The systematic development of attributes and levels for a discrete choice experiment of HIV patient preferences for long-acting antiretroviral therapies in the United States.

INTRODUCTION: Patient preferences for long-acting antiretroviral therapies (LA-ART) should inform development of regimens with optimal adherence and acceptability. We describe a systematic process used to identify attributes and levels for a discrete choice experiment (DCE) designed to elicit preferences for potential LA-ART options in the US. METHODS: Our approach was conducted in four stages: data collection, data reduction, removing inappropriate attributes, and optimizing wording. We started with 8 attributes defining potential LA-ART products based on existing literature and knowledge of products in development. We conducted 12 key informant interviews with experts in HIV treatment. The list of attributes, the set of plausible levels for each attribute, and restrictions on combinations of attribute levels were updated iteratively. RESULTS: Despite uncertainty about which products will become available, key informant discussions converged on 4 delivery modes (infusions and patches were not considered immediately feasible) and 6 additional attributes. Treatment effectiveness and frequency of clinical monitoring were dropped. Oral lead-in therapy was split into two attributes: pre-treatment time undetectable and pre-treatment negative reaction testing. We omitted product-specific systemic and local side effects. In addition to mode, the final set of attributes included: frequency of dosing; location of treatment; pain; pre-treatment time undetectable; pre-treatment negative reaction testing; and late-dose leeway. CONCLUSIONS: A systematic process successfully captured elements that are both feasible and relevant to evaluating the acceptability of potential LA-ART alternatives to patients.

Thirty-day rat toxicity study reveals reversible liver toxicity of mifepristone (RU486) and metapristone.

OBJECTIVE: Mifepristone (RU486) is an oral first-line contraceptive used by hundreds of millions of women, and recently it was tested for anticancer activity in both genders worldwide. We are developing metapristone (the N-monodemethyl RU486) as a potential metastasis chemopreventive. The present acute and 30-d subacute toxicity study aimed at examining and compared in parallel the potential toxicity of the two drugs. METHODS: The single-dose acute toxicity and 30-d subacute toxicity studies were conducted in mice and rats, respectively, by gavaging metapristone or mifepristone at various doses. Blood samples and organs were collected for blood chemistry, hematology and histology analyses. RESULTS: Oral mifepristone (3000 mg/kg) caused 30% and 40% death in female and male mice, respectively, within 15 h post-dosing. In comparison, the same dose of metapristone produced 30% acute death in males only. Thirty-day oral administration of the two drugs to rats (12.5, 50 and 200 mg/kg/day) caused reversible hepatotoxicity that only occurred at 200 mg/kg/day group, evidenced by the elevated liver enzyme activity and liver organ weight. CONCLUSION: The present study, for the first time, reveals reversible hepatotoxicity in rats caused by the 30-d consecutive administration at the high dose, and warns the potential hepatotoxicity caused by long-term administrations of high doses of mifepristone or metapristone in clinical trials but not by the acute single abortion doses.

A simple, efficient, and sensitive method for simultaneous detection of anti-HIV drugs atazanavir, ritonavir, and tenofovir by use of liquid chromatography-tandem mass spectrometry.

In the treatment of HIV infection, a combination of anti-HIV drugs is commonly used in highly active antiretroviral therapy (HAART). One such combination recommended for clinical therapy consists of the two HIV protease inhibitors atazanavir and ritonavir and the HIV nucleotide reverse transcriptase inhibitor tenofovir. The detection of plasma and cell drug concentrations provides an assessment of actual drug exposure and patient compliance. We thus developed a simple, efficient, and sensitive method to simultaneously extract and detect these three drugs in plasma and peripheral blood mononuclear cells. The use of a liquid-liquid extraction followed by protein precipitation provided a simple process, yielding a high recovery rate for all three drugs in plasma (>92%) and in cells (>86%). The liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay was able to detect 0.01, 0.25, and 2.5 pg (2, 50, and 500 pg/ml, respectively) in 5 µl for atazanavir, ritonavir, and tenofovir, respectively. Validation of the method exhibited high precision and accuracy. This method was subsequently applied to a primate study to determine the concentrations of all three drugs in both plasma and cell samples. This validated method can be useful for an evaluation of drug concentrations in biological samples in an efficient and sensitive manner.

Guanfu base A, an antiarrhythmic alkaloid of Aconitum coreanum, Is a CYP2D6 inhibitor of human, monkey, and dog isoforms.

