α-1 Antitrypsin deficiency (AATD) is a genetic disorder characterized by accumulation of misfolded Z α-1 antitrypsin (ZAAT) in hepatocytes, leading to liver injury and metabolic dysfunction. There is no therapy to reduce ZAAT accumulation and restore proteostasis. Pioglitazone activates AMP-activated protein kinase (AMPK), enhance autophagy, and modulate endoplasmic reticulum stress responses, suggesting a potential effect on ZAAT clearance. Our objective is to examine whether pioglitazone can protect against AATD-mediated liver disease. Huh7.5 cells expressing ZAAT (HuhZ) and Pi*Z transgenic mice were used to investigate pioglitazone treatment on hepatic ZAAT accumulation, autophagy activation, and AMPK signaling. Histological, molecular, and metabolic analyses were conducted to assess changes in ZAAT content, autophagy markers, AMPK phosphorylation, and proteostasis. Pioglitazone significantly reduced intracellular ZAAT and decreased lipid droplet accumulation in HuhZ cells. Pioglitazone markedly lowered hepatic ZAAT content in Pi*Z mice, suggesting enhanced degradation. This reduction was mediated through the AMPK pathway, indicated by increased phosphorylation of AMPK and ULK1. Pioglitazone induced autophagy, shown by decreased p62 and increased ATG5 and LC3B-II. This is indicative of enhanced autophagy. Although total hepatic AAT levels were reduced, periodic acid-Schiff with diastase-positive ZAAT aggregates exhibited only a downward trend, suggesting these may be more resistant to clearance. These findings demonstrate pioglitazone reduces hepatic ZAAT accumulation by activating AMPK and inducing autophagy in AATD-associated liver disease, supporting its potential for therapeutic repurposing. As pioglitazone is FDA-approved with benefits for metabolic liver health, further studies are warranted to evaluate efficacy in restoring proteostasis and reducing hepatic ZAAT.NEW & NOTEWORTHY α-1 Antitrypsin deficiency (AATD)-mediated liver disease lacks therapies that reduce hepatic ZAAT accumulation and liver manifestations. We demonstrate that pioglitazone activates AMPK and induces autophagy, leading to decreased ZAAT and improved proteostasis in Pi*Z mouse livers and human hepatocyte models. As an FDA-approved drug with metabolic benefits, pioglitazone holds promise for repurposing in AATD-related liver disease. These findings offer a mechanistic rationale for targeting autophagy to alleviate hepatic injury in protein misfolding disorders.

RATIONALE: Alpha-1 antitrypsin deficiency (AATD) is a rare genetic condition characterized by accumulation of the protein alpha-1 antitrypsin (AAT) in the liver. Low levels of this protein in circulation predispose individuals to lung and liver disease. Patients with AATD-associated lung disease typically have early onset emphysema or bronchiectasis and are often managed with medications for chronic obstructive pulmonary disease and/or augmentation therapy to replace the deficient protein. This study aimed to estimate the economic burden affecting patients, society, and the healthcare system by assessing lifetime direct and indirect costs of AATD patients (PiZZ genotype) receiving augmentation therapy in the United States. METHODS: A literature review was performed, and a cohort-level Markov model was constructed to simulate healthcare resource utilization from diagnosis (age 55) until death in the United States (U.S.). Three health states were modeled: 1) alive on augmentation therapy, 2) post-lung transplant, and 3) death. Utilization and costs were calculated for hospitalizations, outpatient visits, augmentation therapy (Prolastin-C 1000 mg / 20 ml vials; highest U.S. market share), other medications, oxygen, lab tests, and scans. The annual probability of lung transplant was considered, and the costs of lung transplant/post-transplant management were included. Prolastin-C's wholesale-acquisition cost (Red Book) was used, and we ran the model for 5 Prolastin-C vials per week (Base Case) and 6 vials to account for differences in body weight. Other direct costs were sourced from the literature and Medicare fee schedule, while indirect costs were estimated based on productivity losses and early retirement (8.9 years earlier). All costs were adjusted to reflect the commercial payer perspective in 2024 values and adjusted for time value using a 3% discount rate. A scenario analysis of higher mortality was conducted where the Mortality Relative Risk (MRR) was set to 3.2 versus 1.91 in the Base Case. RESULTS: For the Base Case (5 Prolastin-C vials/week), lifetime direct costs were $4,004,870 and indirect costs were $1,296,517, and lifetime overall (direct and indirect) costs were $5,301,387 (undiscounted). In the Base Case, the annual direct cost for patients in the alive on augmentation therapy health state was $203,839. Additional scenarios using a higher mortality relative risk, 6 vials/week, and earlier/older age of diagnosis along with associated costs, can be found in Table 1. CONCLUSIONS: AATD (PiZZ) imposes a significant economic burden, including both direct and indirect costs.

