Hollow-fiber Model Research Articles

Ertapenem's therapeutic potential for Mycobacterium avium lung disease in the hollow fibre model

IntroductionGuideline-based therapy for Mycobacterium avium complex (MAC) pulmonary disease achieves sustained sputum conversion rates in only 43–53% of patients. Repurposing of β-lactam antibiotics such as ertapenem could expedite design of more efficacious regimens, compared to developing new drugs. MethodsWe performed an ertapenem exposure-response study in the hollow fibre system model of intracellular MAC (HFS-MAC). We recapitulated human-like intrapulmonary concentration-time profiles of eight once-daily intravenous doses of ertapenem over 28 days and performed repetitive sampling for drug concentration-time profiles and MAC burden. The % of time concentration persisted above MIC (%TMIC) mediating either 50% or 80% of maximal effect (E50, EC80) were identified. The EC80 was used as target exposure in a 10 000 subject Monte Carlo experiments for ertapenem doses of 1G, 2G, or 4G administered once versus twice daily. ResultsThe ertapenem MIC ranged from 0.5 to 2 mg/L on three occasions. Ertapenem achieved a half-life of 4.04 ± 0.80 h in the HFS-MAC and killed a maximum of 2.17 log10 CFU/mL below day 0. The EC50 was %TMIC of 75.9% (95% confidence interval: 68.43%–86.54%) and the EC80 was %TMIC of 100%. Target attainment probability was >90% for 1G twice daily up to an MIC of 2 mg/L, while for 2G twice daily the susceptibility MIC breakpoint was 4–8 mg/L. ConclusionsErtapenem microbial kill below day 0 burden was better than guideline-based therapy drugs in the HFS-MAC in the past. Ertapenem is a promising drug for novel combination therapies for MAC lung disease.

Abstract 3164: The in vivo Hollow Fiber model is a valuable tool in drug development of selective KRas inhibitors

Abstract The proto-oncogene KRas is a well-described small GTPase that functions as a molecular switch for major physiological signaling pathways involved in cell proliferation, differentiation and survival. It has been shown that activating mutations in KRas are among the most common oncogenic drivers of tumorigenesis. Missense mutations of KRas result in constitutive activation due to impaired hydrolysis of GTP which enhances tumor-promoting downstream signaling pathways. Most KRas mutations are located in exon 2 or 3 including the most frequently altered glycine 12, which is present in most pancreatic cancers as well as in colorectal cancers and lung adenocarcinomas. A panel of cancer cell lines harboring different KRas mutations such as G12C, G12V or G12D was selected to test KRas inhibitors in several cellular assays: phosphorylation assays, 2D and 3D proliferation assays. Applying the cellular pERK AlphaLISA assay showed high selectivity of several inhibitors for specific KRas mutants, while RAF and MEK inhibitors did not show significant selectivity in the tested cancer cell lines. The KRas inhibitors were then examined in a cellular 2D and a 3D spheroid proliferation assay to determine the inhibitory effect on cell growth. These cellular assays revealed a high selectivity for a specific KRas mutant, which was even more pronounced in the 3D format than in the 2D setup. The transferability of the cellular data obtained to in vivo was tested in the Hollow Fiber mouse model. The Hollow Fiber model allows the simultaneous study of multiple cell lines implanted in separated drug-(but not cell-) permeable fibers in a single mouse with a study duration of only 16 days. AMG510 (Sotorasib), a drug approved for tumors with a G12C Kras mutation, and MRTX1133, which is currently in clinical phase and shows activity in tumors with a G12D Kras mutation, were screened for their inhibitory effect on the pancreatic tumor cells MiaPaCa-2 (G12C Kras mutation), AsPC-1 (G12D Kras mutation) and BxPC-3 (wt Kras) in the in vivo Hollow Fiber model in female NMRI nude mice. While the growth of BxPC-3 tumor cells was not affected by either inhibitor, the growth of AsPC-1 tumor cells in particular was selectively and significantly inhibited by MRTX1133. In summary, the in vivo Hollow Fiber model is a fast and cost-effective model that can play an prominent role in drug development as a link between in vitro and in vivo xenograft studies by providing rapid and transferable evidence for in vivo efficacy. Citation Format: Philipp Metzger, Claus-Werner Franzke, Ezgi Dikici, Gregor Sommerkamp, Franziska Fimm-Todt, Jan E. Ehlert, Cynthia Obodozie, Holger Weber. The in vivo Hollow Fiber model is a valuable tool in drug development of selective KRas inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3164.

