Detection of Emerging Ibrutinib Resistance by Flow Cytometry in Patients with Chronic Lymphocytic Leukemia

Purpose: Bruton’s tyrosine kinase inhibitor ibrutinib has revolutionized the treatment of chronic lymphocytic leukemia (CLL). Although ibrutinib is a highly effective drug, during the treatment acquired ibrutinib resistance may occur and its early detection is an important issue. Our aim was to investigate several phenotypic markers on CLL cells to reveal changes in their expression during ibrutinib treatment.Methods: In our study 28 (treatment naive, ibrutinib sensitive, clinically ibrutinib resistant) peripheral blood (PB), and 6 paired PB and bone marrow (BM) samples were examined. The expression of several surface markers (CD69, CD184, CD86, CD185, CD27) was assessed by flow cytometry in each sample. Furthermore, the presence of the BTKC481S resistance mutation was tested using digital droplet PCR. In addition, we investigated the changes of the phenotype of CLL cells during ibrutinib treatment in one patient with acquired ibrutinib resistance.Results: The expression of CD27 decreased during ibrutinib therapy but increased again at the onset of clinical resistance. Expressions of CD69 and CD86 were also elevated at the onset of clinical ibrutinib resistance. The expression of CD86 showed correlation between PB and BM samples. Relapsed cases with high CD86 expression were positive for BTKC481S mutation. Our prospective study showed that the increases in the expression of CD27, CD69 and CD86 were detectable up to several months before the onset of clinical resistance.Conclusion: Our research suggests that the flow cytometric measurements of certain markers, especially CD86, may predict development of ibrutinib resistance, however, confirmatory experiments are still required.

The frequency of HIV-1 infection and surveillance drug-resistant mutations determination among Iranians with high-risk behaviors, during 2014 to 2020

Background and Objectives: Human immunodeficiency virus (HIV) has various transmission routes. Instant antiretroviral therapy (ART) is the recommended treatment for HIV infection. Highly active antiretroviral therapy (HAART) significantly decreases the acquired immunodeficiency syndrome (AIDS) and AIDS-related co-morbidities. Notwithstanding the suit- ability of HAART, the antiretrovirals (ARVs) have adverse effects and antiretroviral drug resistance mutations are reported among those who receive ARVs. In this survey, the abundance of HIV-1 infection in Iranians with high-risk behaviors, and detection of the surveillance drug-resistant mutations (SDRMs) were evaluated. Materials and Methods: This cross-sectional study was conducted on 250 individuals with high-risk behaviors from Sep- tember 2014 to February 2020. HIV-1 Ag/Ab in plasma samples was detected using enzyme immunoassay (EIA) kits. The conserved region of HIV-1 was detected in the plasma samples by real-time polymerase chain reaction (PCR) assay. Further- more, in individuals with positive HIV-1 RNA, HIV-1 viral load testing was performed. After amplification and sequencing of the HIV-1 protease, reverse transcriptase, and integrase genes, surveillance drug resistance mutation (SDRM) and phylo- genetic analysis were determined. Results: Out of the 250 participants with high-risk behaviors, six (2.4%) were infected with HIV-1. According to the phy- logenetic analysis, the CRF35_AD (83.3% or 5/6) was the dominant subtype, followed by CRF01_AE (16.7% or 1/6). In this research, in none of the HIV-1 infected patients, SDRM for protease inhibitors (PIs), nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), and integrase inhibitors (INs) were observed. Nevertheless, in one of the patients, V179L mutation was detected which is a rare non-polymorphic mutation and is listed as a rilpivirine (RPV) -associated resistance mutation. Conclusion: The results of the current survey revealed that 2.4% of people with high-risk behaviors are infected with HIV and the level of drug resistance mutations (DRMs) in these people is very low.

Discovery and Development of Inhibitors of the Plasmodial FNT-Type Lactate Transporter as Novel Antimalarials

Plasmodium spp. malaria parasites in the blood stage draw energy from anaerobic glycolysis when multiplying in erythrocytes. They tap the ample glucose supply of the infected host using the erythrocyte glucose transporter 1, GLUT1, and a hexose transporter, HT, of the parasite’s plasma membrane. Per glucose molecule, two lactate anions and two protons are generated as waste that need to be released rapidly from the parasite to prevent blockage of the energy metabolism and acidification of the cytoplasm. Recently, the missing Plasmodium lactate/H+ cotransporter was identified as a member of the exclusively microbial formate–nitrite transporter family, FNT. Screening of an antimalarial compound selection with unknown targets led to the discovery of specific and potent FNT-inhibitors, i.e., pentafluoro-3-hydroxy-pent-2-en-1-ones. Here, we summarize the discovery and further development of this novel class of antimalarials, their modes of binding and action, circumvention of a putative resistance mutation of the FNT target protein, and suitability for in vivo studies using animal malaria models.

