Malignant pleural effusion (MPE) is common with mesothelioma. We report two cases of extraordinarily viscous MPEs associated with mesothelioma. The viscosity prohibited spontaneous gravity‐dependent drainage via indwelling pleural catheters. Our ex vivo experiments found very high hyaluronic acid (HA) content within the fluid. Treatment of the fluid with hyaluronidase, but not with deoxyribonucleases, significantly reduced fluid viscosity. The results provide proof that HA can contribute to high viscosity of pleural fluid in mesothelioma. Research into strategies of counteracting HA properties in the management of MPEs may provide further insight. We report two cases of extraordinarily viscous malignant pleural effusion (MPE) in mesothelioma patients. Our ex vivo experiments attributed the viscosity to the high hyaluronic acid (HA) content within the fluid, which could be significantly reduced with hyaluronidase. Pleural effusions affect over 90% of patients with malignant pleural mesothelioma. The malignant pleural effusion (MPE) commonly appears serosanguinous or haemorrhagic, and can be evacuated using a range of devices, such as aspiration needles, chest tube, or indwelling pleural catheter (IPC). We report two cases of extraordinarily viscous MPEs in mesothelioma patients. Our ex vivo experiments attributed the viscosity to the high hyaluronic acid (HA) content within the fluid, which could be significantly reduced with hyaluronidase.A 79‐year‐old man presented with a left‐sided pleural effusion which was noted as viscous on the initial therapeutic aspiration. Cytology confirmed malignant mesothelioma. Upon recurrence of the MPE, an IPC  was inserted but the thick fluid prohibited spontaneous drainage. He was referred to our centre for further management. The fluid was partially evacuated by connecting the IPC via a connector to a 60‐mL syringe through which manual suction could be applied. The viscosity of the fluid prohibited usual laboratory biochemical analyses of the fluid. In an attempt to reduce fluid viscosity to clear the remaining fluid, two doses of 5 mg of deoxyribonuclease (DNase) was instilled over two days via the IPC, after which ~300 and ~200 mL of fluid were drained. The underlying lung re‐expanded on chest X‐ray (CXR) and talc slurry pleurodesis was administered via the IPC.Unfortunately, the fluid recurred over the next four weeks, resulting in a large multiloculated effusion associated with breathlessness and fatigue Fig. , necessiThe patient was readmitted two weeks later for ongoing difficulty with IPC drainage (<100 mL each attempt) Fig. . Sample A 79‐year‐old female presenting with a right pleural effusion underwent video‐assisted thoracoscopic pleural biopsy (which showed mesothelioma) and talc poudrage. The fluid recurred after surgery necessitating a therapeutic aspirate which revealed extremely thick exudative pleural fluid. She underwent regular therapeutic aspiration in an outside hospital for her MPE over the next four years (during which time she received various systemic therapy) before she was referred to our centre for IPC insertion. The fluid was described as thick and “jelly‐like” and failed to drain via the IPC. Manual suction via the IPC (Rocket) was required for complete evacuation of the fluid. Talc slurry was administered after lung re‐expansion but the pleurodesis failed.For the following 12 months, drainage was performed (roughly once weekly) via IPC. Sample 3 showed a typical specimen. Standard IPC suction bottles were ineffective to clear the thick fluid. Hence, the patient's carers were taught to perform IPC drainage using manual suction.After one year, IPC drainage failed despite the above‐mentioned measures causing significant breathlessness necessitating a hospital admission. A trial of intrapleural tPA 2.5 mg/DNase 5 mg was successful and drained 1.5 L with significant radiographic clearance. Regular IPC drainage was re‐established. Her mesothelioma continued to progress and she died 10 months later.Pleural fluid viscosity was determined as described by Simpson et al. . HA concWe found that MPE from both patients had very high pleural fluid HA concentrations. Hyaluronidase, but not rhDNase, was effective in reducing the viscosity of the fluid in a dose‐dependent manner Tables .We described two mesothelioma patients with HA‐rich and extremely viscous MPE, limiting fluid drainage. Our laboratory work confirmed that HA, present in high concentrations in these fluids, likely contributed to the high viscosity which could be significantly reduced using hyaluronidase.MPE from mesothelioma contains a vast number of important mediators. Mesothelioma has been known to express HA for over 80 years and HA pleural fluid concentrations are a valuable ancillary test for diagnosis. In our previous study including 96 mesothelioma patients, the pleural fluid HA level has an area under the curve of 0.89 in its diagnostic value of mesothelioma, highly comparable with mesothelin. Pleural fluid HA has also prognostic values . Our expHA is a long polymer of disaccharides up to 20,000 kDa in size produced by hyaluronan synthases, present in abundance in extracellular matrix. It can promote inflammation and angiogenesis—processes involved in MPE formation. HA is approved as intra‐articular treatment for osteoarthritis and for use in ophthalmologic conditions. It is also a common ingredient in skin care products .HA can be degraded by hyaluronidases, a family of enzymes, and by various non‐enzymatic reactions  . HyaluroAt present, clinically available options for viscous MPE are limited. Use of tPA/DNase enhanced the evacuation of thick infected pleural fluid in clinical trials. The regime provided some short‐term benefits in Patient 1, but subsequent administration did not prove effective. Our ex vivo experiments did not find a major role of rhDNase in reducing the viscosity. As tPA is known to stimulate significant production of pleural fluid in healthy and diseased pleura, it is possible that the dilutional effect of this fluid formation helped with initial drainages. Whether tPA itself affects viscosity of MPE is unknown.The composition of pleural fluid may be important to provide an informed clinical decision to ascertain an effective therapy for pleural effusion management. In cases of viscous fluid and high HA levels, strategies to counteract HA production or enhance its breakdown warrant exploration.Appropriate written informed consent was obtained for publication of this case report and accompanying images.Video S1. Highly viscous pleural fluid from Patient 1 (Sample 2) in the tube labelled untreated is shown. Treatment of the same sample with hyaluronidase (100 U/mL for 10 min) significantly reduced the fluid viscosity as shown in the tube labelled treated.Click here for additional data file.


IDH-wildtype GBM. After the resection of the first recurrence, progressive intracranial re-growth was again detected, and chemotherapy was replaced by PD-1 checkpoint inhibition, which led to a complete intracranial remission. Two months later MR-imaging revealed multiple osseous lesions. Biopsy confirmed the GBM origin of the skeleton metastases. Immunophenotyping reflected the effective activation of a peripheral T-cell response, with, however, increase of regulatory T cells during disease progression. WGS sequencing demonstrated distinct genomic alterations of the GBM metastasis, with gains along chromosomes 3 and 9 and losses along chromosome 4, 10, and 11. Mutational analysis showed mutations in potentially immunologically relevant regions. Additionally, we correlated tumour-infiltrating lymphocyte and microglia presence to the occurrence of circulating tumour cells (CTCs) in a larger cohort and found a decreased infiltration of cytotoxic T cells in patients positive for CTCs. This study exemplifies that the tumour microenvironment may dictate the response to immune checkpoint therapy. In addition, our study highlights the fact that despite an effective control of intracranial GBM, certain tumour clones have the ability to evade the tumour-specific T-cell response and cause progression even outside of the CNS.Peripheral metastases of glioblastoma (GBM) are very rare despite the ability of GBM cells to pass through the blood-brain barrier and be disseminated through the peripheral blood. Here, we describe a detailed genetic and immunological characterization of a GBM metastasis in the skeleton, which occurred during anti-PD-1 immune checkpoint therapy. We performed whole genome sequencing (WGS) and 850 K methylation profiling of the primary and recurrent intracranial GBM as well as one of the bone metastases. Copy number alterations (CNA) and mutational profiles were compared to known genomic alterations in the TCGA data base. In addition, immunophenotyping of the peripheral blood was performed. The patient who was primarily diagnosed with  Less than 0.5% of patients develop extracranial GBM tumour manifestation , 31, 32.pothesis , 29, whiThe discovery of circulating tumour cells (CTCs) in GBM has renewed the interest in this discussion. In one of the first studies of its kind, Müller et al. detected CTCs in up to 20% of patients . Cells iGBM metastases have been described to occur at various sites, including bone, lymph nodes, lung and liver, but also to the skin, thus in part challenging the “seed vs soil” hypothesis , 34, 41.In the here presented case, we illustrate the immunological escape of a peripheral GBM metastasis, which formed during intracerebral tumour control with checkpoint inhibition. The molecular and immunological profiling presented in this study provides new insights into potential mechanisms of immune escape and metastatic tumour evolution.IDH1-wildtype GBM WHO °IV with methylated MGMT gene promoter and mesenchymal subtype. Standard adjuvant therapy with combined radio-chemotherapy with temozolomide and 30 × 2 Gy was initiated  T cell compartment , we screened the blood of the patients at multiple time points using GFAP immunostainings to identify CTCs, as previously published . In addin = 116, Fig. +, p = 0.001, t-test) and low cytotoxic T cells , while no difference for CD68+ macrophages/microglia was observed . Copy number alterations (CNA) extracted from the methylation profiling confirmed the WGS analysis with clear amplification of chromosome 7 and deletion of CDKN2A in all 3 specimens which are genomic alterations characteristic of GBM  and 850 K methylation profiling in all three tumour samples. Global genomic methylome confirmed the diagnosis of MGMT-methylated GBM Fig. a. HoweveGBM Fig. b.Fig. 6TPDF52L3 and HIVEP2 were shared between all three tumours. HIVEP2, also known as MIBP2 (c-myc intron binding protein 1), was not only described to control the expression of multiple genes, many of which are involved in brain development, but also to inhibit glioma growth [PIK3CA p.E453Q (COSMIC ID: COSM758), NF1 p.I1679_Y1680del (COSMIC ID: COSM6969872) and CDKN2A/B (p15/16) deletion, further confirming the GBM origin of the metastasis. Additionally, a new mutation was found only in the metastasis in TP53 c.581 T > C p.L194P (COSMIC ID: COSM437527) by sanger sequencing during the initial pathological workup, which could be a possible key driver for tumour formation under anti-PD1 treatment. Among others, the metastasis harboured missense variant mutations in immunologically active genes, such as ANXA5, LGALS14 and FGL1  and the recurrent (0.60 mutations per Mbp) cerebral tumours, as expected for GBM, displayed low tumour mutational burden (TMB), the vertebral metastasis exhibited a 6.5-times higher mutational burden (3.26 mutations per Mbp)  further supports the hypothesis that peripheral immunosurveillance was functionally compromised during the metastatic spread of GBM cells. Potentially relevant is also a recent study by Jiao et al., that demonstrated that the microenvironment of certain organs determines distinct mechanisms of immune escape, as they show that prostate cancer metastasis in the bone utilize TGFβ to restrain TH1 differentiation of T cells in order to escape from immune checkpoint therapy [Recent findings by Congsathidkieth et al. describe