Guanfu base A (GFA) is a novel heterocyclic antiarrhythmic drug isolated from Aconitum coreanum (Lèvl.) rapaics and is currently in a phase IV clinical trial in China. However, no study has investigated the influence of GFA on cytochrome P450 (P450) drug metabolism. We characterized the potency and specificity of GFA CYP2D inhibition based on dextromethorphan O-demethylation, a CYP2D6 probe substrate of activity in human, mouse, rat, dog, and monkey liver microsomes. In addition, (+)-bufuralol 1'-hydroxylation was used as a CYP2D6 probe for the recombinant form (rCYP2D6), 2D1 (rCYP2D1), and 2D2 (rCYP2D2) activities. Results show that GFA is a potent noncompetitive inhibitor of CYP2D6, with inhibition constant Ki = 1.20 ± 0.33 µM in human liver microsomes (HLMs) and Ki = 0.37 ± 0.16 µM for the human recombinant form (rCYP2D6). GFA is also a potent competitive inhibitor of CYP2D in monkey (Ki = 0.38 ± 0.12 µM) and dog (Ki = 2.4 ± 1.3 µM) microsomes. However, GFA has no inhibitory activity on mouse or rat CYP2Ds. GFA did not exhibit any inhibition activity on human recombinant CYP1A2, 2A6, 2C8, 2C19, 3A4, or 3A5, but showed slight inhibition of 2B6 and 2E1. Preincubation of HLMs and rCYP2D6 resulted in the inactivation of the enzyme, which was attenuated by GFA or quinidine. Beagle dogs treated intravenously with dextromethorphan (2 mg/ml) after pretreatment with GFA injection showed reduced CYP2D metabolic activity, with the Cmax of dextrorphan being one-third that of the saline-treated group and area under the plasma concentration-time curve half that of the saline-treated group. This study suggests that GFA is a specific CYP2D6 inhibitor that might play a role in CYP2D6 medicated drug-drug interaction.

Interactions of indocyanine green and lipid in enhancing near-infrared fluorescence properties: the basis for near-infrared imaging in vivo.

Indocyanine green (ICG) is a near-infrared (NIR) contrast agent commonly used for in vivo cardiovascular and eye imaging. For medical diagnosis, ICG is limited by its aqueous instability, concentration-dependent aggregation, and rapid degradation. To overcome these limitations, scientists have formulated ICG in various liposomes, which are spherical lipid membrane vesicles with an aqueous core. Some encapsulate ICG, while others mix it with liposomes. There is no clear understanding of lipid-ICG interactions. Therefore, we investigated lipid-ICG interactions by fluorescence and photon correlation spectroscopy. These data were used to design stable and maximally fluorescent liposomal ICG nanoparticles for NIR optical imaging of the lymphatic system. We found that ICG binds to and is incorporated completely and stably into the lipid membrane. At a lipid:ICG molar ratio of 250:1, the maximal fluorescence intensity was detected. ICG incorporated into liposomes enhanced the fluorescence intensity that could be detected across 1.5 cm of muscle tissue, while free ICG only allowed 0.5 cm detection. When administered subcutaneously in mice, lipid-bound ICG in liposomes exhibited a higher intensity, NIR image resolution, and enhanced lymph node and lymphatic vessel visualization. It also reduced the level of fluorescence quenching due to light exposure and degradation in storage. Lipid-bound ICG could provide additional medical diagnostic value with NIR optical imaging for early intervention in cases of lymphatic abnormalities.

Novel liquid chromatography-tandem mass spectrometry method for simultaneous detection of anti-HIV drugs Lopinavir, Ritonavir, and Tenofovir in plasma.

For HIV infection, anti-HIV drug combinations are typically used as highly active antiretroviral therapy (HAART), intended to maximize viral suppression. Three drugs used frequently in combination are the protease inhibitors lopinavir and ritonavir and the nucleotide reverse transcriptase inhibitor tenofovir. We have successfully developed a simple, efficient, and sensitive method to simultaneously extract and determine the concentrations of lopinavir, ritonavir, and tenofovir in plasma samples. The plasma extractions were performed using a liquid-liquid extraction followed by protein precipitation of the remaining aqueous layer. The collected fractions were combined, dried, and reconstituted in the mobile phase. The drugs were quantified using liquid chromatography coupled with tandem mass spectrometry. The assay was applied to a study of plasma drug levels in two primates (Macaca nemestrina). The bioanalytical assay was optimized and validated to exhibit a high extraction efficiency and good sensitivity and reproducibility. When the assay was applied in a primate study, all three drugs could be detected in plasma within minutes of subcutaneous dosing. This validated assay will be useful for evaluation of drug concentrations in an efficient, selective, and sensitive manner.

