CFAD V. Anacor, IPR2015-01776 ANACOR EX. 2082 - 1/19
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`CFAD v. Anacor, IPR2015-01776 ANACOR EX. 2082 - 1/19
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`

`
`Vo1_.6.2:.Nr9~.z.3902
`
`Contents
`
`
`Leading Article
`
`Angiotensin Converting Enzyme Gene Insertion,’ Deletion
`Polymorphism and Cardiovascular Disease:
`Therapeutic Implications
`T Niu, X Chen, X X1:
`
`977-993
`
`Therapy in Practice Options for Induction Irnmunosuppression in Liver
`Transplant Recipients
`MA] Maser
`
`
`995-1011
`
`Review Articles
`
`Pharmacoeconomics of Influenza Vaccination for Healthy
`Working Adults: Reviewing the Available Evidence
`M] Postma, P Irmsema, MLL van Grmugteri, M—LA Heijnen, IC lager,
`LTW dc long-van den Berg
`
`1013-1024
`
`Mechanisms of Fungal Resistance: An Overview
`MM Balkis, SD Lefdich, PK Mukherjee, MA Ghrmmmm
`
`
`1025-1040
`
`Adis Drug
`Evaluations
`
`Ceftriaxone: An Update of its Use in the Management of
`Commuruty-Acquired and Nosocomjal Infections
`HM Lamb, D Ormmd, L] Scott, DP Figgitt
`
`Esomeprazolez A Review of its Use in the Management of
`Acid-Related Disorders in the US
`LI Scott, C)’ Dimn, G Mallarkcy, M Sharpe
`
`1041-1039
`
`1091-1118
`
`Madison; WI 53705
`
`Dru '5 is indexed in index Mrdicus, Mcdlfiie. EMBASE/Excerpra Medics, Curreirt Cmrlmts/Cliiiiml Medicine, Current Commits/1.{',F¢’ Sci:-uses,
`BIO§IS"”' Database, Iirferiiatirirml Plmrnmceuricnl Abstracts (IPA) and CABS. Lndividual articles are available through the IADONIS
`document delivery service and on-line via the World Wide Web through lngenta. Further details are available from e publisher:
`
`CFAD V. Anacor, |PR2015-01776 ANACOR EX. 2082 - 2/19
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`CFAD v. Anacor, IPR2015-01776 ANACOR EX. 2082 - 2/19
`
`

`
`A D
`
`A Walters Nlunwr Ctrmpilliy
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`DR. Abernathy. Baltimore, MD. USA
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`J.A. Wile, Birntinglmnr, England
`D.J. Zegorelll. New York. NY. USA
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`CFAD v. Anacor, |PFl2015-01776 ANACOR EX. 2082 - 3/19
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`CFAD v. Anacor, IPR2015-01776 ANACOR EX. 2082 - 3/19
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`

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`This material may be protected by Copyright law (Title 17 U.S. Code)
`
`
`
`REWEW ART|C|-E mi2iizii-EE*3§tr?§.iE.E:J§§?3.L%”33
`.
`.-'\i.Il-. Intorne-tlortvzl Lirr.|n:-<3 All right; .r_~-,9-ii.-gtj
`
`Mechanisms of Fungal Resistance
`
`An Overview
`
`Matter M. Brzllris, Steven D. Leitficlt, Pt‘i:Ii?£1l":‘ K. M tikhcrjee and Malmtotid A. G.-'ttm'tt0ttm
`
`Department of Dermatology, Center for Medical Mycology, University Hospitals Research Institute
`and Case Western Reserve University, Cleveland, Ohio, USA
`
`Contents
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`Abstract
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`1. AntitungolSusceptlbilih/Testing
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`2. Mechanisms otAntifungolActIon end Reslstonce.
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`2.1 AzoIe—Bosed Antifungolfitgenis .
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`2.1.1 Modification of the Ei?G1iGene oi the Molecular Leve-1..
`2.1.2 Drug Etflux .
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`2.3 Allylctmines .
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`2.4 Inhibitors of Fungol Cell Woll Synthesis .
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`2.4.1 Inhibitors of Glucctn Synthesis .
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`2.5 Compounds Affecting Protein Synthesis one DNA Replication .
`2.5.1F|uc~/loslne .
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`3. Clinical implications of Antifungol Resistance .
