Alpha-1 antitrypsin deficiency impairs lung antibacterial immunity in mice.

Alpha-1 antitrypsin (AAT) is a major inhibitor of serine proteases in mammals. Therefore, its deficiency leads to protease-antiprotease imbalance and a risk for developing lung emphysema. Although therapy with human plasma-purified AAT attenuates AAT deficiency-related emphysema, its impact on lung antibacterial immunity is poorly defined. Here, we examined the effect of AAT therapy on lung protective immunity in AAT-deficient (KO) mice challenged with Streptococcus pneumoniae. AAT-KO mice were highly susceptible to S. pneumoniae, as determined by severe lobar pneumonia and early mortality. Mechanistically, we found that neutrophil-derived elastase (NE) degraded the opsonophagocytically important collectins, surfactant protein A (SP-A) and D (SP-D), which was accompanied by significantly impaired lung bacterial clearance in S. pneumoniae-infected AAT-KO mice. Treatment of S. pneumoniae-infected AAT-KO mice with human AAT protected SP-A and SP-D from NE-mediated degradation and corrected the pulmonary pathology observed in these mice. Likewise, treatment with Sivelestat, a specific inhibitor of NE, also protected collectins from degradation and significantly decreased bacterial loads in S. pneumoniae-infected AAT-KO mice. Our findings show that NE is responsible for the degradation of lung SP-A and SP-D in AAT-KO mice affecting lung protective immunity in AAT deficiency.

Human plasma-derived alpha1 -proteinase inhibitor in patients with new-onset type 1 diabetes mellitus: A randomized, placebo-controlled proof-of-concept study.

BACKGROUND: While circulating levels of alpha1 -proteinase inhibitor (alpha1 -PI) are typically normal, antiprotease activity appears to be compromised in patients with Type 1 diabetes mellitus (T1DM). Because alpha1 -PI [human] (alpha1 -PI[h]) therapy can inhibit pro-inflammatory mediators associated with ß-cell destruction and reduced insulin production, it has been proposed for T1DM disease prevention. The aim of this study was to evaluate safety, tolerability, and efficacy of intravenous (IV) alpha1 -PI[h] in preserving C-peptide production in newly diagnosed T1DM patients. PARTICIPANTS: Seventy-six participants (aged 6-35 years) were randomized at 25 centers within 3 months of T1DM diagnosis. METHODS: A Phase II, multicenter, partially blinded, placebo-controlled, proof-of-concept study evaluating four dosing regimens of alpha1 -PI[h] (NCT02093221, GTI1302): weekly IV infusions of either 90 or 180 mg/kg, each for either 13 or 26 weeks. Safety and efficacy were monitored over 52 weeks with an efficacy evaluation planned at 104 weeks. The primary efficacy endpoint was change from baseline in the 2-h area-under-the-curve C-peptide level from a mixed-meal tolerance test at 52 weeks. A battery of laboratory tests, including inflammatory biomarkers, constituted exploratory efficacy variables. RESULTS: Infusions were well tolerated with no new safety signals. All groups exhibited highly variable declines in the primary outcome measure at 52 weeks with no statistically significant difference from placebo. Interleukin-6 (IL-6) was reduced from baseline in all alpha1 -PI treatment groups but not the placebo group. CONCLUSION: Pharmacologic therapy with alpha1 -PI[h] is safe, well tolerated, and able to reduce IL-6 levels; however, due to variability in the efficacy endpoint, its effects on preservation of C-peptide production were inconclusive.

Comparison of the liquid and lyophilized formulations of Prolastin®-C for Alpha1-Antitrypsin deficiency: Biochemical characteristics, pharmacokinetics, safety and neoantigenicity in rabbits.

Multiple analytical and preclinical studies were performed to compare the biochemical characteristics, pharmacokinetics (PK), safety and neoantigenicity of a new 5% liquid formulation of Alpha-1 Proteinase Inhibitor (Liquid A1PI, Prolastin®-C Liquid) with the lyophilized version (Lyophilized A1PI, Prolastin®-C). Liquid A1PI and Lyophilized A1PI had similar average mass (~52 kDa), and both forms exhibited glycoform patterns consistent with the known banding pattern of A1PI (dominated by the M6 and M4 bands, including deconvoluted masses). Both Liquid A1PI and Lyophilized A1PI yielded average percent purity values ranging from 96% to 99% and had active content ranging from 53  mg/mL to 59  mg/mL. The PK profile of Liquid A1PI was similar to Lyophilized A1PI. Safety assessments in rabbits showed good tolerability and no test article-related changes in mortality, clinical signs, clinical pathology, body weight, food consumption, or urinalysis parameters. Following immunodepletion of antibodies that recognize Lyophilized A1PI, there were no significant differences in the anti-drug titers among animals immunized with Lyophilized A1PI and Liquid A1PI (p > 0.05), indicating that no antibodies to neoantigens were generated. Liquid A1PI and Lyophilized A1PI have similar profiles with respect to biochemical characteristics, PK, safety and neoantigenicity.

