Selection Of Resistant Mutants Research Articles

Identification of Elongation Factor G as the Conserved Cellular Target of Argyrin B

Argyrins, produced by myxobacteria and actinomycetes, are cyclic octapeptides with antibacterial and antitumor activity. Here, we identify elongation factor G (EF-G) as the cellular target of argyrin B in bacteria, via resistant mutant selection and whole genome sequencing, biophysical binding studies and crystallography. Argyrin B binds a novel allosteric pocket in EF-G, distinct from the known EF-G inhibitor antibiotic fusidic acid, revealing a new mode of protein synthesis inhibition. In eukaryotic cells, argyrin B was found to target mitochondrial elongation factor G1 (EF-G1), the closest homologue of bacterial EF-G. By blocking mitochondrial translation, argyrin B depletes electron transport components and inhibits the growth of yeast and tumor cells. Further supporting direct inhibition of EF-G1, expression of an argyrin B-binding deficient EF-G1 L693Q variant partially rescued argyrin B-sensitivity in tumor cells. In summary, we show that argyrin B is an antibacterial and cytotoxic agent that inhibits the evolutionarily conserved target EF-G, blocking protein synthesis in bacteria and mitochondrial translation in yeast and mammalian cells.

Diversity of Individual Dynamic Patterns of Emergence of Resistance to Quinolones in Escherichia coli From the Fecal Flora of Healthy Volunteers Exposed to Ciprofloxacin

Emergence of quinolone-resistant Escherichia coli (QREC) is an increasing clinical challenge mostly originating in fecal microbiota. The dynamics of the emergence of QREC in feces from individuals exposed to ciprofloxacin is unknown. A total of 48 healthy volunteers received oral ciprofloxacin for 14 days. Fecal specimens were collected on days 0, 8, 14, and 42. Subpopulations of QREC were detected on selective agar, genetically characterized, and compared with quinolone-susceptible E. coli (QSEC) strains collected on different days. On day 42, 34 subjects carried QSEC, and 14 carried QREC. Of the 14 who carried QREC, 9 carried quinolone-susceptible E. coli on day 0, 1 carried E. coli with a lower level of quinolone resistance on day 0, and 4 carried E. coli with similar levels of resistance and RAPD-genotypes on days 0 and 42. No plasmid acquisition and no selection of resistant mutants from the initial microbiota was evidenced in any case. In QREC emerging under ciprofloxacin pressure in the fecal microbiota, no proof of selection of quinolone-resistant mutants from the initial microbiota was evidenced, suggesting that QREC strains on day 42 were either present at undetectable levels in the initial microbiota or that exogenous acquisition of QREC strains occurred. Clinical Trials Registration. NCT00190151.

Activity of delafloxacin against methicillin-resistant Staphylococcus aureus: resistance selection and characterization

To determine the potential for delafloxacin to select for resistant mutants in methicillin-resistant Staphylococcus aureus (MRSA), including isolates with existing mutations in the quinolone resistance determining region (QRDR). Susceptibility testing by broth microdilution was performed on 30 MRSA clinical isolates. For four of these isolates, the presence or absence of mutations in the QRDR was characterized. Resistance selection was performed on these four isolates by spreading cells on drug-containing agar plates followed by incubation for 48 h. Resistance frequencies and mutant prevention concentrations (MPCs) were calculated for each; PCR amplification and sequencing were performed using standard methods to characterize mutations in the QRDR. Growth rate analysis was performed and relative fitness was determined. Delafloxacin demonstrated potent in vitro activity against this set of MRSA isolates, with MICs of 0.008-1 mg/L and an MIC(50) and MIC(90) of 0.03 and 0.5 mg/L, respectively. Spontaneous delafloxacin resistance frequencies for the MRSA strains were 2 × 10(-9) to <9.5 × 10(-11). Delafloxacin MPCs were one to four times the MIC for any isolate, lower than those of comparator quinolones. Some delafloxacin-selected mutants showed a fitness cost when co-cultured with the parent strain. Delafloxacin demonstrates excellent antibacterial potency and exhibits a low probability for the selection of resistant mutants in MRSA. Although mutants can be selected at low frequencies in vitro from quinolone-resistant isolates, delafloxacin MICs and MPCs remain low and a fitness cost can be observed. Consequently delafloxacin warrants further investigation for the potential treatment of drug-resistant MRSA infections.