Tapcin, an In Vivo Active Dual Topoisomerase I/II Inhibitor Discovered by Synthetic Bioinformatic Natural Product (Syn-BNP)-Coupled Metagenomics.

DNA topoisomerases are attractive targets for anticancer agents. Dual topoisomerase I/II inhibitors are particularly appealing due to their reduced rates of resistance. A number of therapeutically relevant topoisomerase inhibitors are bacterial natural products. Mining the untapped chemical diversity encoded by soil microbiomes presents an opportunity to identify additional natural topoisomerase inhibitors. Here we couple metagenome mining, bioinformatic structure prediction algorithms, and chemical synthesis to produce the dual topoisomerase inhibitor tapcin. Tapcin is a mixed p-aminobenzoic acid (PABA)-thiazole with a rare tri-thiazole substructure and picomolar antiproliferative activity. Tapcin reduced colorectal adenocarcinoma HT-29 cell proliferation and tumor volume in mouse hollow fiber and xenograft models, respectively. In both studies it showed similar activity to the clinically used topoisomerase I inhibitor irinotecan. The study suggests that the interrogation of soil microbiomes using synthetic bioinformatic natural product methods has the potential to be a rewarding strategy for identifying potent, biomedically relevant, antiproliferative agents.

Tapcin, an In Vivo Active Dual Topoisomerase I/II Inhibitor Discovered by Synthetic Bioinformatic Natural Product (Syn‐BNP)‐Coupled Metagenomics

AbstractDNA topoisomerases are attractive targets for anticancer agents. Dual topoisomerase I/II inhibitors are particularly appealing due to their reduced rates of resistance. A number of therapeutically relevant topoisomerase inhibitors are bacterial natural products. Mining the untapped chemical diversity encoded by soil microbiomes presents an opportunity to identify additional natural topoisomerase inhibitors. Here we couple metagenome mining, bioinformatic structure prediction algorithms, and chemical synthesis to produce the dual topoisomerase inhibitor tapcin. Tapcin is a mixed p‐aminobenzoic acid (PABA)‐thiazole with a rare tri‐thiazole substructure and picomolar antiproliferative activity. Tapcin reduced colorectal adenocarcinoma HT‐29 cell proliferation and tumor volume in mouse hollow fiber and xenograft models, respectively. In both studies it showed similar activity to the clinically used topoisomerase I inhibitor irinotecan. The study suggests that the interrogation of soil microbiomes using synthetic bioinformatic natural product methods has the potential to be a rewarding strategy for identifying potent, biomedically relevant, antiproliferative agents.

Clofazimine as a substitute for rifampicin improves efficacy of Mycobacterium avium pulmonary disease treatment in the hollow-fiber model.

Mycobacterium avium complex pulmonary disease is treated with an azithromycin, ethambutol, and rifampicin regimen, with limited efficacy. The role of rifampicin is controversial due to inactivity, adverse effects, and drug interactions. Here, we evaluated the efficacy of clofazimine as a substitute for rifampicin in an intracellular hollow-fiber infection model. THP-1 cells, which are monocytes isolated from peripheral blood from an acute monocytic leukemia patient, were infected with M. avium ATCC 700898 and exposed to a regimen of azithromycin and ethambutol with either rifampicin or clofazimine. Intrapulmonary pharmacokinetic profiles of azithromycin, ethambutol, and rifampicin were simulated. For clofazimine, a steady-state average concentration was targeted. Drug concentrations and bacterial densities were monitored over 21 days. Exposures to azithromycin and ethambutol were 20%-40% lower than targeted but within clinically observed ranges. Clofazimine exposures were 1.7 times higher than targeted. Until day 7, both regimens were able to maintain stasis. Thereafter, regrowth was observed for the rifampicin-containing regimen, while the clofazimine-containing regimen yielded a 2 Log10 colony forming unit (CFU) per mL decrease in bacterial load. The clofazimine regimen also successfully suppressed the emergence of macrolide tolerance. In summary, substitution of rifampicin with clofazimine in the hollow-fiber model improved the antimycobacterial activity of the regimen. Clofazimine-containing regimens merit investigation in clinical trials.

Sarecycline pharmacokinetics/pharmacodynamics in the hollow-fibre model of Mycobacterium avium complex: so near and yet so far.