Safety and pharmacokinetics of Alpha-1 MP (Prolastin®-C) in Japanese patients with alpha1-antitrypsin (AAT) deficiency.

BACKGROUND: Alpha1-Proteinase Inhibitor, Modified Process (Alpha-1 MP) is used for augmentation therapy in alpha1-antitrypsin deficiency (AATD), an extremely rare disease in Japan. Weekly doses of 60 mg/kg Alpha-1 MP have been shown to be safe and well tolerated in non-Japanese subjects, but the safety and pharmacokinetics (PK) have not been evaluated in Japanese subjects. The objectives of this study were to evaluate the safety and PK of 60 mg/kg Alpha-1 MP administered by weekly IV infusions over 8 weeks in Japanese subjects with AATD. METHODS: This was a multicenter, open-label trial in Japanese adults aged ≥20 years with AATD. Samples for evaluation of serum alpha1-PI concentration and PK parameters were collected at 10 time points until the seventh day after the last dose at Week 8: immediately before dosing, immediately after dosing (time 0), and 0.25, 2, 4, 8, 24, 48, 120, and 168 hours after dosing. RESULTS: Four subjects were analyzed. The median tmax was 0.534 h. Mean ± SD values for t½, Cmax, and AUC0-7days were 150.4 ± 36.18 h, 174.2 ± 30.51 mg/dL, and 14,913.2 ± 1633.45 mg*h/dL, respectively. Mean trough concentration at week 8 was 55.4 ± 7.23 mg/dL. Alpha-1 MP therapy was safe, with no serious adverse events or deaths reported. Two treatment-emergent adverse events of fatigue in one subject were considered to be possibly related. CONCLUSIONS: The PK and safety of Alpha-1 MP in Japanese subjects with AATD were consistent with the Alpha-1 MP profile in non-Japanese subjects (ClinicalTrials.gov: NCT02870309; JAPIC CTI: JapicCTI-163160).

Hepatic-targeted RNA interference provides robust and persistent knockdown of alpha-1 antitrypsin levels in ZZ patients.

BACKGROUND & AIMS: Alpha-1 antitrypsin deficiency (AATD) is a genetic disorder causing pulmonary and liver disease. The PiZ mutation in AAT (SERPINA1) results in mis-folded AAT protein (Z-AAT) accumulating in hepatocytes, leading to fibrosis and cirrhosis. RNAi-based therapeutics silencing production of hepatic Z-AAT might benefit patients with AATD-associated liver disease. This study evaluated an RNAi therapeutic to silence production of AAT. METHODS: Part A of this double-blind first-in-human study randomized 54 healthy volunteers (HVs) into single dose cohorts (two placebo: four active), receiving escalating doses of the investigational agent ARC-AAT from 0.38 to 8.0 mg/kg or placebo. Part B randomized 11 patients with PiZZ (homozygous for Z-AAT) genotype AATD, who received up to 4.0 mg/kg of ARC-AAT or placebo. Patients with baseline FibroScan® >11 kPa or forced expiratory volume in one second (FEV1) <60% were excluded. Assessments included safety, pharmacokinetics, and change in serum AAT concentrations. RESULTS: A total of 36 HVs received ARC-AAT and 18 received placebo (part A). Seven PiZZ individuals received ARC-AAT and four received placebo (part B). A dose response in serum AAT reduction was observed at doses ≥4 mg/kg with similar relative reductions in PiZZ patients and HVs at 4 mg/kg and a maximum reduction of 76.1% (HVs) vs. 78.8% (PiZZ) at this dose. The time it took for serum AAT to return to baseline was similar for HV and PiZZ. There were no notable differences between HV and PiZZ safety parameters. The study was terminated early because of toxicity findings related to the delivery vehicle (ARC-EX1) seen in a non-human primate study. CONCLUSION: PiZZ patients and HVs responded similarly to ARC-AAT. Deep and durable knockdown of hepatic AAT production based on observed reduction in serum AAT concentrations was demonstrated. LAY SUMMARY: Accumulation of abnormal proteins in the livers of patients with alpha-1 antitrypsin deficiency may lead to decreased liver function and potentially liver failure. Therapeutics targeting the production of these abnormal proteins may be used to prevent or treat liver disease in patients with alpha-1 antitrypsin deficiency. CLINICAL TRIAL REGISTRATION NUMBER: NCT02363946.