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`3.1 Aniifungo|Dose Administration .
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`3.2 DevelopmentotNewAntifungct1s
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`3.3 Prevention ono ControlotfitntifungolResistance
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`AbSl'l'ClCl'
`
`The increased use of antifungal agents in recent years has resulted in the
`development of resistance to these drugs. The significant clinical implication of
`resistance has led to heightened interest in the study of anti ft: nga] resistance from
`different angles. In this article we discuss antifungal susceptibility testing. the
`mode of action. of antifungals and mechanisms of resistance.
`Antifungatls are grouped into five groups on the basis of their site of action:
`(_i) &1?_Ulf.‘S. which inhibit the synthesis of ergosterol (the main fungal sterol); [ii]
`polyenes. which bind to fungal membrane sterol. resulting in the formation of
`aqueous pores through which essential cytoplasmic materials leak out; (iii) al~
`lylamines, which block ergosterol biosynthesis. leading to accumulation ofsqua-
`lene (which is toxic to the cells); (iv) candins (inhibitors ofthe fungal cell wall}.
`which function by inhibiting the syntliesis of[3 l.3—glucan {the major structural
`polymer of the cell wall): and iv) flucytosine. wltich inhibits macromolecular
`synthesis.
`Dit'l'crt':nt mechanism:-I contribute to the resistance ofontifungal agents. These
`mechanisnts include tnodifieation of ERGH gene at the molecular level (gene
`mutation. conversion and overexpression). oiterexpression ofspecific drug el'l'lu.\t
`
`CFAD V. Anacor, |PR2015-01776 ANACOR EX. 2082 - 4/19
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`CFAD v. Anacor, IPR2015-01776 ANACOR EX. 2082 - 4/19
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`
`mas
`Bnllris cl nl.
`
`pumps. alteration in stem] biosynthesis. and reduction in the intracellular con-
`centration of target enzymes. Approaches to prevent and control the emergertce
`of antifungal resistance include prudent use of antifungals. treatment with the
`appropriate anti fungal and conducting surveillance studies to determine the fre-
`quency of resistance.
`
`The increased inc.idence of fungal infections.
`particularly in patients with impaired immune
`function, has sumtnoned the need for more effec-
`
`tive antifungals to replace many of the existing
`agents. which are not optimal against emerging
`fungal infections. exhibit host toxicity or have a
`high propensity to induce the development ot' mi-
`crobial resistance. The major contributing factors
`for the development of fungal resistance are con-
`sidered to be the extensive and prolonged use of
`antifungal agents. Forexarnple. resistance to fluco-
`nazole is especially common in patients infected
`with HIV who require long-term, prophylactic
`therapy to prevent a vtuiety of opportunistic fungal
`infections to which they are susceptible. Indeed. in
`one investigation, 33% ofpalients with AIDS were
`found to have fluconazole-resistant strains of Can-
`
`rfirfti ril'bi't-ans in their oral cavities.“' Nonetheless.
`the good safety profile. bioavailability and clinical
`effectiveness of flttconazole has led to its contin-
`
`ued use in patients with cancer and neutropenia.
`and in bone marrow transplant recipients.
`The significant clinical ramifications of anti-
`fungal resistance have led to heightened interest
`and concern. Current researclt efforts in this area
`
`are directed at identifying the molecular mecha-
`nisms responsible for resistance. developing more
`effective drugs and improving methods to detect
`resistance when it occurs. Although the results of
`this work have substantially increased our under-
`standing of fungal resistance. especially at the mo-
`lecular level. more remain to be addressed. The
`
`apparent fact that fungal resistance mechanisms
`will constantly evolve in response to the use ofnew
`drugs highlights the importance of identifying new
`resistance genes. developing safer and more effec-
`tive drugs. and implementing novel strategies to
`detect. treat and prevent infections caused by resis-
`tant fungi.
`
`The past decade has witnessed a significant in-
`crease in the number of pathogenic fungi exhibit-
`ing resistance. to antifungal agents. Such resistance
`has important implications for morbidity. mortal-
`ity and healthcare costs in hospitals. as well as in
`the community at large.