The role of resistance in HCV treatment

The recent development of small molecule compounds that directly inhibit the viral life cycle represents a major milestone for the treatment of chronic hepatitis C virus (HCV) infection. These new drugs that are collectively termed direct-acting antivirals (DAA) include a range of inhibitors of the non-structural (NS) 3/4A protease, NS5B polymerase and NS5A protein. Two NS3/4A protease inhibitors (boceprevir and telaprevir) in combination with pegylated interferon and ribavirin have now been approved for the treatment of chronic HCV genotype 1 infection and cure rates could be increased by 20–30%. However, the majority of DAAs is still in early clinical development. The rapid replication rate of HCV, along with the error-prone polymerase activity leads to a high genetic diversity among HCV virions that includes mutants with reduced susceptibility to DAA-therapy. These resistance-associated variants often occur at very low frequencies. However, during DAA-based treatment, rapid selection of resistance mutations may occur, eventually leading to viral break-through. A number of variants with different levels of resistance have been described in vitro and in vivo for virtually all DAAs. We review the parameters that determine DAA resistance as well as the clinical implications of resistance testing. In addition, the most recent literature and conference data on resistance profiles of DAAs in clinical development and future strategies to avoid the emergence of viral resistance are also discussed.

Risk factors for raltegravir resistance development in clinical practice

To investigate the best conditions of raltegravir use to avoid the selection of resistance mutations in the three main genetic pathways: 148, 155 and 143. A total of 161 patients failing on raltegravir with two consecutive HIV-1 viral loads >20 copies/mL were studied. Ten parameters [HIV-1 RNA and CD4 at baseline and failure, genotypic sensitivity score (GSS) of treatment associated with raltegravir, protease inhibitors used, time spent on raltegravir, subtype, sex and age] were tested in univariate and multivariate logistic regression analyses and compared with the emergence of resistance mutations to raltegravir at failure. Phenotypic susceptibility to raltegravir was studied in 16 patients without the main resistance mutations to raltegravir at failure. At raltegravir failure, 46/161 patients (28.6%) had integrase resistance mutations, whereas 115/161 (71.4%) had no resistance mutations. High HIV-1 viral load level at failure (OR = 2.81, 95% CI 1.8-4.6, P < 0.001) and low GSS of treatment associated with raltegravir (OR = 11.6, 95% CI 4.5-36.4, P < 0.001) were independently associated with the selection of raltegravir mutations. The percentages of patients with integrase resistance mutations were 7.7% (6/78) versus 48.1% (40/83) when viral load is ≤200 or >200 copies/mL and 47.5% (39/82) versus 8.9% (7/79) when GSS is <2 or ≥2. Among patients without main resistance mutations, two patients showed raltegravir phenotypic resistance, one naturally with F121Y at baseline and the other acquiring G118R at failure. Our results show that to avoid the selection of raltegravir resistance mutations, patients have to be treated with at least two active drugs in combination with raltegravir and to maintain a viral load ≤200 copies/mL.

Harnessing the power of human tumor-derived cell lines for the rational design of cancer therapies

In 2002, a mutated constitutively active form of the proto-oncogene BRAF kinase (BRAFV600E) was discovered in ~50% of patients with melanoma. Just last year, the FDA approved an inhibitor and a companion genetic test to treat metastatic melanomas that are positive for this mutation. The rational selection of drugs based on specific genetic variation(s) within a tumor represents the advent of personalized cancer therapeutics. Clearly, this is the way of the future but much work remains to be carried out – a fact that is also illustrated by the BRAFV600E case study. While regression of BRAFV600E tumors in a vast majority of metastatic melanoma patients treated with BRAF inhibitors is encouraging vis-a-vis efficacy of targeted cancer therapeutics, the response is transient and the tumors inevitably recur. In vitro studies have shown that resistance also arises when BRAF inhibitors are combined with therapeutics targeted to other members of the MAP kinase (MAPK) pathway. This can probably be attributed to the selection of resistant mutants from the extensive genetic diversity of tumors within a single patient with metastatic melanoma (Yancovitz et al., 2012). One potential strategy to prevent such recurrence is to hit multiple targets with a cocktail of drugs that are rationally selected to match the complete complement of oncogenic mutations within the heterogeneous tumor cell population. Rational design of such drug cocktails will undoubtedly benefit from understanding of the assorted mechanisms by which resistance develops: additional mutations, epigenetic changes, modification of drug metabolism or clearance, or switching to alternative pathways. However, for a targeted approach to be effective, it will ultimately have to be paired with technologies to comprehensively genotype the entire diversity of mutations within the tumor cell population (Baslan et al., 2012; Navin et al., 2011).

