P EPTIDES AS R ECOGNITION AND S ENSING E LEMENTS

XXV CICLO DEL DOTTORATO DI RICERCA IN SCIENZE e TECNOLOGIE CHIMICHE e FARMACEUTICHE

PEPTIDES AS RECOGNITION AND S^ENSING E^LEMENTS

CHIM/06

Ph.D. program Director Prof. Mauro Stener Ph.D. Student

Silvia Pavan

Thesis Supervisor Dr. Federico Berti

Anno Accademico 2011/2012

one must still have chaos within oneself, to give birth to a dancing star. ” ¹

1 Nietzsche F (1885) Thus Spoke Zarathustra

Table of Contents

Table of Contents!...i

Abstract - Riassunto!...v

List of Abbreviations!...ix

1. Introduction!...1

1.1 Drug Monitoring of Efavirenz...! 3

! 1.1.1 Efavirenz!...3

! 1.1.2 Drug Monitoring!...3

1.2 Recognition and Sensing Elements!...11

! 1.2.1 Biosensors!...11

! 1.2.2 Molecular Recognition Elements Biosensors!...13

! ! 1.2.2.1 Receptors!...13

! ! 1.2.2.2 Antibodies!...13

! ! 1.2.2.3 Lectins!...14

! ! 1.2.2.4 Aptamers!...14

! ! 1.2.2.5 Peptide Nucleic Acids!...16

! ! 1.2.2.6 Supramolecular Architectures!...17

1.3 Short Peptides as Biosensor Transducers!...21

! 1.3.1 Designed Synthetic Peptides!...22

! 1.3.2 Short Peptides from Random Phage Display!...25

! 1.3.3 Peptide Receptors for Ligand Sensing!...27

! 1.3.4 Peptide Ligands for Receptor Sensing!...28

! ! 1.3.4.1 Antimicrobial and Cell-Penetrating Peptides!...28

! ! 1.3.4.2 Antigens...! 30

! ! 1.3.4.3 Enzyme Substrates and Inhibitors !...30

1.4 Molecularly Imprinted Polymers!...39

! 1.4.1 Molecular Imprinting!...39

! 1.4.2 Strategies of Molecular Imprinting...! 40

! 1.4.3 Optimization of the Polymer Structure!...42

! 1.4.4 Target...! 43

! 1.4.5 Highlighted Applications!...44

1.5 Human Serum Albumin!...51

! 1.5.1 Miniaturized Natural Receptors...! 51

! 1.5.2 Human Serum Albumin!...52

! 1.5.3 Structure and Properties!...53

! 1.5.4 Binding Sites!...54

! ! 1.5.4.1 Site I!...55

! ! 1.5.4.2 Site II!...57

! ! 1.5.4.3 Other Binding Sites!...58

! 1.5.5 HSA Binding Interactions...! 58

! 1.5.6 HSA Structural Properties!...59

! ! 1.5.6.1 Flexibility!...59

! ! 1.5.6.2 Conformational Equilibrium...! 59

! 1.5.7 HSA Spectroscopic Features!...60

! ! 1.5.7.1 UV Spectral Properties...! 60

! ! 1.5.7.2 Circular Dichroism...! 60

! ! 1.5.7.3 Fluorescence!...61

2. Aim of Research!...67

3. Results and Discussion!...71

3.1 Synthesis of Efavirenz Derivates!...73

! 3.1.1 Design of Efavirenz Analogues!...73

! 3.1.2 Preparation of EFV!...75

! 3.1.3 First Generation Analogue of EFV...! 77

! 3.1.4 Second Generation Analogue of EFV!...78

! 3.1.5 Third Generation Analogue of EFV!...79

3.2 De novo Artificial Receptors!...81

! 3.2.1 Design of Binder Peptides!...81

! 3.2.2 Computational Tools!...83

! ! 3.2.2.1 Docking...! 84

! ! 3.2.2.2 Replica Exchange!...84

! ! 3.2.2.3 Optimizing the Length of the Peptide...! 85

! 3.2.3 Synthesis of YYGW!...86

