Intracellular Pharmacokinetics Research Articles

New rhodol-sulforaphane conjugates as innovative isothiocyanate-based cytotoxic agents for cancer cells

In search of semisynthetic derivatives with increased antitumor activity, we condensed sulforaphane (SFR) with rhodol, a fluorophore platform capable of modifying the intracellular trafficking and pharmacokinetics of the linked molecules. The two tested derivatives, namely MG28 and MG46, showed a far higher, as compared to SFR, cytotoxicity toward cancer cells. Apoptotic cell death was preceded by the extensive generation of DNA lesions, which were repaired relatively slowly and caused formation of micronuclei. Unlike SFR, rhodol-SFR conjugates’ DNA lesions resulted from direct interactions with nuclear DNA. Overall, MG28 and MG46 exhibit a remarkable cytotoxic effect, which is the likely consequence of their direct and intense DNA damaging activity, i.e., a novel and peculiar mechanism arising from the conjugation of the parental rhodol and SFR. Considering that a wide number of clinically used drugs kill cancer cells by inducing DNA damage, MG could represent a new and promising chance in antitumor chemotherapy.

Intracellular Pharmacokinetics of Activated Drugs in a Prodrug-Enzyme-Ultrasound System: Evaluations on ZD2767P+CPG2+US.

Intracellular pharmacokinetics (PK) of activated drugs is a window to understanding the pharmacodynamics of prodrug-enzyme-ultrasound therapy. Herein PK of ZD2767D (i.e., activated drug) in the ZD2767P+CPG2+US system on A549, A549/DDP, SKOV3, and SKOV3/DDP cells were evaluated (A549/DDP and SKOV3/DDP were cisplatin-resistant sublines). The noncompartment model under extravascular input mode was deemed appropriate for evaluating drug level vs time curves; Cmax, AUClast, MRTlast, Vz, and Cl can reflect the PK feature, but t1/2, AUCinf, and MRTinf were irrational; higher accumulation and slower elimination characterized the PK mechanism of ZD2767P+CPG2+US; enhanced permeability and retention effect can be assessed with Cmax, AUClast, MRTlast, and tlast; ultrasound equivalently modulated Cmax and AUClast in sensitive and resistant cells. The experimental design and dose proportionality were discussed.

Development of biotin decorated Olaparib loaded cationic lipopolymeric hybrid nanoparticle and evaluation of its anticancer effect and pharmacokinetics for triple negative breast cancer

Olaparib is a Poly(ADP-ribose) polymerase inhibitor, established for treatment of various cancer. Owing to its poor bioavailability, shorter biological half-life and requirement of frequent dosing of high oral dose, it displays lower therapeutic response and undesired toxicity. Thus, to address these issues, in the present work, a surface charge modified biotinylated lipo-polymeric nanoparticle system for Olaparib was prepared using single-step emulsification followed by solvent evaporation method to enhance the intracellular uptake and pharmacokinetics in biological system. The resulting Olaparib loaded lipo-polymeric hybrid nanoparticle exhibited an average particle size of <150 nm with a surface zeta potential of less than +30 mV. The release profile of Olaparib loaded lipo-polymeric nanoparticle (OLA-LPHNs, St@OLA-LPHNs and St/Biotin@OLA-LPHNs) indicated a controlled type drug release pattern in comparison to pure Olaparib. In-vitro cytotoxicity of olaparib loaded nanoparticle demonstrated an improvement in IC50 of Olaparib by 7.6-fold times in St/Biotin@OLA-LPHNs in 4T1 cell line against free Olaparib. Subsequently, blank nanoparticle showed no cellular death which indicates no significant toxicity of nanocarrier. The apoptosis profile showed that St/Biotin@OLA-LPHNs induced significant apoptosis compared to all other groups i.e., OLA-LPHNs, St@OLA-LPHNs and Olaparib. An improved pharmacokinetic profile was observed in Olaparib loaded lipo-polymeric nanoparticle. Moreover, hematological and histological data revealed that the developed nanoparticles are safe and biocompatible to biological system. In conclusion, in the present study, the developed Olaparib loaded lipo-polymeric nanoparticles demonstrated great potential for controlled release and have opened avenues for developing an effective formulation for improving anticancer effect against triple negative breast cancer.