On the rapidity of antibiotic resistance evolution facilitated by a concentration gradient

The rapid emergence of bacterial strains resistant to multiple antibiotics is posing a growing public health risk. The mechanisms underlying the rapid evolution of drug resistance are, however, poorly understood. The heterogeneity of the environments in which bacteria encounter antibiotic drugs could play an important role. E.g., in the highly compartmentalized human body, drug levels can vary substantially between different organs and tissues. It has been proposed that this could facilitate the selection of resistant mutants, and recent experiments support this. To study the role of spatial heterogeneity in the evolution of drug resistance, we present a quantitative model describing an environment subdivided into relatively isolated compartments with various antibiotic concentrations, in which bacteria evolve under the stochastic processes of proliferation, migration, mutation and death. Analytical and numerical results demonstrate that concentration gradients can foster a mode of adaptation that is impossible in uniform environments. It allows resistant mutants to evade competition and circumvent the slow process of fixation by invading compartments with higher drug concentrations, where less resistant strains cannot subsist. The speed of this process increases sharply with the sensitivity of the growth rate to the antibiotic concentration, which we argue to be generic. Comparable adaptation rates in uniform environments would require a high selection coefficient (s>0.1) for each forward mutation. Similar processes can occur if the heterogeneity is more complex than just a linear gradient. The model may also be applicable to other adaptive processes involving environmental heterogeneity and range expansion.

Evaluating the Effect of Early Versus Late ARV Regimen Change if Failure on an Initial Regimen: Results From the AIDS Clinical Trials Group Study A5095

The current goal of initial antiretroviral (ARV) therapy is suppression of plasma human immunodeficiency virus (HIV)-1 RNA levels to below 200 copies per milliliter. A proportion of HIV-infected patients who initiate antiretroviral therapy in clinical practice or antiretroviral clinical trials either fail to suppress HIV-1 RNA or have HIV-1 RNA levels rebound on therapy. Frequently, these patients have sustained CD4 cell counts responses and limited or no clinical symptoms and, therefore, have potentially limited indications for altering therapy which they may be tolerating well despite increased viral replication. On the other hand, increased viral replication on therapy leads to selection of resistance mutations to the antiretroviral agents comprising their therapy and potentially cross-resistance to other agents in the same class decreasing the likelihood of response to subsequent antiretroviral therapy. The optimal time to switch antiretroviral therapy to ensure sustained virologic suppression and prevent clinical events in patients who have rebound in their HIV-1 RNA, yet are stable, is not known. Randomized clinical trials to compare early versus delayed switching have been difficult to design and more difficult to enroll. In some clinical trials, such as the AIDS Clinical Trials Group (ACTG) Study A5095, patients randomized to initial antiretroviral treatment combinations, who fail to suppress HIV-1 RNA or have a rebound of HIV-1 RNA on therapy are allowed to switch from the initial ARV regimen to a new regimen, based on clinician and patient decisions. We delineate a statistical framework to estimate the effect of early versus late regimen change using data from ACTG A5095 in the context of two-stage designs. In causal inference, a large class of doubly robust estimators are derived through semiparametric theory with applications to missing data problems. This class of estimators is motivated through geometric arguments and relies on large samples for good performance. By now, several authors have noted that a doubly robust estimator may be suboptimal when the outcome model is misspecified even if it is semiparametric efficient when the outcome regression model is correctly specified. Through auxiliary variables, two-stage designs, and within the contextual backdrop of our scientific problem and clinical study, we propose improved doubly robust, locally efficient estimators of a population mean and average causal effect for early versus delayed switching to second-line ARV treatment regimens. Our analysis of the ACTG A5095 data further demonstrates how methods that use auxiliary variables can improve over methods that ignore them. Using the methods developed here, we conclude that patients who switch within 8 weeks of virologic failure have better clinical outcomes, on average, than patients who delay switching to a new second-line ARV regimen after failing on the initial regimen. Ordinary statistical methods fail to find such differences. This article has online supplementary material.