that intracranial GBM is able to suppress the peripheral immune response by sequestration of lymphocytes in the bone marrow via a S1P1-mediated mechanism . While t therapy .IDH1 mutated anaplastic astrocytoma WHO °III [+ T cell and more specific CD8+ cytotoxic T-cell infiltration in patients being positive for CTCs indicates a direct link between the tumour-specific immune response, and potentially also immunocompetence, and the occurrence of tumour cells in the circulation.Although CTCs and sometimes even CTC clusters can be found in a large subset of patients, extracranial metastatic outgrowth are very rare in GBM , 31, 39.WHO °III . It is uEven though not reaching the strict definition of a hypermutated genotype, defined by more than 10 mutations per mega base pair, the increased tumour mutational burden of the extracranial GBM metastasis presented in our case, might explain why multiple metastasis were able to form. Although a generally hypermutated phenotype was suggested to favor recognition by tumour-specific immune cells due to the presence of new tumour-specific antigens . It is uANXA5, LGALS14 and FGL1, potentially affecting the tumour-specific immune response of the GBM metastasis. ANXA5, which also displayed a CNA loss, encodes the protein Annexin V, which is involved in stabilization of the T cell receptor (TCR) and peptide-MHC interactions during formation of an immunological synapse [LGALS14) is expressed mainly by the placenta and was shown to be able to induce apoptosis of activated T lymphocytes [FGL1 encodes for a protein called fibrinogen-like protein 1, which was recently described to be highly expressed in many cancers and to inhibit antigen-specific T cell activation through the interaction with the known checkpoint molecule Lag-3 [Another hypothesis favors the occurrence of immunological escape variants by increased prevalence of somatic mutations in the human leukocyte antigen (HLA) class I region . The HLA synapse . Galectile Lag-3 . Increasle Lag-3 . AlthougTaken together, the comprehensive molecular and immunological profiling of this unique case of extracranial GBM metastases gives new insights into potential mechanisms of immune escape of GBM. We postulate that the combination of functional impairment of the peripheral immune system, as reflected by a steady increase of exhaustion markers, such as KLRG1 and CD57, and the occurrence of a metastasis with an increased mutational burden, enabled the extracranial dissemination and disease progression while intracranial GBM could be controlled by checkpoint inhibition.Clinical data and images were analysed after confirmation of peripheral GBM metastasis by two pathologists. The patient signed an informed consent. This study was approved by the local ethics council of the Hamburg chamber for physicians and was performed in accordance with the Helsinki declaration of 1975.Routine histological and immunohistochemical analysis , CD3 , CD8 ) was performed on paraffin embedded sections using a microtome and automatic staining system .C-blocking, samples were stained in flow-cytometry staining buffer (eBioscience) for 45 min at room temperature with the antibody cocktails, washed, and resuspended in buffer prior to analysis. Analysis was performed on a BD LSRFortessa flow-cytometer and using the FACSDiva software (Becton Dickinson).Peripheral blood was collected into EDTA-containing tubes. Ficoll gradient (PromoCell) centrifugation was performed for the isolation of PBMCs. PBMCs were used after being frozen in RPMI/10% DMSO. Flow-cytometric analysis was performed on PBMCs using multicolor antibody staining as described previously . In addiFormalin fixed paraffin embedded tissue part of an already published cohort in which the peripheral blood was tested for the presence of CTCs,  were sta6 PBMCs) was isolated using the genomic DNA miniprep kit . For tumour tissue representative tumour areas were identified from using light microscopy. If necessary, macrodissection was performed. For the metastatic tissue WGS, gDNA was amplified after isolation from paraffin-embedded tissue using the PicoPLEX v2 kit (Takara Bio) as described before [Genomic DNA from fresh frozen or paraffin-embedded tumour tissue or peripheral immune cells  . Strelka.0 . Strelogy.org . StatistAdditional file 1. Supplemental Figure 1.Additional file 2. Supplemental Table 1.


Fluid strategy is the key to the successful management of patients with sepsis. However, previous studies failed to consider individualized treatment strategy, and clinical trials typically included patients with sepsis as a homogeneous study population. We aimed to develop sequential decision rules for managing fluid intake in patients with sepsis by using the dynamic treatment regimen (DTR) model. A retrospective analysis of the eICU Collaborative Research Database comprising highly granular data collected from 335 units at 208 hospitals was performed. The DTR model used a backward induction algorithm to estimate the sequence of optimal rules. 22,868 patients who had sepsis according to the Acute Physiology and Chronic Health Evaluation (APACHE) IV diagnosis group were included. Optimal fluid management  strategy were developed on the Day 1, 3 and 5 after ICU admission according to current states and treatment history. Important determinants of optimal fluid strategy included mean blood pressure, heart rate, previous urine output, previous fluid strategy, ICU type and mechanical ventilation. Different functional forms such as quadratic function and interaction terms were used at different stages. The proportion of subjects being inappropriately treated with liberal fluid strategy  increased from day 1 to 5 (19.3% to 29.5%). The survival time could be significantly prolonged had all patients been treated with optimal fluid strategy . With a large volume of sepsis data, we successfully computed out a sequence of dynamic fluid management strategy for sepsis patients over the first 5 days after ICU admission. The decision rules generated by the DTR model predicted a longer survival time compared to the true observed strategy, which sheds light for improving patient outcome with the aim from computer-assisted algorithm. The results showed that EGDT did not significantly reduce mortality rate as compared with the usual care group. Thus, the optimal fluid strategy remains largely unknown. One important limitation of these trials is that they included all sepsis patients without considering between-subject heterogeneity. As a matter of fact, there has been large body of evidence showing that sepsis is highly heterogenous and different subphenotypes can have distinct responses to fluid administration4. Latent profile analysis and hierarchical clustering are two major techniques to identify subphenotypes of sepsis5, which however are limited by two factors. First, the capacity to establish a causal relationship between treatment and subphenotypes is insufficient. Second, most studies used cross-sectional data when modeling the subphenotypes. We should aware that fluid management in a sepsis patient is a dynamic process over entire treatment course; we should not solely focus on the initial fluid strategy but ignore the importance at later stages. There was evidence that de-resuscitation  at later phase of sepsis is beneficial6. Therefore, we need to adjust our fluid management strategy according to the patient’s changing state and treatment history.Management of critically ill patients with sepsis is imperative as sepsis can attack all human organs leading to a fatal consequence in a short period of time. Among multiple aspects for saving the life of patients with sepsis, we believe that fluid management strategy remains essential to the successful treatment because the cardiac and renal functions, which play important roles in the physiological regulation of fluid balance, are usually impaired in sepsis. Numerous studies have been conducted to explore an optimal fluid strategy. A trio of multinational trials named Protocolized Care for Early Septic Shock (ProCESS), Australasian Resuscitation in Sepsis Evaluation (ARISE), and Protocolized Management in Sepsis (ProMISe) aimed to investigate whether early goal directed therapy (EGDT) could improve the mortality outcome7. RL has been used to determine the optimal fluid treatment strategy in sepsis10. However, these results are difficult to interpret due to the black-box algorithms, which significantly prohibited their use in clinical practice. Dynamic treatment regimen (DTR) method borrows the idea of RL, but simplifies the functional forms of the multi-dimensional feature space, which can help clinicians to better understand the decision rules 11. This study aimed to optimize fluid treatment strategy  by using DTR during the course of sepsis. Instead of using cross-sectional data on one single phase, we applied DTR model on three different days  over the first week of treatment with a purpose of examine dynamic nature of the fluid management on sepsis treatment. We hypothesized that the optimal fluid treatment strategy recommended by DTR would be better than the treatment actually received in terms of survival outcome.Reinforcement learning (RL) is a well-developed algorithm of machine learning focusing on how a computing system gives a response based on environmental inputs so the system can maximize cumulative reward12. Patients with sepsis were included for our analysis. Sepsis was defined according to the sepsis-2.0 definition13. Sepsis-2.0 was defined when a patient had suspected or documented infection plus 2 of the SIRS criteria including temperature 3 or > 10% band. Sepsis was further categorized by infection sites including cutaneous, gastrointestinal, pulmonary, urinary tract, other location and unknown location14.The present study utilized the eICU Collaborative Research Database, which is a multi-center intensive care unit (ICU) database with high granularity data for over 200,000 admissions to ICUs monitored by eICU Programs across the United States2/FiO2 (P/F) ratio, daily urine output and the use of mechanical ventilation (MV).The primary outcome was patient status  at hospital discharge, which was considered as time to event survival data. Subjects who were discharged alive were considered as censored. We further defined the survival time of the patient from ICU admission to expiration. Patients who discharged alive were considered as censored. The state (feature space) of patients at each stage  was constructed by variables including heart rate (HR), mean blood pressure (mBP), respiratory rate (RR), Glasgow coma scale (GCS), body temperature, creatinine, lactate, hemoglobin, bilirubin, use of vasopressor, platelet count, PaOTotal fluid intake was calculated as the sum of fluid intake for a 24-h interval. We assumed that the fluid strategy was determined at the beginning of each interval. Fluid intake was also normalized by the body weight. We defined liberal and restricted fluid administration as ≥ 40 ml/kg/day and < 40 ml/kg/day, respectively. This cutoff point was chosen according to the distribution of the fluid intake in the study population so that both categories would have balanced number of observations.CBCgrps package in R15.Descriptive statistics were analyzed conventionally using the 16. We defined three stages on day 1, 3 and 5 after ICU entry and recorded the survival times within each stage. For the first stage, the survival time The DTR model estimated a sequence of treatment strategy that maximized the survival time across stages of clinical interventionThe DTR model used a backward induction algorithm to estimate the sequence of optimal rules. In the first step, the optimal stage 3 decision rule (day 5) was estimated by modeling the counterfactual survival time in stage 3 . The significance level of 0.05 was not reached on day 4, but the survivors still had less fluid intake than non-survivors . The amount of fluid intake decreased gradually from day 1 to 10 from 38.88 (IQR: 17.63–71.52) to 27.88 (IQR: 14.12–56.71) ml/kg/day , subjects should receive liberal fluid administration if they satisfied the following condition:Otherwise, they