A highly sensitive stem-loop RT-qPCR method to study siRNA intracellular pharmacokinetics and pharmacodynamics.

Small interfering RNA (siRNA) is a powerful tool for sequence-specific silencing of disease-related genes. In this study, we established and validated a stem-loop reverse transcription-quantitativepolymerase chain reaction (RT-qPCR) method applicable for both chemically unmodified and modified siRNA, aiming to elucidate mechanistic intracellular pharmacokinetic and pharmacodynamic (PK/PD) properties of siRNA. We conducted a comprehensive evaluation of factors affecting intracellular siRNA quantification. Our study revealed that immobilization-based siRNA extraction introduced high variation, making it unsuitable for absolute quantification. Conversely, direct cell lysis followed by stem-loop RT-qPCR demonstrated excellent reproducibility, with a quantification range from 0.0002 to 20 femtomole (fmole) for unmodified siRNA and 0.02 to 20 fmole for modified siRNA. The design of a 6-bp overlapping RT primer facilitated the distinction of full-length antisense from its 3'-metabolites, and pre-annealing of antisense to RT primer enhanced sensitivity and reproducibility. Differences in siRNA loss during storage and sample processing were noted among microcentrifuge tubes from various manufacturers. Endogenous miR-16 served as a reference for normalizing cytoplasmic siRNA, while protein concentration post-immunoprecipitation lysis was used to normalize RNA-induced silencing complex (RISC)-loaded siRNA levels. This method successfully enabled a detailed characterization of the time profiles of cytoplasmic and RISC-loaded siRNA, advancing the in vitro-in vivo translation of siRNA therapeutics.

Pharmacokinetics and pharmacodynamic evaluation of hyaluronic acid-modified imatinib-loaded PEGylated liposomes in CD44-positive Gist882 tumor-bearing mice

To develop a PEGylated and CD44-targeted liposomes, enabled by surface coating with hyaluronic acid (HA) via amide bond to improve the efficacy of imatinib mesylate (IM), for tumor-targeted cytoplasmic drug delivery. HA was covalently grafted on DSPE-PEG2000-NH2 polymer. HA-modified or unmodified PEGylated liposomes were prepared with ethanol injection method, and the stability, drug release, and cytotoxicity of these liposomes were studied. Meanwhile, intracellular drug delivery efficiency, antitumor efficacy, and pharmacokinetics were also investigated. Ex vivo fluorescence biodistribution was also detected by small animal imaging. In addition, endocytosis mechanism was also explored HA-coated PEGylated liposomes (137.5 nm ± 10.24) had a negative zeta potential (−29.3 mV ± 5.44) and high drug loading (27.8%, w/w). The liposomes were stable with cumulative drug leakage (<60%) under physiological conditions. Blank liposomes were nontoxic to Gist882 cells, and IM-loaded liposomes had higher cytotoxicity to Gist882 cells. HA-modified PEGylated liposomes were internalized more effectively than non-HA coating via CD44-mediated endocytosis. Besides, the cellular uptake of HA-modified liposomes also partly depends on caveolin-medicated endocytosis and micropinocytosis. In rats, both liposomes produced a prolonged half-life of IM (HA/Lp/IM: 14.97h; Lp/IM: 11.15h) by 3- to 4.5-folds compared with the IM solution (3.61h). HA-decorated PEGylated liposomes encapsulated IM exhibited strong inhibitory effect on tumor growth in Gist882 cell-bearing nude mice and formation of 2D/3D tumor spheroids. The Ki67 immunohistochemistry result was consistent with the above results. IM-loaded PEGylated liposomes modified with HA exerted the excellent anti-tumor effect on tumor-bearing mice and more drugs accumulated into the tumor site.

Drug content on anticancer efficacy of self-assembling ketal-linked dextran-paclitaxel conjugates.