Hepatitis B Virus and HIV Infection

Approximately 5 to 10% of human immunodeficiency virus- (HIV-) infected persons worldwide have chronic hepatitis B virus (HBV). The management of these patients merits special attention. They experience a faster progression to cirrhosis and more frequent liver-related death than HBV-monoinfected individuals. For this reason, therapy for both HIV and HBV is a priority in most cases. Some antivirals (i.e., tenofovir, lamivudine, emtricitabine) are active against both viruses and should be part of the antiretroviral treatment choice. However, drugs such as entecavir, telbivudine, or adefovir are active against HBV and may display some residual activity against HIV, occasionally leading to the selection of resistance mutations in the HIV polymerase, as is clearly shown with entecavir. Thus, they should be used only in the context of potent antiretroviral treatment. In this review, the authors will provide updated information on the natural history of HIV/HBV coinfected patients, when and which drugs should be used in treatment, and the concern about selection of drug resistance and vaccine escape mutants.

Comparative mutant prevention concentration and mechanism of resistance to veterinary fluoroquinolones in Staphylococcus pseudintermedius

The problem of antibacterial drug resistance is increasing worldwide, in part due to the therapeutic concentrations currently used based on the minimal inhibitory concentration (MIC) as a measure of potency are often the very concentrations required to selectively enrich the resistant mutant portion of the population. A mutant prevention concentration (MPC)-based dosing strategy is suggested to improve the therapeutic outcome based on the MIC. Our aim was to investigate the MPC and mechanism of resistance to various fluoroquinolones using recent Staphylococcus pseudintermedius isolates from canine pyoderma. The broth microdilution method for MIC and a series of agar plates containing different concentrations of fluoroquinolones were inoculated with ∼10(10) colony-forming units of the bacterial culture for MPC were used. PCR was used to identify mutation in the resistant isolates. The rank order of potency based on MIC and MPC was ciprofloxacin = enrofloxacin ≥ marbofloxacin > difloxacin ≥ orbifloxacin. Integrating our data with reported pharmacokinetic data at the recommended dose ranges revealed that only high doses of ciprofloxacin, enrofloxacin and marbofloxacin could achieve a maximal plasma concentration (C(max)) greater than the MPC of 90% of isolates (C(max)/MPC(90)). The overall rank of potency against S. pseudintermedius, based on C(max)/MIC, C(max)/MPC, the area under concentration-time curve (AUC)/MIC and AUC/MPC values, was in decreasing order: enrofloxacin > ciprofloxacin ≥ marbofloxacin ≥ orbifloxacin = difloxacin. Sequencing of the quinolone resistant determining region of gyrA, gyrB, grlA and grlB of resistant strains showed a base-pair substitution in both gyrA and gyrB that resulted in Ser-84 to Leu and Ser-80 to Arg amino acid changes, respectively. High doses of ciprofloxacin, enrofloxacin and marbofloxacin could minimize the selection of resistant mutants, whereas the possibility of selecting mutants with the conventional doses of difloxacin and orbifloxacin, and low clinical doses of all fluoroquinolones, seems high.

Decreased serum linezolid concentrations in two patients receiving linezolid and rifampicin due to bone infections

Methicillin-resistant Staphylococcus is a common cause of orthopaedic implant infections. In such cases, rifampicin is the antibiotic of choice, but it should not be administered alone to avoid the selection of resistant mutants. Linezolid has activity against resistant staphylococci and a high oral bioavailability; therefore, it could be a good option for combining with rifampicin. We describe 2 patients admitted to our hospital due to orthopaedic implant infections, who received combination therapy with linezolid and rifampicin. In both cases, the trough serum concentration of linezolid during rifampicin treatment was below the minimum inhibitory concentration required to inhibit the growth of 90% of organisms (MIC(90)) for staphylococci, but increased after rifampicin withdrawal. This finding suggests an interaction between rifampicin and linezolid, and a possible explanation is discussed.

2009H1N1 Influenza in Immunocompetent and Immunocompromised Patients

The treatment and prevention of influenza virus infection has been associated with two major challenges that are exemplified by 2009H1N1 as well as previous pandemics. The constant evolutionary pressure on the influenza virus can result in novel strains which may not be optimally protected by vaccines or currently approved antivirals. Selection for antiviral resistance mutations can occur over time. Prolonged virus shedding, selection for resistance mutations, and cross resistance, can be exacerbated and problematic in immunocompromised patients. This review will cover 2009H1N1 infection in immuno-competent and immunocompromised individuals. Influenza continues to be a major public health concern and these challenges make influenza a suitable area for new vaccine and antiviral approaches.