should receive restricted fluid administration in order to achieve a better survival outcome. Subscripts of variables in the equation denote the ICU days. Positive coefficient of a variable means that the presence or increase in the variable favors liberal fluid administration. The rule indicated that liberal fluid administration was more likely to be beneficial to patients from cardiac surgery ICU (CSICU) , NeuroICU  and surgical ICU (SICU) ; but was harmful to patients from medical ICU (MICU) , Med-Surg ICU . Higher HR mandated more fluid administration . Patients on MV also required more fluid intake . The impact of mBP was a parabola opens down with the line of symmetry at mBP = 62.1 mmHg. Clinical interpretation was that more fluid intake was warranted for patients with mBP < 62.1 mmHg; while more restricted fluid administration was preferred with increasing mBP if mBP > 62.1 mmHg.The condition for liberal fluid administration on day 3 (stage 2) was:This rule indicated that more urine output  in previous days mandated liberal fluid administration on day 3. The mBP on day 3 did not follow a parabolic function . In this case, higher mBP mandated less fluid administration.The condition for liberal fluid administration on day 5 (stage 3) was:There was a significant interaction  between day 3 and day 4 treatment strategy for determining the day 5 treatment strategy. The interaction indicated that if liberal fluid administration was given on day 3, restricted fluid administration was more likely to be beneficial on day 5 if liberal fluid balance was also given on day 4  and modeling the decision rule (blip function) with generalized linear model. The resultant DTR model was clinically interpretable and could easily guide clinical practice, which was an improvement over other less accessible RL algorithms.Conventionally, fluid administration was guided by a variety of biomarkers reflecting the circulatory status such as serum lactate, ScvO2, and capillary refill time. The 2016 version of the surviving sepsis bundle recommended maintaining MAP > 65 mmHg and normalization of lactate25. The mBP was an important determinant of fluid strategy and it was shown to be statistically significant in our blip function. However, the mapping from  mBP to fluid strategy was not similar in early versus later stage. On the first day, a parabolic function was fit with the turning point at 62.1 mmHg, which was very close to the 65 mmHg as recommended in the SSC guideline. At the later stages (Day 3 and 5), we found that previous urine output and treatment strategy were important determinants of current fluid strategy. This novel finding indicated that the optimal treatment strategy must take previous responses (e.g. urine output in response to fluid administration) into consideration.The appropriate selection of feature variables for the blip function was essential for applying the DTR model. Although the SSC guideline recommended targeting lactate clearance as the resuscitation guide, lactate was not statistically significant in the blip function, and thus we excluded this variable. CVP was not included in the DTR model in our study because it has been documented less useful for assessing fluid status22. This may lead to unsatisfactory results of previous trials. We argued that fluid management should be carried out during the entire disease course. Our DTR model correctly captured the clinical variables over the dynamic process of sepsis and provide a sequential decision rules to maximize the survival time. Our result showed that the proportion of subjects being inappropriately treated with liberal fluid strategy increased from Day 1 to 5 . More recently, the concept of de-resuscitation  after hemodynamic stabilization has received more and more attentions26. There was evidence that negative fluid balance achieved with de-resuscitative measures resulted in lower mortality6. These studies also highlighted the importance of careful fluid management in later phase after hemodynamic stabilization.Another strength of this study was that we calculated fluid strategy over 5 days after ICU admission. The majority of previous trials focused on the first 6 or 12 h to investigate the effect of fluid resuscitation strategy27. However, the study was not a prospective study in which screening criteria could be prospectively collected. In the dataset, there was missing data on required items for the definition of sepsis-3.0. For example, the sepsis-3.0 definition requires an acute increase in the SOFA score, which means that we must have information for the baseline SOFA score to implement the Sepsis-3.0 definition. In fact, the database did not contain such complete information for the implementation of sepsis-3.0 criteria.There are limitations in the current study. For example, the study population of sepsis was based on sepsis-2.0 definition, which may identify different population than that identified by using the most updated Sepsis-3.0 criteriaIn conclusion, the study successfully computed out a sequence of dynamic fluid management strategy for sepsis patients over the first 5 days after ICU admission with a large volume of electronic healthcare data. The decision rules on day 1, 3 and 5 adopted different functions of covariates and treatment histories. The optimal treatment strategy generated by the DTR model could significantly improve the survival outcome as compared with the actual fluid strategy. The decision rules developed in the study require further validation in prospective cohorts.


BRAF-mutant melanoma patients respond to BRAF inhibitors and MEK inhibitors (BRAFi/MEKi), but drug-tolerant cells persist, which may seed disease progression. Adaptive activation of receptor tyrosine kinases (RTKs) has been associated with melanoma cell drug tolerance following targeted therapy. While co-targeting individual RTKs can enhance the efficacy of BRAFi/MEKi effects, it remains unclear how to broadly target multiple RTKs to achieve more durable tumour growth inhibition.The blockage of adaptive RTK responses by the new BET inhibitor (BETi), PLX51107, was measured by RPPA and Western blot. Melanoma growth was evaluated in vitro by colony assay and EdU staining, as well as in skin reconstructs, xenografts and PDX models following BRAFi, MEKi and/or PLX51107 treatment.Treatment with PLX51107 limited BRAFi/MEKi upregulation of ErbB3 and PDGFR-β expression levels. Similar effects were observed following BRD2/4 depletion. In stage III melanoma patients, expression of BRD2/4 was strongly correlated with ErbB3. PLX51107 enhanced the effects of BRAFi/MEKi on inhibiting melanoma growth in vitro, in human skin reconstructs and in xenografts in vivo. Continuous triple drug combination treatment resulted in significant weight loss in mice, but intermittent BETi combined with continuous BRAFi/MEKi treatment was tolerable and improved durable tumour inhibition outcomes.Together, our data suggest that intermittent inhibition of BET proteins may improve the duration of responses following BRAFi/MEKi treatment in BRAF-mutant melanoma. Thus, alternative approaches to broadly inhibit adaptive responses to BRAFi/MEKi in melanoma are needed.A major mechanism that enables tumour cells to tolerate BRAF/MEK pathway inhibition is adaptive upregulation and/or activation of receptor tyrosine kinases (RTKs).18 BET proteins are epigenetic readers that bind to acetylated histones at promoters, enhancers, and super-enhancers,20 and regulate the expression of genes related to cell proliferation and apoptosis, such as MYC and BCL2.17 BET inhibitors (BETi), including JQ1 and OTX015, competitively displace BRD2/3/4 from acetylated histones, resulting in the suppression of BRD/BET-dependent genes in breast and ovarian cancer models.22 JQ1 also reduces the expression of RTKs and inhibits tumour cell growth and survival.18 Thus, BET inhibition may prevent RTK upregulation induced as an adaptive response to BRAF/MEK inhibition, and in doing so, enhances the effects of the targeted agents.24 While BETi have produced promising results in several malignancies, they have also been associated with toxicities and remain to be fully tested in BRAF-mutant melanoma.26Bromodomain and extra-terminal domain (BET) protein family, including BRD2, BRD3, and BRD4, regulates transcriptional programmes.Here, we tested the role of BRD/BET proteins in the upregulation of RTKs and resistance to BRAFi/MEKi therapy in BRAF V600E melanoma models. We utilised a new BETi, PLX51107, and an intermittent schedule to improve the efficacy of the BRAFi/MEKi combination while minimising toxicity. We show that PLX51107 treatment reduces expression of ErbB3 and PDGFR-β. The effects were associated with BRD2 and BRD4 expression. PLX51107 enhanced the effects of BRAFi/MEKi on melanoma cell growth in monolayer cultures and in a three-dimensional (3D) human skin reconstruct model. Using an intermittent schedule, we showed that tumour reoccurrence following drug withdrawal was reduced in tumour-bearing mice treated with intermittent BETi with continuous BRAFi/MEKi compared with BRAFi/MEKi alone. Together, our data suggest that inhibition of BET proteins improves the efficacy of BRAFi/MEKi in BRAF-mutant melanoma.27 All cells were grown with 1% penicillin/streptomycin (100 U/mL) added to all media at 37 °C in a humidified incubator supplemented with 5% CO2. Cells were routinely assayed for mycoplasma contamination with MycoScope Kit (Genlantis).A375 cells were cultured in Dulbecco's modified Eagle’s medium  supplemented with 10% foetal bovine serum . M238 cells were cultured in RPMI 1640 medium (GIBCO) supplemented with 10% FBS and 2 mM L-glutamine (GIBCO). 1205Lu cells were cultured in MCDB 153 medium containing 20% Leibovitz-L15 medium, 2% FBS, 0.2% sodium bicarbonate, and 5 μg/mL insulin. All melanoma cell lines used in this study were validated as BRAF V600 mutants by Sanger sequencing. In addition, STR analysis was performed for the cell lines described above. Primary culture of normal keratinocytes (HFK), normal melanocytes (HFM), and normal fibroblasts (HFF) were isolated from human neonatal foreskin samples and cultured as previously described.5/well) were seeded in six-well plates in normal growth media. The next day, cells were treated with either dimethyl sulfoxide (DMSO) or BRAFi/MEKi  ± increasing doses of the BETi JQ1  or PLX51107  for 24 h. Lysates from three independent experiments were processed and analysed, as previously described.28 Reverse-phase protein array (RPPA) data were used to determine antibodies that were significantly different between BRAFi/MEKi ± BETi-treated groups for each cell line. Comparisons were performed by the two-sample t-test method with 1000 permutations and assumed unequal variance. Antibodies with a p-value < 0.05 were considered significant. Statistical calculations were performed in Matlab® (v2015b) using the mattest function. Data points are shown as averages of three experimental replicates.Cells (2.0 × 10BRD2 siRNA (GAAAAGAUAUUCCUACAGA), BRD4 siRNA (UGAGAAAUCUGCCAGUAAU), and negative control siRNA (siCONTROL Non-Targeting siRNA #1 UAGCGACUAAACACAUCAAUU) were purchased from Dharmacon Inc. . Cells were transfected with chemically synthesised siRNAs at a final concentration of 20 nM using Lipofectamine RNAiMAX .Cells were washed twice in cold PBS and lysed with Laemmli sample buffer with β-mercaptoethanol. Proteins were resolved by SDS-PAGE and transferred to PVDF membranes. After blocking in 5% BSA, membranes were incubated with the indicated primary antibodies overnight at 4 °C, followed by incubation with peroxidase-coupled secondary antibodies. Immunoreactivity was detected using horseradish peroxidase (HRP)-conjugated secondary antibodies  and chemiluminescence substrate  on a Versadoc imaging system (Bio-Rad). The primary antibodies used were as follows: phospho-ErbB3 , ErbB3 (#4754), PDGFR-β (#3169), AXL (#4939), BRD2 (#5848), BRD4 (#13440), phospho-ERK1/2 , ERK1/2 (#9102), and GAPDH (#2118) from Cell Signaling Technology ; ERK2 (sc-1647) antibody was purchased from Santa Cruz Biotechnology Inc. .5/well) were plated in six-well plates and treated, as