Poor sustained sputum culture conversion rates with the standard-of-care therapy highlight the need for better drugs to treat Mycobacterium avium complex pulmonary disease (MAC-PD). To determine the pharmacokinetics/pharmacodynamics (PK/PD)-optimized exposure of sarecycline and its potential role in treating MAC-PD. We performed MIC studies with MAC ATCC 700898 and 19 clinical isolates and test-tube static concentration-response studies. A dynamic hollow-fibre system model of intracellular MAC (HFS-MAC) study was performed mimicking six human-equivalent sarecycline dose concentration-time profiles to identify the PK/PD optimal exposure of sarecycline for MAC kill. The inhibitory sigmoid maximal effect (Emax) model was used for PK/PD analysis. The sarecycline MIC of MAC ATCC 700898 was 1 mg/L, while the MIC for the 19 clinical strains ranged between 32 and >256 mg/L. The concentration mediating 50% of Emax (EC50) was similar between intracellular and extracellular MAC. In the HFS-MAC, all six sarecycline doses killed intracellular MAC, with an Emax of 1.0 log10 cfu/mL below Day 0 burden (stasis). The sarecycline EC80 (optimal) exposure was identified as AUC0-24/MIC = 139.46. Sarecycline demonstrated anti-MAC Emax in the HFS-MAC model better than ethambutol but worse than omadacycline (>5 log10 cfu/mL below stasis) in HFS-MAC. However, since currently approved highest oral sarecycline dose achieves an AUC0-24 of 48.2 mg·h/L and MAC MICs are >32 mg/L, the target AUC0-24/MIC of 139.46 is unlikely to be achieved in patients.

The role of rifampicin within the treatment of Mycobacterium avium pulmonary disease.

Rifampicin is recommended for the treatment of Mycobacterium avium complex pulmonary disease alongside azithromycin and ethambutol. We evaluated the azithromycin-ethambutol backbone with and without rifampicin in an intracellular hollow fiber model and performed RNA sequencing to study the differences in adaptation. In an in vitro hollow fiber experiment, we simulated epithelial lining fluid pharmacokinetic profiles of the recommended 3-drug (rifampicin, ethambutol, and azithromycin) or a 2-drug (ethambutol and azithromycin) treatment. THP-1 cells infected with M. avium ATCC700898 were exposed to these regimens for 21 days. We determined intra- and extra-cellular bacterial load- and THP-1 cell densities on days 0, 3, 7, 14, and 21, alongside RNA sequencing. The emergence of macrolide resistance was studied by inoculating intra- and extra-cellular fractions of azithromycin-containing Middlebrook 7H10 agar plates. Complete pharmacokinetic profiles were determined at days 0 and 21. Both therapies maintained stasis of both intra- and extra-cellular bacterial populations for 3 days, whilst regrowth coinciding with the emergence of a macrolide-resistant subpopulation was seen after 7 days. THP-1 cell density remained static. Similar transcriptional profiles were observed for both therapies that were minimally influenced by exposure duration. Transcriptional response was slightly larger during 2-drug treatment. Rifampicin did not add to the antimycobacterial effect to the 2-drug therapy or suppression of emergence resistance. RNA transcription was not greatly altered by the addition of rifampicin, which may be due to strong transcriptional influence of azithromycin and host cells. This questions the role of rifampicin in the currently recommended therapy. These findings should be confirmed in clinical trials.

Meropenem-vaborbactam restoration of first-line drug efficacy and comparison of meropenem-vaborbactam-moxifloxacin versus BPaL MDR-TB regimen

