Academic literature on the topic 'Therapeutic drug monitoring'

Therapeutic drug monitoring (TOM) focuses on measurement of drugs with narrow therapeutic windows. Inappropriate concentrations of these drugs can elicit either an absence of therapeutic response or toxic effects. Microneedles (MN) have previously been studied for their ability to painlessly penetrate the stratum corneum, allowing transdermal delivery of various compounds. Interest in MN technology to sample analytes is increasing due to their minimally-invasive characteristics when compared to conventional needles. . . To overcome problems associated with the use of conventional needles, this study aimed to identify the most suitable polymeric MN array design and utilise this, for the first time, as a sampling device capable of imbibing large amounts of fluid for TOM. Hydrogel-forming MN arrays were fabricated from blends of poly(methyl vinyl ether-eo-maleic acid) crosslinked with poly(ethylene glycol) both with and without pore-forming agents. These MN were used in combination with reverse iontophoresis and hygroscopic Iyophilised tablets to further enhance fluid uptake by polymeric matrices. Theophylline, a bronchodilator with a narrow therapeutic window, was chosen as the model drug of the study. A high-performance liquid chromatography (HPLC) method was developed and validated for detection of theophylline. This study proved the capacity of hydrogel-forming MN to take up theophylline, in vitro and in vivo, and release it in water solution for HPLC quantification. Theophylline was successfully detected and quantified from 12 of 24 MN after 1 h insertion in the back of rats dosed with 10 mg theophylline/kg body mass. While this study posed certain challenges, including problems optimising HPLC methodology and difficulties designing a method to extract theophylline in vivo, the potential of MN as a novel in vivo TOM technology with significant clinical potential was proven. Ideally, given the findings of this study, future work will focus on the use of hydrogel-forming MN for TOM in human volunteers.

Apixaban is a novel oral anticoagulant that prevents clotting by directly inhibiting Factor Xa in the coagulation cascade. Due to its different pharmacokinetics, previous standards for testing anticoagulant concentrations are ineffective at measuring apixaban. In this study, Hyphen Biomed Biophen Direct Xa Inhibitor and Biophen Heparin chromogenic kits from Aniara Diagnostica were used along with a NanoDrop™ One/OneC Microvolume UV-Vis Spectrophotometer to see if either of these kits provide acceptable precision and accuracy for the quantification of apixaban in plasma samples, as well as if there is a significant difference in these two kits at varying concentrations of apixaban. Apixaban is a novel oral anticoagulant that prevents clotting by directly inhibiting Factor Xa in the coagulation cascade. Due to its different pharmacokinetics, previous standards for testing anticoagulant concentrations are ineffective at measuring apixaban. In this study, Hyphen Biomed Biophen Direct Xa Inhibitor and Biophen Heparin chromogenic kits from Aniara Diagnostica were used along withused witha NanoDrop™ One/OneC Microvolume UV-Vis Spectrophotometer to see if either of these kits provide acceptable precision and accuracy for the quantification of apixaban in plasma samples,as well as and to evaluate if there is a significant difference in these two kits at varying concentrations of apixaban.