indicated. At the end of the experiment, cells were detached using 2 mM EDTA and washed twice with cold PBS. Samples were stained with Zombie NIR , for 10 min at room temperature in the dark. Following two washes with FACS buffer (PBS with 1% FBS and 0.5% sodium azide), cells were stained with ErbB3-PE  for 30 min at room temperature. Cells were then fixed with BD Cytofix/Cytoperm fixation/permeabilisation solution kit  and analysed on BD FACSCelesta Flow Cytometer (BD Biosciences) using FlowJo software . Data points are shown as averages of three experimental replicates.Cells (2.0 × 103/well) were seeded in six-well plates. Cells were then treated with either DMSO or dabrafenib, 50 nM + trametinib, 5 nM ± JQ1  or PLX51107  three times per week, for 3 weeks, as indicated. Cells were then washed with PBS and stained with 0.2% crystal violet in 10% buffered formalin for 20 min. Subsequently, wells were washed and air-dried. Plates were scanned, and pictures were taken with a Nikon Eclipse Ti inverted microscope with NIS-Elements AR 3.00 software .Cells (8 × 105/well) were seeded in six-well plates. Cells were then treated with the drug of interest for 48 h. The thymidine analogue EdU was added at a final concentration of 10 μmol/L for the final 16 h. EdU incorporation was measured using the Click-iT Plus EdU Alexa Fluor 647 Flow Cytometry Assay Kit (Thermo Fisher Scientific) and was utilised as per the manufacturer’s instructions. EdU staining was analysed on BD FACSCelesta Flow Cytometer (BD Biosciences) using FlowJo software . Data points are shown as averages of three experimental replicates.Cells . PLX51107 was a gift from Plexxikon Inc. .3/well) were seeded in twelve-well plates. Phase-contrast images were taken every 2 h using an IncuCyte® live cell imager . Cells were treated three times a week of treatment of choice, and cell confluence of the cultures was measured using IncuCyte software over 3 weeks in culture.For real-time analysis of cell viability, cells (5 × 1027 A375 and 1205Lu melanoma cells were incorporated into the epidermis at the time of keratinocyte seeding (one melanoma cell to three keratinocytes). After 24 h of submersion, skin reconstructs were then transferred to the air–liquid interface and maintained for 2 weeks in culture medium composed of DMEM/Ham’s F-12 medium mixture (3:1) supplemented with 10% FBS, 0.1 nM cholera toxin, 5 μg/mL insulin, 5 μg/mL apo-transferrin, 0.4 μg/mL hydrocortisone-21, and 0.5 ng/mL epidermal growth factor (EGF).The skin reconstruct model was performed with minor changes from Sandri et al.After the keratinocyte cell stratification and melanoma invasion period (14 days), skin reconstructs were fixed with 10% buffered formalin at 4 °C and histologically processed for inclusion in paraffin. Sections of 5 μm were stained with haematoxylin/eosin (H&E) and analysed for their morphology. The quality of the skin reconstruct was considered satisfactory when a differentiated and stratified epidermis similar to human epidermis was observed. For immunohistochemical analysis, skin reconstructs were stained for pan-cytokeratin , and Ki-67 . The photomicrographs were obtained by optical microscopy, using a Nikon Optiphot microscope , and analysed using the NIS-Elements software (Nikon).6/flank/animal). When tumours were palpable (50–100 mm3), mice were randomly divided into four cohorts and fed vehicle, BRAFi/MEKi (PLX4720 200 ppm + PLX2695 7 ppm), BETi (PLX51107 90 ppm) or BRAFi/MEKi/BETi-laced chows  in a scheduled treatment, as indicated. All cohorts had six animals . For BRAF-mutant PDX TJUMEL41 experiments, highly immunodeficient NOD-SCID IL2Rγnull (NSG) mice  were used. BRAF-mutant human melanoma biopsy (PDX TJUMEL41) was collected and processed as previously described.29 Briefly, NSG mice were anaesthetised with isoflurane inhalation. The area was scrubbed with ethanol, shaved, and a small incision (~5 mm) was made. Human melanoma tissues  were then implanted subcutaneously, and the incisions were closed with wound clips. Mice were observed until they  awoke from anaesthesia, and the wound clips were removed after 7 days. Animals were monitored for any sign of pain such as anorexia, dehydration, hunched posture, failure to groom, excessive redness, and swelling at the incision site. A dose of carprofen or buprenorphine was administered pre-op and as needed from animal monitoring until staple removal. When tumours were palpable, mice were randomly sorted into four cohorts and treated as indicated. All cohorts had seven animals , except for the BETi group . Tumour volume measurements, animal behaviour, and survival were monitored every 2–3 days once treatments began. Digital calliper measurements were used to calculate tumour volumes using the formula: volume = (length × width2)/2. When approximately 20% of body weight loss was observed, DietGel 76 A (Scanbur) was administrated. Animals were sacrificed when tumours reached ~650 mm3, for 1205Lu and A375 xenografts, or ~1.5 cm3, for PDX models. Animals that progressed in weight loss were also sacrificed and included in overall survival results. We used CO2 inhalation as our euthanasia method, performed in accordance with the AVMA Panel on Euthanasia for adult animals. This information can also be found in items 7, 8, and 10 of ARRIVE checklist  were used. Seven-week-old nude mice were injected with human melanoma cells ,30 while the RNA-seq V2- normalised gene expression and mutation call data were retrieved from the latest Broad GDAC Firehose data run . Spearman’s correlation analysis was performed using the corr function in Matlab®. In vitro data were expressed as mean ± standard deviation and statistically analysed using Student’s t-test  with p < 0.05. Means and standard deviations were calculated using experimental triplicates from three separate assays. In vivo statistical analysis is described in item 9 of ARRIVE checklist  were collected from The Cancer Proteome Atlas  or 2 days on/5 days off BETi (2 + 5BETi) would prolong BRAF/MEK targeting effects . We utilised intermittent scheduling of 4 days on/3 days off BETi combined with continuous BRAFi/MEKi (BRAFi/MEKi/4 + 3BETi) in 7-day cycles for 4 cycles followed by continuous BRAFi/MEKi until 66 days of treatment Fig. . After dDue to observed effects in vitro , we tested a reduced 2 days on/5 days off schedule of BETi combined with continuous BRAFi/MEKi (BRAFi/MEKi/2 + 5BETi) in 7-day cycles for 4 cycles. At this time, BETi was removed and animals were maintained in either vehicle or BRAFi/MEKi chow. Again, we investigated regrowth of disease after cessation of treatment. BRAFi/MEKi/2 + 5BETi led to a 33% CR without affecting weight loss or the animals presenting any symptoms of drug toxicity Fig. . Importa20 Importantly, we show that an intermittent dosing schedule of PLX51107 was tolerable and prolonged the growth inhibition achieved by continuous BRAFi/MEKi treatment.The duration of responses to targeted inhibitors in BRAF-mutant melanoma is limited by rapid adaptations that select for drug-tolerant cell subpopulations. Here, we utilised a new BETi, PLX51107, in combination with BRAFi/MEKi in BRAF-mutant melanoma models to broadly suppress adaptive upregulation of RTKs in response to targeted therapy. The choice of those melanoma cell lines was based on previous studies where adaptive upregulation of ErbB3 and PDGFR-β was observed following BRAF targeting, and those RTKs were associated with resistance to targeted therapy.38 and may be activated by growth factors derived from the stromal microenvironment.13 We show that the next-generation BETi, PLX51107, inhibited BRAFi/MEKi-induced upregulation of two RTKs, ErbB3 and PDGFR-β. These findings are conceptually similar to evidence that the short-term inhibition of BET proteins using JQ1, I-BET151, or I-BET762 reduced lapatinib resistance that was associated with adaptive kinome expression in triple-negative breast cancer models.39 In these contexts, BETi reduced adaptive RTK expression followed by RTK-associated focal adhesion kinase, Src family kinase phosphorylation and AKT re-activation.40 BETi preferentially inhibit targeted therapy-induced gene expression, since JQ1 downregulated ~8% of all expressed genes when used alone but suppressed 27% of lapatinib-induced genes in combination treatment.42 Together, these data suggest that BETi will be useful to suppress adaptive RTK upregulation and enhance tumour inhibitory effects by BRAFi/MEKi therapy.Multiple RTKs are upregulated in BRAF-mutant melanoma cells following BRAFi/MEKi43 Our data show that BETi enhanced the cell cycle arrest induced by BRAFi/MEKi after 48 h of treatment, consistent with studies that showed that BRD/BET proteins regulate expression of p21(WAF1/Cip1) and c-Myc.44 Furthermore, the concomitant targeting of PI3K with JQ1 resulted in potent tumour growth arrest and apoptosis in ovarian cancer cells, compared with either agent alone.45 Although BRAF/MEK targeting reduced ERK1/2 pathway activation and cell survival, therapy-tolerant cell subpopulations may persist and seed disease relapse.46 Residual cells have presented a mesenchymal-like cell shape, prominent actin stress fibres, high invasiveness, low proliferation, and neural crest cell markers;46 targeting these drug-tolerant cells using BETi may avoid adaptive response and delay melanoma relapse.48 Blockage of adaptive BRD/BET-induced upregulation of ErbB3/PDGFR-β following BRAFi/MEKi therapy may contribute to the effects of BETi on delayed tumour recurrence, since those RTKs were closely associated with targeted therapy resistance and poor survival in BRAF-mutant melanoma patients.49 However, we do not rule out a role for other BETi targets. In addition to BRAFi/MEKi-associated RTK downregulation, BETi also altered the cell cycle, contributing to tumour growth inhibition.BETi also regulate the expression of targets other than RTKs. BETi were first identified as a c-Myc and histone modification-targeting therapy.50 Using a continuous triple-schedule combination in mice produced enhanced toxicities, limiting the efficacy of therapy in vivo. Thus, we tested and optimised an intermittent dosing schedule of BETi to enhance BRAFi/MEKi treatment in vivo while maintaining tolerability. While focusing on maximising the short-term effect of BRAFi/MEKi, we also assessed the ability to delay/prevent tumour relapse after cessation of BRAFi/MEKi treatment by monitoring residual disease. Importantly, we observed that BETi following BRAFi/MEKi treatment delayed tumour relapse. We tested several intermittent schedules of BETi to prolong BRAFi/MEKi effects. We observed that an intermittent schedule of 2 days on followed by 5 days off BETi in the presence of BRAFi/MEKi presented less toxicity compared with 4 days on followed by 3 days off BETi schedule in the presence of BRAFi/MEKi, and was more effective than the alternative schedule of 7 days on BRAFi/MEKi alone followed by 7 days on BETi alone. This latter schedule may have been less effective due to the rapid BRAFi/MEKi-associated upregulation of RTKs,51 which may require BETi to be administered at the same time of BRAFi/MEKi for efficacy. Although our data showed that BETi enhanced the efficacy of targeted therapies in two different xenografts and one PDX model, a limitation is the small number of animals used in our study. Altogether, our data suggest that co-targeting of BRD/BET proteins in concert with BRAF/MEK inhibitors is likely to promote more durable responses of targeted therapy for BRAF-mutant melanoma in vivo.We evaluated the therapeutic effects of BETi/BRAFi/MEKi in vivo using human melanoma cell xenografts and PDX models. Continuous dosing of BRAFi/MEKi/BETi was toxic for tumour-bearing mice, and both BRAFi/MEKi and BETi cause toxicities in patients.Supplementary Figures