Meropenem in combination with β-lactamase inhibitors (BLIs) and other drugs was tested to identify alternative treatment regimens for multidrug-resistant tuberculosis (MDR-TB). The following were performed: (1) MIC experiments; (2) static time-kill studies (STKs) with different BLIs; and (3) a hollow fibre model system of TB (HFS-TB) studies with meropenem-vaborbactam combined with human equivalent daily doses of 20 mg/kg or 35 mg/kg rifampin, or moxifloxacin 400 mg, or linezolid 600 mg vs. bedaquiline-pretonamid-linezolid (BPaL) for MDR-TB. The studies were performed using Mycobacterium tuberculosis (M. tuberculosis) H37Rv and an MDR-TB clinical strain (named M. tuberculosis 16D) that underwent whole genome sequencing. Exponential decline models were used to calculate the kill rate constant (K) of different HFS-TB regimens. Whole genome sequencing revealed mutations associated with resistance to rifampin, isoniazid, and cephalosporins. The meropenem-vaborbactam MIC of M. tuberculosis was H37Rv 2mg/L and > 128 mg/L for M. tuberculosis 16D. Relebactam and vaborbactam improved both the potency and efficacy of meropenem in STKs. Meropenem-vaborbactam alone failed to kill M. tuberculosis 16D but killed below day 0 burden when combined with isoniazid and rifampin, with the moxifloxacin combination being the most effective and outranking bedaquiline and pretomanid. In the HFS-TB, meropenem-vaborbactam-moxifloxacin and BPaL had the highest K (log10 cfu/mL/day) of 0.31 (95% CI 0.17-0.58) and 0.34 (95% CI 0.21-0.56), while meropenem-vaborbactam-rifampin (35 mg/kg) had a K of 0.18 (95% CI 0.12-0.25). The K for meropenem-vaborbactam-moxifloxacin-linezolid demonstrated antagonism. Adding meropenem-vaborbactam could potentially restore the efficacy of isoniazid and rifampin against MDR-TB. The meropenem-vaborbactam-moxifloxacin backbone regimen has implications for creating a new effective MDR-TB regimen.

Fiber-orientation independent component of R2* obtained from single-orientation MRI measurements in simulations and a post-mortem human optic chiasm.

The effective transverse relaxation rate (R2*) is sensitive to the microstructure of the human brain like the g-ratio which characterises the relative myelination of axons. However, the fibre-orientation dependence of R2* degrades its reproducibility and any microstructural derivative measure. To estimate its orientation-independent part (R2,iso*) from single multi-echo gradient-recalled-echo (meGRE) measurements at arbitrary orientations, a second-order polynomial in time model (hereafter M2) can be used. Its linear time-dependent parameter, β1, can be biophysically related to R2,iso* when neglecting the myelin water (MW) signal in the hollow cylinder fibre model (HCFM). Here, we examined the performance of M2 using experimental and simulated data with variable g-ratio and fibre dispersion. We found that the fitted β1 can estimate R2,iso* using meGRE with long maximum-echo time (TEmax ≈ 54 ms), but not accurately captures its microscopic dependence on the g-ratio (error 84%). We proposed a new heuristic expression for β1 that reduced the error to 12% for ex vivo compartmental R2 values. Using the new expression, we could estimate an MW fraction of 0.14 for fibres with negligible dispersion in a fixed human optic chiasm for the ex vivo compartmental R2 values but not for the in vivo values. M2 and the HCFM-based simulations failed to explain the measured R2*-orientation-dependence around the magic angle for a typical in vivo meGRE protocol (with TEmax ≈ 18 ms). In conclusion, further validation and the development of movement-robust in vivo meGRE protocols with TEmax ≈ 54 ms are required before M2 can be used to estimate R2,iso* in subjects.

Preclinical testing of antimicrobials for cystic fibrosis lung infections: current needs and future priorities.

A workshop was held by the PIPE-CF strategic research centre to consider preclinical testing of antimicrobials for cystic fibrosis (CF). The workshop brought together groups of people from the CF community to discuss current challenges and identify priorities when developing CF therapeutics. This paper summarizes the key points from the workshop from the different sessions, including talks given by presenters on the day and round table discussions. Currently, it is felt that there is a large disconnect throughout the community, with communication between patients, clinicians and researchers being the main issue. This leads to little consideration being given to factors such as treatment regimes, routes of administration and side effects when developing new therapies, that could alter the day-to-day lifestyles of people living with CF. Translation of numerical data that are obtained in the laboratory to successful outcomes of clinical trials is also a key challenge facing researchers today. Laboratory assays in preclinical testing involve basing results on bacterial clearance and decrease in viable cells, when these are not factors that are considered when determining the success of a treatment in the clinic. However, there are several models currently in development that seek to tackle some of these issues, such as the organ-on-a-chip technology and adaptation of a hollow-fibre model, as well as the development of media that aim to mimic the niche environments of a CF respiratory tract. It is hoped that by summarizing these opinions and discussing current research, the communication gap between groups can begin to close.