`The study of antifungal resistance has lagged
`behind that of antibacterial resistance for several
`
`reasons. Perhaps most importantly, fungi were not
`considered as important pathogens until relatively
`recently.l3--‘l For example. the annual death rate as
`a result of candidiasis remained constant from
`1950 to about l9’FU. Since 1970. this rate increased
`
`dramatically in conjunction with the frequent and
`often indiscriminate use of broad-spectrurrl anti-
`bacterial agents. the common use ofindwelling in-
`travenous devices and the rise in the number of
`
`immunocompromised individuals as a result of
`advances in cancer treatment and the spread of
`AIDS."” These developments and the associated
`increase in fungal infections'“’' have intensified
`the search for new. more efficacious agents with
`improved safety profiles to combat serious fungal
`infections.
`
`For nearly 30 years, amphotericin B was the
`sole drug available for the treatment of serious fun-
`gal infections. Although amphotericin B exhibits
`superior clinical effectiveness. relative to azole anti-
`fungals in the treatment of systemic candidiasis. its
`narrow therapeutic indent and significant nephro—
`toxicity has limited the overall utility of this drug.
`The approval of the imidazole- and triazole—based
`antifungals in late 1980s and early [9903 was an
`important step that greatly advanced the ability to
`safely and effectively treat local and systemic fun-
`gal infections. The high safety profile of the tri-
`azolcs, particularly tluconazole. led to their exten-
`sive. sonietirnes prophylactic. use. Indeed. since its
`launch. fluconazole has been used to treat in excess
`
`ra Ar_1|-5 Interncltlonal Limited. All algl-its reserved
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`Drugs 2002: C32 UP
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`CFAD v. Anacor, |PFt2015-01776 ANACOR EX. 2082 - 5/19
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`CFAD v. Anacor, IPR2015-01776 ANACOR EX. 2082 - 5/19
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`

`
`[U27
`Mechanisms of Fungal Resistance
`
`of 16 million patients, including over 300 000 pa-
`tients with AIDS in the US alone.'7'9l As expected.
`the extensive use offluconazole has resulted in the
`
`development of resistance. particularly in this
`AIDS population as described in section 3.
`Impressive strides have been made in elucidat-
`ing the molecular basis of antifungal drug resis-
`tance, especially in the last 5 years. This review
`provides an update on antifungal resistance mech-
`anisms with brief comments on clinical relevance.
`
`The aim is to provide an understanding of fungal
`resistance mechanisms that is accessible to clini-
`
`cians who prescribe antifungal drugs. and mem-
`bers of the scientific community who may wish to
`study them in the future.
`
`1. Antitungcil Susceptibility Testing
`
`Initially. the concept of fungal minimum inhib-
`itory concentration (MIC) testing was irrelevant
`because no alternative to amphotericin B existed.
`With the introduction of flucytosine (5-FC) in the
`1970s and the azoles iii the 19805. the concept of
`MIC testing became timely as an aid to selecting
`the most appropriate drug. At the beginning of the
`files! century, the growing significance of fungal
`disease.
`the expanding availability of antit'ungal
`drugs and the development of fungal resistance.
`make the need for relevant MIC data urgent.
`In 1983. the National Committee for Clinical
`
`Laboratory Standards {NCCLS} established a sub-
`commirte.e to standardise fungal MIC determina-
`tion. Rex et al.'"’l summarise the complexity of
`such standardisation by pointing out that varia-
`tions in inoculum size and preparation, incubation
`time and temperature. media. and endpoint deter-
`mination can cause MIC determinations to vary
`more than 50 {Jill}-fold. Multiple groups of re-
`searchers worldwide were challenged to agree on
`
`standards that would generate reproducible MIC
`data in the range of normal serum drug concentra-
`tions and were sensitive enough to detect organ-
`isms with truly different drug susceptibilities. As
`a result of this work. in 1997 the NCCLS adopted
`the M27 protocol for the susceptibility testing of
`yeasts."“
`Despite its adoption by the NCCLS, M2? end-
`points can be difticult to interpret for some drugs.
`For amphotericin B. there is a sharp transition from
`visible growth to no visible growth at the MIC and
`the endpoint is readily apparent. For the azoles, in
`particular,
`there is a prominent trailing effect.
`which results in growth at all drug concentrations
`regardless of susceptibility. Therefore. determina-
`tjon of the MIC depends on a difficult visual as-
`sessment of50 to 30% reduction in growth relative
`to the drug-free control.