Sodium–glucose cotransporter 2 (SGLT2) inhibitors are an antihyperglycemic drug with diuretic action. We recently reported that the SGLT2 inhibitor dapagliflozin ameliorates extracellular volume expansion with a mild increase in urine volume. However, the impact of the pretreatment extracellular volume status on the body fluid response to SGLT2 inhibitors remains unclear.Thirty-six diabetic kidney disease (DKD) patients were treated with dapagliflozin. The body fluid volume, including intracellular water (ICW), extracellular water (ECW) and total body water (TBW), were measured on baseline and day 7 using a bioimpedance analysis (BIA) device. The ECW/TBW and ECW were used as markers of the extracellular volume status. For a comparison, the extracellular volume status responses to loop diuretic furosemide (n = 16) and vasopressin V2 receptor antagonist tolvaptan (n = 13) were analyzed.p = 0.006). Only with dapagliflozin treatment (not furosemide or tolvaptan treatment) was the baseline ECW/TBW significantly correlated with the changes in the ECW/TBW  and ECW .The body weight, brain natriuretic peptide and body fluid parameters measured by a BIA  were significantly decreased for 1 week after dapagliflozin administration. The change in the ECW/TBW in the high-ECW/TBW group  was significantly higher than in the low-ECW/TBW group (− 2.1 ± 0.4 vs. − 0.5 ± 0.4%, The pretreatment extracellular volume status predicts the body fluid response to the SGLT2 inhibitor dapagliflozin in DKD patients. The diminished extracellular fluid reduction effect of dapagliflozin in patients without severe extracellular fluid retention may contribute to maintaining a suitable body fluid status. Recexcretion , 6, refl [+ in a :1 ratio We recently reported that the SGLT2 inhibitor dapagliflozin predominantly decreased the extracellular volume with a mild increase in the urine volume in DKD patients with fluid retention , 8. ImpoOn the other hand, we previously showed that the SGLT2 inhibitor ipragliflozin does not affect the body fluid volume  in euvolemic animal models , 13, andBased on these findings, we hypothesized that SGLT2 inhibitors decrease the body fluid volume in patients with fluid retention but show no such decrease in fluid volume for patients without fluid retention. We therefore examined the correlation between the pretreatment extracellular volume status and the body fluid response to the SGLT2 inhibitor dapagliflozin in DKD patients.2 or the presence of proteinuria. In addition to the 14 patients enrolled in a previous study [p = 0.082), as we aimed to examine the effect of dapagliflozin among a variety of extracellular fluid statuses. Among these 36 patients, 12 received a renal biopsy, and 7 had other types of renal diseases  in addition to DKD. The exclusion criteria were a history of renal replacement, current dialysis, type 1 diabetic mellitus or active malignancy.This study prospectively enrolled 36 DKD patients treated with dapagliflozin at Jichi Medical University  and Nasu Minami Hospital  between February 2016 and June 2019. DKD was defined as a combination of diabetes and an estimated glomerular filtration rate (eGFR) < 60 mL/min/1.73 mThe SGLT2 inhibitor dapagliflozin (5 mg/day) was administered in addition to the baseline medication. The patients with loop diuretic furosemide  and vasopressin V2 receptor antagonist tolvaptan , which partially include data from our previous study (2 newly added patients treated with furosemide and 1 patient treated with tolvaptan) , were coThis study was conducted in accordance with the ethical principles of the Declaration of Helsinki. The study protocol and amendments were approved by the independent ethics committees of Jichi Medical University  and Nasu Minami Hospital . All patients provided their written informed consent to participate in this study. The study was registered in the University Hospital Medical Information Network Clinical Trial Registry (UMIN-CTR) Clinical Trial (UMIN registration number: 000029863).Blood samples and body weight data were collected before dosing on days 0 and 7 after the administration of dapagliflozin. The eGFR was calculated using the Modification of Diet in Renal Disease study equation coefficients modified for Japanese patients . The staThe body fluid volume was measured using a BIA device with eight tactile electrodes  before dosing at baseline and day 7 after dapagliflozin administration (3 h or later), similar to our previous studies , 18–20. r). p values of < 0.05 were considered to indicate statistical significance. The statistical analyses were performed using the JMP Pro 14.2.0 software program .The data were expressed as the mean ± standard error. Paired or unpaired t-tests were used to compare two variables, as appropriate. A one-way analysis of variance was used to compare the parameters with a normal distribution  among three groups. The Shapiro–Wilk test was used to check the normality of the distribution. Non-normally distributed data were presented as the median and interquartile range and were analyzed with Wilcoxon’s rank-sum test. The correlations among clinical parameters were analyzed using Pearson’s correlation (+ significantly decreased from baseline to 1 week after dapagliflozin administration (Table p = 0.023).Table r = − 0.593, p < 0.001), BNP  and HbA1c . The hemoglobin, hematocrit, HbA1c and serum albumin were positively and significantly correlated with the absolute change in the ECW/TBW, whereas the BNP, uric acid, ECW, and ECW/TBW were negatively and significantly correlated with the absolute change in the ECW/TBW  and the absolute change in the ECW/TBW  were significantly higher in the patients receiving diuretic therapy than in those without any diuretic therapy.Next, we examined the correlation between the baseline clinical parameters and ECW/TBW and the absolute change in the ECW/TBW, a marker of extracellular fluid volume . The basBW Table . In contBW Table . The basp = 0.006)   Fig. .Fig. 1AbFinally, we compared the fluid response to the SGLT2 inhibitor dapagliflozin, loop diuretic furosemide and vasopressin V2 receptor antagonist tolvaptan. The baseline body weight was similar among the three groups Table . The basThis study shows that the fluid response to dapagliflozin varies depending on the baseline extracellular volume status in DKD patients. In more detail, dapagliflozin decreases the extracellular volume in patients with fluid retention, while not showing any such decrease in the extracellular volume in those without fluid retention. Until recently, several studies including ours have shown the effects of SGLT2 inhibitors on the body fluid status. In our previous studies of DKD patients with fluid retention, dapagliflozin predominantly decreased the ECW with a transient mild increase in urine volume , 8. Rece+ and glucose reabsorption in the late proximal tubule. SGLT1, which is located in the distal parts of the proximal tubule and mediates the transport of glucose together with Na+ (in a 1:2 ratio) [+ reabsorption in the late proximal tubule. Further studies with genetic or pharmacological SGLT1 inhibition during SGLT2 inhibition will be necessary in order to determine whether or not SGLT1 is involved in compensatory mechanisms of body fluid homeostasis during SGLT2 inhibition.Several mechanisms by which SGLT2 inhibitors maintain the homeostasis of body fluid volume have been proposed. Our recent study in non-diabetic euvolemic rats showed that SGLT2 inhibition with ipragliflozin induced a sustained glucose-driven diuretic and natriuretic tone, but homeostatic mechanisms—including compensatory increases in fluid and food intake—are activated to stabilize body fluid volume . Further2 ratio) , is acti2 ratio) . TherefoIs the homeostatic mechanism of body fluid volume by dapagliflozin due to a class effect of SGLT2 inhibitors? In our present and previous studies, neither dapagliflozin nor ipragliflozin decreased the extracellular fluid volume in patients without severe fluid retention (lower ECW/TBW patients) or in euvolemic rats , 13. In + and glucose reabsorption in the early proximal tubule, which may activate sufficient and reasonable compensatory mechanisms in the more distant tubules to maintain body fluid balance. However, furosemide inhibits the Na+–K+–Cl− cotransporter on the luminal site at the thick ascending limb of the loop of Henle, which is distal from the proximal tubule. Tolvaptan inhibits the absorption of water by selectively binding to the V2 receptor in the collecting duct, which is located on the most distal side of the tubule. When furosemide and tolvaptan inhibit the Na+ and fluid absorption at the thick ascending limbs or collecting duct, the compensatory Na+ and/or fluid reabsorption in more distant tubules may be weak or insufficient. Thus, unlike furosemide and tolvaptan, the SGLT2 inhibitor dapagliflozin is not merely a diuretic agent that generally uniformly reduces the body fluid volume regardless of the baseline fluid status; the fluid response to dapagliflozin varies depending on the baseline volume fluid status. In this regard, SGLT2 inhibitors exert different diuretic actions from loop diuretics and vasopressin V2 receptor antagonists. In addition, this pharmacological action of SGLT2 inhibitor may induce a novel clinical benefit that may support the safety (low risk of fluid shortage) of administering SGLT2 inhibitors to patients without fluid retention.In contrast to the SGLT2 inhibitor dapagliflozin, the body fluid responses to the loop diuretic furosemide and vasopressin V2 receptor antagonist tolvaptan did not clearly vary depending on the pretreatment extracellular volume status. The baseline ECW/TBW values in the furosemide and tolvaptan treatment groups were not significantly correlated with the changes in the ECW/TBW or ECW, respectively. Furthermore, furosemide and tolvaptan decreased the ECW in almost all patients, regardless of differences in the baseline ECW/TBW values, although dapagliflozin induced a positive change in the ECW in several patients with lower baseline ECW/TBW values. SGLT2 inhibitors suppress NaIn the present study, dapagliflozin decreased the extracellular volume expansion in patients with higher baseline BNP levels, suggesting that SGLT2 inhibitors may ameliorate the extracellular volume expansion particularly effectively in patients with heart failure. A recent guideline of the European Society of Cardiology and European Association for the Study of Diabetes for diabetes, pre-diabetes and cardiovascular diseases recommends SGLT2 inhibitors be administered to reduce the risk of heart failure hospitalization . The cur2), dapagliflozin did not reduce the plasma glucose levels for 1 week. However, the change in the extracellular volume status induced by dapagliflozin was greater in patients with a lower eGFR than in those with higher values. Therefore, the strength of the diuretic action and the change in the extracellular fluid status induced by SGLT2 inhibitors may not be simply determined by the GFR-dependent filtered glucose and Na+ levels but may be mainly regulated by the compensatory reabsorption of Na+, glucose and fluid in more distant tubules, as we recently reported [The baseline eGFR was weakly but positively correlated with the change in the ECW/TBW after the administration of dapagliflozin, suggesting that SGLT2 inhibitors can ameliorate body fluid retention even in patients with a reduced renal function. This result is quite different from the strength of glycemic control using SGLT2 inhibitors, in which the glucose-lowering effect decreases with a declining eGFR . In the reported .Several limitations associated with the present study warrant mention. First, this study had a relatively small sample size. Second, the relationship between the long-term body fluid response to SGLT2 inhibitors and the clinical outcomes is unclear, as this study had a short-term follow-up. Further follow-up studies are needed to evaluate this relationship. Third, the number of patients without fluid retention is small compared with the number of patients with fluid retention. Fourth, this was not a randomized control study, and a control group treated with vehicle was lacking.The pretreatment extracellular volume status predicts the body fluid response to the SGLT2 inhibitor dapagliflozin in DKD patients. These data suggest that the diminished extracellular fluid reduction effect of dapagliflozin in patients without extracellular fluid retention may contribute to maintaining a suitable body fluid status.