Rifaximin therapy and hepatic encephalopathy: Pros and cons

Hepatic encephalopathy (HE) is the second most common major complication in cirrhotics and it significantly impacts quality of life. Therapeutic approaches for HE treatment and prevention mainly continue to rely on ammonia-lowering strategies and non-absorbable disaccharides are currently considered the cornerstone therapy. Non-absorbable antibiotics, such as neomycin and paramomycin, are effective in treatment of acute HE episodes but their prolonged use for recurrence prevention is hampered by possible side-effects. To overcome these limitations, rifaximin use has been proposed. Rifaximin has been shown to be not superior to non-absorbable disaccharides for either HE treatment or prevention, with a similar incidence of side-effects. Cirrhosis significantly increases rifaximin absorption and this could be a cause for concern. Following long-term rifaximin therapy, Clostridium difficile colitis has been observed and Candida albicans has been isolated from 20% of patients. In addition, selection of resistant mutants of both Gram-negative and -positive bacteria in the gastrointestinal tract cannot be definitely ruled out. Electrolyte alterations (sodium and potassium) have been reported during rifaximin therapy, a warning for its long-term use in cirrhotics. Moreover, a potential interference with vitamin K production should be considered which could further impair the already altered clotting status of these patients. The therapeutic cost of rifaximin is markedly higher than non-absorbable disaccharides. While waiting for further safety data, caution should be used to limit the use of rifaximin therapy for a very short-term period in selected HE cirrhotics not responding to non-absorbable disaccharides.

Peptidomimetic Escape Mechanisms Arise via Genetic Diversity in the Ligand-Binding Site of the Hepatitis C Virus NS3/4A Serine Protease

It is a challenge to develop direct-acting antiviral agents that target the nonstructural protein 3/4A protease of hepatitis C virus because resistant variants develop. Ketoamide compounds, designed to mimic the natural protease substrate, have been developed as inhibitors. However, clinical trials have revealed rapid selection of resistant mutants, most of which are considered to be pre-existing variants. We identified residues near the ketoamide-binding site in x-ray structures of the genotype 1a protease, co-crystallized with boceprevir or a telaprevir-like ligand, and then identified variants at these positions in 219 genotype-1 sequences from a public database. We used side-chain modeling to assess the potential effects of these variants on the interaction between ketoamide and the protease, and compared these results with the phenotypic effects on ketoamide resistance, RNA replication capacity, and infectious virus yields in a cell culture model of infection. Thirteen natural binding-site variants with potential for ketoamide resistance were identified at 10 residues in the protease, near the ketoamide binding site. Rotamer analysis of amino acid side-chain conformations indicated that 2 variants (R155K and D168G) could affect binding of telaprevir more than boceprevir. Measurements of antiviral susceptibility in cell-culture studies were consistent with this observation. Four variants (ie, Q41H, I132V, R155K, and D168G) caused low-to-moderate levels of ketoamide resistance; 3 of these were highly fit (Q41H, I132V, and R155K). Using a comprehensive sequence and structure-based analysis, we showed how natural variation in the hepatitis C virus protease nonstructural protein 3/4A sequences might affect susceptibility to first-generation direct-acting antiviral agents. These findings increase our understanding of the molecular basis of ketoamide resistance among naturally existing viral variants.

Multiple Low Level Mutations Identifies Imatinib Resistant CML Patients At Risk of Poor Response to Second-Line Inhibitor Therapy, Irrespective of the Resistance Profile of the Mutations