`Furthermore. the relevance. or pharmacody—
`namic correlate. of fungal MIC values is not yet
`established. The concept of an MIC has proven
`useful in guiding antibacterial therapy; however. it
`is more accurate to think of the MIC as a predictor
`of failure rather than ofsuccess. Recovery from an
`infection is dependent on many patient—, drug— and
`organism-related factors. of which the MIC is only
`one.
`
`The ability of MIC values to predict antifungal
`therapeutic failure is far from universal and it is
`critical to remember this when evaluating in wire
`antifungal susceptibility data. NCCLS has estab-
`lished interpretive breakpoints for fluconazole,
`itraconazole and llucytositte (‘table I} by examin-
`ing all pertinent animal and lium-an data. and at-
`tempting to define an MIC above which therapeu-
`tic failure with that drug is likely.1“l It is important
`to emphasise the relatively arbitrary nature of all
`breakpoint determinations and the absence of con-
`
`Table l. Tentative interpretive guidelines for susceptibility testing in vitro of Candida species
`
`Antitungal agent
`Flueonazole
`Itraconazole
`
`Susceptible
`58
`$Ct.125
`
`Susceptible-dose dependent‘
`16-32
`0.25—O.5
`
`Resistant
`264
`21
`
`Flucytosine
`$4
`8-1 5
`215
`
`a
`For tlucytosina. the old is-rm ‘intennedlate susceptibility‘ is used by the NCCLS.
`
`1;‘ Actls international Llmlted. All rights reserved.
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`Drugs IQ: 62 (it)
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`
`Bntkis at at’
`1028
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`trolled. evaluative prospective trials. The existent
`breakpointdata correlate most strongly for oesoph-
`ageal canclidiasis in patients with HIV. and must
`be interpreted cautiously in otherclinical scenarios
`since the prediction of therapeutic efficacy based
`on an MIC is supported by fewer data.‘ '31 Now that
`the interpretive breakpoints for antifungal suscep-
`tibility of Candida spp. are available, the Mycosis
`Study Group has recommended their use in the
`management of patients with eandidaemia. Rou-
`tine antifungal susceptibility testing should not be
`performed and should be reserved for those not
`responding to therapy. and to infections by non-
`albicans species. {eg Cziiiclidri gt.-snliratu}.
`For atnpholericin B. some investigators have
`begun to use an MIC cut-off of<l mgi'L as suscep~
`tible but this is on the basis of one study. the results
`of which have not been reproduced.'”l Indeed. a
`major limitation seems to be a clustering of nearly
`all M27 MIC determinations for amphotericin B
`around 0.5 to l mgi'I_, suggesting that this protocol
`may be relatively insensitive for amphotericin B.
`despite its clear endpoint.“ 3'
`MIC‘. deterrnination and interpretation against
`filamentous fungi such as Aspcrgfl.-‘its’ spp.
`lags
`even further behind that of yeasts. Currently. the
`NCCLS has proposed the M38-P protocol for MIC
`determination against filamentous fungi.l‘5l It
`is
`essentially a variation of the M2’? protocol but has
`not yet been successful in generating clinically
`useful MIC values.l ”'l This delay may be attributed
`to di fficulty establishing reliable endpoints that de-
`termine the MIC. and the low numbers of patients
`from whom it is easy to diagnose infection and to
`culture such organisins.