Omadacycline pharmacokinetics/pharmacodynamics in the hollow fiber model and clinical validation of efficacy to treat pulmonary Mycobacterium abscessus disease

BackgroundGuideline-based therapy (GBT) for pulmonary Mycobacterium abscessus (Mab) disease achieves sustained sputum culture conversion (SSCC) rates of 30%; this is reflected by poor efficacy of GBT in the hollow fiber system model of Mab (HFS-Mab), which killed ∼1.22 log10 CFU/mL. This study was performed to determine which clinical dose of omadacycline, a tetracycline antibiotic, should be used in combination therapy to treat pulmonary Mab disease for relapse-free cure. MethodsFirst, omadacycline intrapulmonary concentration-time profiles of seven daily doses were mimicked in the HFS-Mab model and exposures associated with optimal efficacy were identified. Second, 10,000 subject Monte-Carlo simulations were performed to determine whether oral omadacycline 300 mg/day achieved these optimal exposures. Third, a retrospective clinical study on omadacycline vs. primarily tigecycline-based salvage therapy was conducted to assess rates of SSCC and toxicity. Fourth, a single patient was recruited to validate the findings. ResultsOmadacycline efficacy in the HFS-Mab was 2.09 log10 CFU/mL at exposures achieved in >99% of patients on 300 mg/day omadacycline. In the retrospective study of omadacycline 300 mg/day-based combinations vs. comparators, SSCC was achieved in 8/10 vs. 1/9 (P=0.006), symptom improvement in 8/8 vs. 5/9 (P=0.033), toxicity in 0 vs. 9/9 (P<0.001), and therapy discontinuation due to toxicity in 0 vs. 3/9 (P<0.001) cases, respectively. In one prospectively recruited patient, omadacycline 300 mg/day salvage therapy achieved SSCC and symptom-resolution in 3 months. ConclusionBased on the preclinical and clinical data, omadacycline 300 mg/day in combination regimens could be appropriate for testing in Phase III trials in patients with Mab pulmonary disease.

All Roads Lead to Rome: Enhancing the Probability of Target Attainment with Different Pharmacokinetic/Pharmacodynamic Modelling Approaches.

In light of rising antimicrobial resistance and a decreasing number of antibiotics with novel modes of action, it is of utmost importance to accelerate development of novel treatment options. One aspect of acceleration is to understand pharmacokinetics (PK) and pharmacodynamics (PD) of drugs and to assess the probability of target attainment (PTA). Several in vitro and in vivo methods are deployed to determine these parameters, such as time-kill-curves, hollow-fiber infection models or animal models. However, to date the use of in silico methods to predict PK/PD and PTA is increasing. Since there is not just one way to perform the in silico analysis, we embarked on reviewing for which indications and how PK and PK/PD models as well as PTA analysis has been used to contribute to the understanding of the PK and PD of a drug. Therefore, we examined four recent examples in more detail, namely ceftazidime-avibactam, omadacycline, gepotidacin and zoliflodacin as well as cefiderocol. Whereas the first two compound classes mainly relied on the 'classical' development path and PK/PD was only deployed after approval, cefiderocol highly profited from in silico techniques that led to its approval. Finally, this review shall highlight current developments and possibilities to accelerate drug development, especially for anti-infectives.

Potency of the novel PolC DNA polymerase inhibitor CRS0540 in a disseminated Listeria monocytogenes intracellular hollow-fibre model.

BackgroundListeriosis is an orphan disease, which is nevertheless fatal in immunocompromised people. CRS0540 is a novel PolC DNA polymerase inhibitor that has demonstrated good in vitro and in vivo activity against Listeria monocytogenes.MethodsRodent-to-human allometry projection-based human population pharmacokinetics of CRS0540 were used for all studies. CRS0540 pharmacokinetics/pharmacodynamics studies in an intracellular hollow-fibre system model of disseminated listeriosis (HFS-Lister) examined the effect of eight treatment doses, administered daily over 7 days, in duplicate units. Total bacterial burden versus AUC/MIC exposures on each day were modelled using the inhibitory sigmoid Emax model, while CRS0540-resistant bacterial burden was modelled using a quadratic function. Ten thousand-subject Monte Carlo simulations were used to predict an optimal clinical dose for treatment.ResultsThe mean CRS0540 intracellular/extracellular AUC0–24 ratio was 34.07 (standard error: 15.70) as measured in the HFS-Lister. CRS0540 demonstrated exposure-dependent bactericidal activity in the HFS-Lister, with the highest exposure killing approximately 5.0 log10 cfu/mL. The free drug AUC0–24/MIC associated with 80% of maximal kill (EC80) was 36.4. Resistance emergence versus AUC/MIC was described by a quadratic function, with resistance amplification at an AUC/MIC of 54.8 and resistance suppression at an AUC/MIC of 119. Monte Carlo simulations demonstrated that for the EC80 target, IV CRS0540 doses of 100 mg/kg achieved PTAs of >90% at MICs up to 1.0 mg/L.ConclusionsCRS0540 is a promising orphan drug candidate for listeriosis. Future PK/PD studies comparing it with penicillin, the standard of care, could lead to this drug as a new treatment in immunocompromised patients.