! 3.2.4 Peptide Syntheses on Solid Phase!...87

! ! 3.2.4.1 Synthesis on Solid Phase of YYGW-NH2...! 89

! ! 3.2.4.2 Synthesis on Solid Phase of FFWPPFLNVW-COOH!...90

! 3.2.5 Peptide Characterization!...91

! ! 3.2.5.1 NMR of FFWPPFLNVW!...91

! ! 3.2.5.2 Circular Dichroism of FFWPPFLNVW!...96 Table of Contents

ii

! ! 3.2.5.3 Fluorescence of FFWPPFLNVW !...98

! ! 3.2.5.4 NMR of YYGW!...98

! ! 3.2.5.5 Circular Dichroism of YYGW!...101

! ! 3.2.5.6 Fluorescence of YYGW !...101

! 3.2.6 Peptide - EFV Interaction!...102

! ! 3.2.6.1 NMR spectroscopy!...102

! ! 3.2.6.2 Circular Dichroism!...107

! ! 3.2.6.3 Fluorescence Spectroscopy !...107

! 3.2.7 Selectivity for EFV...! 111

! 3.2.8 Improvements in de novo Peptide Design!...112

! 3.2.9 Cyclo-pepHA!...112

! 3.2.10 Further Perspectives for de novo Peptide Receptors!...107

3.3 Molecularly Imprinted Polymers!...123

! 3.3.1 Design of Molecularly Imprinted Polymers!...123

! 3.3.2 Synthesis of Functional Monomers!...124

! 3.3.3 Cooperative Monomers!...125

! 3.3.4 Synthesis of Molecularly Imprinted Polymers!...128

! 3.3.5 Characterization of the Polymers!...132

! ! 3.3.5.1 NMR!...132

! ! 3.3.5.2 Dynamic Laser Light Scattering!...134

! 3.3.6 Binding Properties!...137

! ! 3.3.6.1 ITC...! 137

! ! 3.3.6.2 HPLC!...139

3.4 A Binder Peptide from a Natural Host: Human Serum Albumin!...145

! 3.4.1 HSA100 Identification and Optimization!...145

! 3.4.2 Expression of HSA100!...147

! 3.4.3 Structural Characterization of GST-HSA100!...148

! ! 3.4.3.1 LC-MS/MS and Peptide Sequencing!...148

! ! 3.4.3.1 Circular Dichroism!...148

! 3.4.4 Small Molecule Binding Activity of GST-HSA100!...150

! ! 3.4.4.1 Fluorescence!...150

! ! 3.4.4.2 ELISA Assay!...152

! 3.4.5 Biosensing Application: SPR !...154

! 3.4.6 Towards a Library of HSA100-based Receptors!...161

! ! 3.4.6.1 Structural Analysis!...161

! ! 3.4.6.2 HSA100 Site-directed Mutagenesis!...162

! ! 3.4.6.3 Binding Activity of the Mutant Clones!...163

4. Experimental Procedures!...169

4.1 Experimental Procedures!...171

! 4.1.1 Instrumentation !...171

! 4.1.2 Materials and General Methods!...172

! 4.1.3 Experimental Procedures!...172

! 4.1.4 Peptide Synthesis!...197

! 4.1.5 De novo Artificial Peptides Measurements!...205

! ! 4.1.5.1 NMR spectroscopy...! 205

! ! 4.1.5.2 Fluorescence spectroscopy!...207

... ! ! 4.1.5.3 CD Spectroscopy! 208 ! 4.1.6 Molecularly Imprinted Polymers Synthesis and Measurements!...209

! ! 4.1.6.1 Nanogels Synthesis!...209

! ! 4.1.6.2 Dynamic Laser Light Scattering!...211

! ! 4.1.6.3 UV-visible Spectroscopy!...211

! ! 4.1.6.4 ITC...! 211

! ! 4.1.6.5 RP-HPLC!...212

! 4.1.7 GST-HSA100 and Clones Measurements!...213

! ! 4.1.7.1 Fluorescence Spectroscopy!...213

! ! 4.1.7.2 CD Spectroscopy...! 213

! ! 4.1.7.3 SPR!...213

5. Conclusions!...217

Acknowledgements!...221

! !