La chemoterapia consiste nell’impiego di uno o di una combinazione di farmaci antitumorali per il trattamento del cancro. Tuttavia tali farmaci sono caratterizzati da una famacocinetica molto variabile e da una elevata tossicità che porta alla comparsa di molti effetti indesiderati nei pazienti, diminuendo l’efficienza della terapia. In questo contesto l’impiego del “Therapeutic Drug Monitoring” (TDM) risulta particolarmente importante in quanto permette per lo sviluppo di terapie personalizzate per i pazienti aumentando l’efficienza della terapia e la qualità della vita dei pazienti. Questo progetto è finalizzato alla sintesi e allo sviluppo di sensori, basati su nanoparticelle polimeriche ad impronta molecolare, per i farmaci antitumorali: sunitinib, paclitaxel, SN38 e irinotecano, suo profarmaco. Tali nanoparticelle solubili sono state ottenute mediante polimerizzazione radicalica ad elevata diluizione utilizzando diversi monomeri funzionali: N-acriloil-tirosina metil estere, N-acriloil triptofano metil estere, 4-vinilpiridina e 7-acrilossicumarina. La reazione è stata effettuata lasciando interagire i monomeri funzionali e il farmaco mediante interazioni deboli (legami idrogeno, π-staking, interazioni di Van der Waals) in DMSO e dopo l’aggiunta dell’acrilamide come co-monomero, dell’N,N’-etilenebisacrilamide come crosslinker e dell’iniziatore radicalico azobisisobitironitrile (AIBN) la polimerizzazione è stata ottenuta scaldando a 70°C per 4 giorni. La molecola templato è stata rimossa mediante dialisi prima in una miscela di metanolo e acido acetico, e poi in acqua. Le particelle ottenute dopo liofilizzazione, sono state caratterizzate mediante 1H-NMR, Nanosigh e Dynamic Laser Ligh Scattering. La capacità di legame e la selettività dei polimeri è stata studiata mediante test di recupero utilizzando un metodo HPLC per la quantificazione del farmaco catturato. Mentre le proprietà fluorescenti di alcuni dei polimeri sintetizzati sono state utilizzate per studiare le affinità di legame dei polimeri a basse concentrazioni di farmaco. Il polimero contenente cumarina e specifico per il sunitinib è stato utilizzato come sensore fluorescente per lo sviluppo di un test di validazione in DMSO:plasma. Il sensore ha mostrato un’accuratezza del 15%, una precisione del 10% e una buona robustezza. Inoltre due diversi sensori fluorescenti sono stati sviluppati per la quantificazione dell’irinotecano in metanolo:plasma. Il primo sensore si basa sul quenching della fluorescenza intrinseca dell’irinotecano, mentre il secondo è un polimero molto fluorescente contenente un monomero funzionale con un gruppo naftalimidico la cui fluorescenza è spenta in seguito all’interazione con l’irinotecano. Inoltre un test fluorimetrico e colorimetrico è stato sviluppato per quantificare il paclitaxel mediante la tecnica del “dye displacement”. Il test infatti, si basa sulla competizione tra il farmaco libero ed il farmaco legato covalentemente al DABCYL per il legame ad un polimero ad imprinting molecolare contenente EDANS come monomero funzionale fluorescente. Infatti il DABCYL è sia un colorante rosso, sia un FRET quencher dell’EDANS, perciò il suo legame nel polimero porta ad una variazione del colore e della fluorescenza del polimero. Infine un test colorimetrico per la quantificazione dell’irinotecano è stato sviluppato utilizzando un polimero ad imprinting molecolare contenente l’acido 2-acrilamido-2-metil propansolfonico come monomero funzionale. Il test si basa sul legame del colorante: anilina gialla nei siti di legame del polimero rimasti liberi dopo interazione con i campioni di farmaco. L’interazione tra il colorante e l’acido solfonico nel polimero genera un cambio di colore da giallo a rosso.<br>Chemotherapy consists in cancer treatment by the administration of a single or a combination of anticancer drugs. However chemotherapy agents are often characterized by an high variability of pharmacokinetic among patients and an high toxicity that leads to the appearance of many side effects decreasing the therapy efficiency. Therefore the therapeutic drug monitoring (TDM) become useful to develop personalized therapies for patients in order to increase the efficiency of the therapy and patient compliance. This project is aimed on the synthesis and development of specific sensors based on molecularly imprinted polymeric nanoparticles, to be applied in TDM of the anticancer drugs: sunitinib, paclitaxel, SN38 and its prodrug irinotecan. Soluble nanoparticles were obtained by high dilution radical polymerization with different functional monomers: N-acryloyl-tyrosine methyl ester, N-acryloyl-tryptophan methyl ester, 4-vinyl pyridine or 7-acryloyloxy-coumarin. The reactions were carried out allowing the functional monomer to interact with the drug by weak interactions (H-bonds, -staking and Van der Waals) in DMSO and after the addition of the co-monomer