`The development of the new class of echino-
`candin antifungals raises new issues in susceptibil-
`ity testing. Data is accumulating to show that the
`developed NCCLS methodologies are not suitable
`for measuring the antifungal susceptibility ofthese
`agents. Consequently, several investigators are at-
`tempting to develop alternative assays that may be
`useful for susceptibility testing of fungi to ec|iino-
`candins. Recently our group used a 2.3-Bis (2-
`tnethoxy-4—nitro-5-sulfophenyl"J-5-|tpl1enyl~amino'J
`
`carbonyl]—2H—tetrazo|ium hydroxide [XTTI-based
`assay to evaluate the effects of rnulunducantlin (an
`echinocandin-like compound) on A.rpcrgiHi.i.v
`fiirnigttius in vitro and compared this technique
`with the microdilution assay performed by the
`NCCLS M38-P method. Our data showed that. in
`
`contrast to the NCCLS methodology. which does
`not predict the activity in viva. the XTT-based as-
`say showed that/t.fimiignru.v is susceptible to mul-
`undocandin. This indicates that the XTT-based as-
`
`say might be useful for determination of the
`susceptibilities of moulds to echinocandinl ' 7'l
`
`2. Mechanisms of Antifungal Action
`and Resistance
`
`2.1 Azole-Based Antifungol Agents
`
`The azoles. including the imidazoles lTketocon-
`azole and miconazole) and the triazoles ("fluen-
`
`nazole. itraconazole. voriconazole. posaconazole
`and ravuconazole] function by inhibiting lanosterol
`l4ot~dcmethy|ase. a key. cytochrome P450 (CYP}—
`dependent enzyme in the ergosterol biosynthetic
`pathway that participates in the multistep conver-
`sion of lanosterol to ergosterol {figure 1). Ergos—
`terol is a necessary sterol important for maintain-
`ing the structural
`integrity of the fungal cell
`rnernbrane.{'“l Inhibition of Mot-deinetliylase leads
`to depletion of ergosterol, which leads to the for-
`mation of membranes with altered structure and
`
`function. and accumulation of sterol precursors.
`especially l=l-(1—mt:tl1yl fecostcrol and l40t—mcthyl-
`ergosta—8.24(28}-dien-3l3,6ot—diol. Accumulation
`of the latter diol has been associated with growth
`arrest
`in Snt't‘huroni_\‘ces c'ci‘e*w's'ine and C. at‘-
`bi'cmi.~;.l '94” Although azoles are usually effective
`against different Cat-tditfn species. they tend to be
`less active against the emerging pathogen Candida
`.‘l.Tl'.£S£’i (although the new triazoles. e.g. voricon-
`azole. have potent activity against this species).
`Mammalian cholesterol biosynthesis is also af-
`fected by azoles at the stage of l4ot-demethylation:
`however.
`the dose required to produce the same
`degree of inhibition is much higher than that re-
`quired for l"ungi.'33‘“| Human stcro! biosynlltesis
`
`u‘. Actls International Ltmlled All rights reserved
`
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`
`I ll'.".‘.l
`Mcchanisnis of Fungal Resistance
`
`
`l
`
`’ terbinaline
`
`4.14-Dirnnthylzynioaterol
`lluconazole
`itraconazole
`vorieonazole
`
`itraconazole
`voricoriazole
`
` flueonazole
`
`
`
`
`
`al..m' using biochemical and molecular tech-
`niques showed that a dramatic decrease in fluco—
`nazole susceptibility occurred in isolate l3.l2“l
`Comparison of the DNA sequences of ERG.-‘l ho—
`mologs from this azo|e~i'csistant isolate and a sen-
`sitive C. nlbic-or:.r strain revealed a point mutation
`(R4671-C} in the resistant isolate that results in the
`
`
`
`replacement of arginine (R) for lysine (K) at posi-
`tion 467.l29l Since this mutation is in close proxim-
`ity to a cysteine that participates in the coordina—
`tion of the iron atom in the heme cofactor of the
`
`
`
`Fig. ‘I. Ergosterol biosynthetic pathway in tungi.l5l
`
`is most prominently effected by ketoconazole.
`Buttke and Chapmanlfil showed that ketoconazole
`inhibited the incorporation of "‘C acetate into cho-
`lesterol. with a resultant accumulation of “C
`
`this inhibition was af-
`lanosterol. Importantly.
`fected by drug concentrations obtained therapeuti-
`cally. Another study reported that ketoconazole
`specifically inhibited the intracellular transport of
`low-density Iipoprotein cholesterol. In addition.
`ketoconazole also had a general effect on choles-
`terol movement.l~"’l The potent ability of ketocon-
`azolc to inhibit mammalian cholesterol could. in
`
`part, explain the high toxicity of this azole. which
`limits its clinical utility.
`
`2. I. I Modification of the EEG] 1 Gene or
`the Molecular level
`
`The gene encoding l-4-oz-demethylase is cur-
`rently referred to as ERG}! in all fungi from which
`it has been cloned. Mutation, gene conversion and
`overespression of ERG}.-' have been investigated
`to determine if these genetic modifications can
`contribute to the development of antifungal resis-
`lance.