Rationale. Alpha-1-antitrypsin deficiency (AATD) is a genetic condition that increases the risk for emphysema. Alveolar integrity relies on the ability of AT2 cells to proliferate and differentiate into AT1 cells. In AATD, AT2 cell proliferation, differentiation, and survival are impaired, and senescence is increased, likely due to a lack of pro-survival anti-protease AAT. WNT signaling is implicated in these processes and impaired in aging-related diseases, including pulmonary fibrosis and COPD associated with smoking. In AT2 cells, WNT/β-catenin-dependent signaling promotes differentiation and alveolar regeneration, and, when compromised, it leads to alveolar loss. We hypothesize that decreased canonical WNT signaling in murine Pi[asterisk]Null and Pi[asterisk]Z AT2 cells leads to poor proliferation and differentiation leading to early senescence, and emphysema-like airspace enlargement. Methods. Lungs and AT2 cells from young and old Pi[asterisk]Null, Pi[asterisk]Z or wild-type mice, and plasma from AATD and non-smokers individuals were analyzed by western blotting, q-RT-PCR, immunofluorescence, or 4.0SomaScan-platform. Primary AT2 cells were used to derive alveolar epithelial organoids or AT1-like monolayer and treated with CHIR99021 (WNT signaling activator). Proliferation and differentiation were assessed by colony-forming efficiency and immunofluorescence. Airspace enlargement was measured by Flexivent and stereologic morphometry. Results. Pi[asterisk]Null and Pi[asterisk]Z mice developed spontaneous emphysema-like airspace enlargement as measured by inspiratory capacity, static compliance, and mean linear intercept, and alveolar surface area (ANOVA 1-way, p<0.05 vs. wild-type). The senescence marker p16 was increased in young Pi[asterisk]Null and Pi[asterisk]Z lung vs. wild-type mice. In turn, β-catenin, involved in canonical WNT signaling, was decreased in young and aged Pi[asterisk]Null vs. wild-type mice. Interestingly, Pi[asterisk]Null and Pi[asterisk]Z mice have less SPC+AT2 cells with lower Axin2 (p<0.001, 1-way ANOVA, vs. wild-type), WNT-related protein. Pi[asterisk]Null and Pi[asterisk]Z AT2-organoids exhibited reduced proliferation (CFE) and differentiation (RAGE staining) compared to wild-type. CHIR99021 rescued Axin2 expression in old AT2-Pi[asterisk]Null differentiated into AT1-like monolayers and decreased Cdkn2a expression (p21, senescence marker) vs. control conditions. Moreover, plasma levels of SASPs-related proteins (uPA, FGF7 and MIP1a) are increased and WNT-related proteins (WNT3A and WISP1) are decreased in young ZZ vs. MZ individuals (Mann-Whitney test, p<0.05). Lastly, p16 was increased in Z-AATD lung vs. healthy individuals (p<0.05, t-student). Conclusions. Impaired WNT signaling in AT2 cells and lungs of young Pi[asterisk]Null and Pi[asterisk]Z mice, is associated with early senescence, decreased AT2 proliferation and differentiation, and spontaneous emphysema-like airspace enlargement. WNT signaling could become a therapeutical target in AATD, being already under investigation for COPD-emphysema. Funding agencies: A1F endorsed professorship, 1R01HL166828 (KAS).