!

Table of Contents

iv

University of Trieste

Peptides as Recognition and Sensing Elements

PhD Thesis

PhD candidate: Silvia Pavan Supervisor: Dr. Federico Berti

Abstract

! Antiretroviral therapy (ART) is the recommended treatment of people infected with HIV and involves taking a combination of three or more anti-HIV medications from at least two different drug classes every day to suppress HIV replication. Efavirenz (EFV) is a potent and selective non- nucleoside inhibitor of the HIV-1 reverse transcriptase, used for over a decade as an essential component of antiretroviral therapy, due to its relatively long half-life that allows convenient once- daily dosing. High intrapatient variability in EFV plasma is a clinical problem demanding a rapid measure of pharmacokinetic parameters on each patient to improve the compliance and efficiency of the therapy and to limit the appearance of resistant mutant strains¹.

! The aim of this project has been the design and development of new peptidic artificial receptors for EFV to be developed as recognition and sensing elements in biosensor systems for therapeutic drug monitoring.

! A novel computational tool was implemented for designing short peptides (between 5 and 15 amino acids) with high affinity for a desired small target molecule. Starting from a poly-alanine, this new algorithm had selected a decapeptide (FFWPPFLNVW) with a theoretical binding energy of "12.1 kcal/mol for experimental validation². The peptide was synthesized by solid-phase techniques, and its structure was studied by NMR. The sequential chemical shifts assignments were provided by ¹H,¹H TOCSY and ¹H,¹H NOESY experiments. Circular dichroism data and the range of JH!-NH coupling constants suggested a #-turn secondary structure. Fluorescence quenching measurements gave a ligand-protein dissociation constant value KD = 64 nM $ "14.7 kcal/mol for the dissociation constant of the EFV-peptide complex and this is in good agreement with the theoretical value obtained with the algorithm. ¹H,¹H NOESY and the variation of proton chemical shifts in the titration of EFV to a peptide solution confirmed the intermolecular contacts of the EFV-peptide complex, described by the theoretical model.

! For biosensor purpose this short peptide was incorporated in molecularly imprinted polymers to increase the solubility in water and selectivity for EFV. A set of imprinting nanoparticles with a combination of peptidic functional monomers was currently tested with several techniques (UV, HPLC and ITC) to compare their re-binding capacity towards our target and their selectivity.

! Another approach to develop a peptidic receptor for EFV was based on miniaturizing a natural binder of this molecule. We have identified a 101-amino-acid polypeptide derived from the sequence surrounding the IIA binding site of human serum albumin. Codon usage optimization was used to express a GST fusion protein in E. coli in high yields. This fusion protein retains its structural integrity, its catalytic activity, its ability to direct the stereochemical outcome of a diketone reduction, and its binding capacity to warfarin and EFV³. Notably, this newly cloned polypeptide represents a valuable starting point for the construction of libraries of binders and catalysts with

improved proficiency. Three libraries of clones were designed by site directed mutagenesis and fast screening was performed with surface plasmon resonance experiments. Then novel synthetic pathways were pursued to yield two EFV analogues with a spacer arm placed on different position of EFV molecule and with a suitable free amine group for immobilizing on a SPR golden chip.

! This work contributes to design artificial peptidic receptors for small molecules and to advance several methodologies to define their interaction and the structure of ligand-peptide complex. Our peptides developed toward the highly-used HIV-drug EFV are worthwhile in biosensor devices. Prospectively electrochemical assays with the albumin-derived fragment and solid-phase extraction cartridges, exploiting our short peptides incorporated within imprinted nanoparticles, may be applied for therapeutic drug monitoring of EFV in patient plasma.