Physiologically based Pharmacokinetic Model Validated to Enable Predictions Of Multiple Drugs in a Long-acting Drug-combination Nano-Particles (DcNP): Confirmation with 3 HIV Drugs, Lopinavir, Ritonavir, and Tenofovir in DcNP Products.

Drug-Combination Nanoparticles (DcNP) are a novel drug delivery system designed for synchronized delivery of multiple drugs in a single, long-acting, and targeted dose. Unlike depot formulations, slowly releasing drug at the injection site into the blood, DcNP allows multiple-drug-in-combination to collectively distribute from the injection site into the lymphatic system. Two distinct classes of long-acting injectables products are proposed based on pharmacokinetic mechanisms. Class I involves sustained release at the injection site. Class II involves a drug-carrier complex composed of lopinavir, ritonavir, and tenofovir uptake and retention in the lymphatic system before systemic access as a part of the PBPK model validation. For clinical development, Class II long-acting drug-combination products, we leverage data from 3 nonhuman primate studies consisting of nine PK datasets: Study 1, varying fixed-dose ratios; Study 2, short multiple dosing with kinetic tails; Study 3, long multiple dosing (chronic). PBPK validation criteria were established to validate each scenario for all drugs. The models passed validation in 8 of 9 cases, specifically to predict Study 1 and 2, including PK tails, with ritonavir and tenofovir, fully passing Study 3 as well. PBPK model for lopinavir in Study 3 did not pass the validation due to an observable time-varying and delayed drug accumulation, which likely was due to ritonavir's CYP3A inhibitory effect building up during multiple dosing that triggered a mechanism-based drug-drug interaction (DDI). Subsequently, the final model enables us to account for this DDI scenario.

Inoculation of Pan02 cells produces tumor nodules in mouse pancreas: Characterization of a novel orthotopic pancreatic ductal adenocarcinoma tumor model for interventional studies.

Preclinical models of cancer are vital for assessing and predicting efficacies and toxicities of novel treatments prior to testing in human subjects. Current pancreatic tumor models exhibit variable growth rates, unpredictable tumor size after implantation in non-native tissues, or require surgical implantation. Surgical implantation in the pancreas may produce not only unpredictable tumor uptake but could also elicit additional inflammatory responses. In searching for a pancreatic carcinoma cell that can be introduced into a mouse via simple injection, we found that Pan02, a murine ductal pancreatic adenocarcinoma derived from a pancreatic lesion of a C57BL/6 mouse, inoculated peritoneally can consistently produce pancreatic tumors. This intraperitoneal, but not intravenous, introduction of Pan02 cells leads to the attachment and growth of Pan02 in the pancreas before spreading to other tissues. Time-course tissue analysis indicates that the Pan02 cells first find, infiltrate, and grow within the pancreas, producing a pancreatic tumor model. This model appears to mimic pancreatic cancer development in humans and is the first reported use of Pan02 cells to produce orthotopic pancreatic and metastatic neoplasms in a mouse model without the need for tumor implantation within matrices or survival surgeries. This orthotopic pancreatic tumor model, with consistent tumor uptake, synchronized tumor development and survival, and predictable outcomes may enable and accelerate the preclinical evaluation of treatment candidates for pancreatic cancer.

Design and Characterization of a Novel Venetoclax-Zanubrutinib Nano-Combination for Enhancing Leukemic Cell Uptake and Long-Acting Plasma Exposure.