Neutrophilic inflammation and a high level of free α-defensins are main features of chronic airway inflammation in alpha-1 antitrypsin-deficient (AATD) individuals. Despite the antimicrobial activities of α-defensins by direct bacterial killing and by modulation of immune responses, AATD individuals are paradoxically burdened by recurrent exacerbation triggered by bacterial infections, frequently with nontypeable Haemophilus influenzae (NTHi). Previous studies demonstrated that high, rather than low α-defensin level could modulate the local pro-inflammatory milieu of bronchial epithelial cells and macrophages promoting chronic inflammation and lower pathogen phagocytosis. IgG-mediated phagocytosis and NTHi adherence, engulfment and phagocytosis were measured in human alveolar macrophages and monocyte-derived macrophages (MDM) isolated from patients with AATD and from healthy individuals. A high concentration of free α-defensins induced NTHi adherence to MDMs but decreased IgG-mediated phagocytosis by MDMs. The decreased phagocytosis was associated with TLR4 activation, downstream signaling via NF-κB p65 and marked increased secretion of inflammatory cytokines, CXCL8, IL-1b, and TNFα by the α-defensin-treated and NTHi-infected MDMs. Exogenous AAT treatment and TLR4 inhibitor decreased TNFα expression in α-defensin-treated cells. Dampening the downstream effects of a high concentration of α-defensins may render AAT and TLR4 inhibitors as potential therapies to decrease NTHi colonization and increase its clearance by phagocytosis in AATD individuals.

BACKGROUND & AIMS:Alpha-1 antitrypsin deficiency-associated liver disease (AATD-LD) remains underrecognized despite its significant contribution to morbidity and mortality in adults with the PiZZ genotype. Lack of standardized definitions, diagnostic criteria, and staging impedes timely diagnosis and therapeutic development. To address these gaps, a multi-disciplinary expert panel convened under the auspices of the American Gastroenterological Association in collaboration with the American Association for the Study of Liver Diseases, European Association for the Study of the Liver, and Alpha-1 Foundation to develop consensus recommendations for nomenclature, diagnosis, staging, and clinical trial endpoints in AATD-LD. METHODS:An international multi-disciplinary expert consensus synthesized data from recent epidemiologic, histopathologic, and noninvasive biomarker studies related to AATD-LD. Expert opinion was integrated with published evidence to establish case definitions, staging algorithms, and clinical trial endpoint criteria. RESULTS:AATD-LD is defined by the presence of liver enzyme (ie, aspartate aminotransferase, alanine aminotransferase, or γ-glutamyl transferase) elevations-which may be episodic-and/or liver fibrosis (≥F2) in adults with AATD, particularly those with the PiZZ genotype. Liver stiffness measurement by means of noninvasive elastography (eg, vibration-controlled transient elastography and magnetic resonance elastography) was identified as the preferred method for staging fibrosis, with liver stiffness measurement ≥8 kPa by means of vibration-controlled transient elastography as a threshold for clinically significant fibrosis. Aspartate aminotransferase-to-platelet ratio (<0.5) and fibrosis-4 index (<1.3) (low risk for advanced liver disease) were endorsed for risk stratification, although their sensitivity is limited. A foundational, tiered algorithm was developed to guide longitudinal monitoring and staging, incorporating serial noninvasive testing and timely referral for liver transplantation evaluation. For clinical trial design, adults with F2-F4 fibrosis were recommended for inclusion in phase 3 trials; earlier stages (eg, F1) may be appropriate for phase 1-2 safety studies and patients with cirrhosis may be excluded, depending on therapeutic class. Preservation of pulmonary function was not deemed necessary for inclusion in trials of therapies that increase AAT levels via DNA or RNA editing. The consensus primary efficacy endpoint is a ≥1-stage improvement in fibrosis. CONCLUSIONS:These consensus statements provide a unified framework for diagnosing, staging, and studying AATD-LD. Broad adoption will improve disease recognition, optimize clinical management, and facilitate therapeutic development.

