Reduced Serum Alkaline Phosphatase Research Articles

Safety and efficacy of HK-660S in patients with primary sclerosing cholangitis: A randomized double-blind phase 2a trial.

A clinical unmet need persists for medications capable of modulating the progression of primary sclerosing cholangitis (PSC). This study aimed to assess the clinical feasibility of HK-660S (beta-lapachone) in PSC. In this multicenter, randomized, double-blind, placebo-controlled, parallel-group phase 2 trial, participants were assigned in a 2:1 ratio to receive either 100 mg of HK-660S or a placebo twice daily for 12 weeks. The primary outcomes were the reduction in serum alkaline phosphatase (ALP) levels and the percentage of participants showing improvements in PSC severity, as determined by magnetic resonance cholangiopancreatography (MRCP) with the Anali score. Secondary endpoints included changes in liver stiffness and adverse events. The analysis included 21 patients, 15 receiving HK-660S, and six receiving a placebo. Improvements in the Anali score were observed in 13.3% of the HK-660S group, with no improvements in the placebo group. HK-660S treatment resulted in a 15.2% reduction in mean ALP levels, compared to a 6.6% reduction in the placebo group. A stratified ad-hoc analysis based on baseline ALP levels showed a statistically significant response in the HK-660S group among those with ALP levels greater than twice the upper limit of normal, with a 50% responder rate (p = 0.05). Additionally, 26.7% of the HK-660S group showed improvements in the enhanced liver fibrosis score, with no improvements in the placebo group. HK-660S was generally well-tolerated. HK-660S is well-tolerated among patients with PSC and may improve bile duct strictures, decrease serum ALP levels, and reduce liver fibrosis. (cris.nih.go.kr, Number KCT0006590).

Vascular adhesion protein-1 blockade in primary sclerosing cholangitis: Open-label, multicenter, single-arm, phase II trial.

Primary sclerosing cholangitis is a progressive inflammatory liver disease characterized by biliary and liver fibrosis. Vascular adhesion protein-1 (VAP-1) is important in the inflammatory process driving liver fibrosis. We evaluated the safety and efficacy of VAP-1 blockade with a monoclonal antibody (timolumab, BTT1023) in patients with primary sclerosing cholangitis. BUTEO was a prospective, single-arm, open-label, multicenter, phase II trial, conducted in 6 centers in the United Kingdom. Patients with primary sclerosing cholangitis aged 18-75 years had an alkaline phosphatase value of >1.5 times the upper limit of normal. The dose-confirmatory stage aimed to confirm the safety of timolumab through the incidence of dose-limiting toxicity and sufficient trough levels of circulating antibody to block VAP-1 function. The primary outcome of the dose-expansion portion of the trial was patient's response to timolumab at day 99, as measured by a reduction in serum alkaline phosphatase by 25% or more from baseline to day 99. Twenty-three patients were recruited: 7 into the initial dose-confirmatory stage and a further 16 into an expansion stage. Timolumab (8mg/kg) was confirmed to be safe for the duration of administration with sufficient circulating levels. Only 2 of the 18 evaluable patients (11.1%) achieved a reduction in alkaline phosphatase levels of 25% or more, and both the proportion of circulating inflammatory cell populations and biomarkers of fibrosis remained unchanged from baseline. The BUTEO trial confirmed 8mg/kg timolumab had no short-term safety signals and resulted in sufficient circulating levels of VAP-1 blocking timolumab. However, the trial was stopped after an interim assessment due to a lack of efficacy as determined by no significant change in serum liver tests.

Predictive value of serum alkaline phosphatase, tumor-specific growth factor, and macrophage migration inhibitory factor for the efficacy of immunotargeted therapy in osteosarcoma patients.

To assess the predictive value of serum alkaline phosphatase (ALP), tumor-specific growth factor (TSGF), and macrophage migration inhibitory factor (MIF) for the efficacy of combined immunosuppressive and targeted therapy in osteosarcoma (OS). We retrospectively analyzed clinical data from 161 OS patients treated at Xi'an Honghui Hospital from October 2020 to October 2022. Patients received 12 weeks of therapy with interferon-α (IFN-α) and bevacizumab. Serum levels of ALP, TSGF, and MIF were measured before and after treatment. Based on treatment efficacy, patients were categorized into effective and ineffective groups. Both univariate and logistic regression analyses were utilized to evaluate the influence of these biomarkers on therapy outcomes. A significant reduction in serum ALP, TSGF, and MIF levels post-treatment was found (all P<0.001). Higher pre-treatment levels of these biomarkers were associated with less effective outcomes (P<0.001). Pre-treatment levels of ALP, TSGF, and MIF are significant independent predictors of response to immunotargeted therapy in OS patients, suggesting their potential role in guiding treatment strategies.

Novel Treatments for PXE: Targeting the Systemic and Local Drivers of Ectopic Calcification.

