of 37 antibiotics. Meropenem and piperacillin are the most prevalent antibiotics being common in six and five analytical methods, respectively, while the others are shared in two to four methods (Table 4). The enormous popularity of meropenem and piperacillin is not unexpected. These broad spectrum antibiotics are primarily used as a first line of treatment in ICU patients followed by narrower spectrum antibiotics when the type of infection and resistance are identified [102]. Secondly, the levels of these antibiotics are recurrently found to vary extensively, in critically ill patients, which classify them as important targets for TDM (Table 5). Table 5 presents a quick overview about the current reported variability of the trough levels and maximum levels of the most studied antibiotics as observed in ICU patients and therefore, emphasize the clinical relevance of their selection for TDM. A more extensive ranking of the different antibiotics is beyond the scope of this review as published surveys show that the type and extent of use of antibiotics differ between countries, type of infection and known resistance [107], [108]. Nevertheless, a brief analysis of the current use of antibiotics in Germany shows that β-lactams are widely used in ICU in University hospitals [107]. The median RDD/100 of the different classes of β-lactams is as follows: carbapenem (3.7), extended-spectrum penicillin (5.9), third/fourth generation cephalosporin (4.3), first/second generation cephalosporin (8.0), aminopenicillin/β-lactamase inhibitors (6.5), and narrow-spectrum penicillin (1.9). Among the other prescribed antibiotics fluoroquinolones have the highest RDD/100 (8.0) which continues to be prescribed mainly to elderly people [107]. To have an overview about the global use of antibiotics readers are referred to a recent survey supplied by the Center for Disease Dynamics (https://cddep.org/sites/default/files/swa_2015_final.pdf).

Conclusions and future prospects

Overcoming the pathophysiological changes affecting the PK/PD target attainment of the current antimicrobial therapy in ICU patients is the focus of intense investigations with the main intention of improving clinical outcome. This incorporates ensuring that the required antibiotic level is maintained to penetrate the site of infection, prevent resistance, and avoid toxicity-related to antibiotic accumulation. This is particularly significant for cancer patients suffering from severe infections, found to be associated with their increased mortality rate. Furthermore, an increase in the awareness regarding potential antineoplastic-antibiotic interactions would spare cancer patients from the occurrence of additional unnoticed reasons for toxicity. To improve the clinical outcome from the current therapy regimen TDM seems to offer a basis on which clinicians can adjust and personalize the treatment. When sample throughput is important such as in TDM the development of a reliable method with short analysis time becomes essential. Immunoassays, which are easy to perform and can be accessible worldwide, are not available for all classes of antibiotics and attempts to establish immunoassays for β-lactams have proved to be difficult due to the reactivity or degradation of the β-lactam ring. Fortunately, the ongoing advances in the available analytical methods render the application of these techniques for TDM of β-lactams and many other antibiotics more feasible. LC-MS/MS method, through accurate and rapid antibiotics detection along with the availability of isotopically labeled internal standards that increase the fidelity of the analysis, has become the method of choice. Different analytical methods have been published, yet, due to the different characteristics of β-lactams and other antibiotics used in ICU, the establishment of one single method is not possible for the time being. Additional concern is the reliability of the estimation of the free pharmacologically drug concentration from the total detected concentrations. Yet, more studies are needed in that direction to rule out the best strategy to be used in the future. There is no doubt that with the ongoing advancement in analytical method developments many of the encountered difficulties will be overcome and will add more to our understanding in improving the clinical outcome from the antimicrobial therapy of ICU/cancer patients. Furthermore, both TDM and an increase in the collaboration between clinicians, pharmacists and general practitioners as well as the use of computer-based medication prescription systems in hospitals will help to avoid and notice the frequently unnoticed drug-drug interactions.