Up to date, the results have been published in the following articles:

Pavan S, Berti F (2012) Short peptides as biosensor transducers. Anal Bioanal Chem 402(10):

3055-3070

Hong R, Pavan S, Benedetti F, Tossi A, Savoini A, Berti F, Laio A (2012) Designing Short Peptides with High Affinity for Organic Molecules: A Combined Docking, Molecular Dynamics, And Monte Carlo Approach. J Chem Theor Comput 8:1121-1128

Luisi I, Pavan S, Fontanive G, Tossi A, Benedetti F, Savoini A, Maurizio E, Sgarra R, Sblattero D, Berti F (2013) An albumin-derived Peptide scaffold capable of binding and catalysis. PLoS One 8(2):e56469

Further papers will be submitted in a short time.

Abstract

vi

Università di Trieste

Peptidi: Elementi Attivi nel Riconoscimento Molecolare

Tesi di dottorato

Dottoranda: Silvia Pavan Relatore: Dr. Federico Berti

Riassunto

! Nella cura di persone affette da AIDS, l"attuale terapia di prima scelta è quella antiretrovirale (ART) che prevede l"assunzione giornaliera combinata di tre o più farmaci anti-HIV, appartenenti ad almeno due diverse classi per sopprimere la replicazione del virus. Efavirenz (EFV) è un potente inibitore non nucleosidico della trascrittasi inversa del virus HIV-1. É usato da oltre un decennio come componente essenziale della terapia antiretrovirale in quanto possiede un tempo di emivita sufficientemente elevato da permetterne un"unica somministrazione giornaliera.

L"alta variabilità della concentrazione plasmatica di EFV nel singolo paziente, e da paziente a paziente, rappresenta un grave problema clinico. Una rapida misura dei suoi parametri farmacocinetici in ciascun paziente migliorerebbe la collaborazione, l"efficacia della terapia e ridurrebbe la comparsa di ceppi virali resistenti¹.

! L"obiettivo di questo lavoro è la progettazione e lo sviluppo di nuovi recettori peptidici artificiali per EFV da poter utilizzare come elementi attivi nel riconoscimento molecolare in sistemi biosensoristici per il dosaggio di farmaci.

! É stato applicato un innovativo strumento computazionale per “disegnare” piccoli peptidi (lunghi dai 5 ai 15 amminoacidi) che presentino alta affinità per una piccola molecola target. A partire da una poli-alanina, questo nuovo algoritmo ha selezionato un decapeptide (FFWPPFLNVW) con una teorica energia di legame di #12.1 kcal/mol da validare sperimentalmente². Il peptide è stato sintetizzato su fase solida e la sua struttura è stata studiata con tecniche NMR. L"assegnazione ordinata degli spostamenti chimici dei protoni è stata effettuata con esperimenti NMR ¹H,¹H TOCSY e ¹H,¹H NOESY. I dati di dicroismo circolare e il calcolo delle constanti di accoppiamento JH!-NH concordano nell"attribuire al decapeptide una struttura secondaria di tipo $-turn. Le misure di quenching della fluorescenza hanno fornito una costante di dissociazione ligando-proteina pari a KD = 64 nM % #14.7 kcal/mol per la dissociazione del complesso EFV-peptide, concordando con il valore teorico ottenuto con l"algoritmo. Esperimenti NMR ¹H,¹H NOESY e la variazione degli spostamenti chimici dei protoni, a seguito di una titolazione di EFV ad una soluzione di peptide, hanno confermato i contatti intermolecolari già descritti dal modello teorico.

! Con lo scopo di creare sensori biomimetici, questo peptide è stato incorporato in polimeri ad imprinting molecolare in modo da aumentarne la solubilità in acqua e la selettività per EFV. Un set di nanoparticelle con combinazioni di diversi monomeri funzionali peptidici sono attualmente in fase di studio tramite varie tecniche (UV, HPLC e ITC) per determinare la loro capacità di catturare EFV e la loro selettività per il nostro target.