Alpha-1 antitrypsin treatment of new-onset type 1 diabetes: An open-label, phase I clinical trial (RETAIN) to assess safety and pharmacokinetics.

OBJECTIVE: To determine the safety and pharmacokinetics of alpha-1 antitrypsin (AAT) in adults and children. RESEARCH DESIGN AND METHODS: Short-term AAT treatment restores euglycemia in the non-obese mouse model of type 1 diabetes. A phase I multicenter study in 16 subjects with new-onset type 1 diabetes studied the safety and pharmacokinetics of Aralast NP (AAT). This open-label, dose-escalation study enrolled 8 adults aged 16 to 35 years and 8 children aged 8 to 15 years within 100 days of diagnosis, to receive 12 infusions of AAT: a low dose of 45 mg/kg weekly for 6 weeks, followed by a higher dose of 90 mg/kg for 6 weeks. RESULTS: C-peptide secretion during a mixed meal, hemoglobin A1c (HbA1c), and insulin usage remained relatively stable during the treatment period. At 72 hours after infusion of 90 mg/kg, mean levels of AAT fell below 2.0 g/L for 7 of 15 subjects. To identify a plasma level of AAT likely to be therapeutic, pharmacodynamic ex vivo assays were performed on fresh whole blood from adult subjects. Polymerase chain reaction (PCR) analyses were performed on inhibitor of IKBKE, NOD1, TLR1, and TRAD gene expression, which are important for activation of nuclear factor-κB (NF-κB) and apoptosis pathways. AAT suppressed expression dose-dependently; 50% inhibition was achieved in the 2.5 to 5.0 mg/mL range. CONCLUSIONS: AAT was well tolerated and safe in subjects with new-onset type 1 diabetes. Weekly doses of AAT greater than 90 mg/kg may be necessary for an optimal therapeutic effect.

α1-Antitrypsin infusion for treatment of steroid-resistant acute graft-versus-host disease.

Corticosteroid resistance after acute graft-versus-host disease (SR-aGVHD) results in high morbidity and mortality after allogeneic hematopoietic cell transplantation. Current immunosuppressive therapies for SR-aGVHD provide marginal effectiveness because of poor response or excessive toxicity, primarily from infection. α1-Antitrypsin (AAT), a naturally abundant serine protease inhibitor, is capable of suppressing experimental GVHD by downmodulating inflammation and increasing ratios of regulatory (Treg) to effector T cells (Teffs). In this prospective multicenter clinical study, we sought to determine the safety and response rate of AAT administration in SR-aGVHD. Forty patients with a median age of 59 years received intravenous AAT twice weekly for 4 weeks as first-line treatment of SR-aGVHD. The primary end point was overall response rate (ORR), the proportion of patients with SR-aGVHD in complete (CR) or partial response by day 28 without addition of further immunosuppression. Treatment was well tolerated without drug-related adverse events. A significant increase in serum levels of AAT was observed after treatment. The ORR and CR rates by day 28 were 65% and 35%, respectively, and included responses in all aGVHD target organs. At day 60, responses were sustained in 73% of patients without intervening immunosuppression. Infectious mortality was 10% at 6 months and 2.5% within 30 days of last AAT infusion. Consistent with preclinical data, correlative samples showed an increase in ratio of activated Tregs to Teffs after AAT treatment. These data suggest that AAT is safe and may be potentially efficacious in treating SR-aGVHD. This trial was registered at www.clinicaltrials.gov as #NCT01700036.

Bioequivalence of a Liquid Formulation of Alpha1-Proteinase Inhibitor Compared with Prolastin®-C (Lyophilized Alpha1-PI) in Alpha1-Antitrypsin Deficiency.