Although polymer-drug conjugates (PDCs) show great promise as versatile drug delivery systems, no antitumor PDCs based on small-molecule drugs are currently on the market, partly because of the lack of validated design principles for PDCs. High drug content is thought to be essential for devising highly efficacious PDCs based on poorly soluble antitumor drugs, but this has not been well validated. Therefore, revisiting the relationship between drug content and PDC performance is vital. In this study, we synthesized four dextran-paclitaxel (PTX) conjugates (designated as DKPs) with different drug contents by linking dextran and PTX via an acid-responsive ketal, and we used the conjugates to construct self-assembled DKP nanoparticles (NPs) for antitumor therapy. We focused on how PTX content influenced the hydrolysis kinetics, cytotoxicity, cellular uptake and intracellular hydrolysis, pharmacokinetics, biodistribution, and antitumor efficacies of the DKP NPs. We found that DKP NPs with lower PTX content showed accelerated drug release and increased tumor accumulation, and consequently enhanced antitumor efficacy. In 4T1-Luc and Panc02-Luc cancer models, the NPs showed considerably improved therapeutic efficacy than the micellar formulation of PTX that is currently in clinical use. Our results indicate that DKP NPs with lower PTX content possess greater antitumor potential, and our findings offer new insights for the connection of drug content-formulation-bioactivity relationship in the rational design of PDC prodrugs.

LC-QQQ-MS based intracellular quantification of bictegravir in peripheral blood mononuclear cells and plasma

Most antiretrovirals (ARVs) have intracellular therapeutic target sites and therefore, their plasma concentration may be misleading when relating to their efficacy or toxicity. A bioanalytical method for quantification of the ARV drug bictegravir (BTG) in its target site peripheral blood mononuclear cells (PBMCs) is not available till date. This is the first time to establish a sufficiently sensitive mass spectrometry-based bioanalytical method to quantify BTG in both rat PBMCs and plasma. The developed method was validated over the range of 1 ng/ml to 100 ng/ml and 0.005 ng–10ng/sample for plasma and PBMCs, respectively. For PBMCs, average accuracy and precision at four quality control levels were found to be 93.30%–110.00% and 6.52%–8.25%, respectively. Plasma and intracellular pharmacokinetics of BTG was evaluated by the developed method in rats and a lack of accumulation of BTG in the PBMCs was observed. Pearson correlation coefficient data analysis indicated a moderated correlation between plasma and PBMC concentration of BTG. Therefore, it will be beneficial to include a quantification plan for BTG in its actual therapeutic target site during all its future research and development work. This reported method can be useful for site-specific monitoring of BTG in research laboratories and pharmaceutical industries.

Doravirine and Rilpivirine Intra Cellular Accumulation in the Clinical Setting

Background: Doravirine (DOR) and Rilpivirine (RPV) are the NNRTIs currently most used in the clinical setting, in dual and triple drug regimens (2DR and 3DR). Intracellular (IC) Pharmacokinetics (PK) of these drugs has not been fully elucidated. Our aim was to compare plasma PK and IC accumulation in real-life experienced patients (pts). Methods: Pts on DOR- and RPV-including Antiretroviral (ARV) regimen were considered. DOR and RPV plasma and IC (PBMCs) concentrations were measured at 12 (37%) (T12) and 24 ± 4 hours (63%) (T24) after last drug intake by means of UHPLC-MSMS validated methods. Results: 90 pts (65% on 3DR and 35% on 2DR) were included: 52% on DOR- and 48% on RPV-containing ARV. RPV IC/plasma ratio was significantly higher than DOR IC/plasma ratio: 6.034 (4.878-7.186) vs. 1.479 (1.256-1.702) (p=0.001) independently from timing T12 (p=0.003) and T24 (p&lt;0.001). RPV in 3DR resulted to have higher plasma and IC accumulation compared to 2DR. Linear and significative correlations between DOR and RPV plasma and IC concentrations were found (+0.749, p&lt;0.001 and +0.733, p&lt;0.001). No significative correlation between overall DOR and RPV PK and creatinine, BMI or age or difference by gender was found. Conclusion: RPV proved to accumulate in PBMCs at a higher degree as compared to DOR: RPV and DOR IC levels were 498% and 50% higher than in plasma. RPV showed an IC PBMC/plasma ratio 3-fold higher than DOR. Potential explanation could rely on the higher lipophilicity of RPV. Clinical significance of these data needs to be investigated in further studies.

Ultrasonically Enhanced ZD2767P-Carboxypeptidase G2 Deactivates Cisplatin-Resistant Human Lung Cancer Cells.