Background Alpha-1 antitrypsin (AAT)-deficient individuals have a greater risk for developing COPD than individuals with normal AAT levels. Methods This was a double-blind, randomised, parallel group, placebo-controlled trial to examine the safety and tolerability of “Kamada-AAT for Inhalation” (inhaled AAT) in subjects with AAT deficiency, and to explore its effect on AAT and biomarkers in the lung epithelial lining fluid (ELF). 36 patients with severe AAT deficiency were randomised 2:1 to receive 80 mg or 160 mg inhaled AAT or placebo once daily for 12 weeks. The primary outcomes were AAT and antineutrophil elastase capacity (ANEC) in bronchoalveolar lavage and plasma after treatment. Secondary outcomes included safety, levels of normal M-type AAT in the plasma and concentrations of AAT, neutrophil elastase (NE), AAT–NE complexes and neutrophil count in the ELF. Results 12 weeks of active treatment significantly increased AAT, ANEC and AAT–NE complexes in the ELF. Mean antigenic AAT levels in the ELF were restored to 5.2±2.3 μM in the 80 mg arm and to 17.7±2 μM in the 160 mg arm. Both doses significantly restored AAT antiprotease activity within the lung and reduced NE levels. M-specific AAT levels in plasma increased in a dose-dependent manner. A clinically meaningful reduction in ELF neutrophil % was observed in the 80 mg arm. AAT for inhalation was well tolerated. Conclusions Inhaled AAT restores protease–antiprotease homoeostasis and may represent a safe and effective therapy.

Alpha-1 antitrypsin (AAT) deficiency is an autosomal codominant disorder caused by SERPINA1 gene mutations. PI*Z and PI*S mutations commonly underlie this deficiency, but rarer homozygous PI*Null (Q0) mutations may result in a complete loss of AAT. Such rare mutations lead to severe AAT deficiency and early onset of lung disease. We present a case of a 35-year-old female never-smoker born to consanguineous parents who developed severe panlobular emphysema and end-stage respiratory insufficiency requiring lung transplantation. Subsequent genetic testing identified her as homozygous for a novel c.82del mutation-here named Q0Bani-Yas based on the region of the primary carrier's origin-which resulted in undetectable levels of the AAT protein.

Alpha-1 antitrypsin deficiency (AATD) is a rare genetic disorder caused by accumulation of misfolded α-1 antitrypsin within hepatocytes. AATD patients are prone to develop liver disease that remains undiagnosed until the late stages of the disease. Due to challenges in manipulating the α-1 antitrypsin genes in mice, determining a true loss of function of α-1 antitrypsin in previous AATD mouse models has been challenging. Here, we report generation and liver characterization of a new humanized transgenic mouse model for AATD with a background of a CRISPR-Cas9 generated SERPINA1-null mouse. Male and female transgenic mice for normal (Pi*M) and mutant (Pi*Z) variants of human α-1 antitrypsin at 4-6 months of age were subjected to this study. The accumulation of human α-1 antitrypsin in the hepatocytes and fibrotic features of the liver were monitored by performing an in vivo study. We demonstrate a strong liver phenotype satisfying clinically relevant manifestations of liver pathology associated with AATD, including hepatic accumulation of human α-1 antitrypsin globules, liver deposition of extracellular matrix proteins, hepatic ER stress, and liver fibrosis in Pi*Z mice, in addition to mild systemic inflammation. In addition to major phenotypic criteria of AATD-associated liver fibrosis, accompanying single-nucleus RNA-seq data demonstrate activation of pathways associated with liver metabolic changes, inflammation, and regeneration. Data from this study suggest our humanized transgenic AATD mouse model could provide a suitable model to study α-1 antitrypsin loss of function, replicate the pathophysiology of AATD associated liver disease, and evaluate therapeutic reagents against this disease. ### Competing Interest Statement The authors have declared no competing interest.