Leukemia remains incurable partly due to difficulties in reaching and maintaining therapeutic drug concentrations in the target tissues and cells. Next-generation drugs targeted to multiple cell checkpoints, including the orally active venetoclax (Bcl-2 target) and zanubrutinib (BTK target), are effective and have improved safety and tolerability compared to conventional, nontargeted chemotherapies. However, dosing with a single agent frequently leads to drug resistance; asynchronous coverage due to the peak-and-trough time-course of two or more oral drugs has prevented drug combinations from simultaneously knocking out the respective drugs' targets for sustained leukemia suppression. Higher doses of the drugs may potentially overcome asynchronous drug exposure in leukemic cells by saturating target occupancy, but higher doses often cause dose-limiting toxicities. To synchronize multiple drug target knockout, we have developed and characterized a drug combination nanoparticle (DcNP), which enables the transformation of two short-acting, orally active leukemic drugs, venetoclax and zanubrutinib, into long-acting nanoformulations (VZ-DCNPs). VZ-DCNPs exhibit synchronized and enhanced cell uptake and plasma exposure of both venetoclax and zanubrutinib. Both drugs are stabilized by lipid excipients to produce the VZ-DcNP nanoparticulate (d ~ 40 nm) product in suspension. The VZ-DcNP formulation has enhanced uptake of the two drugs (VZ) in immortalized leukemic cells (HL-60), threefold over that of its free drug counterpart. Additionally, drug-target selectivity of VZ was noted with MOLT-4 and K562 cells that overexpress each target. When given subcutaneously to mice, the half-lives of venetoclax and zanubrutinib were extended by approximately 43- and 5-fold, respectively, compared to an equivalent free VZ. Collectively, these data suggest that VZ in VZ-DcNP warrant consideration for preclinical and clinical development as a synchronized and long-acting drug-combination for the treatment of leukemia.

A novel formulation enabled transformation of 3-HIV drugs tenofovir-lamivudine-dolutegravir from short-acting to long-acting all-in-one injectable.

OBJECTIVE: To develop an injectable dosage form of the daily oral HIV drugs, tenofovir (T), lamivudine (L), and dolutegravir (D), creating a single, complete, all-in-one TLD 3-drug-combination that demonstrates long-acting pharmacokinetics. DESIGN: Using drug-combination-nanoparticle (DcNP) technology to stabilize multiple HIV drugs, the 3-HIV drugs TLD, with disparate physical-chemical properties, are stabilized and assembled with lipid-excipients to form TLD-in-DcNP . TLD-in-DcNP is verified to be stable and suitable for subcutaneous administration. To characterize the plasma time-courses and PBMC concentrations for all 3 drugs, single subcutaneous injections of TLD-in-DcNP were given to nonhuman primates (NHP, M. nemestrina ). RESULTS: Following single-dose TLD-in-DcNP , all drugs exhibited long-acting profiles in NHP plasma with levels that persisted for 4 weeks above predicted viral-effective concentrations for TLD in combination. Times-to-peak were within 24 hr in all NHP for all drugs. Compared to a free-soluble TLD, TLD-in-DcNP provided exposure enhancement and extended duration 7.0-, 2.1-, and 20-fold as AUC boost and 10-, 8.3-, and 5.9-fold as half-life extension. Additionally, DcNP may provide more drug exposure in cells than plasma with PBMC-to-plasma drug ratios exceeding one, suggesting cell-targeted drug-combination delivery. CONCLUSIONS: This study confirms that TLD with disparate properties can be made stable by DcNP to enable TLD concentrations of 4 weeks in NHP. Study results highlighted the potential of TLD-in-DcNP as a convenient all-in-one, complete HIV long-acting product for clinical development.

U.S. patient preferences for long-acting HIV treatment: a discrete choice experiment.

INTRODUCTION: Recent advances in long-acting antiretroviral therapy (LA-ART) could provide new options for HIV treatment and reduce adherence barriers, if regimens are acceptable to patients. We elicited preferences for key attributes of potential LA-ART regimens among people with HIV (PWH) in the United States, focusing on four treatment modes (oral tablets, subcutaneous injections, intramuscular injections, and implants), product characteristics and location of administration. METHODS: A discrete choice experiment was conducted among PWH aged ≥18 years recruited from HIV clinics in Washington State and Atlanta, Georgia from March 2021 to June 2022. Participants responded to 17 choice scenarios, each with three options: two systematically generated hypothetical LA-ART regimens and a constant opt-out (their current daily oral treatment). LA-ART regimen descriptions included treatment mode, pain, dosing frequency, location, pre-treatment time with undetectable viral load, pre-treatment negative reaction testing and "late-dose leeway" (i.e. flexibility or forgiveness in timing the next dose). We used conditional logistic regression, with an interaction between treatment mode and pain, to estimate preference weights for all attribute levels. RESULTS: Seven hundred participants (350 at each site) enrolled, with median age 51 years (range 18-73); 70% identified as cisgender male, 24% as cisgender female and 6% as non-binary or transgender. LA oral tablets were the only mode preferred over current daily oral treatment, with annual implants and injections the next most preferred LA-ART option. Longer time between doses was preferred, and administration at home was preferred to clinics, which were preferred to pharmacies. Attributes with less impact on preferences included oral lead-in treatment to achieve viral suppression or test for negative reactions and late-dose leeway around the prescribed dosing interval. Participants in Atlanta were more likely to prefer their current daily oral ART than participants from Seattle. CONCLUSIONS: PWH in the United States may soon have several options for LA-ART. Our results suggest that LA oral tablets will be preferred by many patients over their current daily oral treatment, while implants and injections with longer duration may be acceptable to some. Future research should investigate sources of preference heterogeneity and actual uptake of and adherence to LA-ART products, when available.