! Un altro approccio per sviluppare recettori peptidi per EFV ha coinvolto la riduzione della sequenza di un recettore naturale di questa molecola. Abbiamo identificato un polipeptide di 101

amminoacidi, che deriva dalla sequenza nell!intorno del sito di legame IIA dell!albumina umana.

Grazie all!ottimizzazione dei codoni, il peptide è stato espresso, con elevate rese, in fusione con la proteina GST in E. coli. Questa proteina di fusione ha dimostrato di mantenere la sua integrità strutturale, la sua attività catalitica, la sua abilità di controllare la sterochimica della riduzione di dichetoni, e la capacità di legare warfarin ed EFV³. É da notare che questo polipeptide, clonato per la prima volta, rappresenta un prezioso punto di partenza per la costruzione di librerie di binders e catalizzatori, che potrebbero presentare miglioramenti nelle sue proprietà. Sono state progettate tre librerie di cloni da generare tramite mutagenesi sito specifica ed è stato ottimizzato un screening veloce dei cloni tramite esperimenti SPR. Sono state seguite nuove vie sintetiche per ottenere due analoghi di EFV con uno spaziatore posizionato in diversi punti della molecola EFV, e con un!ammina libera adatta per immobilizzare i due nuovi derivati di EFV su un chip d!oro di SPR.

" Questo lavoro ha contribuito alla progettazione di recettori artificiali per piccole molecole ed all!ottimizzazione di varie tecniche per definire la loro interazione e la struttura del complesso ligando-peptide. I nostri peptidi sviluppati verso il farmaco EFV, ampiamente usato in terapia anti- HIV, sono adatti a dispositivi biosensoristici. In futuro, misure elettrochimiche con il frammento di albumina o cartucce d!estrazione in fase solida, sfruttando i piccoli peptidi incorporati in nanopolimeri ad imprinting molecolare, potrebbero esser utilizzati per il dosaggio del farmaco EFV nel plasma dei pazienti.

Finora, i risultati sono stati pubblicati nei seguenti articoli:

Pavan S, Berti F (2012) Short peptides as biosensor transducers. Anal Bioanal Chem 402(10):

3055-3070

Hong R, Pavan S, Benedetti F, Tossi A, Savoini A, Berti F, Laio A (2012) Designing Short Peptides with High Affinity for Organic Molecules: A Combined Docking, Molecular Dynamics, And Monte Carlo Approach. J Chem Theor Comput 8:1121-1128

Luisi I, Pavan S, Fontanive G, Tossi A, Benedetti F, Savoini A, Maurizio E, Sgarra R, Sblattero D, Berti F (2013) An albumin-derived Peptide scaffold capable of binding and catalysis. PLoS One 8(2):e56469

Ulteriori articoli saranno inviati a breve.

"

Abstract

viii

1 Maggiolo F (2009) Efavirenz: a decade of clinical experience in the treatment of HIV. J Antimicrob Chemother 64:910-928

2 Hong R, Pavan S, Benedetti F, Tossi A, Savoini A, Berti F, Laio A (2012) Designing Short Peptides with High Affinity for Organic Molecules: A Combined Docking, Molecular Dynamics, And Monte Carlo Approach. J Chem Theor Comput 8:1121-1128

3 Luisi I, Pavan S, Fontanive G, Tossi A, Benedetti F, Savoini A, Maurizio E, Sgarra R, Sblattero D, Berti F (2013) An albumin-derived Peptide scaffold capable of binding and catalysis. PLoS One 8(2):e56469