Efficacy of Dipterocarpus alatus oil combination with Rhinacanthus nasutus leaf and Garcinia mangostana pericarps against canine demodicosis

Background and Aim: Canine demodicosis is a skin disease that is a major global health problem in dogs. Ivermectin is a drug of choice for treatment, but it may cause toxicity in dogs carrying multidrug resistance mutation-1 gene mutations. Hence, alternative herbal medicines are used instead of the drug, such as Dipterocarpus alatus oil (YN oil), Rhinacanthus nasutus leaf (WC), and Garcinia mangostana pericarps (MG) extracts. This study aimed to determine the efficacy of D. alatus oil, R. nasutus leaf, and G. mangostana pericarp extracts on canine demodicosis in vivo. Materials and Methods: Twenty-five mixed-breed dogs with localized demodicosis were examined. Dogs were diagnosed with demodicosis through deep skin scraping and screened with the inclusion criteria. Five dogs of each group were treated in five treatment groups (ivermectin, YN oil, YN oil+WC, YN oil+MG, and YN oil+WC+MG) for 1 month. The individual dogs were clinically evaluated, and the dermatological lesions were monitored daily for 60 days. Results: Dermatological lesion improvement was predominantly observed in the group of dogs treated with YN oil+WC. This was evidenced by the disappearance of the hyperpigmentation and lichenification on day 28 post-treatment and alopecia on day 56 post-treatment. Moreover, no allergic or clinical signs were observed during treatment. Conclusion: YN oil+WC is an alternative herbal medicine that could be used for the treatment of localized canine demodicosis.

Detection of emerging ibrutinib resistance by flow cytometry in patients with chronic lymphocytic leukemia

Background: Bruton ’ s tyrosine kinase inhibitor ibrutinib has revolutionized the treatment of chronic lymphocytic leukemia (CLL). Although ibrutinib is a highly effective drug, continuous treatment is required to maintain remission, which may lead to acquired ibrutinib resistance. Early detection of acquired resistance preceding clinical disease progression is an important issue. This is why our aim was to investigate several phenotypic markers on CLL cells to reveal changes in their expression during ibrutinib treatment in sensitive and clinically resistant patients. Materials and methods: In our study 28 (treatment naive, ibrutinib sensitive, clinically ibrutinib resistant) peripheral blood (PB), and 6 paired PB and bone marrow (BM) samples from CLL patients were examined. The expression of several surface markers (CD69, CD184, CD86, CD185, CD27) was assessed by flow cytometry in each sample. Furthermore, the presence of the BTK C481S resistance mutation was tested using digital droplet PCR (ddPCR) in samples from ibrutinib sensitive and resistant cases. In addition, we investigated the changes of CLL cells ’ phenotype during ibrutinib treatment in one patient with acquired ibrutinib resistance. Results: We found that the expression of CD27 decreased during ibrutinib therapy but increased again at the onset of clinical resistance. Expressions of CD69 and CD86 were also elevated at the onset of clinical ibrutinib resistance. Furthermore, the expression of CD86 showed correlation between PB and BM samples. Relapsed cases with high CD86 expression were positive for BTK C481S mutation. In addition, our prospective study showed that the increases in the expression of CD27, CD69 and CD86 were detectable up to several months before the onset of clinical resistance. Conclusion: Our research suggests that the flow cytometric measurements of certain markers, especially CD86, may predict development of ibrutinib resistance, however, confirmatory experiments are still required. Monitoring CD86 expression on peripheral blood CLL cells during ibrutinib treatment may become a potential new method to detect acquired ibrutinib resistance in the near future.

Larotrectinib (Vitrakvi)

CADTH reimbursement reviews are comprehensive assessments of the clinical effectiveness and cost-effectiveness, as well as patient and clinician perspectives, of a drug or drug class. The assessments inform non-binding recommendations that help guide the reimbursement decisions of Canada's federal, provincial, and territorial governments, with the exception of Quebec. This review assesses larotrectinib (Vitrakvi); 25 mg and 100 mg capsules (as larotrectinib sulphate) and 20 mg/mL oral solution (as larotrectinib sulphate). Indication: For the treatment of adult and pediatric patients with solid tumours that: have an NTRK gene fusion without a known acquired resistance mutation are metastatic or where surgical resection is likely to result in severe morbidity have no satisfactory treatment options

High Throughput Drug Discovery in S. Cerevisiae: the Characterisation of FC-592 and FC-888