Viral dissemination and immune activation modulate antiretroviral drug levels in lymph nodes of SIV-infected rhesus macaques.

Introduction and methods: To understand the relationship between immunovirological factors and antiretroviral (ARV) drug levels in lymph nodes (LN) in HIV therapy, we analyzed drug levels in twenty-one SIV-infected rhesus macaques subcutaneously treated with daily tenofovir (TFV) and emtricitabine (FTC) for three months. Results: The intracellular active drug-metabolite (IADM) levels (TFV-dp and FTC-tp) in lymph node mononuclear cells (LNMC) were significantly lower than in peripheral blood mononuclear cells (PBMC) (P≤0.005). Between Month 1 and Month 3, IADM levels increased in both LNMC (P≤0.001) and PBMC (P≤0.01), with a steeper increase in LNMC (P≤0.01). The viral dissemination in plasma, LN, and rectal tissue at ART initiation correlated negatively with IADM levels at Month 1. Physiologically-based pharmacokinetic model simulations suggest that, following subcutaneous ARV administration, ART-induced reduction of immune activation improves the formation of active drug-metabolites through modulation of kinase activity and/or through improved parent drug accessibility to LN cellular compartments. Conclusion: These observations have broad implications for drugs that need to phosphorylate to exert their pharmacological activity, especially in the settings of the pre-/post-exposure prophylaxis and efficacy of antiviral therapies targeting pathogenic viruses such as HIV or SARS-CoV-2 replicating in highly inflammatory anatomic compartments.

Development and characterization of transgenic mouse models for conditional gene knockout in the blood-brain and blood-CSF barriers.

For many CNS acting drugs, penetration into the central nervous system (CNS) is limited by the blood-CNS-barriers. In an effort to quantitate the role of the protein components that make up the blood-CNS-barriers, we created transgenic mice that allow conditional gene knockout using Cre/loxP technology. We targeted the expression of Cre-recombinase to the choroid plexus (the blood-cerebral spinal fluid barrier) using the lymphotropic papovavirus control region (LPVcr) and to brain endothelium (the blood-brain-barrier) using the proximal promoter region of the human von Willebrand Factor gene (hVWF-f). We verified that LPVcr restricts expression to the choroid plexus in adult mice by using the LPVcr to drive n-LacZ expression in transgenic mice. The LPV-Cre and hVWF-Cre plasmids were then constructed and tested for Cre-recombinase function in vitro, and subsequently used to create transgenic mice. The resulting transgenic mice were characterized for cell-type specific Cre-mediated endonuclease activity by crossing them with transgenic mice containing a loxP-flanked-LacZ/EGFP dual reporter gene Z/EG. The dual Cre-Z/EG transgenic offspring were evaluated for the location of EGFP mRNA expression by reverse transcriptase PCR and for protein expression by immunohistochemistry. Immunohistochemistry for EGFP verified expression in the target cells, and no ectopic expression outside of the expected cell types. The LPV-Cre.0607 transgenic line expressed functional Cre only in the choroid plexus and hVWF-Cre.1304 line in brain endothelium.

Elucidation of the time course of adenosine deaminase APOBEC3G and viral infectivity factor vif in HIV-2(287) -infected infant macaques.