The prodrug-enzyme regimen ZD2767P+CPG2 is limited by low efficacy. Here, ultrasound was used to modulate ZD2767P+CPG2 (i.e., ZD2767P+CPG2+US) against cisplatin-resistant human lung cancer cells. A549 and A549/DDP (resistant subline) cells received ZD2767P+CPG2 or ZD2767P+CPG2+US. Either ZD2767P+CPG2 or ZD2767P+CPG2+US led to cell death and apoptosis, and ZD2767P+CPG2+US produced stronger effects; comet assays revealed that these two means directly caused DNA double-strand break. Z-VAD-fmk and/or ferrostatin-1 increased the cell survival percentage, and Z-VAD-fmk decreased the apoptosis percentage. The level of transferrin was increased in treated cells, but those of ferroportin and glutathione peroxidase 4 (GPX4) were reduced, with higher intracellular levels of reactive oxygen species and of iron. Intracellular pharmacokinetics of ZD2767D (activated drug) indicated that the peak level, area under the drug level vs. time curve, and mean residence time in ZD2767P+CPG2+US were higher than those in ZD2767P+CPG2. Both ZD2767P+CPG2 and ZD2767P+CPG2+US were effective on xenograft tumors in nude mice; inhibitory rates were 39.7% and 63.5% in A549 tumors and 50.0% and 70.1% in A549/DDP tumors, respectively. A higher apoptosis level and a lower GPX4 level were noted in tumors receiving treatments. No severe adverse events were observed. These data demonstrated that ZD2767P+CPG2+US deactivated cancer cells via apoptosis and ferroptosis pathways, being a candidate therapy for cisplatin-resistant lung cancer.

Doxorubicin Loaded Dextran-coated Superparamagnetic Iron Oxide Na-noparticles with Sustained Release Property: Intracellular Uptake, Phar-macokinetics and Biodistribution Study.

Due to the short biological half-life and serious side effects (especially for heart and kidney), the application of Doxorubicin (Dox) in clinical therapy is strictly limited. To overcome these shortcomings, a novel sustained release formulation of doxorubicin-loaded dextran-coated superparamagnetic iron oxide nanoparticles (Dox-DSPIONs) was prepared. The purpose of this study was to evaluate the intracellular uptake behavior of Dox-DSPIONs and to investigate their pharmacokinetics and biodistribution properties. Confocal laser scanning microscopy was employed to study the intracellular uptake and release properties of Dox from Dox-DSPIONs in SMMC-7721 cells. Simple high-performance liquid chromatography with fluorescence detection (HPLC-FLD) method was established to study the pharmacokinetics and biodistribution properties of Dox-DSPIONs in vivo after intravenous administration and compared with free Dox. Intracellular uptake experiment indicated that Dox could be released sustainedly from Dox-DSPIONs over time. The pharmacokinetics parameters displayed that the T1/2and AUC0-24h of Dox-DSPIONs were higher than those of free Dox, while the Cmax of Dox-DSPIONs was significantly lower than that of free drug. The biodistribution behaviors of the drug were altered by Dox-DSPIONs in mice, which showed obvious liver targeting, and significantly reduced the distribution of the drug in the heart and kidney. Dox-DSPIONs have the sustained-release property in vitro and in vivo, which could significantly prolong blood circulation time, improve bioavailability, and reduce the side effects of Dox. Therefore, the novel formulation of the Dox-DSPIONs has the potential as a promising drug delivery system in cancer therapy.

Determination of the effect of berberine on epirubicin concentration in MCF-7 cells by LC-MS/MS: The mechanism of synergism explained by intracellular pharmacokinetics

This study investigated the inhibitory effect of epirubicin combined with berberine on MCF-7 cell proliferation and explored intracellular pharmacokinetics. A CCK-8 assay was used to detect the inhibitory effect of epirubicin alone and in combination with berberine on MCF-7 cell proliferation in vitro. A rapid and sensitive LC-MS/MS method was developed and validated to detect epirubicin in MCF-7 cells. After incubation with epirubicin and berberine at different time points, MCF-7 cells were lysed and precipitated by acetonitrile to extract the analyte. Chromatographic separation was performed on a C18 column. Daunorubicin was selected as the internal standard. The analytical method was applied to determine the epirubicin concentration in MCF-7 cells. Berberine was found to enhance the inhibitory effect of epirubicin (1 μg/mL and 2 μg/mL) on the proliferation of MCF-7 cells (P < 0.05). LC-MS/MS analysis revealed that berberine significantly increased epirubicin concentration in MCF-7 cells at 8, 12, and 24 h (P < 0.05). The results suggest that berberine may enhance the inhibitory effect of epirubicin on cell proliferation by increasing the concentration of epirubicin in MCF-7 cells. The assay provides a basis for research on the intracellular pharmacokinetics of epirubicin and develops analytical methods for other cell-targeted drugs.