BACKGROUND & AIMS:Homozygous ZZ alpha-1 antitrypsin (AAT) deficiency produces mutant AAT (Z-AAT) proteins in hepatocytes, leading to progressive liver fibrosis. We evaluated the safety and efficacy of an investigational RNA interference therapeutic, fazirsiran, that degrades Z-AAT messenger RNA, reducing deleterious protein synthesis. METHODS:This ongoing, phase 2 study randomized 40 patients to subcutaneous placebo or fazirsiran 25, 100, or 200 mg. The primary endpoint was percent change in serum Z-AAT concentration from baseline to week 16. Patients with fibrosis on baseline liver biopsy received treatment on day 1, at week 4, and then every 12 weeks and had a second liver biopsy at or after weeks 48, 72, or 96. Patients without fibrosis received 2 doses on day 1 and at week 4. RESULTS:At week 16, least-squares mean percent declines in serum Z-AAT concentration were -61%, -83%, and -94% with fazirsiran 25, 100, and 200 mg, respectively, vs placebo (all P < .0001). Efficacy was sustained through week 52. At postdose liver biopsy, fazirsiran reduced median liver Z-AAT concentration by 93% compared with an increase of 26% with placebo. All fazirsiran-treated patients had histologic reduction from baseline in hepatic globule burden. Portal inflammation improved in 5 of 12 and 0 of 8 patients with a baseline score of >0 in the fazirsiran and placebo groups, respectively. Histologic meta-analysis of histologic data in viral hepatitis score improved by >1 point in 7 of 14 and 3 of 8 patients with fibrosis of >F0 at baseline in the fazirsiran and placebo groups, respectively. No adverse events led to discontinuation, and pulmonary function tests remained stable. CONCLUSIONS:Fazirsiran reduced serum and liver concentrations of Z-AAT in a dose-dependent manner and reduced hepatic globule burden. (ClinicalTrials.gov, Number NCT03945292).

INTRODUCTION:Altered complement component 3 (C3) activation in patients with alpha-1 antitrypsin (AAT) deficiency (AATD) has been reported. To understand the potential impact on course of inflammation, the aim of this study was to investigate whether C3d, a cleavage-product of C3, triggers interleukin (IL)-1β secretion via activation of NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome. The objective was to explore the effect of AAT augmentation therapy in patients with AATD on the C3d/complement receptor 3 (CR3) signalling axis of monocytes and on circulating pro-inflammatory markers. METHODS:Inflammatory mediators were detected in blood from patients with AATD (n=28) and patients with AATD receiving augmentation therapy (n=19). Inflammasome activation and IL-1β secretion were measured in monocytes of patients with AATD, and following C3d stimulation in the presence or absence of CR3 or NLRP3 inhibitors. RESULTS:C3d acting via CR3 induces NLRP3 and pro-IL-1β production, and through induction of endoplasmic reticulum (ER) stress and calcium flux, triggers caspase-1 activation and IL-1β secretion. Treatment of individuals with AATD with AAT therapy results in decreased plasma levels of C3d (3.0±1.2 µg/mL vs 1.3±0.5 µg/mL respectively, p<0.0001) and IL-1β (115.4±30 pg/mL vs 73.3±20 pg/mL, respectively, p<0.0001), with a 2.0-fold decrease in monocyte NLRP3 protein expression (p=0.0303), despite continued ER stress activation. DISCUSSION:These results provide strong insight into the mechanism of complement-driven inflammation associated with AATD. Although the described variance in C3d and NLRP3 activation decreased post AAT augmentation therapy, results demonstrate persistent C3d and monocyte ER stress, with implications for new therapeutics and clinical practice.