`
`Analysis of a series of C. nlbicnns clinical iso-
`lates (17 strains obtained from the same patient
`over a 2-year period) described by Redding et
`
`en2yn1e.l-“'1 it has been suggested that this mutation
`causes structural or functional changes associated
`with the herne.l3'l Preliminary studies indicate that
`R46'r'K by itself can confer azole resistance by re—
`ducing the affinity of the enzyme for flucon-
`azole.'-"El
`
`A similar point mutation T3l5A. in which thre-
`onine (T) was replaced with alanine. {A} at position
`3 l 5. was generated in the ERGH gene from a lab-
`oratory strain of C.
`t'i'ffJt('(£Fi‘.S’.L33l This particular
`mutation was selected on the basis of the architec-
`
`ture of the active site of the enzyme. which is po-
`sitioned directly above the heme -:ofactor.l ml Stud-
`ies of this mutated version of the C‘.
`iilhi'i;-rms
`
`ERGH gene in the genetically tractable yeast Stic-
`t‘litu‘oin_\-‘t‘c.i‘ C6’-l‘t’1-'l.§‘l‘t1'£’ showed that its altered en-
`zyme product was less active and had a reduced
`affinity for fluconazole.
`Other investigators have reported additional
`mutations that play a role in azolc resistance. Re-
`cently Edlind et a|.l-“*1 cloned and sequenced the A.
`fiiiiiigarus CYP sterol
`l40t-demcthylase {C}’P5l J
`gene and reported that the resistance ofthis organ-
`ism to azoles may be dtie to mutation of He 30!
`residue, which corresponds to C. o!'hr'cnn.r Thr 315
`residue, to an Ala residue in resistant strains.
`Existing exclusively as a diploid organism. C‘.
`olbicans harbours two copies ofeach gene. Allelic
`differences between copies of a gene are common
`among clinical C. r1t'i‘Jr‘.cnn.s isolates. The ability of
`C. olbicnns to preferentially replace one allele of
`a gene for another may contribute to or enhance
`azole resistance. Indeed. analysis of ERGH in re-
`sistant isolates showed that all allelic clifferences
`
`at Adts lnramotioriol Limited. All rights reserved.
`
`Drugs 2002: fl (F)
`
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`

`
`103-U
`
`Iinilris ct nl.
`
`present in sensitive strains were absent and that
`both copies contained the R-16?l( rnutation.[3‘-‘I Re-
`sistant strains harbouring two copies of ERG}!
`containing the R46'i'K mutation are more resistant
`to azoles compared with strains with a single mu-
`tated allele.‘-“l Since portions of the gene encoding
`homoserine kinase (THRIJ, which is positioned
`immediately downstream of ERGH. also lacked
`allelic variation, this suggests that the gene conver-
`sion event resulted in the loss ofallelic variation at
`the ERG}! locus.
`
`Overexpression of ERG.-'1 has been docu-
`mented in clittical isolates of C. glabrara and C.
`albirrrtiis.l3“~35l In C. tttbit-mi.r isolates exhibiting
`this phenotype. the level of ERGU expression was
`only increased 5-ft1ld.'33~3"33l Furtherrnore. other
`resistance mechanisms. such as the R4-6'.r'K muta-
`
`tion and overexpression of genes encoding drug
`efflux pumps {see section 2.1.3) are invariably
`present in strains that overe.~:p1‘css ERG.-‘I. Thus.
`the contribution of ERGH overexpression to azole
`resistance is not clear. The scarcity of clinical iso-
`lates in which overexpression ol’ERGH has been
`observed. together with the finding that other re-
`sistance mechanisms may be operative in the same
`strain, suggests that overexpression of ERGH
`plays only a limited role in clinical resistance to
`azoles. Azole-resistant C. ot'ht'cans and Cr_vptococ-
`(‘l'.t.5' neofortimns clinical isolates may also originate
`as a result of mutation to other genes in the ergos-
`terol biosynthetic pathway. namely ERG}? and
`ERG.i.l3"’3"‘37l These genes encode enzymes [C-8
`sterol isomerase and C-5 sterol desaturase. respec-
`tively) that function downstream of ERG! I. As de-
`scribed in section 2.1. inhibition of 140-demcthy|—
`ase results in the accumulation of the diol
`that
`
`arrests fungal growth. C. olbiazvnts erg3 strains that
`continue to grow in the presence of l4ot—¢.leme1'hyl-
`ase inhibitors have been shown to do so by block-
`ing the synthesis of the toxic tliol. presumably by
`the activity of the defective C‘-5 sterol desatur—
`ase.-.13"
`
`2. L2 Drug Efflux
`Evidence implicating drug efflux as a mecha-
`nism of resistance in Cnitdida species continues to
`
`mount. This mechanism is believed to be the prom-
`inent mechanism responsible for the resistance
`phenotype observed in clinical isolates. Parkinson
`et al.[-‘El compared pre-treatment (azole-susceptible}
`and post—treatment (azole-resistant) isolates of C.