Abstract 111▪▪This icon denotes a clinically relevant abstractSpecific imatinib resistant BCR-ABL1 mutations confer clinical resistance to nilotinib (NIL; Y253H, E255K/V, T315I, F359V/C) and/or dasatinib (DAS; V299L, T315I/A, F317L/I/V/C). Therefore, mutation analysis is recommended for CML patients (pts) after imatinib failure to facilitate selection of appropriate therapy. However, around 40% of chronic phase (CP) pts without these NIL/DAS resistant mutations also fail second line inhibitor therapy. For imatinib resistant pts without these mutations at the time of commencing NIL/DAS therapy (switchover) we investigated whether sensitive mutation analysis could identity pts at risk of poor response to subsequent therapy.Switchover samples of 220 imatinib resistant pts (DAS n=131, NIL n=89) were analysed by direct sequencing (detection limit 10–20%) and sensitive, high throughput mass spectrometry (mass spec; Sequenom MassARRAY, detection limit 0.05–0.5%), which detects 31 common BCR-ABL1 mutations (approximately 89% of mutations detected in pts receiving imatinib). We previously demonstrated that mass spec could detect NIL/DAS resistant mutations at switchover in an extra 9% of pts compared to sequencing and that these low level resistant mutations were associated with subsequent failure of these inhibitors (Parker et al, JCO. 2011 In Press). Therefore, for the current analysis, pts with NIL/DAS resistant mutations detected by either method (n=45) were excluded since response is already known to be poor in these cases.In the switchover samples of the remaining 175 pts, 159 mutations were detected in 86 pts by mass spec, but just 108 mutations were detected in 89 pts by sequencing. Thirteen rare mutations detected by sequencing were not included in the mass spec assay design. Mass spec detected all other mutations detected by sequencing, plus an additional 64 low level mutations. Multiple NIL/DAS sensitive mutations (≥2 mutations) were detected at switchover in more of the 175 pts by mass spec (34/175, 19%; 2–9 mutations per pt) than sequencing (16/175, 9%; 2–3 mutations per pt), P=.009. We divided pts into 2 groups; those with multiple mutations detected by mass spec at switchover (n=34) and those with 0/1 mutation (n=141), and investigated the impact of multiple mutations on response to subsequent NIL/DAS therapy. Pts with 0 or 1 mutation, and similarly pts with 2 or >2 mutations, were grouped together, as no difference in response was observed. The median follow up for CP, accelerated phase and blast crisis pts was 17 (2–33), 18 (1–33) and 3 (1–27) mo, and the frequency of multiple mutations was 18%, 24% and 18%, respectively. During follow up, multiple mutations at switchover was associated with lower rates of complete cytogenetic response (CCyR; 21% vs 50%, P=.003, Fig 1A) and major molecular response (MMR; 6% vs 31%, P=.005, Fig 1B), and a higher incidence of acquiring new NIL/DAS resistant mutations detectable by sequencing (56% vs 25%, P=.0009, Fig 1C). At 18 mo, the failure-free survival rate (European LeukemiaNet recommendations) for CP pts with multiple mutations at switchover was 33% compared to 51% for CP pts with 0 or 1 mutation (P=.26, Fig 1D).The number of mutations detected per pt by mass spec at switchover (max of 9, 8 of 86 pts with mutations had ≥4, 9%) far exceeded the number concurrently detected by sequencing (max of 3). This suggests that mass spec detected a pool of subclonal mutants, each with a small survival advantage after imatinib therapy that was insufficient for their clonal predominance. Multiple low level mutations may be a marker of an increased propensity for subsequent selection of resistant mutations, possibly driven by genetic instability, demonstrating the advantage of a sensitive multiplex mutation assay.In conclusion, sensitive mutation analysis identified a poor-risk subgroup with multiple mutations that were not identified by sequencing. This subgroup represented 15.5% of the total cohort (34/220), who would not otherwise be classified as being at risk of poor response on the basis of their mutation status. These pts did not have NIL/DAS resistant mutations at switchover; however, they had a lower incidence of CCyR and MMR, and higher incidence of acquiring new NIL/DAS resistant mutations during NIL/DAS therapy compared to pts with 0 or 1 mutation. This poor-risk subgroup may warrant closer monitoring or experimental approaches to reduce the high risk of kinase inhibitor failure after imatinib resistance. [Display omitted] Disclosures:Hughes:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Ariad: Honoraria, Membership on an entity's Board of Directors or advisory committees. Branford:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Honoraria, Research Funding; Ariad: Research Funding.