List of Abbreviations

Å ! Ångström

AcCN! acetonitrile

ACCP! activatable cell-penetrating peptide

AFM! atomic force microscopy

AIBN! 2,2"-azobisisobutyronitrile

AMP! antimicrobial peptide

ART! antiretroviral therapy

Asn! asparagine

Boc! tert-butoxycarbonyl

bm ! backbone monomer

BMPs! bacterial magnetic particles

bp! base pair

br ! broad signal

BSA! bovine serum albumin

°C ! centigrade degree

CE! capillary electrophoresis

cl! cross-linker

CM! monomer concentration

CMPF! 3-carboxy-4-methyl-5-propyl-2-furanepropanoic acid

CNS! central nervous system

CPP! cell-penetrating peptide

1H-¹H COSY! Proton-Proton homonuclear Correlation Spectroscopy

d! doublet

DCM ! dichloromethane

dd ! doublet of doublets

DE ! diethyl ether

DIEA ! N,N-diisopropylethylamine

DMF! dimethylformamide

DMSO! dimethyl sulfoxide

dmso-d6! deuterated dimethyl sulfoxide

DODT! 3,6-dioxa-1,8-octanedithiol

dt ! double of triplets

ddt ! doublet of doublet of triplets

EDC! 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide

EDC-Cl ! N-(3-Dimethylaminopropyl)-N"-ethylcarbodiimide hydrochloride

EFV! efavirenz

ELISA! enzyme-linked immunosorbent assay

ESI! electrospray ionization

F! phenylalanine

Fc! ferrocene

fm! functional monomer

Fmoc! 9-fluorenylmethyloxycabonyl

FRET! Förster resonance energy transfer

G! glycine

gCOSY! gradient COrrelation SpectroscopY

gHSQCAD! gradient Heteronuclear Single-Quantum with ADiabatic

Gly! glycine

GST! glutathione S-transferase

HIV! human immunodeficiency virus

HOBt ! 1-hydroxybenzotriazole hydrate HPLC! high-performance liquid chromatography

HPLC-UV! high performance liquid chromatography with ultraviolet detection

HSA! human serum albumin

Hz! hertz

I! initiator

IR! infrared

ITC! Isothermal Titration Calorimetry

°K! Kelvin degree

L! leucine

Leu! leucine

LC/MS! liquid chromatography with mass spectrometry detection LC-MS/MS! liquid chromatography with tandem mass spectrometry detection

LFM! AFM-based lateral force microscopy

LOD! limit of detection

MS! mass spectrometry

N ! asparagine

NHS! N-hydroxysuccinimide

NMM ! N-methylmorpholine

m ! multiplet

MALDI-TOF-MS! matrix-assisted laser desorption/ionization time-of-flight mass

! spectrometry

MHz ! megahertz

MIP! molecularly imprinted polymer

mp ! melting point

MPA! mercaptoproprionic acid

MS ! mass spectrometry

NMP ! N-methyl-2-pyrrolidone

NMR! nuclear magnetic resonance

NNRTI! non-nucleoside reverse transcriptase inhibitor

1H-¹H NOESY! Nuclear Overhauser Effect Spectroscopy

P! proline

PCR! polymerase chain reaction

PE! petroleum ether

Phe! phenylalanine

PNA! peptide nucleic acid

PPFs! plasma polymerized films

ppm! parts per million

Pro! proline

PyBop ! benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate List of Abbreviations

x

q ! quartet

qt ! quartet of triplets

QCM! quartz crystal microbalance

QD! quantum dot

QDs-DDTC ! quantum dots with dopamine dithiocarbamate

Rf ! retardation factor

RP-HPLC! reverse phase high-performance liquid chromatography

rpm! revolutions per minute

rt ! room temperature

RT! reverse transcriptase

s! singlet

SAM! self-assembled monolayer

SAR ! structure–activity relationship

SELEX! Systematic Evolution of Ligands by EXponential enrichment SNAP-25! synaptosomal-associated protein 25