Penicillin G concentrations required for prophylaxis against Group A Streptococcus infection evaluated using a hollow fibre model and mathematical modelling.

BackgroundAcute rheumatic fever (ARF), an autoimmune reaction to Group A Streptococcus (Streptococcus pyogenes; Strep A) infection, can cause rheumatic heart disease (RHD). New formulations of long-acting penicillins are being developed for secondary prophylaxis of ARF and RHD.ObjectivesTo evaluate the penicillin G concentrations required to suppress growth of Strep A.MethodsBroth microdilution MIC and MBC for Strep A strains M75611024, M1T15448 and M18MGAS8232 were determined. All strains were studied in a hollow fibre model (initial inoculum 4 log10 cfu/mL). Constant penicillin G concentrations of 0.008, 0.016 and 0.05 mg/L were examined against all strains, plus 0.012 mg/L against M18MGAS8232. Viable counts were determined over 144 h. Subsequently, all penicillin G-treated cartridges were emptied, reinoculated with 5 log10 cfu/mL and counts determined over a further 144 h. Mathematical modelling was performed.ResultsMIC and MBC were 0.008 mg/L for all strains; small subpopulations of M75611024 and M1T15448, but not M18MGAS8232, grew at 1× MIC. Following the first inoculation, 0.008 mg/L achieved limited killing and/or stasis against M75611024 and M1T15448, with subsequent growth to ∼6 log10 cfu/mL. Following both inocula, concentrations ≥0.016 mg/L suppressed M75611024 and M1T15448 to <1 log10 cfu/mL from 6 h onwards with eradication. Concentrations ≥0.008 mg/L suppressed M18MGAS8232 to <1 log10 cfu/mL from 24 h onwards with eradication after both inoculations. Mathematical modelling well described all strains using a single set of parameter estimates, except for different maximum bacterial concentrations and proportions of bacteria growing at 1× MIC.ConclusionsIn the absence of validated animal and human challenge models, the study provides guidance on penicillin G target concentrations for development of new penicillin formulations.

Repurposing Cefazolin-Avibactam for the Treatment of Drug Resistant Mycobacterium tuberculosis.

Background: While tuberculosis (TB) is curable and preventable, the most effective first-line antibiotics cannot kill multi-drug resistant (MDR) Mycobacterium tuberculosis (Mtb). Therefore, effective drugs are needed to combat MDR-TB, especially in children. Our objective was to repurpose cefazolin for MDR-TB treatment in children using principles of pharmacokinetic/pharmacodynamics (PK/PD). Methods: Cefazolin minimum inhibitory concentration (MIC) was identified in 17 clinical Mtb strains, with and without combination of the β-lactamase inhibitor, avibactam. Next, dose-ranging studies were performed using the intracellular hollow fiber model of TB (HFS-TB) to identify the optimal cefazolin exposure. Monte Carlo experiments were then performed in 10,000 children for optimal dose identification based on cumulative fraction of response (CFR) and Mtb susceptibility breakpoint in three age-groups. Results: Avibactam reduced the cefazolin MICs by five tube dilutions. Cefazolin-avibactam demonstrated maximal kill of 4.85 log10 CFU/mL in the intracellular HFS-TB over 28 days. The % time above MIC associated with maximal effect (EC80) was 46.76% (95% confidence interval: 43.04–50.49%) of dosing interval. For 100 mg/kg once or twice daily, the CFR was 8.46 and 61.39% in children <3 years with disseminated TB, 9.70 and 84.07% for 3–5 years-old children, and 17.20 and 76.13% for 12–15 years-old children. The PK/PD-derived susceptibility breakpoint was dose dependent at 1–2 mg/L. Conclusion: Cefazolin-avibactam combination demonstrates efficacy against both drug susceptible and MDR-TB clinical strains in the HFS-TB and could potentially be used to treat children with tuberculosis. Clinical studies are warranted to validate our findings.