BACKGROUND: Although the interactions of cellular cytidine deaminase A3G and viral infection factor (vif) of human immunodeficiency virus (HIV) were reported, regulation of A3G after in vivo HIV infection and disease progression is not known. METHODS: Time courses of plasma virus, CD4(+) T lymphocyte Macaca levels, and concentrations of A3G and vif transcripts were determined in infant macaques infected with HIV-2(287) . These in vivo results were compared with those collected in vitro in HIV-2-infected T cells. RESULTS: Human immunodeficiency virus-infected macaques exhibited plasma viremia (≥10(8) copies/ml) followed by a precipitous CD4(+) T-cell (from 40-70 to ≤5%) decline. An initial increase in A3G transcripts coincides with early increases in virus and vif RNA. As virus load continues to increase, A3G RNA decreases but recovers at a later phase as virus level stabilizes. Pearson correlation analysis revealed strong interactions of A3G-CD4, vif-CD4, and A3G-vif. CONCLUSIONS: There is a time-dependent A3G and vif RNA interaction throughout the course of HIV infection.

Physiologically Based Pharmacokinetic Modeling of 3 HIV Drugs in Combination and the Role of Lymphatic System after Subcutaneous Dosing. Part 2: Model for the Drug-combination Nanoparticles.

We previously developed a mechanism-based pharmacokinetic (MBPK) model to characterize the PK of a lymphocyte-targeted, long-acting 3 HIV drug-combination nanoparticle (DcNP) formulation of lopinavir, ritonavir, and tenofovir. MBPK describes time-courses of plasma drug concentration and has provided an initial hypothesis for the lymphatic PK of DcNP. Because anatomical and physiological interpretation of MBPK is limited, in this Part 2, we report the development of a Physiologically Based Pharmacokinetic (PBPK) model for a detailed evaluation of the systemic and lymphatic PK of drugs associated with DcNP. The DcNP model is linked to the PBPK model presented earlier in Part 1 to account for the disposition of released free drugs. A key feature of the DcNP model is the uptake of the injected dose from the subcutaneous site to the adjacent lymphoid depot, routing through the nodes within and throughout the lymphatic network, and its subsequent passage into the blood circulation. Furthermore, the model accounts for DcNP transport to the lymph by lymphatic recirculation and mononuclear cell migration. The present PBPK model can be extended to other nano-drug combinations that target or transit through the lymphatic system. The PBPK model may allow scaling and prediction of DcNP PK in humans.

Physiologically Based Pharmacokinetic Modeling of 3 HIV Drugs in Combination and the Role of Lymphatic System after Subcutaneous Dosing. Part 1: Model for the Free-Drug Mixture.

Drug-combination nanoparticles (DcNP) allow the formulation of multiple HIV drugs in one injectable. In nonhuman primates (NHP), all drugs in DcNP have demonstrated long-acting pharmacokinetics (PK) in the blood and lymph nodes, rendering it suitable for a Targeted Long-acting Antiretroviral Therapy (TLC-ART). To support the translation of TLC-ART into the clinic, the objective is to present a physiologically based PK (PBPK) model tool to control mechanisms affecting the rather complex DcNP-drug PK. Two species contribute simultaneously to the drug PK: drugs that dissociate from DcNP (Part 1) and drugs retained in DcNP (Part 2, presented separately). Here, we describe the PBPK modeling of the nanoparticle-free drugs. The free-drug model was built on subcutaneous injections of suspended lopinavir, ritonavir, and tenofovir in NHP, and validated by external experiments. A novelty was the design of a lymphatic network as part of a whole-body PBPK system which included major lymphatic regions: the cervical, axillary, hilar, mesenteric, and inguinal nodes. This detailed/regionalized description of the lymphatic system and mononuclear cells represents an unprecedented level of prediction that renders the free-drug model extendible to other small-drug molecules targeting the lymphatic system at both the regional and cellular levels.

Challenges and opportunities in metastatic breast cancer treatments: Nano-drug combinations delivered preferentially to metastatic cells may enhance therapeutic response.