acrylamide, the crosslinker N,N’-ethylene bisacrylamide, and the radical initiator azobisisobutyronitrile (AIBN) the polymerization was achieved heating at 70°C for 4 days. The template was removed by dialysis first in methanol:acetic acid mixture and after in water. After the freeze-drying the polymers were characterized by 1H-NMR, Nanosight, and Dynamic Laser Light Scattering. The polymers binding capabilities and selectivity were investigated by rebinding tests using an HPLC method for the quantification of drug captured. while the fluorescence properties of some of these polymers were exploited to study the polymers binding affinities at low drug concentrations. The polymer containing coumarin and imprinted with sunitinib was used as fluorescence sensor to set up a validation test in DMSO:plasma mixture. The system showed an accuracy of 15%, a precision of 10% and a good robustness. Two different fluorescence sensors were also developed for irinotecan able to quantify the drug in methanol:plasma mixtures. The first sensor is based on the quenching of the intrinsic fluorescence of irinotecan, while the second is an highly fluorescence polymer containing a functional monomer with a naphthalimide mojety whose fluorescence is quenched upon interaction with the drug. Moreover the dye displacement technique was used to set up a fluorescent and colorimetric test for paclitaxel quantification. The test is based on the competition between the free paclitaxel and the drug covalently linked to DABCYL dye, for the binding in to an imprinted polymer containing EDANS fluorescent functional monomer. Since DABCYL is both a red dye and a FRET quencher of EDANS, its binding into the polymer gives a change in the polymer fluorescence and colour. Finally a colorimetric test for irinotecan quantification was developed using an imprinted polymer containing 2-acrylamido-2-methylpropane sulfonic acid as functional monomer. The test is based on the binding of aniline yellow dye in to the remaining free binding sites of the polymer after treatment with drug samples. The interaction between the dye and the sulfonic acid in to the polymer gives a change of colour from yellow to red.

Considerato che la maggior parte dei farmaci antitumorali risulta caratterizzata da un'alta variabilità interindividuale nelle concentrazioni plasmatiche, che si riflette sull'efficacia del trattamento, durante il progetto di dottorato qui descritto sono state sviluppate tecniche per il loro monitoraggio terapeutico (TDM). In primo luogo, è stato sviluppato, validato e cross-validato un metodo LC-MS/MS per la quantificazione di imatinib (IMA) e del suo metabolita attivo, norimatinib (norIMA), in pazienti affetti da tumore stromale gastrointestinale utilizzando la tecnica del dried blood spot (DBS). Il DBS consente di ridurre costi e tempi di campionamento e migliorare la compliance dei pazienti, poiché l’analisi viene effettuata tramite una goccia di sangue capillare depositata su carta. Da questa gli analiti vengono estratti con MeOH acidificato e l'estratto viene iniettato in un sistema LC (configurato con una cromatografia 2D per la pulizia on-line del campione), accoppiato ad un API-4000QT. Il metodo ha mostrato una buona linearità (R2> 0,996) nei range di 50-7500 ng/mL e 10-1500 ng/mL per IMA e norIMA. La precisione e l’accuratezza intra-day sono state, rispettivamente, ≤3.1% e tra 88.9-112.8%, mentre quelle inter-day erano ≤6,6% e tra 95.7-104.3%, per entrambi gli analiti. Sono stati valutati inoltre: l'influenza dell'ematocrito (Hct), del volume depositato e dell'omogeneità del campione sull’analisi; la correlazione tra la concentrazione in DBS da prelievo venoso e da finger-prick (differenza tra -12 e 3.8%) e la stabilità dei DBSs (fino a 16 mesi). Il metodo è stato applicato per la quantificazione di 67 campioni DBSs di pazienti. Le concentrazioni in DBS, normalizzate per Hct e per un fattore di correzione che prescinde dall’Hct, correlano con quelle plasmatiche. Parte del lavoro di questo progetto è stato anche dedicato allo sviluppo di strategie alternative a LC-MS/MS per favorire ulteriormente l’applicazione del TDM. In particolare, è stata eseguita la sintesi di polimeri (molecularly imprinted polymers - MIPs), con l'obiettivo futuro di applicarli come recettori in un sistema di rilevamento fluorimetrico per IMA. I MIPs sono stati sintetizzati sfruttando l'approccio non covalente e la polimerizzazione radicale ad alta diluizione. Tramite questa tecnica, i MIPs ottenuti, sintetizzati in DMSO e con acido metacrilico come monomero funzionale, presentano dimensioni nanometriche (dati acquisiti tramite dynamic light scattering). I tests di rebinding hanno dimostrato che solo 2 MIPs sono stati in grado di legare IMA con una buona specificità (rispetto ai corrispondenti polimeri non-imprinted) e selettività. È stato infine sviluppato e validato un metodo LC-MS/MS