SPE! solid-phase extraction

SPR! surface plasmon resonance

SPRI! surface plasmon resonance imaging

t! triplet

TAT! transactivator of transcription

TFA ! trifluoroacetic acid

TMEDA ! tetramethylethylenediamine

THF ! tetrahydrofuran

TIPS! triisopropylsilane

TLC ! thin layer chromatography

1H-¹H TOCSY! Total Correlation Spectroscopy

TNT! 2,4,6-trinitrotoluene

Trp! tryptophan

Tyr! tyrosine

UV! ultraviolet

V! valine

Val! valine

Y! tyrosine

W! tryptophan

1.1 Drug Monitoring of Efavirenz

1.1.1 Efavirenz

! Current treatments for human immunodeficiency virus (HIV) infection are designed to interfere with the viral replication by inhibiting either HIV protease or HIV reverse transcriptase (RT). Antiretroviral therapy (ART) is the recommended treatment of people infected with HIV and involves taking a combination of three or more anti-HIV medications from at least two different drug classes every day to suppress HIV replication. ART is used in order to reduce the likelihood of the virus developing resistance and has prolonged the survival of HIV-infected individuals and to improve their quality of life¹.

! Efavirenz (EFV), marketed by Bristol-Myers Squibb with the brand name Sustiva®, is one of the FDA-approved drugs used in the treatment of patients infected with HIV. EFV is a potent and selective non-nucleoside inhibitor of the HIV-1 reverse transcriptase, used for over a decade as an essential component of antiretroviral therapy². Efavirenz is currently used worldwide in first-line regimens, coformulated with emtricitabine and tenofovir, due to the convenience of a single pill taken once daily and resultantly high treatment adherence³.

! Peak EFV plasma concentrations are reached by 5 h following single oral doses. The time to peak plasma concentrations is !3-5 h and steady-state plasma concentrations of EFV are reached in 6-7 days. The bioavailability of a single 600 mg dose of EFV hard capsules in is increased by 17%-22% by food⁴.

EFV is highly bound (!99.5%-99.75%) to human plasma proteins, predominantly albumin.

! EFV is extensively metabolized in humans by CYP2B6 to inactive hydroxy metabolites, 8-hydroxyefavirenz, which is subsequently hydroxylated to 8,14-di-hydroxyefavirenz by the same enzyme².These metabolites are excreted predominantly in urine as glucuronides and to some extent as sulphate conjugates⁵. The elimination half-life of EFV after single oral doses is 55–76 h⁴. EFV is a well-known victim and perpetrator of drug– drug interactions and induces its own metabolism and clearance⁶. Central nervous system side effects (including dizziness, insomnia, impaired concentration, somnolence, and abnormal dreams) occur commonly and a relatively low genetic barrier to HIV-1 resistance is reported^{2, 4}.

! Although HIV infection is generally well controlled by EFV-containing regimens, a number of patients develop resistance to the drug. In these patients, the K103N mutation is present in over 90% of the RT sequences examined⁷. In an e!ort to develop new generation drug with improved

activity against K103N and other resistant mutants, SAR (structure–activity relationship) investigations were launched to describe the e!ect of varying the aromatic substitution pattern of EFV, of modifying and replacing the acetylenic side chain⁸.

! The emergence of EFV-resistant HIV mutants could be a result of repeated exposure to ineffective of sub-therapeutic drug level due to the high intra- and inter-patient variability in plasma EFV concentrations⁹. Moreover, 20-40% of patients receiving EFV reported central nervous system side effects that are associated high EFV plasma levels. A plasma therapeutic range of 1–4

"g/mL has been established for EFV¹⁰ and its pharmacokinetics vary between individuals and populations causing variation in drug concentrations¹¹. Factors reported to be associated with interpatient variability in EFV concentration include gender, ethnicity¹² and genetic polymorphisms, while autoinduction¹³ and adherence may contribute to both inter- and intrapatient variability¹⁴.