Background and Aims: Alpha-1 antitrypsin deficiency (AATD) is a genetic disorder associated with liver disease, ranging from fibrosis to hepatocellular carcinoma. The disease remains asymptomatic until its final stages when liver transplantation is the only available therapy. Biomarkers offer an advantage for disease evaluation. The presence of microRNAs (miRNAs) in plasma extracellular vesicles (EVs) presents a noninvasive approach to assess the molecular signatures of the disease. In this study, we aimed to identify miRNA biomarkers to distinguish molecular signatures of the liver disease associated with AATD in AATD individuals. Methods: Using small RNA sequencing and qPCR, we examined plasma EV miRNAs in healthy controls (n = 20) and AATD patients (n = 17). We compared the EV miRNAs of AATD individuals with and without liver disease, developing an approach for detecting liver disease. A set of miRNAs identified in the AATD testing cohort was validated in a separate cohort of AATD patients (n = 45). Results: We identified differential expression of 178 EV miRNAs in the plasma of the AATD testing cohort compared to controls. We categorized AATD individuals into those with and without liver disease, identifying 39 differentially expressed miRNAs. Six miRNAs were selected to test their ability to discriminate liver disease in AATD. These were validated for their specificity and sensitivity in an independent cohort of 45 AATD individuals. Our logistic model established composite scores with threeand four-miRNA combinations, achieving areas under the curve of 0.737 and 0.751, respectively, for predicting AATD liver disease. Conclusions: We introduce plasma EV-derived miRNAs as potential biomarkers for evaluating AATD liver disease. Plasma EV-associated miRNAs may represent a mo lecular signature of AATD liver disease and could serve as valuable tools for its detection and monitoring.

Liver fibrosis associated with increased mortality is caused by activation of hepatic stellate cells and excessive production and accumulation of extracellular matrix in response to fibrotic insults. It has been shown that in addition to liver inflammation, systemic inflammation also contributes to liver fibrogenesis. A deeper understanding of mechanisms that control liver fibrotic response to intra- and extra-hepatic inflammation is essential to develop novel clinical strategies against this disease. Extracellular vesicles (EV) have been recognized as immune mediators that facilitate activation of hepatic stellate cells. In inflammatory diseases, activated neutrophils release neutrophil elastase (NE) bound to EV, which has been identified as a significant contributor to inflammation by promoting immune cell activation. Here, we aimed to explore the role of inflammation derived plasma EV-associated NE in liver fibrogenesis and its potential mechanisms. We show EV-associated NE induces activation, proliferation and migration of hepatic stellate cells by promoting activation of the ERK1/2 signaling pathway. This effect did not occur through EV without surface NE, and Sivelestat, a NE inhibitor, inhibited activation of the ERK1/2 signaling pathway mediated by EV-associated NE. Moreover, we found plasma EV-associated NE increases deposition of collagen1 and α-smooth muscle actin in the liver of a mouse model of liver fibrosis (Mdr2-/-). Notably, this effect does not occur in control mice without preexisting liver disease. These data suggest that EV-associated NE is a pro-fibrogenic factor for hepatic stellate cell activation via the ERK1/2 signaling pathway in pre-existing liver injuries. Inhibition of the plasma EV-associated NE in inflammatory conditions may be a therapeutic target for liver fibrosis in patients with inflammatory diseases.