This study evaluated the bioequivalence, safety, and immunogenicity of a new liquid formulation of human plasma-derived alpha1-proteinase inhibitor, Liquid Alpha1-PI, compared with the Lyophilized Alpha1-PI formulation (Prolastin®-C), for augmentation therapy in patients with alpha1-antitrypsin deficiency (AATD). In this double-blind, randomized, 20-week crossover study, 32 subjects with AATD were randomized to receive 8 weekly infusions of 60 mg/kg of Liquid Alpha1-PI or Lyophilized Alpha1-PI. Serial blood samples were drawn for 7 days after the last dose followed by 8 weeks of the alternative treatment. The primary endpoint was bioequivalence at steady state, as measured by area under the concentration versus time curve from 0 to 7 days (AUC0-7 days) postdose using an antigenic content assay. Bioequivalence was defined as 90% confidence interval (CI) for the ratio of the geometric least squares (LS) mean of AUC0-7 days for both products within the limits of 0.80 and 1.25. Safety and immunogenicity were assessed. Mean alpha1-PI concentration versus time curves for both formulations were superimposable. Mean AUC0-7 days was 20 320 versus 19 838 mg × h/dl for Liquid Alpha1-PI and Lyophilized Alpha1-PI, respectively. The LS mean ratio of AUC0-7 days (90% CI) for Liquid Alpha1-PI versus Lyophilized Alpha1-PI was 1.05 (1.03-1.08), indicating bioequivalence. Liquid Alpha1-PI was well tolerated and adverse events were consistent with Lyophilized Alpha1-PI. Immunogenicity to either product was not detected. In conclusion, Liquid Alpha1-PI is bioequivalent to Lyophilized Alpha1-PI, with a similar safety profile. The liquid formulation would eliminate the need for reconstitution and shorten preparation time for patients receiving augmentation therapy for AATD.

Investigation of the Influence of Protein-Losing Enteropathy on Monoclonal Antibody Pharmacokinetics in Mice.

Protein losing enteropathy (PLE), which is characterized by substantial loss of plasma proteins into the gastrointestinal (GI) tract, is a complication of a variety of GI diseases, including inflammatory bowel disease. Clinical studies have found that the clearance of monoclonal antibodies (mAb) is often increased in subjects with diseases known to cause PLE; however, direct relationships between PLE and mAb pharmacokinetics have not been demonstrated. This study employed a murine model of colitis to examine the influence of PLE on mAb pharmacokinetics. Mice were given dextran sodium sulfate (DSS, 2% w/v) supplemented tap water as drinking source for 6 days to induce colitis and PLE. Mice were then intravenously injected with 8C2, a murine IgG1 mAb. 8C2 plasma concentrations were measured up to 14 days post injection. Fecal alpha-1-antitrypsin (A1AT) clearance was measured as biomarker for PLE. DSS-treated mice developed PLE of clinically relevant severity. They also showed a transient increase in 8C2 plasma clearance and a decrease in 8C2 plasma exposure. The area under the 8C2 plasma concentration-time curve for the length of the study (AUC0-14d) reduced from 1368 ± 255 to 594 ± 224 day µg/ml following DSS treatment (p = 0.001). A quantitative relationship between A1AT clearance and 8C2 clearance was obtained via population pharmacokinetic modeling. DSS treatment substantially increased 8C2 clearance and reduced 8C2 exposure. Increased mAb plasma clearance was highly correlated with A1AT fecal clearance, suggesting the possible utility of A1AT fecal clearance as a mechanistic biomarker to predict the pharmacokinetics of therapeutic antibodies.

Additional N-glycosylation in the N-terminal region of recombinant human alpha-1 antitrypsin enhances the circulatory half-life in Sprague-Dawley rats.

Glycosylation affects the circulatory half-lives of therapeutic proteins. However, the effects of an additional N-glycosylation in the unstructured region or the loop region of alpha-1 antitrypsin (A1AT) on the circulatory half-life of the protein are largely unknown. In this study, we investigated the role of an additional N-glycosylation site (Q4N/D6T, Q9N, D12N/S14T, A70N, G148T, R178N, or V212N) to the three naturally occurring N-glycosylation sites in human A1AT. A single-dose (445 µg/kg) pharmacokinetic study using male Sprague-Dawley rats showed that, among the seven recombinant A1AT (rA1AT) mutants, Q9N and D12N/S14T showed the highest serum concentration and area under the curve values, as well as similar circulatory half-lives that were 2.2-fold higher than plasma-derived A1AT and 1.7-fold higher than wild-type rA1AT. We further characterized the Q9N mutant regarding the N-glycan profile, sialic acid content, protease inhibitory activity, and protein stability. The results indicate that an additional N-glycosylation in the flexible N-terminal region increases the circulatory half-life of rA1AT without altering its protease inhibitory activity. Our study provides novel insight into the use of rA1AT for the treatment of emphysema with an increased injection interval relative to the clinically used plasma-derived A1AT.

Inhaled alpha1-proteinase inhibitor therapy in patients with cystic fibrosis.