Pseudoxanthoma elasticum (PXE) is a heritable multisystem ectopic calcification disorder. The gene responsible for PXE, ABCC6, encodes ABCC6, a hepatic efflux transporter regulating extracellular inorganic pyrophosphate (PPi), a potent endogenous calcification inhibitor. Recent studies demonstrated that in addition to the deficiency of plasma PPi, the activated DDR/PARP signaling in calcified tissues provides an additional possible mechanism of ectopic calcification in PXE. This study examined the effects of etidronate (ETD), a stable PPi analog, and its combination with minocycline (Mino), a potent inhibitor of DDR/PARP, on ectopic calcification in an Abcc6-/- mouse model of PXE. Abcc6-/- mice, at 4 weeks of age, before the development of ectopic calcification, were treated with ETD, Mino, or both for 18 weeks. Micro-computed tomography, histopathologic examination, and quantification of the calcium content in Abcc6-/- mice treated with both ETD and Mino revealed further reduced calcification than either treatment alone. The effects were associated with reduced serum alkaline phosphatase activity without changes in plasma PPi concentrations. These results suggest that ETD and Mino combination therapy might provide an effective therapeutic approach for PXE, a currently intractable disease.

Saroglitazar improves nonalcoholic fatty liver disease and metabolic health in liver transplant recipients.

NAFLD is common after liver transplantation (LT) and is associated with an increased metabolic burden. Currently, there is a paucity of investigations into the treatment of post-LT NAFLD. In the present study, we evaluated the safety and efficacy of saroglitazar, a novel dual peroxisome proliferator-associated receptor α/γ agonist, on the treatment of post-LT NAFLD and metabolic burden. This is a phase 2A, single-center, open-label, single-arm study in which patients with post-LT NAFLD received saroglitazar magnesium 4mg daily for 24 weeks. NAFLD was defined by a controlled attenuation parameter ≥264dB/m. The primary endpoint was the reduction in liver fat as measured by MRI proton density fat fraction (MRI-PDFF). Secondary MRI-based metabolic endpoints included visceral adipose tissue, abdominal subcutaneous adipose tissue volumes, muscle fat infiltration, and fat-free muscle volume. Saroglitazar treatment led to a reduction in MRI-PDFF from 10.3±10.5% at baseline to 8.1±7.6%. A relative 30% reduction from baseline MRI-PDFF value was noted in 47% of all patients and 63% of patients with baseline MRI-PDFF >5%. Reduction in serum alkaline phosphatase was an independent predictor of MRI-PDFF response. Saroglitazar did not decrease fat-free muscle volume nor increase muscle fat infiltration, but did lead to a mild increase in visceral adipose tissue and abdominal subcutaneous adipose tissue. The study drug was well tolerated and a mild nonsignificant increase in serum creatinine was noted. Saroglitazar did not affect the weight. The study provides preliminary data demonstrating the safety and metabolic benefits of saroglitazar in LT recipients and underscores the importance of future studies to establish its efficacy after LT.

Adjunct Fenofibrate Up-regulates Bile Acid Glucuronidation and Improves Treatment Response For Patients With Cholestasis.

Accumulation of cytotoxic bile acids (BAs) during cholestasis can result in liver failure. Glucuronidation, a phase II metabolism pathway responsible for BA detoxification, is regulated by peroxisome proliferator–activated receptor alpha (PPARα). This study investigates the efficacy of adjunct fenofibrate therapy to up‐regulate BA‐glucuronidation and reduce serum BA toxicity during cholestasis. Adult patients with primary biliary cholangitis (PBC, n = 32) and primary sclerosing cholangitis (PSC, n = 23), who experienced an incomplete response while receiving ursodiol monotherapy (13‐15 mg/kg/day), defined as serum alkaline phosphatase (ALP) ≥ 1.5 times the upper limit of normal, received additional fenofibrate (145‐160 mg/day) as standard of care. Serum BA and BA‐glucuronide concentrations were measured by liquid chromatography–mass spectrometry. Combination therapy with fenofibrate significantly decreased elevated serum ALP (−76%, P < 0.001), aspartate transaminase, alanine aminotransferase, bilirubin, total serum BAs (−54%), and increased serum BA‐glucuronides (+2.1‐fold, P < 0.01) versus ursodiol monotherapy. The major serum BA‐glucuronides that were favorably altered following adjunct fenofibrate include hyodeoxycholic acid–6G (+3.7‐fold, P < 0.01), hyocholic acid–6G (+2.6‐fold, P < 0.05), chenodeoxycholic acid (CDCA)–3G (−36%), and lithocholic acid (LCA)–3G (−42%) versus ursodiol monotherapy. Fenofibrate also up‐regulated the expression of uridine 5′‐diphospho‐glucuronosyltransferases and multidrug resistance–associated protein 3 messenger RNA in primary human hepatocytes. Pearson’s correlation coefficients identified strong associations between serum ALP and metabolic ratios of CDCA‐3G (r2 = 0.62, P < 0.0001), deoxycholic acid (DCA)‐3G (r2 = 0.48, P < 0.0001), and LCA‐3G (r2 = 0.40, P < 0.001), in ursodiol monotherapy versus control. Receiver operating characteristic analysis identified serum BA‐glucuronides as measures of response to therapy. Conclusion: Fenofibrate favorably alters major serum BA‐glucuronides, which correlate with reduced serum ALP levels and improved outcomes. A PPARα‐mediated anti‐cholestatic mechanism is involved in detoxifying serum BAs in patients with PBC and PSC who have an incomplete response on ursodiol monotherapy and receive adjunct fenofibrate. Serum BA‐glucuronides may serve as a noninvasive measure of treatment response in PBC and PSC.