Mechanisms of Drug Resistance Against JAK2 Inhibitors

Abstract 1426 The discovery of activating somatic mutations of JAK2 kinase as a contributing factor in the pathogenesis of myeloproliferative neoplasms (MPNs) —polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (MF)—prompted the development of JAK2-targeted therapies for these diseases1–3. Therapy with JAK2 inhibitors (INCB018424 and TG101348) induces rapid and marked reductions in spleen size and can lead to remarkable improvements in constitutional symptoms and overall quality of life suggesting that therapeutic targeting of JAK2 holds great promise for the treatment of JAK2 mediated disorders. It is likely that these two clinical agents will soon be approved by the FDA for the treatment of these diseases. Given our experience in development of resistance in tyrosine kinase inhibitor therapy (TKI) especially in the context of BCR/ABL targeting by Gleevec, we anticipate that resistance to JAK2 inhibitors will develop as well. To understand the mechanism of resistance against JAK2 targeted therapies, we performed mutagenesis studies and in vitro screening for resistance as previously reported for selection of resistance mutation in BCR/ABL (Azam et. al. (2003), Cell and Azam et. al. (2006), PNAS.). We sequenced the resistant clones isolated from INCB018424 resistant screening and identified 125 different amino substitutions spanning across FERM, SH2, pseudokinase and kinase domains. Frequency of resistant mutations demonstrated several major hot-spots clustered in the FERM, pseudokinase and kinase domain. We observed sixty different mutations in the kinase domain that clustered into eight major hot-spots with several minor-spots in regulatory regions including the hinge region, catalytic-loop, activation-loop and in the helical region of c-lobe. Structural modeling of these mutations suggests that the JAK2 kinase, like ABL kinase, is subject to auto-inhibited regulatory mechanisms and selects for drug resistant mutations from allosteric domains (FERM, SH2 and Pseudokinase domain). We anticipate that these results will be valuable in diagnosis of clinical resistance and in the identification and design of next generation small molecule JAK2 inhibitors Disclosures: No relevant conflicts of interest to declare.

Detection and quantification of the K103N mutation in HIV reverse transcriptase by pyrosequencing

Prolonged treatment of human immunodeficiency virus (HIV)–infected patients with nonnucleoside reverse transcriptase inhibitors (NNRTIs) might result in the selection of resistant mutants, the most frequent being the K103N mutation in reverse transcriptase. Resistance mutations are routinely detected by Sanger sequencing of the whole viral population, which does not detect sequence variants with frequencies below 20%. We have developed a pyrosequencing approach for the analysis of codon 103 of the HIV reverse transcriptase gene in the circulating viral population that detects variants below the limit of conventional sequencing. The method was tested with samples from 5 controls (not exposed to NNRTIs), 6 from patients exposed to NNRTIs and having a K103N mutant virus population detected by conventional sequencing, and 9 from patients previously exposed to NNRTIs that had a wild-type virus population by conventional sequencing. In 7 of 9, samples the mutation could not be detected by either the standard assay or pyrosequencing, while in 2 samples persistence of the mutation could be detected by pyrosequencing. The method might be of practical use in detecting minority variants of HIV in the clinical setting, in epidemiological studies with large numbers of samples, or as a complement to more complex approaches.

Pharmacokinetics of Antiretroviral Drugs in Anatomical Sanctuary Sites: The Male and Female Genital Tract

HIV resides within anatomical 'sanctuary sites', where local drug exposure and viral dynamics may differ significantly from the systemic compartment. Suboptimal antiretroviral concentrations in the genital tract may result in compartmentalized viral replication, selection of resistant mutations and possible re-entry of wild-type/resistant virus into the systemic circulation. Therefore, achieving adequate antiretroviral exposure in the genital tract has implications for the prevention of sexual and vertical transmission of HIV. Penetration of antiretrovirals in the genital tract is expressed by accumulation ratios derived from the measurement of drug concentrations in time-matched seminal plasma/cervicovaginal fluid and plasma samples. Penetration varies by gender and may be drug (as opposed to class) specific with high interindividual variability. Concentrations in seminal plasma are highest for nucleoside analogues and lowest for protease inhibitors and efavirenz. Seminal accumulation of newer agents, raltegravir and maraviroc, is moderate (rank order of accumulation is nucleoside/nucleotide reverse transcriptase inhibitors [lamivudine/zidovudine/tenofovir/didanosine > stavudine/abacavir] > raltegravir > indinavir/maraviroc/nevirapine >> efavirenz/protease inhibitors [amprenavir/atazanavir/darunavir > lopinavir/ritonavir > saquinavir] > enfuvirtide). In the female genital tract, the nucleoside analogues exhibit high accumulation ratios, whereas protease inhibitors have limited penetration; however, substantial variability exists between individuals and study centres. Second generation non-nucleoside reverse transcriptase inhibitor etravirine, and maraviroc and raltegravir, demonstrate effective accumulation in cervicovaginal secretions (rank order of accumulation is nucleoside/nucleotide reverse transcriptase inhibitor [zidovudine/lamivudine/didanosine > emtricitabine/tenofovir] > indinavir > maraviroc/raltegravir/darunavir/etravirine > nevirapine/abacavir > protease inhibitors [amprenavir/atazanavir/ritonavir] > lopinavir/stavudine/efavirenz > saquinavir).