Here we devise the first QBF system where  strategies are built into the proof and are piecewise constructed by simple operations along with the derivation. This has several advantages: (1) lines of our calculus have a clear semantic meaning as they are accompanied by semantic objects; (2) partial strategies are represented succinctly (in contrast to some previous approaches); (3) our calculus has strategy extraction by design; and (4) the partial strategies allow new sound inference steps which are disallowed in previous central QBF calculi such as Q-Resolution and long-distance Q-Resolution. The last item (4) allows us to show an exponential separation between our new system and the previously studied reductionless long-distance resolution calculus. Our approach also naturally lifts to dependency QBFs (DQBF), where it yields the first sound and complete CDCL-style calculus for DQBF, thus opening future avenues into CDCL-based DQBF solving.Strategy extraction is of great importance for quantified Boolean formulas (QBF), both in solving and proof complexity. So far in the QBF literature, strategy extraction has been algorithmically performed  Proof complexity investigates the resources for proving logical theorems, focussing foremost on the minimal size of proofs needed in a particular calculus. Since its inception the field has enjoyed strong connections to computational complexity cf. , 20) and and20]) During the past decade, proof complexity has emerged as a key tool to model and analyse advances in the algorithmic handling of hard problems such as SAT and beyond. While traditionally perceived as a computationally hard problem, SAT solvers have been enormously successful in tackling huge industrial instances , 56 and quantified Boolean formulas (QBF), which is dependency QBFs (DQBF), which is even The algorithmic success story of solving has not stopped at SAT, but is currently extending to even more computationally complex problems such as mentclass2pt{minimcomplete , 22, vercomplete , synthescomplete , 40 and complete .huge advances in QBF solving. While some of the main innovations in SAT solving, including the development of conflict-driven clause learning (CDCL), revolutionised SAT in the late 1990s .We conclude this subsection by showing that the number of lines really is the correct size measure for Merge Resolution. The justification lies in the fact that the domain of the merge map at line u over Let i; (2) if The claim follows straightforwardly from three observations: (1) each The soundness of sense of . This meu over X with domains h be the functions computed by Let Let Let let X consistent with let for each for each  and u satisfies (ii) then u satisfies (i) and For the inductive step, let p-simulation of reductionless We show the completeness of p-simulates reductionless X and universal variables Y, and let Let i to the falsifying u-literal for l is the falsifying u-literal for if Both are established trivially when u-literals for Suppose that u over u be left of x. If u-literal for one of By Proposition This completes the induction. Since For each With soundness and completeness established by Lemma In this section we exponentially separate entclass1pt{minimasquared equality formulas) The squared equality family is the QBF family whose  Let path from a clause C in for each (The lower-bound proof is based upon two facts: (1) every total existential assignment corresponds to a path, all of whose clauses are consistent with the assignment Lemma ; (2) evees Lemma . It is tA be a clause with A occurs.Let P is clearly a path in A, for each We describe a procedure that constructs a sequence The second lemma is more technical, and its proof more involved. The proof works directly on the definition of path, the rules of reductionless C for which either Let R in p-resolvent if there exist earlier clauses Put l be the least integer such that Let We prove the lemma by showing that either C on P, if for each clause each R in for each We proceed to show that every row in r  in f(S) is the image of at most two distinct symmetrical clauses. Hence, By Lemma Close inspection of the lower-bound proof reveals that particular resolution steps are blocked due to the appearance of merged literals in the antecedents (see the proof of claim (1) of Lemma Here we construct short The squared equality family has Let Stage one. For each L as an alias for C be the clause, U be the merge map for V be the merge map for L. These U and V are the only merge maps in Letting L can be derived by resolution from L and L; to see this, note that U is clearly isomorphic to U and V is trivially consistent with V (their domains are disjoint), therefore L can be derived by resolution from L and L.The next L can be derived by resolution from L and L, since V is clearly isomorphic to V  is trivially consistent with U (disjoint domains).The final Stage two. We now show how U is isomorphic to U  and Herbrand-form (H-form) . To dateWe will recall S-form and H-form DQBFs, their semantics, and the transformation operation that relates them.S-form dependency quantified Boolean formula (DQBF) is a formula of the formAn g for H-form DQBF is the obvious dual to S-form, namely a formula of the formAn S-form DQBF is true if and only if it has a Skolem-function model. A Skolem-function model h for T defined below. those which have a Skolem-function model, but whose transform has no Herbrand-function countermodel.(B)those which have no Skolem-function model, but whose transform does have an Herbrand-function countermodel.(C)those which have a Skolem-function model, and whose transform also has an Herbrand-function countermodel.(D)those which have no Skolem-function model, nor does their transform have an Herbrand-function countermodel.All QBFs are either type A or B. Type C and D are classes of DQBFs whose semantic properties are markedly different from QBF.But this is not the case in general for DQBF. The authors of  partitioGiven what we know about the semantics of DQBF, we pose the following question: What is the impact of the existence of type C and D DQBFs on the transfer of solving techniques from QBF? We argue that the impact is indeed visible in theoretical models of solving. Moreover, it forms a decent explanation for the results that we have seen there.Figure The main message of Fig. Expansion-based (D)QBF calculi prove the non-existence of Skolem functions, whereas CDCL-based (D)QBF calculi prove the existence of Herbrand functions.Such an explanation could scarcely be sought in the QBF realm, where the non-existence of a Skolem-function model and the existence of an Herbrand-function countermodel are equivalent. One really needs to consider the behaviour of type C and D formulas to understand that these two things are not equivalent for DQBF.A reasonable explanation for this goes as follows:Whereas our statement is not the kind that can be proved as a theorem, there appears good reason to promote it as a credible hypothesis, since it explains the situation depicted in Fig Expansion-based systems prove that the universal expansion of a (D)QBF  is unsatisfiable. Satisfying assignments for the expansion are in one-one correspondence with Skolem-function models, so a proof of unsatisfiability is a proof of the non-existence of Skolem functions. Thus, the expansion systems T-transformed formulas, which is an H-form DQBF. We know that there exist type C S-form DQBFs that are true, but whose transform also has Herbrand functions, and type D S-form DQBFs that are false, but whose transform does not have Herbrand functions. In the former case we could expect to refute a true formula (unsoundness), in the latter case we find false formulas that we cannot refute (incompleteness). This is precisely what we see in Fig. Moreover, if CDCL-based systems prove the existence of Herbrand functions, we should expect to see difficulties lifting them to S-form DQBF, because the rules of these systems implicitly work on the orm DQBF , whereasentclass1pt{minimaNote that We suggest, then, that it is worthwhile to investigate further the use of H-form DQBF as an input encoding for CDCL-based DQBF solving. At least for theoretical models, this is yet to be investigated. Here we undertake the first such investigation, and we get some positive results: Merge Resolution lifts naturally to a sound and complete CDCL-based refutational proof system on H-form DQBF.It should be noted that a resolution system for DQBF called Fork Resolution  was showIn this section, we show that To lift (c)Weakening. There exists an integer By ‘existential superclause’ it is meant that For consistency with the QBF definition, we introduce an equivalent notation for H-form DQBF. We write the quantifier prefix of the H-form DQBFWeakening is, in a clear sense, the simplest rule with which one extends Soundness of Let u satisfies (i) then u satisfies (ii) then We add an additional case to the inductive step in the proof of Lemma Completeness, on the other hand, cannot be established with an analogue of Theorem h. For each X. The merge maps are constructed by merging over the pivot x iff guarantees that they are isomorphic.We give an overview of the construction. Let As merge maps essentially represent the structure of resolution steps in the subderivation, it is no surprise that the merge maps in our construction also have a full binary tree structure. This structure is captured by the following definition.binary tree merge map) A binary tree merge map for a variable u over a sequence of variables M with domain . Merge maps detail precisely how the Herbrand functions are encoded in the resolution structure of long-distance proofs. One could say that such refutations are ‘proving the existence of Herbrand functions’. For QBF, this is of course equivalent to proving the non-existence of Skolem functions, but that does not carry over to DQBF  solving paradigm. Our discussion in Sect. It seems natural, then, to suggest Merge Resolution as the underlying resolution engine in a CDCL-based solver for Herbrand-form DQBF. Conceiving such an implementation would require some work; for example, one would need to store partial strategies with learned clauses, and carry out an efficient isomorphism test. Isomorphism is an easy way to determine the equivalence of two Boolean functions, but in general it seems unlikely that two equivalent functions will have identical representations. This points towards efficient (approximate) equivalence testing as the key to a successful implementation of Whereas the decision problem for S-form DQBF is known to be mentclass2pt{minimmentclass2pt{minim