Depth of Organ Response in Newly Diagnosed AL Amyloidosis Is Associated with Improved Survival: Improved Outcome Discrimination with Graded Organ Response

Introduction: Organ recovery following anti-plasma cell therapy is associated with an improved outcome in patients with AL amyloidosis. However, unlike hematological response criteria, organ response criteria are graded only as present or absent, without taking into consideration the depth of organ response. Monoclonal antibodies targeting the amyloid deposits are emerging as a potential therapeutic modality to enhance organ recovery. Therefore, more exact grading of organ response may help in selection of patients who may benefit from immune-mediated therapies. Methods: Patients were included if they had biopsy-proven systemic AL amyloidosis seen within 90 days of diagnosis, between 2000 and 2015. Patients had to have involvement of at least one assessable organ (heart, kidney or liver) and documentation of organ response per consensus criteria. Time to organ response and time to best organ response (defined as the lowest level of biochemical dysfunction, prior to hematological progression or institution of second line therapy) were calculated from date of first line therapy. Results: Four hundred and sixteen patients are included. Patients had a median of 1 assessable organ for response [interquartile range (IQR) 1-2]. Summary of organ response parameters is presented in Table 1. One hundred and eighty two patients were evaluable for cardiac response. Time from treatment initiation to achievement of cardiac response was 9.4 months (IQR 4.7-15.9). The median maximal reduction in NT-proBNP from baseline was 75% (IQR 62-88%) with a median nadir level of 544 pg/mL. The median time to maximal cardiac response was 24.1 months (IQR 15.6-40.5). The lower the NT-proBNP trough level achieved, the better the survival, both if assessed by percentage reduction from baseline as well as the nadir level. However, in patients with a baseline NT proBNP >2000 pg/mL survival was improved when the lowest NT proBNP was below 500 pg/mL rather than a >90% reduction. Two-hundred and eighty six patients were evaluable for renal response. Time to reduction of 30% and 50% in urinary protein was 5.9 and 10.9 months, respectively. Median maximal reduction in urinary protein from baseline was 92% (IQR 78-98%), to a median nadir value of 0.2 gr/24-h (IQR 0.1-1.1). Median time to maximal reduction in urinary protein was 30.1 (IQR 17.7-48.7) months. In patients with renal and no cardiac involvement (n=147), the greater the urinary protein reduction, the better the overall survival. Fifty eight patients were evaluable for hepatic response. The median time to hepatic response was 13 (IQR 4.3-30.5) months. The median maximal reduction in serum alkaline phosphatase (AP) was 78% (IQR 65-89%) with a median nadir level of 84 U/L. The median time to maximal hepatic response was 39.2 (IQR 20.4-58.7) months. Patients with hepatic response who achieved greater than 75% reduction in baseline AP had improved survival compared to patients with hepatic response at a 50-75% reduction in AP (median survival 179 vs 90.8 months, respectively). Conclusion: Grading the depth of organ response provides better prognostic information for patients with newly diagnosed AL amyloidosis. In all organs (heart, kidney, liver), the deeper the organ response, the better the survival. This should lead to a refinement in organ response criteria to include increasing depths of organ response rather than a dichotomous outcome of response versus no response. Deep organ responses are more common in patients with renal and hepatic involvement than cardiac involvement. [Display omitted] DisclosuresDispenzieri:Celgene, Millenium, Pfizer, Janssen: Research Funding. Kapoor:Takeda, Celgene and Amgen: Research Funding. Russell:Imanis Life Sciences: Equity Ownership; Vyriad: Equity Ownership. Kumar:Celgene, Millennium/Takeda, Onyx, AbbVie, Janssen, Sanofi, Novartis, Amgen, Genentech, Merck, Oncopeptides, Roche, Skyline Diagnostics: Research Funding; Celgene, Millennium, BMS, Onyx, Janssen, Noxxon, AbbVie, Amgen, Merck, Oncopeptides, Skyline Diagnostics, Takeda: Consultancy; Skyline: Honoraria. Gertz:Millennium: Consultancy, Honoraria; Celgene, Novartis, Smith-Kline, Prothena, Ionis, Amgen: Honoraria.

A phase 1b study of novel immunogenic cell death inducer PT-112 plus PD-L1 inhibitor avelumab in metastatic castrate-resistant prostate cancer (mCRPC) patients.