BACKGROUND: Inhaled alpha1-proteinase inhibitor (PI) is known to reduce neutrophil elastase burden in some patients with CF. This phase 2a study was designed to test inhaled Alpha-1 HC, a new aerosolized alpha1-PI formulation, in CF patients. METHODS: We performed a randomized, double-blind, placebo-controlled study and evaluated the safety of 100 or 200mg of inhaled Alpha-1 HC once daily for 3 weeks in subjects with CF. Thirty adult subjects were randomized in a 2:1 ratio to receive Alpha-1 HC or placebo. RESULTS: Drug delivery was confirmed by a dose-dependent increase in the sputum alpha1-PI. Seven (20.0%) of the 35 adverse events in the 100-mg dose group, 3 (13.0%) of 23 in the 200-mg dose group, and 4 (14.3%) of 28 in the placebo group were drug-related in these subjects. One serious adverse event occurred in 1 subject within each group. CONCLUSIONS: Alpha-1 HC inhalation was safe and well tolerated.

Lung Deposition of Alpha1-Proteinase Inhibitor (Human) (A1-PI[H]) Inhalation Solution Using Two Inhalation Modes of the I-neb Adaptive Aerosol Delivery (AAD) System in Healthy Subjects and Subjects with Cystic Fibrosis.

BACKGROUND: In cystic fibrosis (CF) patients, inhalation of alpha1-proteinase inhibitor (A1-PI) can prevent or slow down persistent infections and reduce the massive ongoing inflammation and excessive levels of NE that destroy the airway epithelium, leading to progressive loss of pulmonary function and death. It is essential for an efficient treatment with inhaled A1-PI that an adequate and reproducible dose is deposited within all regions of the lung. The I-neb AAD System provides two inhalation modes: the Target Inhalation Mode (TIM) and the Tidal Breathing Mode (TBM). Both were compared in this study for their efficiency to deliver A1-PI to the lungs. METHODS: This was a randomized, open label, cross-over study to investigate the lung deposition of A1-PI in 6 healthy subjects (HS) and 15 CF subjects. The primary endpoint was to evaluate the total lung deposition relative to filling dose of A1-PI inhalation solution using the I-neb AAD System in TIM and in TBM. The main secondary endpoints were extra-thoracic deposition, exhaled drug fraction, nebulizer residue, C/P ratio, and variance of pixel counts. Additional exploratory endpoints were total treatment time and the inhalation time. Radiolabeling was performed considering GMP using a commercially available sterile labeling kit. Radiolabeling was validated using NGI data acquired by gamma scintillation and UV spectrometry. RESULTS AND CONCLUSIONS: The intrapulmonary deposition (mean ± SD) in CF subjects was 47.0% ± 6.6% and 46.7% ± 10.3% in TIM and TBM, respectively, and in healthy subjects, 50.0% ± 6.7% and 54.8% ± 7.0% in TIM and TBM, respectively. TIM resulted in an approximately 40% lower treatment time (HS 6.4 min vs. 10.3 min, CF 5.3 min vs. 10.7 min) and less extra-thoracic deposition compared to TBM, and showed a higher residue of drug in the nebulizer, compared to TBM. In both groups, inhalation of a single dose of 77 mg of A1-PI was efficient, safe, and well tolerated using TIM and TBM.

SPARTA clinical trial design: exploring the efficacy and safety of two dose regimens of alpha1-proteinase inhibitor augmentation therapy in alpha1-antitrypsin deficiency.

BACKGROUND: Alpha1-antitrypsin deficiency (AATD) is an underdiagnosed genetic disorder that results in early-onset emphysema due to low serum levels of alpha1-proteinase inhibitor (alpha1-PI), leading to increased activity of tissue-damaging neutrophil elastase. Clinical outcomes of AATD may be improved by administering alpha1-PI augmentation therapy. Here, we describe the design of the ongoing Study of ProlAstin-c Randomized Therapy with Alpha-1 augmentation (SPARTA), a phase 3 trial designed to evaluate progression of lung tissue loss in patients with severe AATD receiving human alpha1-PI (Prolastin(®)-C) versus placebo, using whole-lung computed tomography (CT) densitometry. STUDY DESIGN: SPARTA is a randomized, placebo-controlled trial assessing the efficacy and safety of two separate doses of Prolastin-C (60 and 120 mg/kg) administered weekly over 3 years in patients aged 18-70 years with a diagnosis of AATD and clinical evidence of pulmonary emphysema. The primary measure of efficacy (change from baseline whole-lung 15th percentile lung density [PD15]) will be determined by CT lung densitometry measured at total lung capacity. Secondary efficacy variables will be the evaluation of severe chronic obstructive pulmonary disease exacerbations, as defined by American Thoracic Society/European Respiratory Society criteria, and PD15 of the basal lung region using CT densitometry. Adverse events will be collected and documented. CONCLUSIONS: The SPARTA trial is designed to evaluate the long-term (3-year) efficacy of 2 separate doses of Prolastin-C for the treatment of emphysema in patients with AATD. Protocol number: GTi1201. CLINICAL TRIALS IDENTIFIER: NCT01983241.