Intracellular islatravir pharmacology differs between species in an in vitro model: implications for preclinical study design.

Islatravir (4'-ethynyl-2-fluoro-2'-deoxyadenosine; EFdA) is a first-in-class nucleoside reverse transcriptase translocation inhibitor (NRTTI) being investigated for HIV treatment and prevention. EFdA is intracellularly phosphorylated to EFdA-triphosphate (EFdA-tp), a competitive substrate of deoxyadenosine-triphosphate (dATP). Thus, translating safety and efficacy findings from preclinical studies relies on the assumption that EFdA's intracellular pharmacology can be extrapolated across species. We investigated how EFdA is phosphorylated across animal species commonly used for preclinical models in drug development to identify those that most closely matched humans. PBMCs were isolated from whole blood of six species (human, rhesus macaque non-human primate (rmNHP), rat, minipig, dog, and rabbit) using Ficoll separation and counted on a haemocytometer by Trypan blue staining. One million live cells were cultured in media supplemented with 10 U/mL human IL-2, 10% FBS and 1% antibiotics and treated with 0, 17, 170, and 1700 nM EFdA (n = 3 replicates per concentration). After 24 h, representative cell counts were derived from untreated control wells (as above), cells were washed in PBS, and lysed with 70:30 methanol:water. EFdA-tp and dATP concentrations were quantified by HPLC-MS/MS and normalized to the representative live cell counts for each species. When compared to human values, EFdA-tp concentrations for each EFdA treatment concentration were lower in all species (rmNHP 1.5-2.1-fold, rat 4.5-15-fold, minipig 37-71-fold, dog and rabbit >100-fold). Additionally, rmNHP and dog PBMCs exhibited significantly higher (7-10-fold; P < 0.001) dATP when compared with human PBMCs. Given intracellular pharmacology differences, these preclinical models may be a conservative estimate of EFdA's intracellular pharmacokinetics and efficacy in humans.

Data-Driven Discovery of Mathematical and Physical Relations in Oncology Data Using Human-Understandable Machine Learning.

For decades, researchers have used the concepts of rate of change and differential equations to model and forecast neoplastic processes. This expressive mathematical apparatus brought significant insights in oncology by describing the unregulated proliferation and host interactions of cancer cells, as well as their response to treatments. Now, these theories have been given a new life and found new applications. With the advent of routine cancer genome sequencing and the resulting abundance of data, oncology now builds an “arsenal” of new modeling and analysis tools. Models describing the governing physical laws of tumor–host–drug interactions can be now challenged with biological data to make predictions about cancer progression. Our study joins the efforts of the mathematical and computational oncology community by introducing a novel machine learning system for data-driven discovery of mathematical and physical relations in oncology. The system utilizes computational mechanisms such as competition, cooperation, and adaptation in neural networks to simultaneously learn the statistics and the governing relations between multiple clinical data covariates. Targeting an easy adoption in clinical oncology, the solutions of our system reveal human-understandable properties and features hidden in the data. As our experiments demonstrate, our system can describe nonlinear conservation laws in cancer kinetics and growth curves, symmetries in tumor’s phenotypic staging transitions, the preoperative spatial tumor distribution, and up to the nonlinear intracellular and extracellular pharmacokinetics of neoadjuvant therapies. The primary goal of our work is to enhance or improve the mechanistic understanding of cancer dynamics by exploiting heterogeneous clinical data. We demonstrate through multiple instantiations that our system is extracting an accurate human-understandable representation of the underlying dynamics of physical interactions central to typical oncology problems. Our results and evaluation demonstrate that, using simple—yet powerful—computational mechanisms, such a machine learning system can support clinical decision-making. To this end, our system is a representative tool of the field of mathematical and computational oncology and offers a bridge between the data, the modeler, the data scientist, and the practicing clinician.