Background and Aims:Alpha-1 antitrypsin deficiency (AATD) is a genetic disorder characterized by the misfolding and accumulation of the mutant variant of alpha-1 antitrypsin (AAT) within hepatocytes, which limits its access to the circulation and exposes the lungs to protease-mediated tissue damage. This results in progressive liver disease secondary to AAT polymerization and accumulation, and chronic obstructive pulmonary disease (COPD) due to deficient levels of AAT within the lungs. Our goal was to characterize the unique effects of COPD secondary to AATD on liver disease and gene expression. Methods:A subcohort of AATD individuals with COPD (n = 33) and AATD individuals without COPD (n = 14) were evaluated in this study from our previously reported cross-sectional cohort. We used immunohistochemistry to assess the AATD liver phenotype, and RNA sequencing to explore liver transcriptomics. We observed a distinct transcriptomic profile in liver tissues from AATD individuals with COPD compared to those without. Results:A total of 339 genes were differentially expressed. Canonical pathways related to fibrosis, extracellular matrix remodeling, collagen deposition, hepatocellular damage, and inflammation were significantly upregulated in the livers of AATD individuals with COPD. Histopathological analysis also revealed higher levels of fibrosis and hepatocellular damage in these individuals. Conclusions:Our data supports a relationship between the development of COPD and liver disease in AATD and introduces genes and pathways that may play a role in AATD liver disease when COPD is present. We believe addressing lung impairment and airway inflammation may be an approach to managing AATD-related liver disease.

Background: Alpha - 1 antitrypsin deficiency (AATD) is characterized by low alpha - 1 antitrypsin (AAT) levels, predisposing individuals to lung disease. The standard of care, plasma - derived AAT (pdAAT), is delivered as weekly infusions to maintain serum AAT concentrations >= 11 mu M (approximate to 50% of those in healthy individuals). INBRX - 101, a recombinant human AAT - Fc fusion protein, was designed to have a longer half - life and achieve higher AAT levels than pdAAT. Methods: In this phase 1 dose - escalation study (N=31), adults with AATD received 1 dose (part 1) or 3 doses (part 2) of 10 (part 1), 40, 80, or 120mg/kg INBRX - 101 every 3 weeks (Q3W) via intravenous infusion. The primary endpoint was safety and tolerability. Secondary endpoints were pharmacokinetics (PK), pharmacodynamics (PD), and immunogenicity of INBRX - 101. Results: INBRX - 101 was well tolerated. Most treatment - emergent adverse events were grade <= 2. In part 2 (n=18; each dose, n=6), dose - related increases in serum functional AAT (fAAT) were observed; mean fAAT levels remained above the 21 mu M target for up to 4 weeks after the final dose in the 120 - mg/kg cohort. Antidrug antibodies had no meaningful impact on PK or PD. INBRX - 101 was detected in pulmonary epithelial lining fluid (PELF) from all patients assessed (n=11), and PELF fAAT increased after dosing. PK/PD modeling projected steady - state serum fAAT >= 21 mu M at 120mg/kg Q3W (average concentration approximate to 43 mu M; trough concentration approximate to 28 mu M) and Q4W (approximate to 34 mu M; approximate to 21 mu M). Conclusion: The favorable safety profile and ability to maintain serum fAAT levels >21 mu M with extended - interval dosing, support a phase 2 trial evaluating Q3W and Q4W dosing of INBRX - 101.

BACKGROUND:Alpha-1 antitrypsin deficiency (AATD) is a genetic disease caused by misfolding and accumulation of mutant alpha-1 antitrypsin (ZAAT) in the endoplasmic reticulum of hepatocytes. Hepatic ZAAT aggregates acquire a toxic gain-of-function that impacts the endoplasmic reticulum which is theorized to cause liver disease in individuals with AATD who present asymptomatic until late-stage cirrhosis. Currently, there is no treatment for AATD-mediated liver disease except liver transplantation. In our study of mitochondrial RNA, we identified that Sirtuin3 (SIRT3) plays a role in the hepatic phenotype of AATD. METHODS:Utilizing RNA and protein analysis in an in vitro AATD model, we investigated the role of SIRT3 in the pathophysiology of AATD-mediated liver disease while also characterizing our novel, transgenic AATD mouse model. RESULTS:We show lower expression of SIRT3 in ZAAT-expressing hepatocytes. In contrast, the overexpression of SIRT3 increases hepatic ZAAT degradation. ZAAT degradation mediated by SIRT3 appeared independent of proteasomal degradation and regular autophagy pathways. We observed that ZAAT-expressing hepatocytes have aberrant accumulation of lipid droplets, with ZAAT polymers localizing on the lipid droplet surface in a direct interaction with Perilipin2, which coats intracellular lipid droplets. SIRT3 overexpression also induced the degradation of lipid droplets in ZAAT-expressing hepatocytes. We observed that SIRT3 overexpression induces lipophagy by enhancing the interaction of Perilipin2 with HSC70. ZAAT polymers then degrade as a consequence of the mobilization of lipids through this process. CONCLUSIONS:In this context, SIRT3 activation may eliminate the hepatic toxic gain-of-function associated with the polymerization of ZAAT, providing a rationale for a potential novel therapeutic approach to the treatment of AATD-mediated liver disease.