Effects of Prolastin C (Plasma-Derived Alpha-1 Antitrypsin) on the acute inflammatory response in patients with ST-segment elevation myocardial infarction (from the VCU-alpha 1-RT pilot study).

Alpha-1 antitrypsin (AAT) has broad anti-inflammatory and immunomodulating properties in addition to inhibiting serine proteases. Administration of human plasma-derived AAT is protective in models of acute myocardial infarction in mice. The objective of this study was to determine the safety and tolerability of human plasma-derived AAT and its effects on the acute inflammatory response in non-AAT deficient patients with ST-segment elevation myocardial infarction (STEMI). Ten patients with acute STEMI were enrolled in an open-label, single-arm treatment study of AAT at 60 mg/kg infused intravenously within 12 hours of admission and following standard of care treatment. C-reactive protein (CRP) and plasma AAT levels were determined at admission, 72 hours, and 14 days, and patients were followed clinically for 12 weeks for the occurrence of new onset heart failure, recurrent myocardial infarction, or death. Twenty patients with STEMI enrolled in previous randomized trials with identical inclusion and/or exclusion criteria, but who received placebo, served as historical controls. Prolastin C was well tolerated and there were no in-hospital adverse events. Compared with historical controls, the area under the curve of CRP levels was significantly lower 14 days after admission in the Prolastin C group (75.9 [31.4 to 147.8] vs 205.6 [78.8 to 410.9] mg/l, p = 0.048), primarily due to a significant blunting of the increase occurring between admission and 72 hours (delta CRP +1.7 [0.2 to 9.4] vs +21.1 [3.1 to 38.0] mg/l, p = 0.007). Plasma AAT levels increased from admission (149 [116 to 189]) to 203 ([185 to 225] mg/dl) to 72 hours (p = 0.005). In conclusion, a single administration of Prolastin C in patients with STEMI is well tolerated and is associated with a blunted acute inflammatory response.

Safety and pharmacokinetics of 120 mg/kg versus 60 mg/kg weekly intravenous infusions of alpha-1 proteinase inhibitor in alpha-1 antitrypsin deficiency: a multicenter, randomized, double-blind, crossover study (SPARK).

Augmentation therapy with the approved dose of 60 mg/kg weekly intravenous (IV) alpha-1 proteinase inhibitor (alpha1-PI), achieves a trough serum level of 11 µM in individuals with alpha-1 antitrypsin deficiency (AATD), yet this is still below the level observed in healthy individuals. This study assessed the safety and pharmacokinetic profile of weekly infusions of a 120 mg/kg dose of alpha1-PI in 30 adults with AATD. Subjects with symptomatic, genetically determined (genotypes PI*ZZ, PI*Z(null), PI*(null)(null) or PI*(Z)Mmalton) AATD were randomly assigned to weekly infusions of 60 or 120 mg/kg alpha1-PI (Prolastin-C®) for 8 weeks before crossing over to the alternate dose for 8 weeks. Adverse events (AEs) (including exacerbations), vital signs, pulmonary function tests, and laboratory assessments were recorded. Pharmacokinetic measurements included AUC0-7days, Cmax, trough, tmax, and t1/2, based on serum alpha1-PI concentrations. In total for both treatments, 112 AEs were reported, with exacerbation of COPD being the most frequent, consistent with the subjects' diagnoses. Mean steady-state serum alpha1-PI concentrations following 120 mg/kg weekly IV alpha1-PI were higher than with the 60 mg/kg dose and mean trough concentrations were 27.7 versus 17.3 µM, respectively. Dose proportionality was demonstrated for AUC0-7days and Cmax, with low inter-subject variability. The 120 mg/kg alpha1-PI weekly dose was considered to be safe and well tolerated, and provided more favorable physiologic alpha1-PI serum levels than the currently recommended 60 mg/kg dose. The effect of this dosing regimen on slowing and/or preventing emphysema progression in subjects with AATD warrants further investigation.