Good response to methotrexate is associated with a decrease in the gene expression of ABCG2, a drug transporter, in patients with rheumatoid arthritis

Objectives Methotrexate (MTX) is used as an anchor drug in the treatment of rheumatoid arthritis (RA), although more than a half of the patients with RA require additional treatments. We designed a prospective study involving two medical centers in Japan to examine the association between the expression of MTX-related genes including a drug transporter ATP-binding cassette sub-family G member 2 (ABCG2) gene and the clinical response to MTX in MTX-naive patients with RA. Methods The primary endpoint of this study was good response based on the European League Against Rheumatism (EULAR) response criteria by Disease Activity Score using 28-joint count (DAS28). We evaluated the association between the baseline expression of six genes involved in the intracellular pharmacokinetics of MTX, including ABCG2, as well as their temporal changes, and the clinical response at week 12 from the initiation of MTX. Results Based on the clinical response at 12 weeks after the initiation of MTX, 24 patients were classified into good responders (n = 9) and non-good responders (n = 15; 10 moderate responders and 5 non-responders) groups. A univariate logistic regression analysis of the baseline gene expression levels to predict the EULAR good response at week 12 showed a significant association with ABCG2 expression alone. Furthermore, the rate of baseline expression of ABCG2 mRNA above the cut-off value determined using a receiver operating characteristic curve was higher in good responders than in non-good responders (p = .012). Moreover, ABCG2 expression decreased in almost all good responders, but not in non-good responders, after MTX treatment for 12 weeks (median −76% vs. +41% from baseline, respectively; p = .011). The ABCG2 expression level did not correlate with DAS28 at baseline or week 12. Conclusions Our study revealed that good response to MTX is associated with a decrease in the expression of ABCG2 in patients with RA.

In vitro antibiotic activity against intraosteoblastic Staphylococcus aureus: a narrative review of the literature.

Staphylococcus aureus – a major aetiological agent of bone and joint infection (BJI) – is associated with a high risk of relapse and chronicity, in part due to its ability to invade and persist in non-professional phagocytic bone cells such as osteoblasts. This intracellular reservoir protects S. aureus from the action of the immune system and most antibiotics. To date, the choice of antimicrobial strategies for BJI treatment mostly relies on standard susceptibility testing, bone penetration of antibiotics and their ‘antibiofilm’ activity. Despite the role of intracellular persistent S. aureus in the development of chronic infection, the ability of antibiotics to target the S. aureus intraosteoblastic reservoir is not considered in therapeutic choices but might represent a key determinant of treatment outcome. This review provides an overview of the intracellular pharmacokinetics of antistaphylococcal drugs used in the treatment of BJI and of their ability to target intraosteoblastic S. aureus. Thirteen studies focusing on the intraosteoblastic activity of antibiotics against S. aureus were reviewed, all relying on in vitro models of osteoblast infection. Despite varying incubation times, multiplicities of infection, bacterial strains, and the types of infected cell lines, rifamycins and fluoroquinolones remain the two most potent antimicrobial classes for intraosteoblastic S. aureus eradication, consistent with clinical data showing a superiority of this combination therapy in S. aureus orthopaedic device-related infections.

Relationship between Plasma and Intracellular Concentrations of Bedaquiline and Its M2 Metabolite in South African Patients with Rifampin-Resistant Tuberculosis.