Purpose: The aim of this study was to evaluate the combined effect, acacia gum(AG)/ hydroxypropylmethylcellulose (HPMC), on biopharmaceutical performances of floating tablets of metformin hydrochloride (MTH) prepared by thermoplastic granulation using stearic acid. Methods: We have prepared the matrixes using AG/HPMC as a polymer combination. This combination of polymers which represents 15% of the total mass of tablet was used at various ratios 3:1, 1:1, 1:3, with two viscosity grade of HPMC (k15M and k100M). The developed matrixes have been evaluated for their pharmacotechnical and biopharmaceutical properties. Results: In addition to the satisfactory physical characteristics of matrixes, it was revealed that the Fc3 and Fc6 formulations with AG/HPMC k15M and AG/HPMC k100M respectively, at ratio, 1:3 were considered the most performance. These formulations have shown swelling, fast flotation, 360 and 480 seconds respectively, and remained floating on the surface of the medium for more than 24 hours, with the matrix integrity, while F1, containing only AG, did not show swelling and did not float. In addition, extendedin vitro release (>8 hours) with decreased dissolved MTH rates was demonstrated for Fc3 and Fc6 matrixes, 95% and 91% respectively, compared to F1 where MTH dissolution was complete after 2 hours. The drug release from the highest-performance matrixes (Fc3 and Fc6) was found to follow Korsmeyer-Peppas’s model. The mechanism drug release was controlled by diffusion and erosion. Conclusion: The AG/HPMC combination was suitable as a polymer matrix to improve the in vitro biopharmaceutical properties of MTH compared to AG. The developed floating matrices have showed fast flotation, total flotation time of more than 24 hours, matrix integrity and drug release extended (>8 hours) with significantly decreased drug dissolution rate (92% and 80%). However, in the same study, it was noted that among the studied hydrophilic polymers, the natural polysaccharide, acacia gum (AG), did not give any effective result. This is why in this work; we proposed to improve the biopharmaceutical performances of this polymer. In this perspective and according to the literature, the combination of hydrophilic polymers seems to be an interesting approach. Several studies aiming to develop sustained-release MTH floating tablets, have used this strategy based on the combination of hydrophilic polymers.Metformin hydrochloride (MTH) is a drug with a short half-life and a narrow absorption window in the upper gastrointestinal tract. In addition, this drug belongs to the biopharmaceutical classification system, BCS III. In this case, the development of floating tablets is a real alternative to extend the gastric residence time of MTH. Indeed, this makes it possible to sustain and prolong its release and, consequently, to improve its bioavailability in the upper part of the small intestine. In this perspective, several manufacturing techniques allow the design of such systems. Djebbar et al2 have developed a matrix-controlled drug delivery system of metformin HCl by wet granulation using various grades of hydrophilic polymers, hydroxypropylmethylcellulose  and polyacrylate polymers (Eudragit RL100 and Eudragit RS100). It was shown that various grades of HPMC at suitable concentration combined with polyacrylate polymers could be used effectively to modify the release rates. The formulation F6 with polymer combination (26%HPMC k200M-Eudragit RS100) could sustain the drug release for 12 hours. In the same perspective, Nha et al3 have developed new metformin-based sustained-release floating systems by wet granulation using polymer combinations for the prolonged release and absorption. It was concluded from the evaluation of these systems that the combination of HPMC k15M and HPMC k100M with the ratio of 90:260 w/w and the amount of NaHCO3 and citric acid at 65 mg and 13 mg, respectively, benefits the floating sustained release tablet formulation with a lower floating lag time to 1 minute. The same observations were reported in the work of Bahri-Najafi et al4 with the aim of developing a floating drug delivery system based on MTH. It has been exhibited that F7 formulation (300HPMC k4M–50PVP) is a promising system revealing excellent floating properties and sustained drug release characteristics (MDT=5.26h and DE12h49.88%). These conclusions are supported by Senjoti et al.5 who have developed sustained-release floating tablets of metformin HCl based on swelling and effervescence. The developed tablets contained an effervescent agent (NaHC03), a polymer combination (HPMC-PEO) and a swelling enhancer (SSG). The study has showed that flotation properties and drug release profile of developed tablets were influenced by the amount of HPMC, PEO, NaHCO3 and SSG. The optimized tablets of the study have showed remained floating for more than 24 hours with a floating lag time of less than 4 minutes and sustained-release of 12 hours. In addition, accelerated stability study (40°C and 75% RH) revealed that tablets from optimized formulation were stable for three months without any major changes in assay, dissolution profile, floating lag time, and other physical properties. Finally, the authors propose the HPMC-PEO, polymer combination, for being exploited in the future as a swelling polymer for floating systems to effectively increase the residence time of the drug in the stomach. Similarly, Patel et al6 have developed by direct compression technique effervescent floating tablets of metformin using a combination of polymers. The study revealed that the prepared formulation F2 using kappa carrageenan in combination with HPMC k15M is best in terms of showing excellent floating properties, extended adhesion periods and sustained drug release characteristics.Roy et al1 in the present investigation, it is proposed to prepare matrix-tablets of metformin based on AG in combination with HPMC, using a simple solvent-free manufacturing process, the thermoplastic granulation. Therefore, the aim of this work was to evaluate the combined effect of AG–HPMC on the biopharmaceutical properties of MTH: in vitro flotation, swelling kinetics and in vitro drug release kinetics of MTH. The study has carried out on AG and HPMC at two viscosity grades, HPMC k15M and HPMC k100M. The AG–HPMC polymers combination, whose total polymer concentration represents 15% of the tablet’s mass, was evaluated in three ratios: 3:1, 1:1 and 1:3.The polymer combination approach for designing floating tablets appears to provide promoter systems exhibiting excellent floating properties and satisfactory extended release characteristics of MTH. However, it should be noted that these systems are often prepared either by wet granulation or direct compression. The development of floating MTH tablets by thermoplastic granulation in the presence of a natural polysaccharide, AG combined with HPMC, was not investigated. With this in mind and in order to improve the performance of MTH floating tablets based on AG,®, Royaume-Uni), HPMC k100M , AG , sodium bicarbonate (SB) , microcrystalline cellulose pH 102 (CMC) , Talc  and magnesium stearate (Mg Stearate) .1Metformin hydrochloride (MTH) , Stearic acid (SA) , HPMC: HPMC k15M (Colorcon1The combination of two hydrophilic polymers (AG–HPMC) in three ratios of 3:1, 1:1 and 1:3 was used to prepare the MTH floating tablets. Six formulas were developed with the combination, AG–HPMC, Fc1 to Fc6. It should also be noted that HPMC was used at two viscosity grades (k15M and k100M). The formulas, Fc1 to Fc3 contain AG–HPMC k15M, and Fc4 to Fc6 are based on AG–HPMC k100M. In 1 the melt granulation. In a first step, weighing the drug and previously screened pharmaceutical excipients , melting of stearic acid at 58°C and dry mixing of MTH, CMC, NaHCO3 and combined polymers (AG–HPMC). In a 2nd step, adding this mixture to the molten stearic acid, mixing and cooling the resulting mass. Finally, after solidification of this mass, calibration , lubrication of the obtained granules , then compression using an alternative press (Frogerais Type A 307), equipped with a double set of flat punches and a 12 mm diameter die. Each tablet of 12 mm diameter contains 250 mg of MTH and weights 500 mg.The MTH floating tablets were prepared using a simple process adopted in a previous study,The prepared granules of MTH were evaluated for angle of repose, bulk and tapped densities, Carr’s index and Hausner’s ratio.8All the formulated tablets were evaluated for drug content, weight variation, hardness, friability and thickness.in vitro buoyancy was performed according to the method described by Jiménez-Castellanos et al.9 The time required for the tablet to rise to the surface and float, floating lag time (FLT) expressed of seconds (s) and the total floating time (TFT) expressed of hours (h) were determined. The results represent the average of 3 measures.The The swelling index (SI) percent was calculated by using the following equation:0 = Initial weight of tablet. The swelling characteristics of floating tablets were expressed as % in terms of weight gain as a function of time. The results are the average of three measures.Where, Wt = Weight of tablet at time t, Win vitro dissolution study was performed using the USP II paddle apparatus (Erweka DT 80), under the same operating conditions as those adopted in a previous study.1 The samples (5 mL) were collected from each test at 20 minutes, 40, 60, 90, 2 hours, then, each hour until 8 hours. The removed samples were filtered , then analyzed with a UV-Vis spectrophotometer for MTH at 233 nm (Biochrom WPA Lightwave II). The drug release rate was calculated using calibration curve equation (y=0.0165x).1 The results represent the average of six measures (Mean ± CV). The mechanism of release was determined by fitting the in vitro release data to the various kinetic equations: Zero order, First order, Higuchi and model Korsmeyer-Peppas.21 For the high-performance formulations, the coefficient of determination (R2) and Akaike information criteria (AIC)23 of the dissolution profile corresponding to each model were determined. The highest R2 and the lowest AIC of a model nominated it as the best fitting model. The times required for 25%, 50% and 80% drug release  from these formulas were determined according to the best fit model. The mean dissolution times (MDT) were also calculated.24 Finally, the diffusion exponent, “n” was calculated for characterizing the different release mechanisms of MTH from matrix. Fickian diffusion (n=0.45), non-Fickian diffusion (0.45<n<0.89), case II transport (n=0.89) and super case-II transport (n>0.89).25For evaluating the effect of AG–HPMC, combination on the MTH release, the Zero order: Qt = Q0 + K0tFirst order:Higuchi model: Qt= KH t ½25% = (25/k)1/n; T50% = (50/k)1/n; T80% = (80/k)1/n;TAIC = n × ln (WRSS) + 2p .