First-in-Human Evaluation of the Safety, Tolerability, and Pharmacokinetics of SPR720, a Novel Oral Bacterial DNA Gyrase (GyrB) Inhibitor for Mycobacterial Infections.

ABSTRACTSPR720 (phosphate prodrug of SPR719) is a novel aminobenzimidazole bacterial DNA gyrase (GyrB) inhibitor in development for nontuberculous mycobacterial pulmonary disease (NTM-PD) and pulmonary tuberculosis. SPR719 has demonstrated activity against clinically relevant mycobacteria in vitro and in murine and hollow-fiber infection models. This phase 1 randomized, double-blind, placebo-controlled, single ascending dose (SAD)/multiple ascending dose (MAD) trial evaluated the safety, tolerability, and pharmacokinetics of SPR720/SPR719. A total of 96 healthy volunteers (n = 8/cohort, 3:1 randomization) received SPR720 (or placebo) as single oral doses ranging from 100 to 2,000 mg or repeat total daily doses ranging from 500 to 1,500 mg for 7 or 14 days. SPR720 was well tolerated at daily doses of up to 1,000 mg for up to 14 days. Across SAD/MAD cohorts, the most common adverse events (AEs) were gastrointestinal (nausea, vomiting, and diarrhea) and headache, all of mild or moderate severity and dose dependent. No serious AEs were reported. The median SPR719 Tmax ranged from 2.8 to 8.0 h across cohorts, and the t1/2 ranged from 2.9 to 4.5 h and was shown to be dose independent. Dosing with food decreased SPR719 plasma exposure by approximately 20%. In the MAD cohorts, SPR719 plasma exposure declined approximately 40% between days 1 and 7, suggesting induction of an elimination pathway. However, plasma AUC0–24 was comparable between days 7 and 14. The results of this first-in-human study suggest that predicted therapeutic exposures of SPR719 can be attained with a once-daily oral administration of SPR720. (This study has been registered at ClinicalTrials.gov under registration no. NCT03796910.)

Application of ultra-low concentrations of moderately-hydrophobic chitosan for ultrafiltration membrane fouling mitigation

Membrane fouling is a major obstacle for ultrafiltration (UF) in water treatment. Here, a strategy for fouling mitigation, by applying an ultra-low concentration of moderately-hydrophobic chitosan (MHC) and using a mildly elevated backwash temperature was proposed. The effects of MHC type, concentration, and backwash temperature were investigated in continuous-flow tests using PVDF hollow fiber membranes and model raw water. Compared to the process without MHC, or with the addition of chitosan or conventional coagulant polyaluminum chloride, the MHC-based UF system under optimal conditions (MHC: 0.01 mg/L; backwash water temperature: 40 °C) demonstrated its superiority with reduction of ~55% reversible fouling (RR) and ~80% irreversible fouling (IRR), as well as improved effluent quality. A mechanistic study by experimental analyses and theoretical computations revealed that the improved performance of the MHC-based UF process was attributed to the following: (1) MHC inhibited contaminant accumulation on the membrane through a hydrophilization surface-modification effect on the PVDF; (2) increased backwash temperature promoted fouling release (by destabilizing the cake layer structure) and enhanced the surface-modification effect. The additional cost of MHC dosing and backwash water heating was estimated at ~0.126 Chinese Yuan per ton of produced water, and the environmental risk of applying MHC was lower than that of conventional water treatment chemicals. In summary, the MHC-based process has the potential to be a feasible and cost-effective approach for UF fouling control.

Cefdinir and β-Lactamase Inhibitor Independent Efficacy Against Mycobacterium tuberculosis.