ABSTRACTBedaquiline is recommended for the treatment of all patients with rifampin-resistant tuberculosis (RR-TB). Bedaquiline accumulates within cells, but its intracellular pharmacokinetics have not been characterized, which may have implications for dose optimization. We developed a novel assay using high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) to measure the intracellular concentrations of bedaquiline and its primary metabolite M2 in patients with RR-TB in South Africa. Twenty-one participants were enrolled and underwent sparse sampling of plasma and peripheral blood mononuclear cells (PBMCs) at months 1, 2, and 6 of treatment and at 3 and 6 months after bedaquiline treatment completion. Intensive sampling was performed at month 2. We used noncompartmental analysis to describe plasma and intracellular exposures and a population pharmacokinetic model to explore the relationship between plasma and intracellular pharmacokinetics and the effects of key covariates. Bedaquiline concentrations from month 1 to month 6 of treatment ranged from 94.7 to 2,540 ng/ml in plasma and 16.2 to 5,478 ng/ml in PBMCs, and concentrations of M2 over the 6-month treatment period ranged from 34.3 to 496 ng/ml in plasma and 109.2 to 16,764 ng/ml in PBMCs. Plasma concentrations of bedaquiline were higher than those of M2, but intracellular concentrations of M2 were considerably higher than those of bedaquiline. In the pharmacokinetic modeling, we estimated a linear increase in the intracellular-plasma accumulation ratio for bedaquiline and M2, reaching maximum effect after 2 months of treatment. The typical intracellular-plasma ratios 1 and 2 months after start of treatment were 0.61 (95% confidence interval [CI]: 0.42 to 0.92) and 1.10 (95% CI: 0.74 to 1.63) for bedaquiline and 12.4 (95% CI: 8.8 to 17.8) and 22.2 (95% CI: 15.6 to 32.3) for M2. The intracellular-plasma ratios for both bedaquiline and M2 were decreased by 54% (95% CI: 24 to 72%) in HIV-positive patients compared to HIV-negative patients. Bedaquiline and M2 were detectable in PBMCs 6 months after treatment discontinuation. M2 accumulated at higher concentrations intracellularly than bedaquiline, supporting in vitro evidence that M2 is the main inducer of phospholipidosis.

Conjugation with nanodiamonds via hydrazone bond fundamentally alters intracellular distribution and activity of doxorubicin

Doxorubicin (DOX) has been widely incorporated in various delivery forms for tareted treatment of malignant tumors such as triple-negative breast cancer (TNBC), with numerous studies reporting higher therapeutic efficacy and lower toxicity at the same time. However, little attention has been paid to whether DOX in a delivery form acts with the same actions and processes as in free form at the cellular level. This question was investigated in the present study wherein DOX conjugated with polyglycerol-coated nanodiamonds through the pH-sensitive hydrazone bond (Nano-DOX) was compared with DOX in free form on the 4T1 mouse TNBC model. We first found Nano-DOX to have a distinct intracellular distribution profile from DOX. Internalized Nano-DOX mainly stayed in the lysosomes slowly releasing DOX into the cytoplasm and then the nucleus whereas DOX displayed both nuclear and lysosomal distribution after cell uptake. Next, Nano-DOX was shown to induce endoplasmic reticulum (ER) stress without substantial DNA damage while DOX caused massive DNA damage as well as ER stress. Consequently, Nano-DOX only caused minimal activation of pro-inflammatory signaling mediated by MAPK/ERK, NF-κB and STAT3 as seen in response to DOX-inflicted DNA damage. Consistently, DOX-induced activities of ABC transporters, CXCL-1, GM-CSF and IL-6, which are tumor protective events downstream to the pro-inflammatory signaling, were also minimal in Nano-DOX-treated cancer cells. These findings are compelling proof that a chemotherapy in nano form can have distinct intracellular pharmacokinetics from its free from, which can result in altered cellular effects of the drug. Implications of these findings are discussed with an emphasis on nano-drug design, tumor pharmacology and chemoresistance.

Pharmacokinetic modelling to estimate intracellular favipiravir ribofuranosyl-5'-triphosphate exposure to support posology for SARS-CoV-2.

ObjectivesFavipiravir has discrepant activity against SARS-CoV-2 in vitro, concerns about teratogenicity and pill burden, and an unknown optimal dose. This analysis used available data to simulate the intracellular pharmacokinetics of the favipiravir active metabolite [favipiravir ribofuranosyl-5′-triphosphate (FAVI-RTP)].MethodsPublished in vitro data for intracellular production and elimination of FAVI-RTP in Madin–Darby canine kidney cells were fitted with a mathematical model describing the time course of intracellular FAVI-RTP as a function of favipiravir concentration. Parameter estimates were then combined with a published population pharmacokinetic model in Chinese patients to predict human intracellular FAVI-RTP. In vitro FAVI-RTP data were adequately described as a function of concentrations with an empirical model, noting simplification and consolidation of various processes and several assumptions.ResultsParameter estimates from fittings to in vitro data predict a flatter dynamic range of peak to trough for intracellular FAVI-RTP (peak to trough ratio of ∼1 to 1) when driven by a predicted free plasma concentration profile, compared with the plasma profile of parent favipiravir (ratio of ∼2 to 1). This approach has important assumptions, but indicates that, despite rapid clearance of the parent from plasma, sufficient intracellular FAVI-RTP may be maintained across the dosing interval because of its long intracellular half-life.ConclusionsPopulation mean intracellular FAVI-RTP concentrations are estimated to be maintained above the Km for the SARS-CoV-2 polymerase for 9 days with a 1200 mg twice-daily regimen (following a 1600 mg twice-daily loading dose on day 1). Further evaluation of favipiravir as part of antiviral combinations for SARS-CoV-2 is warranted.