Development and analysis of alpha 1-antitrypsin neoglycoproteins: the impact of additional N-glycosylation sites on serum half-life.

Therapeutic efficacy of glycoproteins is affected by many factors, including molecular size and net charge; both are influenced by the presence and composition of glycan structures. Human alpha 1-antitrypsin (A1AT) was cloned and expressed in human embryonic kidney cells (HEK293) that are capable of mammalian glycosylation. Utilizing PCR-based site-directed mutagenesis, new A1AT variants were created with single, double, or triple additional N-glycosylation sites to the three naturally occurring N-glycosylation sites. Because of the supplementary N-glycans, the A1AT variants exhibited an increased molecular weight. Retention of inhibitory activity was shown via trypsin inhibitory assay. The A1AT variants were treated with PNGase F, and the resulting N-glycans were analyzed by MALDI-TOF mass spectrometry. The N-glycan profile of the recombinant A1AT variants was mostly composed of monofucosylated bi-, tri-, and tetraantennary complex-type N-glycans, with a tendency toward higher antennary structures compared to the wild-type. The relevance of N-glycosylation in A1AT for the circulatory serum half-life was demonstrated in CD1 mice. The A1AT neoglycoprotein with an additional N-glycosylation site at position N123 exhibited a 62% increase in serum half-life. Additionally, using a two-compartment model, the A1AT variants exhibited increased α-phase values, especially N123 (223%) and N201 (255%). The results suggest the recombinant A1AT neoglycoprotein as a serious alternative to A1AT derived from human plasma.

Production and characterization of a novel human recombinant alpha-1-antitrypsin in PER.C6 cells.

Alpha-1-antitrypsin (A1PI) is a proteinase inhibitor of the serpin superfamily and circulates in plasma at about 1-2 g/L. A1PI deficiency in humans often results in organ damage, particularly to the lungs and liver. Current augmentation therapies rely entirely on A1PI isolated from human plasma, thus prompting an evaluation of alternate sources. We have co-expressed recombinant A1PI and α-2,3-sialyltransferase in the human cell line, PER.C6. The requirement for sialyltransferase overexpression in PER.C6 and the essential contribution of sialic acid glycan capping on pdA1PI and recA1PI to prevent rapid A1PI plasma elimination is shown. Using assays to predict high levels of A1PI production and sialylation, stably transfected PER.C6 cells were screened through two rounds of cell cloning to ensure monoclonality. Fed-batch culturing was used to evaluate recA1PI production and cell line characteristics, identifying subclones expressing over 2.5 g/L recA1PI. Cell stability was assessed over 50 generations, verifying subclone stability during continuous culture. Finally, data are presented showing that recA1PI and pdA1PI are equivalent in their ability to block elastase activity in functional cell-based assays and their pharmacokinetic properties. These data show that recombinant human A1PI recovered from PER.C6 cells offers a reliable source of functionally active A1PI for augmentation therapies and, potentially, other diseases.

Encapsulation of alpha-1 antitrypsin in PLGA nanoparticles: in vitro characterization as an effective aerosol formulation in pulmonary diseases.

BACKGROUND: Alpha 1-antitrypsin (α1AT) belongs to the superfamily of serpins and inhibits different proteases. α1AT protects the lung from cellular inflammatory enzymes. In the absence of α1AT, the degradation of lung tissue results to pulmonary complications. The pulmonary route is a potent noninvasive route for systemic and local delivery. The aerosolized α1AT not only affects locally its main site of action but also avoids remaining in circulation for a long period of time in peripheral blood. Poly (D, L lactide-co glycolide) (PLGA) is a biodegradable and biocompatible polymer approved for sustained controlled release of peptides and proteins. The aim of this work was to prepare a wide range of particle size as a carrier of protein-loaded nanoparticles to deposit in different parts of the respiratory system especially in the deep lung. Various lactide to glycolide ratio of the copolymer was used to obtain different release profile of the drug which covers extended and rapid drug release in one formulation. RESULTS: Nonaqueous and double emulsion techniques were applied for the synthesis of nanoparticles. Nanoparticles were characterized in terms of surface morphology, size distribution, powder X-ray diffraction (XRD), encapsulation efficiency, in vitro drug release, FTIR spectroscopy and differential scanning calorimetry (DSC). To evaluate the nanoparticles cytotoxicity, cell cytotoxicity test was carried out on the Cor L105 human epithelial lung cancer cell line. Nanoparticles were spherical with an average size in the range of 100 nm to 1µ. The encapsulation efficiency was found to be higher when the double emulsion technique was applied. XRD and DSC results indicated that α1AT encapsulated in the nanoparticles existed in an amorphous or disordered-crystalline status in the polymer matrix. The lactic acid to glycolic acid ratio affects the release profile of α1AT. Hence, PLGA with a 50:50 ratios exhibited the ability to release %60 of the drug within 8, but the polymer with a ratio of 75:25 had a continuous and longer release profile. Cytotoxicity studies showed that nanoparticles do not affect cell growth and were not toxic to cells. CONCLUSION: In summary, α1AT-loaded nanoparticles may be considered as a novel formulation for efficient treatment of many pulmonary diseases.