-1 with a resolution of 4 cm-1.The characterization of MTH in the prepared matrixes was performed using FTIR spectral analysis (Shimadzu IR Affinity 1SCE) for any change in its physical state or any chemical interaction if any between the drug and the pharmaceutical excipients, particularly the melting agent. Samples of pure drug (MTH), stearic acid (SA), AG, HPMC, sodium bicarbonate (SB) and high-performance floating tablets were scanned in the spectral region of 4000 to 750 cmThe statistical analysis of the drug release kinetics data was carried out with GraphPad Prism 7 software to evaluate the MTH release profiles from developed floating systems. The evaluation was based on comparison between dissolution profiles according to the ratio of the combination (AG–HPMC) and the viscosity grade of the HPMC used in the polymers combination. We also compared the dissolution profiles of the matrices (Fc), developed with AG-HPMC combination, with those of the matrices (F), containing the same polymers separately. A one-factor variance analysis (ANOVA) was applied. The differences were considered significant at p < 0.05 with a 95% confidence interval.1 all the formulations of this study have exhibited good flowability and compressibility properties regardless of the ratio of AG-HPMC, combination. It was noted: angle of repose in the range 33.66 to 35.37°, V10 - V500 between 11 and 16 mL, Carr’s index between 13.20 and 15.73% and Hausner’s ratio between 1.15 and 1.18  obtained by melt granulation with stearic acid have manifested satisfactory and reproducible physical characteristics as shown in in vitro buoyancy study reported in 1 the combined effect of the AG with the HPMC is beneficial. It results in a significant decrease in FLT. This observation is particularly pronounced in the case where the ratio is in favor of the concentration of the HPMC; AG–HPMC (1:3). Indeed, the HPMC at a sufficient concentration in the combination, AG–HPMC is able to form, following its hydration, a gelled barrier sufficiently resistant to the pressures generated by the release of carbon dioxide bubbles and the agitation of the medium. Also, we found that the improvement in buoyancy properties was more pronounced in the case of the high-viscosity grade HPMC. TFT>24 hours for Fc5 compared to TFT<8 hours for Fc2 and FLT=360 seconds for Fc6 compared to FLT=480 seconds for Fc3  for all formulations that have shown swelling have reported in 1 according to the same approach, melt granulation with stearic acid, in the presence of the same polymers, but used separately, F1 (AG), F2 (HPMC k 15M) and F3 (HPMC k100M). It is clear that the MTH release kinetics is slowed and prolonged from the formulations based on polymers combination (Fc), compared to floating matrixes containing only AG (F1) whose kinetics was complete after 2 hours.On The combined effect of AG–HPMC on the dissolution kinetics of metformin HCl was also evaluated according to the ratio of combination (AG–HPMC) 1 and 2a2Case of HPMC k15M: In general, dissolution profiles show significant differences (P <0.05). From ratio (AG–HPMC). It is slowed and extended with the increase in HPMC k15M concentration in the following order: Fc3˃Fc2≥Fc1=F1. The dissolution profiles for Fc1 and Fc2 tablets at the respective ratios (3:1) and (1:1) have indicated complete kinetics after 3 and 5 hours of testing respectively , which did not produce positive results,1 the combination of AG–HPMC k15M with the ratio (1:3) corresponding to the Fc3 formulation, with the highest concentration of HPMC, provided floating tablets whose dissolution kinetics of MTH is slowed and extended. Indeed, it was noted: 25.49% of dissolved drug after 20 minutes, 50% between 2 and 3 hours, 80% around 6 hours and 95.03% after 8 hours of testing (P <0.05); the extended drug release follows the order: Fc3>F1. Result in accordance with the buoyancy and swelling tests. On the other hand, it should be noted that no significant difference was found between the dissolution profiles of the formulation containing AG–HPMC k15M, at the ratio (1:3), Fc3 and of the matrixes prepared with HPMC k 15M used alone, F2 .ectively 1. This iectively . The AG– testing 1. FinallP <0.05). In the same way, the kinetics of MTH is a function of the ratio (AG–HPMC). It is slowed and extended with the increase in HPMC concentration in the combination (AG–HPMC k100M) and follows the following order: Fc6˃Fc5˃Fc4=F1 (2). The dissolution profile of the Fc4 formulation at the ratio (3:1) has indicated complete kinetics after 3 h of testing  of about 90 min. This relatively high release of MTH can be explained by surface erosion of the tablets upon contact with the test medium. The prepared tablets from this formulation have shown complete dissolution after 7 hours , at the ratio of 1:3 has provided floating tablets with significantly slower dissolution kinetics of MTH (>8 hours). Indeed, it was found: 22.83% of dissolved MTH after 20 minutes, 50% between 2 and 3 h, 80% around 6 h and 91.15% at the end of the test (8 hours) (P <0.05). The extended drug release follows the order: Fc6>F1. Result in compliance with the buoyancy and swelling tests. Finally, no significant difference was noted between the dissolution profiles of the formulation containing AG–HPMC k100M, at the ratio (1:3), Fc6 and of the floating tablets based on HPMC k 100M used alone, F3 (P ≥0.05).As for the HPMC k15M, dissolution profiles show significant differences (5˃Fc4=F1 2. The di testing 2. As in  testing . The com 7 hours 2. Howeve8 hours) 2. A sign1 and 2a2). The behavior of MTH floating tablets based on this combination (Fc3 and Fc6) could be explained by the properties of the HPMC which, at high concentration in the polymer combination and thanks to its hydration capacity, quickly forms a resistant gel barrier, trapping induced CO2 bubbles in contact with the H+ medium, thus promoting swelling, flotation of the form and control of drug release (extended release).At the end of this evaluation, it was concluded that the combination (AG–HPMC) at the ratio 1:3 is the most performance. It was demonstrated a fast flotation time and a prolonged total flotation time. In addition, the release of MTH was slowed and extended with approximately 95% and 91% of dissolved drug after 8 hours for Fc3 and Fc6, respectively . We can conclude that the viscosity grade of HPMC, combined with AG, has no effect on the release kinetics of MTH from floating matrixes prepared by melt granulation with stearic acid, unlike the observations made of the buoyancy and swelling tests which have indicated a non-negligible effect of the viscosity grade.In addition to the extended drug release, in vitro release, it emerges:Finally, from the study of On the one hand, the improvement of MTH’s biopharmaceutical performances with the combination AG–HPMC, at 1:3 ratio , unlike the use of the natural polysaccharide, AG, alone (F1). Indeed, it was observed that the extended release follows the order following; Fc3>>>F1 and Fc6>>>F1.On the second hand, despite the demonstrated similarity between the dissolution profiles of Fc3 with F2 and Fc6 with F3, it should be noted that the extended release of MTH is achieved with a low concentration of HPMC (11.25%) in the case of floating tablets Fc3 and Fc6, compared to 15% for tablets F2 and F3, containing HPMC alone.25%, T50% and T80% and the average dissolution time, MDT was also evaluated. The prepared tablets from the high-performance formulations (Fc3 and Fc6) have shown T25%, T50%, T80%, and MDT, delayed and therefore an extended MTH delivery. Indeed, it was registered respectively T25%, T50% and T80% of 0.5157 h-2.1401 h-5.6169 h and of 0.5576 h-2.2750 h-5.9019 h for Fc3 and Fc6 respectively. Also, MDT values equal to 2.6784 h and 2.5821 h for Fc3 and Fc6 respectively were registered. Finally, no significant difference between the dissolution times of the successive fractions of these formulations was observed. This explains the similarity of their dissolution kinetics, thus confirming that the HPMC viscosity grade has a negative effect on the release of MTH from developed floating matrixes in this study.The combined effect of Acacia gum with HPMC (k15M or k100M) on dissolution times, Tin vitro dissolution data of the floating tablets prepared from the performance formulations, Fc3 and Fc6 was analyzed with the different mathematical models of release kinetics. The values R2, k, AIC and n were recorded in in vitro dissolution profiles of Fc3 and Fc6 were satisfied the Korsmeyer-Peppas model with R2value of 0.998 and AIC values of −61.59 and −44.59 respectively. The drug release exponents “n”, for Fc3 and Fc6, are equal to 0.487 and 0.493 respectively. The dissolution of MTH from floating matrixes prepared by melt granulation with stearic acid in the presence of two combined polymers, AG and HPMC, at the ratio (1:3) has followed a non-Fickian transport mechanism  (0.45<n<0.89). The MTH release was therefore controlled by diffusion and erosion . Also, the FTIR spectroscopy did not reveal any interaction between the drug and the excipients. This confirms the non-interaction between MTH and the melting agent (stearic acid). The infrared spectra of floating matrixes have clearly shown the characteristic absorption peaks of MTH; N-H in the region 3400 et 3100 cm050 cm-1 . These r3). Compared to AG as a polymer matrix, the AG–HPMC combination has improved the biopharmaceutical properties. The in vitro evaluation showed that the combined effect (AG–HPMC) resulted in the development of floating matrices with significantly reduced flotation times, 6 to 8 minutes for Fc3 (AG–HPMC k15M) and Fc6 (AG–HPMC k100M) respectively, instead of more than 1 hour for F1 (AG). Also, dissolution kinetics were slowed and extended, >8 hours for Fc3 and Fc6, instead of 2 hours for F1 (AG), with significantly reduced drug release rates, of approximately 95% and 91% for Fc3 and Fc6 respectively. The authors have demonstrated that these performances were dependent on the ratio of the combination (AG–HPMC). On the other hand, no significant influence of HPMC viscosity grade on the release kinetics of MTH has been revealed. The Fc3 and Fc6 formulations based on the AG–HPMC combination, at ratio, 1:3, appear to be the high-performances. Finally, based on the promising results of this study, it can be concluded that the AG–HPMC combination (1:3) can be considered as an interesting alternative to improve the flotation properties of the MTH tablets and to prolong the drug release.MTH floating tablets were developed with the combination of AG–HPMC, by thermoplastic granulation using stearic acid, according to an effervescent approach (NaCONot applicable.The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.The authors would like to thank the pharmaceutical laboratories El-Kendi, the Saïdal Annaba production unit, Inpha-Médis and Biocare for providing free raw materials. We also express our gratitude to the staff of the Saïdal Research and Development Center for their assistance with the study.