per la quantificazione di ribociclib (RIBO), palbociclib (PALBO) e letrozolo (LETRO) in plasma. RIBO e PALBO sono farmaci appartenenti alla famiglia dei CDKIs recentemente approvati per il trattamento del carcinoma mammario in combinazione con LETRO. Il metodo messo a punto risulta adatto per l’applicazione nella pratica clinica, grazie ad una semplice preparazione del campione e ad una rapida analisi (6.5 min). Il metodo risulta lineare (R2 tra 0.992-0.983) nei range di concentrazione di 0.3-250 ng/mL per PALBO, 10-10000 ng/mL per RIBO e 0.5-500 ng/mL per LETRO (che coprono le concentrazioni plasmatiche terapeutiche). La precisione e l’accuratezza intra-day sono state, rispettivamente, ≤3.6% e tra 94.5-112.3% per tutti e gli analiti, mentre quelle inter-day erano rispettivamente ≤7.3% e tra 94.5-112.9%. Il metodo è stato applicato con successo alla quantificazione di campioni plasmatici di pazienti. In conclusione, con lo sviluppo di queste strategie si spera di implementare l’utilizzo del TDM per i farmaci oncologici nella pratica clinica.<br>Despite the continuous progress in drug therapy, most anticancer drugs appear to be characterized by a high interindividual variability in plasma concentrations that is reflected in the efficacy of the treatment. From this arises the need of a personalized approach, so that the drug concentrations in plasma are adequate in each patient. On this basis, during the PhD project reported hereby, different techniques for therapeutic monitoring (TDM) of anticancer drugs were developed. First, a LC-MS/MS method for the quantification of imatinib (IMA) and its active metabolite, norimatinib (norIMA), was developed, validated and cross-validated in patients affected by gastrointestinal stromal tumour. This method allows to perform the quantification directly on a drop of capillary blood, exploiting the dried blood spot (DBS) technique, reducing sampling time, costs and improving patients’ compliance. Analytes were extracted from DBS samples by adding acidified methanol and the extract is injected into a LC system (configured with a 2D chromatography for online cleaning of the sample), coupled with an API-4000QT. The method showed good linearity (R2> 0.996) in the ranges of 50-7500 ng/mL and 10-1500 ng/mL for IMA and norIMA. Intra-day precision and accuracy were ≤3.1% and between 88.9-112.8%, respectively, while inter-day ones were ≤6.6% and between 95.7-104.3 %, for both analytes. Moreover, were also evaluated: the influence of the haematocrit (Hct), of the spot size and of the sample homogeneity on the analysis; the correlation between the concentration in DBS from venous sampling and from finger-prick (% difference between -12 and 3.8%) and the stability of DBSs (up to 16 months). Then, the method was applied for the quantification of 67 DBSs patients’ samples. Good agreement was obtained between IMA and norIMA concentrations found in DBS and plasma samples applying either the Hct normalization or avoiding it, simply multiplying the DBS concentration with a correction factor. Part of the work of this project was also dedicated for the development of alternative strategies for the quantification of anticancer drugs, to promote the application of TDM. In particular, the synthesis of molecularly imprinted polymers (MIPs) was performed, with the future goal of applying them as receptors in a fluorimetric detection system for IMA. MIPs were synthesized using the non-covalent approach and high dilution radical polymerization. Through this synthesis, the MIPs obtained, synthesized in DMSO with methacrylic acid as functional monomer, shown nanometric size (data acquired by dynamic light scattering). The rebinding tests then showed that 2 MIPs in particular were able to bind IMA with a good specificity (compared to the corresponding non-imprinted polymers) and selectivity. Finally, a LC-MS/MS method was developed and validated for the quantification of ribociclib (RIBO), palbociclib (PALBO) and letrozole (LETRO) in human plasma. RIBO and PALBO are drugs belonging to the CDKIs family, recently approved for breast cancer treatment in combination with LETRO. The method developed is suitable for its application in clinical practice, thanks to simple sample preparation and rapid analysis (6.5 min). The method showed a good linearity (R2 between 0.992-0.983) in the concentration ranges of 0.3-250 ng/mL for PALBO, 10-10000 ng mL for RIBO and 0.5-500 ng/mL for LETRO (covering the therapeutic plasma concentrations). Intra-day precision and accuracy were ≤3.6% and between 94.5-112.3% for all and analytes, respectively, while inter-day ones were ≤ 7.3% and 94.5-112.9%. The method has been successfully applied for patients’ plasma samples quantification. In conclusion, with the development of these strategies there is the hope to implement the application of TDM for anticancer drugs in the clinical practice.