Mutant-prevention concentration and mechanism of resistance in clinical isolates and enrofloxacin/marbofloxacin-selected mutants of Escherichia coli of canine origin

The antibacterial activity and selection of resistant bacteria, along with mechanisms of fluoroquinolone resistance, were investigated by integrating the static [MIC or mutant-prevention concentration (MPC)] and in vitro dynamic model approaches using Escherichia coli isolates from diseased dogs. Using the dynamic models, selected E. coli strains and enrofloxacin and marbofloxacin at a range of simulated area under concentration-time curve over a 24 h interval (AUC(24 h))/MIC ratios were investigated. Our results indicated increasing losses in susceptibility of E. coli upon continuous exposure to enrofloxacin and marbofloxacin in vitro. This effect was transferable to other fluoroquinolones, as well as to structurally unrelated drugs. Our results also confirmed an AUC(24 h)/MIC (AUC(24 h)/MPC)-dependent antibacterial activity and selection of resistant E. coli mutants, in which maximum losses in fluoroquinolone susceptibility occurred at simulated AUC(24 h)/MIC ratios of 40-60. AUC(24 h)/MPC ratios of 39 (enrofloxacin) and 32 (marbofloxacin) were considered protective against the selection of resistant mutants of E. coli. Integrating our MIC and MPC data with published pharmacokinetic information in dogs revealed a better effect of the conventional dosing regimen of marbofloxacin than that of enrofloxacin in restricting the selection of resistant mutants of E. coli. Target mutations, especially at codon 83 (serine to leucine) of gyrA, and overexpression of efflux pumps contributed to resistance development in both clinically resistant and in vitro-selected mutants of E. coli. We also report here a previously undescribed mutation at codon 116 of parC in two laboratory-derived resistant mutants of E. coli. Additional studies would determine the exact role of this mutation in fluoroquinolone susceptibility, as well as establish the importance of our findings in the clinical setting.

Efficacy of a new fluoroquinolone, JNJ-Q2, in murine models of Staphylococcus aureus and Streptococcus pneumoniae skin, respiratory, and systemic infections.

The in vivo efficacy of JNJ-Q2, a new broad-spectrum fluoroquinolone (FQ), was evaluated in a murine septicemia model with methicillin-susceptible Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus (MRSA) and in a Streptococcus pneumoniae lower respiratory tract infection model. JNJ-Q2 and comparators were also evaluated in an acute murine skin infection model using a community-acquired MRSA strain and in an established skin infection (ESI) model using a hospital-acquired strain, for which the selection of resistant mutants was also determined. JNJ-Q2 demonstrated activity in the MSSA septicemia model that was comparable to that moxifloxacin (JNJ-Q2 50% effective dose [ED(50)], 0.2 mg/kg of body weight administered subcutaneously [s.c.] and 2 mg/kg administered orally [p.o.]) and activity in the MRSA septicemia model that was superior to that of vancomycin (JNJ-Q2 ED(50), 1.6 mg/kg administered s.c.). In an S. pneumoniae lower respiratory tract infection model, JNJ-Q2 displayed activity (ED(50), 1.9 mg/kg administered s.c. and 7.4 mg/kg administered p.o.) that was comparable to that of gemifloxacin and superior to that of moxifloxacin. In both MRSA skin infection models, treatment with JNJ-Q2 resulted in dose-dependent reductions in bacterial titers in the skin, with the response to JNJ-Q2 at each dose exceeding the responses of the comparators ciprofloxacin, moxifloxacin, linezolid, and vancomycin. Additionally, in the ESI model, JNJ-Q2 showed a low or nondetectable propensity for ciprofloxacin resistance selection, in contrast to the selection of ciprofloxacin-resistant mutants observed for both ciprofloxacin and moxifloxacin. JNJ-Q2 demonstrated activity that was comparable or superior to the activity of fluoroquinolone or antistaphylococcal comparators in several local and systemic skin infection models performed with both S. aureus and S. pneumoniae and is currently being evaluated in phase II human clinical trials.