! Furthermore EFV is one of the anti HIV drugs with poor aqueous solubility (4 µg/ml)¹⁵. The low solubility of the EFV in aqueous medium hinders the absorption and biodistribution of the drug from the gastrointestinal tract¹⁶. The oral bioavailability of the drug is around 40–45% and the interindividual variability observed demands therapeutic drug monitoring¹⁷. Therefore EFV dosage individualization based on plasma concentration monitoring might improve the patient compliance and clinical outcomes, reducing associated toxicities, potential drug-drug and food-drug interactions and the development of resistance.

1.1.2 Drug monitoring

! The development of rapid, accurate, and portable diagnostic tools is very important for the monitoring of drug concentrations in plasma samples from patients affected by chronic diseases. It should be noticed that there is no cure or established primary prevention measure (such as a vaccine) against HIV; a universal “test-and-treat” strategy has been proposed for HIV infected patients and antiretroviral therapy is essentially required for HIV positive patients¹⁸. The main objective of clinical research is to systematically identify and quantify drug molecules in a given sample. Therefore, it is necessary to detect accurately the concentration of anti-HIV drugs in HIV infected human plasma for evaluation of drug pharmacokinetics in clinical trials with efficacy¹⁹. In recent years, the development of facile and rapid analytical methods for detection and quantification of anti-HIV drugs in human plasma has become a popular subject²⁰.

Drug Monitoring of Efavirenz

4

Fig 1.1.1 Structures of EFV and of its main metabolites.

NH Cl O

F3C

O NH

Cl O F3C

O

Efavirenz

OH

NH Cl O

F3C

O OH

OH

8-Hydroxyefavirenz 8,14-Dihydroxyefavirenz

! Indeed a number of methods have been reported for the analysis of EFV alone in biological fluids, and in combination with other drug including high performance liquid chromatography with ultraviolet detection HPLC-UV21 22 23 24, liquid chromatography with mass spectrometry detection LC-MS²⁵, HPLC with fluorescence detection²⁶ and a capillary electrophoresis method²⁷. More recently, different innovative techniques were combined for therapeutic drug monitoring of EFV, using functionalized quantum dots as matrix in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analyses (MALDI-TOF-MS)²⁸ or liquid chromatography with tandem mass spectrometry detection (LC-MS/MS)²⁹. The detection efforts have been extended to its metabolites³⁰ (LC-MS/MS), to its dosage in pharmaceutical formulations (reverse phase HPLC³¹, ¹⁹F-NMR³²) and to its intracellular quantification (HPLC–UV³³, HPLC–

MS³⁴) that may provide further understanding of therapeutic failure, especially where virological rebound occurs despite adequate plasma levels. The impressive number of these papers confirm the worldwide importance of therapeutic EFV monitoring³⁵. However, all these methods are impractical for commercial use because they suffer from a long sample preparation time, or a long assay time, or a lack of an adequate sensitivity. Some of these methods require expensive equipments, that are not available in most laboratories, or involve laborious liquid-liquid sample extraction procedures that negatively affect the accuracy of the results. Furthermore, these methods, as relied on sequential samples analysis, are not suited for screening of large number of specimens. Immunoassays are preferable in the field of clinical analysis because of their applicability for a wide range of analytes, high-throughput, low cost, convenience for screening of a large number of samples, and their specificity for the analyte of interest even in multi-component complex sample matrix such as plasma³⁶. The antibody is the most important key reagent in the development of any immunoassay system. As well, specificity of the antibody to the analyte of interest is the limiting factor in the validity of the assay and the mode to link the small target molecule or hapten to the larger immunogenic carrier protein results the key step for generating highly sensitive and specific antibodies. Several immunoassay attempts were reported with different types of immunogen conjugates between a protein carrier and EFV derivatives³⁷. However, no immunoassay has been commercially available as portable device for use in clinica to date.

! Accordingly, the development of a new alternative analytical technology for the determination of EFV in plasma with adequate sensitivity, improved simplicity, lower cost, and higher throughput is urgently needed.

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