Background: There is renewed interest in repurposing β-lactam antibiotics for treatment of tuberculosis (TB). We investigated efficacy of cefdinir, that withstand the β-lactamase enzyme present in many bacteria, against drug-susceptible and multi-drug resistant (MDR) Mycobacterium tuberculosis (Mtb). Methods: Minimum inhibitory concentration (MIC) experiments were performed with Mtb H37Ra, eight drug-susceptible, and 12 MDR-TB clinical isolates with and without the β-lactamase inhibitor, avibactam at 15 mg/L final concentration. Next, we performed dose-response study with Mtb H37Ra in test-tubes followed by a sterilizing activity study in the pre-clinical hollow fiber model of tuberculosis (HFS-TB) study using an MDR-TB clinical strain. Inhibitory sigmoid Emax model was used to describe the relationship between the drug exposure and bacterial burden. Results: Cefdinir MIC for Mtb H37Ra was 4 and 2 mg/L with or without avibactam, respectively. The MIC of the clinical strains ranged between 0.5 and 16 mg/L. In the test-tube experiments, cefdinir killed 4.93 + 0.07 log10 CFU/ml Mtb H37Ra in 7 days. In the HFS-TB studies, cefdinir showed dose-dependent killing of MDR-TB, without combination of avibactam. The cefdinir PK/PD index linked to the Mtb sterilizing efficacy was identified as the ratio of area under the concentration-time curve to MIC (AUC0–24/MIC) and optimal exposure was calculated as AUC0–24/MIC of 578.86. There was no resistance emergence to cefdinir in the HFS-TB. Conclusion: In the HFS-TB model, cefdinir showed efficacy against both drug susceptible and MDR-TB without combination of β-lactamase inhibitor. However, clinical validation of these findings remains to be determined.

Pharmacodynamic Evaluation of a Single Dose versus a 24-Hour Course of Multiple Doses of Cefazolin for Surgical Prophylaxis.

The optimal perioperative duration for the administration of cefazolin and other prophylactic antibiotics remains unclear. This study aimed to describe the pharmacodynamics of cefazolin for a single 2 g dose versus a 24 h course of a 2 g single dose plus a 1 g eight-hourly regimen against methicillin-susceptible Staphylococcus aureus. Static concentration time–kill assay and a dynamic in vitro hollow-fibre infection model simulating humanised plasma and interstitial fluid exposures of cefazolin were used to characterise the pharmacodynamics of prophylactic cefazolin regimens against methicillin-sensitive Staphylococcus aureus clinical isolates. The initial inoculum was 1 × 105 CFU/mL to mimic a high skin flora inoculum. The static time–kill study showed that increasing the cefazolin concentration above 1 mg/L (the MIC) did not increase the rate or the extent of bacterial killing. In the dynamic hollow-fibre model, both dosing regimens achieved similar bacterial killing (~3-log CFU/mL within 24 h). A single 2 g dose may be adequate when low bacterial burdens (~104 CFU/mL) are anticipated in an immunocompetent patient with normal pharmacokinetics.

Comparison of Rifamycins for Efficacy Against Mycobacterium avium Complex and Resistance Emergence in the Hollow Fiber Model System.

Rifamycins are integral part of the combination regimen for treatment of pulmonary Mycobacterium avium-complex [MAC] infection, but different practitioners prefer different rifamycins. The objective of the study was to compare microbial kill and resistance emergence of rifamycins using principles of pharmacokinetics/pharmacodynamics. First, we identified rifamycin MICs in 20 MAC isolates from patients followed by concentration-response studies in test-tubes. Next, we examined efficacy and resistance suppression of three doses of each rifamycin in the hollow fiber system model of pulmonary MAC [HFS-MAC], mimicking human like concentration-time profile of the drugs. HFS-MAC units were repetitively sampled for total and drug-resistant MAC burden and for drug concentration measurements. Inhibitory sigmoid E max model, linear regression, and analysis of variance was used for data analysis. For rifabutin 90% of isolates had MIC ≤ 0.125 mg/L while for both rifampin and rifapentine this was ≤2.0 mg/L. There was no statistically significant difference (p > 0.05) in maximal kill and effective concentration mediating 50% of the bacterial kill among three rifamycins in the static concentration experiment. In the HFS-MAC, the bactericidal kill (day 0–4) for rifampin was 0.89 (95% Confidence Interval (CI): 0.43–1.35), for rifapentine was 1.05 (95% CI: 0.08–1.23), and for rifabutin was 0.92 (95% CI: 0.61–1.24) log10 CFU/ml, respectively. Rifamycins monotherapy failed after just 4-days of treatment and entire MAC population was drug resistant on day 26 of the study. There was no dose dependent difference in MAC kill or resistance suppression among the three rifamycins tested in the HFS-MAC. Therefore, replacing one rifamycin, due to emergence of drug-resistance, with other may not be beneficial in clinical setting.