SummaryBackgroundAlpha‐1 antitrypsin liver disease (AATLD) occurs in a subset of patients with alpha‐1 antitrypsin deficiency. Risk factors for disease progression and specific pathophysiologic features are not well known and validated non‐invasive assessments for disease severity are lacking. Currently, there are no approved treatments for AATLD.AimsTo outline existing understanding of AATLD and to identify knowledge gaps critical to improving clinical trial design and development of new treatments.MethodsThis report was developed following a multi‐stakeholder forum organised by the Alpha‐1 Antitrypsin Deficiency Related Liver Disease Expert Panel in which experts presented an overview of the available literature on this topic.ResultsAATLD results from a ‘gain of toxic function’ and primarily manifests in those with the homozygous Pi*ZZ genotype. Accumulation of misfolded ‘Z’ AAT protein in liver cells triggers intracellular hepatocyte injury which may ultimately lead to hepatic fibrosis. Male gender, age over 50 years, persistently elevated liver tests, concomitant hepatitis B or C virus infection, and metabolic syndrome, including obesity and type 2 diabetes mellitus, are known risk factors for adult AATLD. While the gold standard for assessing AATLD disease activity is liver histology, less invasive measures with low intra‐ and inter‐observer variability are needed. Measurement of liver stiffness shows promise; validated thresholds for staging AATLD are in development. Such advances will help patients by enabling risk stratification and personalised surveillance, along with streamlining the development process for novel therapies.ConclusionsThis inaugural forum generated a list of recommendations to address unmet needs in the field of AATLD.

Background Alpha-1 antitrypsin deficiency (AATD) is an inherited disease, the common variant caused by a Pi*Z mutation in the SERPINA1 gene. Pi*Z AAT increases the risk of pulmonary emphysema and liver disease. Berberine (BBR) is a nature dietary supplement and herbal remedy. Emerging evidence revealed that BBR has remarkable liver-protective properties against various liver diseases. In the present study, we investigated the therapeutic effects and toxicities of BBR in Pi*Z hepatocytes and Pi*Z transgenic mice. Methods Huh7.5 and Huh7.5Z (which carries the Pi*Z mutation) cells were treated with different concentrations of BBR for 48 hours. MTT was performed for cell viability assay. Intracellular AAT levels were evaluated by western blot. In vivo studies were carried out in wild type, native phenotype AAT (Pi*M), and Pi*Z AAT transgenic mice. Mice were treated with 50 mg/kg/day of BBR or solvent only by oral administration for 30 days. Western blot and liver histopathological examinations were performed to evaluate therapeutic benefits and liver toxicity of BBR. Results BBR reduced intracellular AAT levels in Huh7.5Z cells, meanwhile, no Pi*Z-specific toxicity was observed. However, BBR did not reduce liver AAT load but significantly potentiated liver inflammation and fibrosis accompanying the activation of unfolded protein response and mTOR in Pi*Z mice, but not in wild type and Pi*M mice. Conclusions BBR exacerbated liver inflammation and fibrosis specifically in Pi*Z mice. This adverse effect may be associated with the activation of unfolded protein response and mTOR. This study implicates that BBR should be avoided by AATD patients.