e17025 Background: PT-112 is a novel pyrophosphate-platinum conjugate that induces immunogenic cell death, reaches highest concentrations in bone (osteotropism), and synergizes with immune checkpoint inhibitors (ICIs) in preclinical models. Phase I studies in solid tumors, as monotherapy and in combination with PD-L1 inhibitor avelumab (“PAVE”), as well as in multiple myeloma as monotherapy have demonstrated that PT-112 is well-tolerated and active. We present updated safety and exploratory efficacy findings in a mCRPC sub-population treated with PAVE, including a cohort of patients at a lower-frequency dosing schedule. Methods: A total of 32 mCRPC patients (pts) received 800 mg of avelumab on days 1 and 15 of a 28-day cycle, of whom 18 received PT-112 on days 1, 8 and 15 at 200mg/m2 (n = 17) or 150 mg/m2 (n = 1); a separate cohort of 14 received PT-112 on days 1 and 15 at 300 mg/m2 (n = 13) or 200mg/m2 (n = 1). Results: Median age was 68 (range 47-87) and number of prior lines of therapy (Tx) was 7 (2-12), including prior platinum-containing therapy in 11 (34%) and ICI Tx in 9 pts (28%). Baseline grade 2 anemia was seen in 7 (22%) pts. The most common treatment-related adverse events (TRAEs) were anemia (47%) and thrombocytopenia (41%); 23 pts (72%) had ≥1 grade 3-4 TRAE, with no cases of bleeding, sepsis, or grade 5 TRAEs. Antitumor effects included 8 (25%) pts with a PSA reduction of ≥50% (PSA50); 5 (16%) were maintained through ≥1 follow-up. Of ten pts with RECIST-measurable disease, 3 had tumor volume reductions, one with a confirmed partial response (PR). Twenty-four (75%) pts experienced a reduction in serum alkaline phosphatase (ALP) (median reduction 15%), and improvement in patient-reported pain and quality of life was observed. For the 13 pts given 300 mg/m2 PT-112 bi-weekly, 7 (54%) had prior platinum-containing Tx and 4 (31%) had baseline grade 2 anemia. The most common TRAEs were anemia (69%) and thrombocytopenia (54%), with 11 (85%) pts having ≥1 grade 3-4 TRAE. Four pts (31%) had PSA50 reductions; 3 (23%) were maintained through ≥1 follow-up. Conclusions: PAVE is safe and generally well tolerated in mCRPC patients at the doses and schedules tested, providing meaningful antitumor effects in heavily pre-treated patients, including tumor volume, PSA and ALP reductions. Reductions in ALP may be indicative of anti-cancer activity at bone sites of disease. Although the sample sizes are small, the frequency of confirmed PSA50 responses and of grade 3-4 TRAEs was slightly higher in the group receiving biweekly 300 mg/m2 PT-112, and higher rates of TRAEs may be explained by higher baseline anemia and/or prior platinum Tx. Further clinical investigation of the combination of PT-112 plus ICIs in a less heavily pre-treated mCRPC population is warranted. A phase 2 study of PT-112 monotherapy in mCRPC is ongoing. Clinical trial information: NCT03409458.

Fibrates for Itch (FITCH) in Fibrosing Cholangiopathies: A Double-Blind, Randomized, Placebo-Controlled Trial

Pruritus may seriously impair quality of life in patients with cholestatic diseases such as primary or secondary sclerosing cholangitis (PSC, SSC) and primary biliary cholangitis (PBC). Pharmacologic strategies show limited efficacy and can provoke serious side effects. We hypothesized that bezafibrate, a broad peroxisome proliferator-activated receptor (PPAR) agonist, relieves cholestasis-associated itch by alleviating hepatobiliary injury. The aim of this investigator-initiated FITCH trial (Fibrates for cholestatic ITCH) was to assess effects of bezafibrate on pruritus in patients with PSC, PBC, and SSC. Patients with moderate to severe pruritus (≥5 of 10 on visual analog scale [VAS]) due to PSC, PBC, or SSC were recruited for this double-blind, randomized, placebo-controlled trial between 2016 and 2019. Patients received once-daily bezafibrate (400 mg) or placebo for 21 days. The primary end point was ≥50% reduction of pruritus (VAS; intention-to-treat). Of 74 randomized patients, 70 completed the trial (95%; 44 PSC, 24 PBC, 2 SSC). For the primary end point, bezafibrate led in 45% (41% PSC, 55% PBC) and placebo in 11% to ≥50% reduction of severe or moderate pruritus (P= .003). For secondary end points, bezafibrate reduced morning (P= .01 vs placebo) and evening (P= .007) intensity of pruritus (VAS) and improved the validated 5D-Itch questionnaire (P= .002 vs placebo). Bezafibrate also reduced serum alkaline phosphatase (-35%, P= .03 vs placebo) correlating with improved pruritus (VAS, P= .01) suggesting reduced biliary damage. Serum bile acids and autotaxin activity remained unchanged. Serum creatinine levels tended to mildly increase (3% bezafibrate, 5% placebo, P= .14). Bezafibrate is superior to placebo in improving moderate to severe pruritus in patients with PSC and PBC. Netherlands Trial Register, ID: NTR5436 (August 3, 2015), ClinicalTrials.gov ID: NCT02701166 (March 2, 2016).

S1037 Portal-Based Lymphocytic Infiltration Is Associated With Disease Activity in Patients With Primary Biliary Cholangitis