Site-specific enzymatic polysialylation of therapeutic proteins using bacterial enzymes.

The posttranslational modification of therapeutic proteins with terminal sialic acids is one means of improving their circulating half-life, thereby improving their efficiency. We have developed a two-step in vitro enzymatic modification of glycoproteins, which has previously only been achieved by chemical means [Gregoriadis G, Jain S, Papaioannou I, Laing P (2005) Int J Pharm 300:125-130). This two-step procedure uses the Campylobacter jejuni Cst-II α2,8-sialyltransferase to provide a primer on N-linked glycans, followed by polysialylation using the Neisseria meningitidis α2,8-polysialyltransferase. Here, we have demonstrated the ability of this system to modify three glycoproteins with varying N-linked glycan compositions: the human therapeutic proteins alpha-1-antitrypsin (A1AT) and factor IX, as well as bovine fetuin. The chain length of the polysialic acid addition was optimized by controlling reaction conditions. After demonstrating the ability of this system to modify a variety of proteins, the effect of polysialylation on the activity and serum half-life of A1AT was examined. The polysialylation of A1AT did not adversely affect its in vitro inhibition activity against human neutrophil elastase. The polysialylation of A1AT resulted in a significantly improved pharmacokinetic profile when the modified proteins were injected into CD-1 mice. Together, these results suggest that polysialylated A1AT may be useful for improved augmentation therapy for patients with a deficiency in this protein and that this modification may be applied to other therapeutic proteins.

Pharmacokinetic comparability of Prolastin®-C to Prolastin® in alpha1-antitrypsin deficiency: a randomized study.

BACKGROUND: Alpha1-antitrypsin (AAT) deficiency is characterized by low blood levels of alpha1-proteinase inhibitor (alpha1-PI) and may lead to emphysema. Alpha1-PI protects pulmonary tissue from damage caused by the action of proteolytic enzymes. Augmentation therapy with Prolastin® (Alpha1-Proteinase Inhibitor [Human]) to increase the levels of alpha1-PI has been used to treat individuals with AAT deficiency for over 20 years. Modifications to the Prolastin manufacturing process, incorporating additional purification and pathogen-reduction steps, have led to the development of an alpha1-PI product, designated Prolastin®-C (Alpha1-Proteinase inhibitor [Human]). The pharmacokinetic comparability of Prolastin-C to Prolastin was assessed in subjects with AAT deficiency. METHODS: In total, 24 subjects were randomized to receive 60 mg/kg of functionally active Prolastin-C or Prolastin by weekly intravenous infusion for 8 weeks before crossover to the alternate treatment for another 8 weeks. Pharmacokinetic plasma samples were drawn over 7 days following last dose in the first treatment period and over 10 days following the last dose in the second period. The primary end point for pharmacokinetic comparability was area under the plasma concentration versus time curve over 7 days post dose (AUC0₋7 (days)) of alpha1-PI determined by potency (functional activity) assay. The crossover phase was followed by an 8-week open-label treatment phase with Prolastin-C only. RESULTS: Mean AUC0₋7 (days) was 155.9 versus 152.4 mg*h/mL for Prolastin-C and Prolastin, respectively. The geometric least squares mean ratio of AUC0₋7 (days) for Prolastin-C versus Prolastin had a point estimate of 1.03 and a 90% confidence interval of 0.97-1.09, demonstrating pharmacokinetic equivalence between the 2 products. Adverse events were similar for both treatments and occurred at a rate of 0.117 and 0.078 per infusion for Prolastin-C (double-blind treatment phase only) and Prolastin, respectively (p = 0.744). There were no treatment-emergent viral infections in any subject for human immunodeficiency virus, hepatitis B or C, or parvovirus B19 during the course of the study. CONCLUSION: Prolastin-C demonstrated pharmacokinetic equivalence and a comparable safety profile to Prolastin. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT00295061.