`globmrtrt and showed that the post-treatment" iso-
`late accumulated less fluconazoie than the suscep-
`tible one. The reduced ability ofthe resistant strain
`to accumulate fluconazole was a consequence of
`energy—depende-nt drug efflux.l-“ii In an extension
`of these studies. Hitchcock and coworkers exam-
`ined Lbe mechanism of resistance to azoles in C.
`
`rtlbicatts. C. gl.'rtLu'.:tta and C. krttsei using the fluo-
`rescent dye rhodamine 1'23 ('Rh1?.3). which is
`known to be transported by a number of organisms
`displaying multi-drug resistance {MDR).’3"l Their
`results showed that azole-resistant strains accumu-
`
`lated less Rh.l23 than did azole-susceptible strains.
`In C‘. glnbrrtta. a single efflux pump appears to be
`capable of exporting both Rh]:-13 and iluconazole.
`since accumulation ofthese drugs in this fungus is
`competitive. By contrast. no competition is ob-
`served in C. albicans. suggesting that separate
`pumps are used for each drug.”‘” Drug efflux has
`recently been observed in a laboratory derived C.
`neo_fortnans strain resistant to azoles and polyenes
`and in aA.fmm‘gnrns clinical isolate exhibiting re-
`sistance to itraconazole.l*"-‘*'l
`
`two types of efflux
`least
`Fungi possess at
`pumps; those belonging to the ATP-binding cas-
`sette {ABCJ and the major facilitator IMF] super-
`families of proteins. ABC proteins contain four do-
`tttains. two domains that span the membrane. and
`as their name indicates. two nucleotide binding do-
`mains (NBD} specific for ATFJ3” The only excep-
`tion that has been observed is in members of the
`
`YEF3 subfamily, which lack membrane-spanning
`domainsfl-"J The MF efflux pumps are associated
`with fluconazole resistance.l3'l
`
`The avai Iahility of the complete sequence of the
`genome ot'S. cerevisioe has allowed the number of
`candidate ABC and MF genes to be estimated in
`this model yeast, Thirty genes that encode proteins
`with ABCS and 28 genes that encode. MF eftlux
`pumps were identitied.'“l The 30 candidate ABC
`
`-6- Actls Internotton cal tlrnited All rights rerservect.
`
`D1ugs2D|]E.'oi‘ C-"'t
`
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`

`
`1031
`Mechanisms of Fungal Rcsistartce
`.
`
`genes were grouped into six subfatnilies (PDR5.
`ALDP. CFTRIMRP. MDR, YEF3. and RH) on the
`
`that of the Cdrlp. CDR2 is associated with resis-
`tance to azoles, terbinatine and amorolfine. The
`
`basis of phylogenetic analyses. However. only the
`PDR5. CFTRIMRP and MDR subfamilies contain
`
`concomitant overexpression of CDRI and CDR2
`has been documented in two azote-resistant. clini-
`
`genes that encode proteins known to confer azole
`resistance.l"“l
`
`cal isolates compared to matched sensitive strains.
`However. in one of the resistant strains the level
`
`Efflux pumps belonging to the ABC superfarn-
`ily in C. olbic.:ms' and more recently C. glribrornl“-‘l
`and Aspergr'h'tts nidm'nrt.r"l‘” continue to be identi-
`lied.l“3“‘5l However. the most notable ABC efflux
`
`pumps in Crmdidri spp. are encoded by members
`of the PDR5 subfamily. These ge.nes have been
`named CDR for Cniidfdci drug resistance. and are
`
`the only ones characterised thus far that are asso-
`ciated with azole resistance. The results of several
`
`molecular and genetic studies indicate that at least
`[0 CDR genes exist in the C. rtlbr‘criii.r genome.l-“'3-"5'
`The firs