INTRODUCTION: Primary biliary cholangitis (PBC) is a cholestatic liver disease that results in inflammation of the intrahepatic bile duct and the portal tract. While the liver biopsy is the gold standard in relation to disease activity and fibrosis, histological findings have not been compared to serum alkaline phosphatase (ALP) changes with therapy. This is important as ALP is a well-established serum marker in the assessment of PBC. METHODS: Patients with biopsy-confirmed PBC, received a retrospective medical record review. The histological features were stratified by a GI pathologist into the following categories: 1) lymphocytic infiltration of the portal tract, 2) neutrophilic infiltration of the portal tract, 3) eosinophilic infiltration of the portal tract, 4) plasma cell infiltration of the portal tract, 4) florid duct lesions and bile duct loss, 5) bile flow obstruction, 6) inflammation of the hepatic parenchyma, and 7) hepatic necrosis. The primary endpoint was the non-reduction in ALP over the 1-year period. RESULTS: Of the 217 patients in the study, 173 (79.7%) did not have a reduction in their ALP levels over a 1 year period even though 148 of the 173 patients (85.5%) were undergoing therapy. Of the histological features analyzed in the study, lymphocytic infiltration of the portal tract was associated with the non-reduction in ALP levels (P = 0.02, OR 2.18 95%CI 1.11–4.30). However, other features including neutrophilic infiltration (P = 0.97, OR 0.98 95%CI 0.37–2.59), eosinophilic infiltration (P = 0.27, OR 1.87 95%CI 0.68–5.10), plasma cell infiltration (P = 0.27, OR 1.94 95%CI 0.71–5.28), bile duct damage (P = 0.52, OR 1.24 95%CI 0.64–2.41), hepatic inflammation (P = 0.13, OR 0.58 95%CI 0.28–1.18), and hepatic necrosis (P = 0.17, OR 1.65 95%CI 0.81–3.38) were not significantly associated with disease exacerbation that is demonstrated by the maintenance or elevation in ALP levels. In a multivariate model, the association between lymphocytic infiltration and the non-reduction in ALP remained significant (P = 0.26, aOR 2.31 95%CI 1.11–4.84), despite controlling for age (P = 0.34), gender (P = 0.49), race (P = 0.78), weight (P = 0.80), diabetes (P = 0.03), alcohol use (P = 0.998), and ursodiol therapy (P = 0.88). CONCLUSION: A lack of reduction in serum ALP correlates with disease activity and lymphocytic infiltration of the portal tract in patients with PBC. Hence, patients with these characteristics in biochemical and histological analysis would likely benefit from escalation of therapy and close surveillance.Figure 1.: Multivariate model: lymphocytic infiltration of the portal tract is an independent predictor of PBC disease activity as demonstrated by the non-reduction in the level of alkaline phosphatase.

Effects of phytase and benzoic acid supplementation on growth performance, nutrient digestibility, tibia mineralization and serum traits in male broiler chickens

This study was conducted to investigate the effects of phytase (Phy), benzoic acid (BA) and their interactions on growth performance, nutrients digestibility and tibia mineralization in broiler chickens fed standard or nutrient,s deficient diets. A total of 768 one-d-old male chicks were divided into 8 treatments for 42 d. The treatments were as follows:positive control (PC), PC diet with 0.5 g/kg of BA, PC diet with 500 FTU/kg of Phy, PC diet with 0.5 g/kg of BA and 500 FTU/kg of Phy, negative control diet (NC) with a nutrient deficiency (ND) equal to matrix value of 500 FTU/kg of Phy, NC diet with 0.5 g/kg of BA, NC diet with 500 FTU/kg of Phy, and NC diet with 0.5 g/kg of BA and 500 FTU/kg of Phy. Broiler chickens received NC diets had lower body weight gain (BWG), feed intake (FI) and higher feed conversion ratio (FCR) in the entire experiment (P ≤ 0.05). Dietary ND negatively influenced tibia and toe ash contents, serum, and tibia P contents as well as ileal P, Ca and CP digestibility at d 28 (P ≤ 0.05). FCR of d 1 to 11 and d 12 to 24 improved when Phy was added to NC rather than PC diets (P ≤ 0.05). Phy supplementation did not affect serum P and Ca content at d 28 (P> 0.05), but reduced serum alkaline phosphatase content in broiler chickens fed PC diets (P ≤ 0.05). Phy supplementation increased tibia and toe ash contents at d 28, especially in broiler chickens fed NC diets (P ≤ 0.05). Inclusion of BA did not influence (P> 0.05) tibia ash, tibia Ca and P contents at d 28, but increased toe ash content (P ≤ 0.05).The interaction between BA and the dietary nutrient level was significant for FCR of d 25–42 and d 1–42 (P ≤ 0.05). Broiler chickens fed PC diets with BA had better FCR compared with those fed PC diets without BA atd 25–42 (P ≤ 0.05). Adding Phy to the diets containing BA improved BWG and FI atd 1–11(P ≤ 0.05). In conclusion, Phy was effective in improving some growth performance traits, and bone mineralization especially when added to NC diets. However, BA had a positive effect on FCR of broiler chickens fed PC diets and had no impact on ileal nutrients digestibility, but improved toe ash content.

SO078APABETALONE, A BROMODOMAIN AND EXTRA-TERMINAL (BET) PROTEIN INHIBITOR, REDUCES ALKALINE PHOSPHATASE IN CVD PATIENTS, IN MICE, AND IN CELL CULTURE SYSTEMS