Determination of tigecycline in human lung epithelial cells and polymorphonuclear neutrophils by liquid chromatography/tandem mass spectrometry and its application in a cellular pharmacokinetics study.

In order to characterize the intracellular pharmacokinetic properties of tigecycline, we developed and fully validated a liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for quantification of tigecycline in human lung epithelial (BEAS-2B) cells and polymorphonuclear neutrophils (PMNs). Tetracycline was used as an internal standard and chromatographic separation was achieved on a C18 Hypersil Gold aQ column using two mobile phases, a solution of water (containing 0.1% formic acid) and acetonitrile. The flow rate was 0.4mL/min for 5.0min. Tigecycline drug uptake was evaluated by incubating the BEAS-2B cells and the PMNs for up to 3h at tigecycline concentrations of 1mg/L. The assay was linear over the tested concentration range of 0.01-2mg/L for tigecycline in BEAS-2B cells and PMNs (r2 >0.99). The inter- and inter-day precisions (RSD, %) were <10.02% and the accuracies (%) were within the range of 85-115%. The uptake study showed that after incubation with tigecycline (1mg/L) for 3h at 37°C, the intracellular peak concentration of BEAS-2B cells was 14.44 ± 7.12 mg/L at 1 h, and 41.43 ± 25.66 mg/L in PMNs at 20 min. The mean intracellular concentrations fluctuated in the range of 0.8-14.44 mg/L in BEAS-2B cells and 10.14-41.43 mg/L in PMNs for 1mg/L tigecycline exposure. Validated LC/MS/MS is a simple, rapid, and sensitive method for determining the intracellular concentration of tigecycline, and tigecycline has good penetrations both in human BEAS-2B cells and PMNs. The method can be efficiently used for future studies of the intracellular pharmacokinetics of tigecycline.

Zebularine suppressed gemcitabine-induced senescence and improved the cellular and plasma pharmacokinetics of gemcitabine, augmented by liposomal co-delivery

Chemoresistance is a major factor driving cancer recurrence. This study investigated the potential of zebularine, a dual cytidine deaminase (CDA)/epigenetic inhibitor, to circumvent gemcitabine-resistance in pancreatic cancer using a nanomedicine co-delivery approach. The mRNA expression of key metabolic enzymes, including CDA for gemcitabine deactivation in a gemcitabine-resistant cell line Gr2000 and its parental MIA PaCa-2 was compared using quantitative reverse transcription polymerase chain reaction. A highly gemcitabine-resistant population (HRP) in Gr2000 were characterised for their growth pattern, β-galactosidase activity (a hallmark of senescence) and chemosensitivity to zebularine after isolation. The CDA inhibition effects of zebularine on the intracellular gemcitabine accumulation and pharmacokinetics in rats when co-delivered with pH-sensitive liposomes (pSL) were investigated. Gr2000 had a 3-time upregulated mRNA expression and enzyme activity for CDA. The HRP (28% of bulk Gr2000) were predominately senescent cells which re-proliferated following a growth arrest for a week. Zebularine suppressed the regrowth of senescent cells, meanwhile enhanced cellular gemcitabine concentration by 2-fold. When co-delivered with pSL, zebularine increased cellular gemcitabine concentration by 4-fold, and extended the half-life of gemcitabine in plasma by 22-fold in rats. In conclusion, multiple mechanisms including therapy-induced senescence were identified with gemcitabine-resistance. Co-delivery of zebularine using liposomes could provide multifaceted benefits in gemcitabine therapy for pancreatic cancer treatment.