<p>The discovery and characterisation of novel small molecule drug candidates is a medical priority. Recent advances in synthetic organic chemistry allow the de novo production of diversity oriented synthetic compound libraries and synthetic modification of natural products to provide candidate compounds for screening as potential therapeutics, bioactive agents or genetic probes. Small drugs function through interaction with complex genetic networks and pathways. However, it is difficult to characterise these interactions on a genome wide level to achieve understanding of drug mechanism. Here, discovery based approaches are utilised to achieve system wide parsing of biological mechanism, in an attempt to characterise the action of novel synthetic compounds and natural product derivatives. Chemical genomic analysis allows for such understanding by examining growth profiles of a genomic deletion library of Saccharomyces cerevisiae mutants in the presence of sub-inhibitory concentrations of drug. The gene targets of small molecule compounds can be identified by noting deletion strains which display increased sensitivity, indicating chemical interaction with the associated gene network. In addition, the development and characterisation of resistant mutants can be used to identify putative drug targets. In this strategy, characterisation of the mechanism of resistance gives insight into drug mode-of-action. This study develops a high throughput yeast inhibition assay to identify bioactive compounds from a synthetic organic compound library, and attempts to characterise mechanism of action by establishing a profile of each compound’s interaction with these gene networks; and mapping a resistance mutation to provide evidence of inhibitory mechanism. Two candidate compounds are identified, FC-592 and FC-888. FC-592 displayed cytostatic inhibition. Further, yeast tag microarray homozygous profiling (HOP), chemical structure analysis, and cell-cycle analysis via flow cytometry for this compound provided evidence for a mechanism of poor specificity that targets glycoprotein biosynthesis and the secretory (Sec) pathway, as well as the cell-division cycle (CDC) pathway. Attempts to characterise a mutant resistant to this compound via synthetic genetic array mapping were unsuccessful when the resistance mutation proved to mediate a slow growth phenotype, abrogating the Synthetic Genetic Array Mapping approach utilised. Pending further analysis, it is suggested that this compound could have a role as a genetic probe in future exploration of the Sec and CDC pathways. Chemical structure analysis and a non-specific HOP screen chemigenomic profile suggested that FC-888 is an alkylating agent with a broad affinity for cellular nucleophiles. The compound demonstrates cytotoxic activity, and its efflux is not mediated by the pleiotropic drug resistance (PDR) network. It is suggested that the compound could find utility as a probe dissecting processes related to cellular defence against non-DNA specific alkylation.</p>

High Throughput Drug Discovery in S. Cerevisiae: the Characterisation of FC-592 and FC-888

<p>The discovery and characterisation of novel small molecule drug candidates is a medical priority. Recent advances in synthetic organic chemistry allow the de novo production of diversity oriented synthetic compound libraries and synthetic modification of natural products to provide candidate compounds for screening as potential therapeutics, bioactive agents or genetic probes. Small drugs function through interaction with complex genetic networks and pathways. However, it is difficult to characterise these interactions on a genome wide level to achieve understanding of drug mechanism. Here, discovery based approaches are utilised to achieve system wide parsing of biological mechanism, in an attempt to characterise the action of novel synthetic compounds and natural product derivatives. Chemical genomic analysis allows for such understanding by examining growth profiles of a genomic deletion library of Saccharomyces cerevisiae mutants in the presence of sub-inhibitory concentrations of drug. The gene targets of small molecule compounds can be identified by noting deletion strains which display increased sensitivity, indicating chemical interaction with the associated gene network. In addition, the development and characterisation of resistant mutants can be used to identify putative drug targets. In this strategy, characterisation of the mechanism of resistance gives insight into drug mode-of-action. This study develops a high throughput yeast inhibition assay to identify bioactive compounds from a synthetic organic compound library, and attempts to characterise mechanism of action by establishing a profile of each compound’s interaction with these gene networks; and mapping a resistance mutation to provide evidence of inhibitory mechanism. Two candidate compounds are identified, FC-592 and FC-888. FC-592 displayed cytostatic inhibition. Further, yeast tag microarray homozygous profiling (HOP), chemical structure analysis, and cell-cycle analysis via flow cytometry for this compound provided evidence for a mechanism of poor specificity that targets glycoprotein biosynthesis and the secretory (Sec) pathway, as well as the cell-division cycle (CDC) pathway. Attempts to characterise a mutant resistant to this compound via synthetic genetic array mapping were unsuccessful when the resistance mutation proved to mediate a slow growth phenotype, abrogating the Synthetic Genetic Array Mapping approach utilised. Pending further analysis, it is suggested that this compound could have a role as a genetic probe in future exploration of the Sec and CDC pathways. Chemical structure analysis and a non-specific HOP screen chemigenomic profile suggested that FC-888 is an alkylating agent with a broad affinity for cellular nucleophiles. The compound demonstrates cytotoxic activity, and its efflux is not mediated by the pleiotropic drug resistance (PDR) network. It is suggested that the compound could find utility as a probe dissecting processes related to cellular defence against non-DNA specific alkylation.</p>

Immediate pools of malaria infections at diagnosis combined with targeted deep sequencing accurately quantifies frequency of drug resistance mutations

Antimalarial resistance surveillance in sub-Saharan Africa is often constrained by logistical and financial challenges limiting its breadth and frequency. At two sites in Ghana, we have piloted a streamlined sample pooling process created immediately by sequential addition of positive malaria cases at the time of diagnostic testing. This streamlined process involving a single tube minimized clinical and laboratory work and provided accurate frequencies of all known drug resistance mutations after high-throughput targeted sequencing using molecular inversion probes. Our study validates this method as a cost-efficient, accurate and highly-scalable approach for drug resistance mutation monitoring that can potentially be applied to other infectious diseases such as tuberculosis.