Abstract Background and Aims Elevated serum alkaline phosphatase (ALP) independently predicts major adverse cardiac events (MACE) by contributing to vascular calcification and endothelial dysfunction arising in chronic kidney disease (CKD) and cardiovascular disease (CVD). Apabetalone is an orally active inhibitor of bromodomain and extraterminal (BET) proteins – epigenetic readers that modulate gene expression involved in vascular inflammation and calcification. Here we examined apabetalone’s effects on ALP post-hoc in recent clinical trials, then performed mechanistic studies into apabetalone’s impact on tissue non-specific ALP (TNALP) expression in mice and cell culture. Method Serum ALP was determined in CVD patients in phase 2 trials (3 month ASSERT and 6 month SUSTAIN &amp; ASSURE) and in the phase 3 BETonMACE CVD outcomes trial, including subpopulations with CKD (eGFR&amp;lt;60 mL/min/1.73m2). Apabetalone’s effect on expression of TNALP (gene symbol ALPL) was examined in mice, cultured primary human hepatocytes (PHH), HepaRG, HepG2, vascular smooth muscle cells (VSMCs), and vascular endothelial cells by real-time PCR. TNALP protein levels were assessed by immunoblots and flow cytometry. ALP enzyme activity was measured in enzymatic assays. Results In phase 2 trials, baseline serum ALP independently predicted MACE (hazard ratio [HR] 1.6, 95% CI 1.2-2.2, p=0.001). In the 3 month ASSERT trial, apabetalone dose dependently reduced serum ALP (p&amp;lt;0.001 vs placebo). Prominent reductions in ALP were apparent in patients on apabetalone (n=331) vs placebo (n=166) in combined analysis of the ASSURE &amp; SUSTAIN trials (median % change -11 vs -3.2; p&amp;lt;0.001). In the subset with CKD, patients on apabetalone (n=69) had greater reduction in serum ALP than placebo (n=22; p=0.008). Strikingly, ALP reductions in phase 2 correlated with reduction in MACE (HR 0.58, 95% CI 0.44-0.77, p&amp;lt;0.001). Consistent with phase 2, BETonMACE saw serum ALP reduced by 6.8 U/L with apabetalone (n=1082) vs placebo (n=1070; p&amp;lt;0.001) at 24 weeks. At the conclusion of BETonMACE, fewer MACE occurred in the CKD subgroup with apabetalone (n=124) vs placebo (n=164; HR 0.50 95% CI 0.26-0.96 p=0.032). Neither apabetalone nor statins that control low-density lipoprotein cholesterol inhibited recombinant TNALP enzyme activity, implying that decreased serum ALP activity in patients reflected reduction in TNALP production rather than inhibition of the enzyme. Liver-derived TNALP accounts for ≈50% of circulating ALP. In the liver of mice on high fat diet, apabetalone or JQ1 (BET inhibitors with different chemical scaffolds) reduced Alpl mRNA (p&amp;lt;0.001) with corresponding trends in TNALP activity. In PHH, HepaRG, &amp; HepG2 cells, apabetalone dose dependently suppressed ALPL expression by 60-80%. In HepG2 cells, apabetalone reduced TNALP protein (&amp;gt;55%, p&amp;lt;0.001), enzyme activity (&amp;gt; 40%; p&amp;lt;0.001), and % of TNALP positive cells (15-30%; p&amp;lt;0.001). MZ1, which promotes degradation of BET proteins, downregulated ALPL / TNALP similar to apabetalone. In VSMCs, apabetalone or JQ1 suppressed ALPL gene expression, TNALP protein levels, and enzyme activity, leading to decreases in extracellular calcium deposition. In addition, apabetalone downregulated ALPL expression in human aortic, umbilical vein, and brain microvascular endothelial cells by 50-70%. Conclusion Apabetalone lowers serum ALP in clinical trials, which is consistent with reduced hepatic production of TNALP - the most abundant ALP isoform. Further, apabetalone downregulates ALPL gene expression in vascular cell types while reducing calcification. Together, BET-dependent epigenetic modulation of ALP by apabetalone can affect several pathogenetic processes, and thereby improve cardiovascular outcomes. This study provides insights to the CVD event reductions observed in the CKD subpopulation in the BETonMACE Phase 3 trial.

A randomized, placebo-controlled, phase II study of obeticholic acid for primary sclerosing cholangitis

Background & Aims:Primary sclerosing cholangitis (PSC) is a rare, cholestatic liver disease with no currently approved therapies. Obeticholic acid (OCA) is a potent farnesoid X receptor (FXR) agonist approved for the treatment of primary biliary cholangitis. We investigated the efficacy and safety of OCA in patients with PSC.Methods:AESOP was a phase II, randomized, double-blind, placebo-controlled, dose-finding study. Eligible patients were 18 to 75 years of age with a diagnosis of PSC and serum alkaline phosphatase (ALP) ≥2× the upper limit of normal (ULN) and total bilirubin <2.5× ULN. Patients were randomized 1:1:1 to receive placebo, OCA 1.5–3.0 mg, or OCA 5–10 mg once daily for a 24-week, double-blind phase followed by a 2-year, long-term safety extension (LTSE). Primary endpoints were change in ALP from baseline to week 24, and safety.Results:The intent-to-treat population comprised 76 patients randomized to placebo (n = 25), OCA 1.5–3.0 mg (n = 25), and OCA 5–10 mg (n = 26). At week 24, serum ALP was significantly reduced with OCA 5–10 mg vs. placebo (least-square [LS] mean difference = −83.4 [SE = 40.3] U/L; 95% CI −164.28 to −2.57; p = 0.043). Serum ALP was not significantly reduced with OCA 1.5–3.0 mg vs. placebo at week 24 (LS mean [SE] difference = −78.29 [41.81] U/L; 95% CI −162.08 to 5.50; p = 0.067). Total bilirubin remained comparable to baseline in all groups. The most common treatment-emergent adverse event was dose-related pruritus (placebo 46%; OCA 1.5–3.0 mg 60%; OCA 5–10 mg 67%). Reductions in ALP were maintained during the LTSE, and no new safety signals emerged.Conclusions:Treatment with OCA 5–10 mg reduced serum ALP in patients with PSC. Mild to moderate dose-related pruritus was the most common adverse event.Registration:ClinicalTrials.gov: NCT02177136; EudraCT: 2014-002205-38.