HIV antiretroviral therapy spans several different drug classes, meant to combat various aspects of viral infection and replication. Many authors have argued the benefits of therapeutic drug monitoring (TDM) for the HIV patient including compliance assurance and assessment of appropriate drug concentrations; however, the array of drug chemistries and combinations makes TDM an arduous task. HPLC-UV and LC-MS/MS are both frequent instruments for the quantification of HIV drugs in biological matrices with investigators striving to balance sensitivity and affordability. Plasma, the dominant matrix for these analyses, is prepared using protein precipitation, liquid-liquid extraction or solid-phase extraction depending on the specific complement of analytes. Despite the range of polarities found in drug classes relevant to HIV therapeutics, most chromatographic separations utilize a hydrophobic column (C18 ). Additionally, as the clinically relevant samples for these assays are infected with HIV, along with possible co-infections, another important aspect of sample preparation concerns viral inactivation. Although not routine in clinical practice, many published analytical methods from the previous two decades have demonstrated the ability to conduct TDM in HIV patients receiving various medicinal combinations. This review summarizes the analytical methods relevant to TDM of HIV drugs, while highlighting respective challenges.

2017 - 2018<br>The U.S. Food and Drug Administration defines as Precision or Personalized Medicine (PM) an innovative therapeutic approach that tailors therapy and prevention on patients based on inter-individual variabilities in molecular or environmental features and in lifestyles. The major goals of PM are to maximize treatment efficacy and to reduce cost, toxicities and therapy failure rates by early identification of patients who might benefit or not of a specific treatment. In this scenario, therapeutic drug monitoring (TDM) is an important laboratory tool for PM because of the possibility to measure several drugs and bioactive molecules in human biological matrices. TDM is based on the hypothesis that in the majority of drugs, there is a relationship between administered dose and circulating concentration of unbound fraction - and between this concentration and observed pharmacological effects. TDM is recommended for drugs with significant inter-individual pharmacokinetic variability and an established relationship between blood concentrations and clinical efficacy and/or toxicity. Moreover, TDM is also advisable in special populations such as pregnant women and children. To date, liquid chromatography and immunometric assay are still considered the standard for molecule measurement in biological fluids; however, in recent years, LC tandem mass spectrometry (LC-MS) is gaining popularity because of the possibility of in-depth and multiplexed analysis with high selectivity and specificity. During this Ph.D. program, we developed several high performance LC (HPLC)- and LC-MS/MS-based approaches for TDM of different drugs measured in various types of body fluids and validated according to EMA and FDA guidelines. In particular, we focused on: 1) TDM of hydroxychloroquine (HCQ) blood concentration, a drug with a wide therapeutic window. Our method was validated on a cohort of patients with Systemic Lupus Erythematosus treated with HCQ and blood concentrations were correlated to several clinical parameters, such quality of life. Moreover, TDM of HCQ was also used to monitor treatment adherence in those subjects. 2) TDM of a commonly used chemotherapeutic agent, the 5-fluorouracil (5-FU), which is known to have a narrow therapeutic window and a high toxicity 3) TDM of a new kinase inhibitor, Ruxolitinib, approved for the treatment of myeloproliferative hematologic disorders. 4) TDM of several drugs, such as caffeine and phenobarbital, in newborns who are at particular risk of uncorrected drug dosage. Due to the need to carry out analyses on very-small volume samples, we validated an analytical method using micro-sampling techniques such as the dried blood spot (DBS) sampling combined with LC-MS/MS analysis. [edited by author]<br>XVII n.s. (XXXI ciclo)