Despite advances in breast cancer treatments and related 5-year survival outcomes, metastatic breast cancer cures remain elusive. The current standard of care includes a combination of surgery, radiation therapy and drug therapy. However, even the most advanced procedures and treatments do not prevent breast cancer recurrence and metastasis. Once metastasis occurs, patient prognosis is poor. Recent elucidation of the spatiotemporal transit of metastatic cancer cells from primary tumor sites to distant sites provide an opportunity to integrate knowledge of drug disposition in our effort to enhance drug localization and exposure in cancer laden tissues . Novel technologies have been developed, but could be further refined to facilitate the distribution of drugs to target cancer cells and tissues. The purpose of this review is to highlight the challenges in metastatic breast cancer treatment and focus on novel drug combination and nanotechnology approaches to overcome the challenges. With improved definition of metastatic tissue target, directed localization and retention of multiple, pharmacologically active drugs to tissues and cells of interest may overcome the limitations in breast cancer treatment that may lead to a cure for breast cancer.

Prediction of the potential mechanism of compound gingerol against liver cancer based on network pharmacology and experimental verification.

OBJECTIVES: To explore gingerol's potential mechanism for treating liver cancer using network pharmacology and molecular docking technology and to conduct in-vitro experiments of human liver cancer cell HepG2 to verify important signalling pathways. METHODS: We obtained potential targets of gingerol derivatives (6-gingerol, 8-gingerol and 10-gingerol) from PubChem and SwissTargetPrediction websites and collected related targets for liver cancer with the help of GeneCards. We performed Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis on key targets using the DAVID data platform and combined with Cytoscape 3.7.1 software to construct a component-target-signal pathway interaction map to study its mechanism of action. Subsequently, the components and key proteins were molecularly docked through Autodock Vina software. Finally, the important signal pathways were verified by HepG2 cell in-vitro experiments. KEY FINDINGS: A total of 318 drug targets were screened for gingerol derivatives, and 2509 gene targets related to liver cancer were collected. The Venn diagram showed that there were 104 intersection targets between gingerol derivatives and liver cancer. Module analysis results show that these intersection targets can be divided into 5 modules and 49 nodes. Bioinformatics analysis found that GO obtained 20 important functional items including cancer cell proliferation, protein kinase activity, phosphotransferase activity and kinase activity; KEGG enrichment analysis yielded a total of 20 key signal pathways including the PI3K-Akt signalling pathway. The results of molecular docking show that the binding energy of gingerol derivatives has good binding activity with PI3K and Akt. In-vitro experimental results show that gingerol derivatives and compound gingerol (compound gingerol is composed of 6-gingerol, 8-gingerol and 10-gingerol in a ratio of 7:1.5:1.5) can produce HepG2 cell proliferation inhibition, and each administration group can significantly increase the apoptosis rate of HepG2 cells and the fluorescence intensity of the nucleus and block the cell cycle in the S phase; the results of Western Blot and real-time quantitative PCR show that gingerol derivatives and compound gingerol can down-regulate the expression of Akt and p-Akt and up-regulate the expression of Bax/Bcl-2. And the effect of compound gingerol is more obvious than that of gingerol derivatives. CONCLUSIONS: The results of network pharmacology and experimental validation suggest that gingerol derivatives and compound gingerol can act against liver cancer by acting on the PI3K-Akt signalling pathway.

All-in-one: Accurate quantification, on-site detection, and bioimaging of sulfite using a colorimetric and ratiometric fluorescent probe in vitro and in vivo.

SO2 and its derivatives (SO32-/HSO3-) are used widely in food, beverages, and pharmaceutical production. However, they could induce multiple diseases in respiratory, nervous, and cardiovascular systems. Although several fluorescent probes have been developed for detecting SO32-/HSO3-, reports on rapid fluorescent probes for the on-site detection of SO2 derivatives are scarce. Herein, a colorimetric and ratiometric fluorescent probe 1 based on the intramolecular charge transfer (ICT) was reported. Probe 1 resulted in a 122 nm blue-shift in fluorescent emission and decrement of absorbance at 500 nm upon the addition of sulfite. Therefore, probe 1 could quantify SO32-/HSO3- using both UV-Vis and fluorescent methods (LOD: UV-Vis method 34 nM; fluorescent method 51 nM). Importantly, probe 1 was used for a rapid (60 s) and convenient (1 step, on-site) measurement of the SO2 derivatives in real samples (LOD: 0.47 µM) using smartphone based on the colorimetric method. The SO32-/HSO3--sensing mechanism was confirmed as the Michael addition reaction. Furthermore, the probe was used for the real-time monitoring of SO32-/HSO3- in A549 cells and zebrafish. In summary, an all-in-one fluorescent probe was successfully developed for the accurate quantification, on-site detection, and bioimaging of SO32-/HSO3-.