Prednisolone therapy for chronic hepatitis in English springer spaniels: a prospective study of 12 cases

BackgroundEnglish springer spaniels (ESS) show an increased risk of chronic hepatitis (CH). In a previous study of 68 ESS with CH, in which only one dog received corticosteroids, a median...

Early exfoliation as an indication for evaluation: A case report

Hypophosphatasia (HPP) is a rare genetic skeletal disorder that occurs due to mutation in the ALPL gene. It is characterized by defective bone and teeth mineralization. Odontohypophosphatasia is the mildest form of HPP that affects only the dental tissues, manifests as early exfoliation of teeth, defective cementum formation, reduced alveolar bone height, and enlarged pulp chamber. We report a case of an 8-year-old female who presented only with dental findings of HPP. HPP was suspected due to reduced serum alkaline phosphatase level, reduced alveolar bone height, and enlarged pulp chamber. The cemental defects were identified by the ground section of the exfoliated tooth and measured using Image J software. Thus, we arrived at the final diagnosis of Odontohypophosphatasia.

Alhagi maurorum aqueous extract protects against norfloxacin-induced hepato-nephrotoxicity in rats

ObjectivesWhile the protective effects of Alhagi maurorum have been shown against various ailments, its role against norfloxacin-induced adverse effects has not been studied. The current study was conducted to determine the effect of A. maurorum aqueous extract against norfloxacin-induced side effects in rats. MethodsTwenty-four male albino rats were randomly assigned into four groups, which received normal saline, norfloxacin (50 mg/kg b.wt orally once a day), A. maurorum aqueous extract (300 mg/kg b.wt orally once a day), and norfloxacin with A. maurorum aqueous extract by the same previous mentioned dosages. Blood samples were collected for hematological examination to evaluate liver and kidney function tests. Hepatic and renal tissue samples were obtained to assess antioxidant activity and histopathological examination. ResultsA. maurorum aqueous extract significantly ameliorated norfloxacin-induced elevation in tissue malondialdehyde, and reduction in tissue antioxidant enzymes such as catalase, glutathione peroxidase, and superoxide dismutase activities as well as reduced glutathione concentration. Concurrent administration of A. maurorum aqueous extract with norfloxacin significantly reduced serum alkaline phosphatase, aminotransferases, urea, creatinine, and uric acid and increased RBCs count, Hb concentration, PCV, leucocyte, and monocyte counts compared with the norfloxacin-treated group. Co-administration of A. maurorum aqueous extract with norfloxacin prevented the degenerative changes induced by norfloxacin alone in liver and kidney tissues. The phytochemical profile of the extract showed the presence of carbohydrates, alkaloids, saponins, tannins, phenolics, and flavonoids. ConclusionThese findings indicated that A. maurorum possesses potent antioxidant activities and could be used to attenuate norfloxacin-induced side effects.

Modulatory effects of artichoke (Cynara scolymus L.) leaf extract against oxidative stress and hepatic TNF-α gene expression in acute diazinon-induced liver injury in rats.

Background Diazinon (DZN) causes serious liver damage in both humans and animals. In the present study, the hepatoprotective effects of Cynara scolymus L. leaf extract against DZN-induced liver injury were examined. Methods Forty male rats were divided into five groups. The control group received a normal diet. The DZN group received DZN only (25 mg/kg, po). The DZN + Syl group received DZN (25 mg/kg, po) and silymarin (Syl) (50 mg/kg, po). The DZN + Art group received DZN (25 mg/kg, po) and artichoke (Art) leaf extract (1500 mg/kg, po). The Art group received Art leaf extract only (1500 mg/kg, po). After 15 days, serum tumor necrosis factor α (TNF-α), alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), lipid profile, protein carbonyl content, serum and hepatic malondialdehyde (MDA), hepatic TNF-α gene expression, hepatic catalase (CAT), superoxide dismutase (SOD), and vitamin C (Vit C) were measured and histopathological examination was performed. Results DZN caused a significant elevation in serum ALP, AST, ALT, MDA, TNF-α, protein carbonyl, hepatic MDA, and TNF-α gene expression in the DZN group as opposed to the control group. Also, DZN led to the reduction of hepatic CAT, SOD, and Vit C in the DZN group relative to the control group. The administration of Art extract resulted in not only a significant reduction in serum ALP, AST, ALT, MDA, TNF-α, and protein carbonyl but also an improvement of liver histopathological changes and hepatic CAT and SOD activities as opposed to the DZN group. Conclusions This study confirmed that Art leaf extract has liver protective effects and causes downregulation of oxidative stress in acute DZN-induced liver injury in rats.

MON-496 Bone Mineral Density improvement with Vitamin Dsupplementation in a Case Of Childhood Hypophosphatasia

Hypophosphatasia is associated with defective mineralization of bone with possible teeth involvement with low activity of serum and bone alkaline phosphatase. It is caused by a mutations in the TNSALP gene. It is usually life-threatening when it presents within the first six months of life. It has six subtypes: perinatal lethal, prenatal benign, infantile, childhood, adulthood and odontohypophosphatasia. Childhood hypophosphatasia, defined as onset of symptoms between six months and eighteen years, can present as rickets, pain, decreased mobility, deficits of growth, and fractures. CASE REPORT: A 3-year-old child presented in the Endocrinology clinic due to premature loss of her four lower incisors and x-rays showed significant bone loss in the mandible. She also had reduced serum alkaline phosphatase activity. She was diagnosed with Childhood Hypophosphatasia, which was confirmed by genetic investigation. The molecular study showed that the patient is heterozygous for a c.1133A greater than T (p.Asp378Val) variation in exon 10 of the ALPL gene. The child also had a low Vitamin D level of 23 ng/ml (30-100), the Dexa-scan showed a low bone mineral density with a z-score of -2.4 SD below the mean in the distal femur area, which responded to Vitamin D supplementation and oral calcium therapy and normalized. The child did not have any fractures about 9 years after initial diagnosis. Bone turnover markers including Osteocalcin and N-telopeptide levels also normalized. The premature loss of teeth stopped. The child grew and gained weight as expected. CONCLUSION: Hypophosphatasia is suspected in a child with premature loss of deciduous teeth. Testing for a reduced serum alkaline phosphatase activity should be done. The children can be associated with rickets and low vitamin D. Improvement in low bone mineral density is possible with giving adequate doses of vitamin D, calcium and doing non-contact sports. Monitoring of bone mineral density can be done on a yearly basis.

Humulus lupulus L. Extract Prevents Ovariectomy-Induced Osteoporosis in Mice and Regulates Activities of Osteoblasts and Osteoclasts.

To systematically evaluate the protective effects of Humulus lupulus L. extract (HLE) on osteoporosis mice. In vivo experiment, a total of 35 12-week-old female ICR mice were equally divided into 5 groups: the sham control group (sham); the ovariectomy with vehicle group (OVX); the OVX with estradiol valerate [EV, 0.2 mg/(kg•d)] the OVX with low- or high-dose HLE groups [HLE, 1 g/(kg•d) and 3 g/(kg•d)], 7 in each group. Treatment began 1 week after the ovariectomized surgery and lasted for 12 weeks. Bone mass and trabecular bone mircoarchitecture were evaluated by micro computed tomography, and bone turnover markers in serum were evaluated using enzyme-linked immunosorbent assay (ELISA) kits. In vitro experiment, osteoblasts and osteoclasts were treated with HLE at doses of 0, 4, 20 and 100 µg/mL. Biomarkers for bone formation in osteoblasts and bone resorption in osteoclasts were analyzed. Compared with the OVX group, HLE exerted bone protective effects by the increase of estradiol (P<0.05), the improvement of cancellous bone structure, bone mineral density (P<0.01) and the reduction of serum alkaline phosphatase (ALP), tartrate resistant acid phosphatase (TRAP), bone gla-protein, c-terminal telopeptides of type I collagen (CTX-I) and deoxypyridinoline levels (P<0.01 for all). In vitro experiment, compared with the control group, HLE at 20 µg/mL promoted the cell proliferation (P<0.01), and increased the expression of bone morphogenetic protein-2 and osteopontin levels in osteoblasts (both P<0.05). HLE at 100 µg/mL increased the osteoblastic ALP activities, and HLE at all dose enhanced the extracellular matrix mineralization (both P<0.01). Furthermore, compared with the control group, HLE at 20 µg/mL and 100 µg/mL inhibited osteoclastic TRAP activity (P<0.01), and reduced the expression of matrix metalloproteinase-9 and cathepsin K (both P<0.05). HLE may protect against bone loss, and have potentials in the treatment of osteoporosis.

Genetic engineering a large animal model of human hypophosphatasia in sheep

The availability of tools to accurately replicate the clinical phenotype of rare human diseases is a key step toward improved understanding of disease progression and the development of more effective therapeutics. We successfully generated the first large animal model of a rare human bone disease, hypophosphatasia (HPP) using CRISPR/Cas9 to introduce a single point mutation in the tissue nonspecific alkaline phosphatase (TNSALP) gene (ALPL) (1077 C > G) in sheep. HPP is a rare inherited disorder of mineral metabolism that affects bone and tooth development, and is associated with muscle weakness. Compared to wild-type (WT) controls, HPP sheep have reduced serum alkaline phosphatase activity, decreased tail vertebral bone size, and metaphyseal flaring, consistent with the mineralization deficits observed in human HPP patients. Computed tomography revealed short roots and thin dentin in incisors, and reduced mandibular bone in HPP vs. WT sheep, accurately replicating odonto-HPP. Skeletal muscle biopsies revealed aberrant fiber size and disorganized mitochondrial cristae structure in HPP vs. WT sheep. These genetically engineered sheep accurately phenocopy human HPP and provide a novel large animal platform for the longitudinal study of HPP progression, as well as other rare human bone diseases.