Sensitivity of Pharmacokinetics to Differences in the Particle Size Distribution for Formulations of Locally Acting Mometasone Furoate Suspension-Based Nasal Sprays.

To assess bioequivalence of locally acting suspension-based nasal sprays, the U.S. FDA currently recommends a weight-of-evidence approach. In addition to in vitro and human pharmacokinetic (PK) studies, this includes a comparative clinical endpoint study to ensure equivalent bioavailability of the active pharmaceutical ingredient (API) at the site of action. The present study aimed to assess, within an in vitro/in vivo correlation paradigm, whether PK studies and dissolution kinetics are sensitive to differences in drug particle size for a locally acting suspension-based nasal spray product. Two investigational suspension-based nasal formulations of mometasone furoate (MF-I and MF-II; delivered dose: 180 µg) differed in API particle size and were compared in a single-center, double-blind, single-dose, randomized, two-way crossover PK study in 44 healthy subjects with oral charcoal block. Morphology-directed Raman spectroscopy yielded volume median diameters of 3.17 µm for MF-I and 5.50 µm for MF-II, and dissolution studies showed that MF-II had a slower dissolution profile than MF-I. The formulation with larger API particles (MF-II) showed a 45% smaller Cmax and 45% smaller AUC0-inf compared to those of MF-I. Systemic bioavailability of MF-I (2.20%) and MF-II (1.18%) correlated well with the dissolution kinetics, with the faster dissolving formulation yielding the higher bioavailability. This agreement between pharmacokinetics and dissolution kinetics cross-validated both methods and supported their use in assessing potential differences in slowly dissolving suspension-based nasal spray products.

Systematic Evaluation of the Effect of Formulation Variables on In Vitro Performance of Mometasone Furoate Suspension-Metered Dose Inhalers.

The therapeutic benefits of metered dose inhalers (MDIs) in pulmonary disorders are mainly driven by aerosol performance, which depends on formulation variables (drug and excipients), device design, and patient interactions. The present study provides a comprehensive investigation to better understand the effect of formulation variables on mometasone furoate (MF) suspension-based MDI product performance. The effects of MF particle size (volume median diameter; X50) and excipient concentration (ethanol and oleic acid, cosolvent, and surfactant, respectively) on selected critical quality attributes (delivered dose (DD), fine particle dose of particles lesser than 5 µm (FPD < 5), ex-throat dose and median dissolution time (MDT)) were studied. Eight MF-MDI formulations (one per batch) were manufactured based on a reduced factorial design of experiment (DOE) approach, which included relevant formulation levels with varying X50 (1.1 and 2 µm), concentration of ethanol (0.45, 0.9, 1.8, and 3.6%w/w), and oleic acid (0.001 and 0.025%w/w). The in vitro evaluation of these MF-MDI formulations indicated the importance of drug particle's X50, oleic acid, and ethanol canister concentration as critical formulation variables governing the performance of MF suspension-based MDI products. The effect of these formulation variables on DD, FPD < 5, ex-throat dose, and MDT was subsequently utilized to develop empirical relationships linking formulation factors with effects on in vitro performance measures. The developed strategy could be useful for predicting MF-MDI product performance during MDI product development and manufacturing. The systematic DOE approach utilized in this study may provide insights into the understanding of the formulation variables governing the MF-MDI product performance.

Can Pharmacokinetic Studies Assess the Pulmonary Fate of Dry Powder Inhaler Formulations of Fluticasone Propionate?

In the context of streamlining generic approval, this study assessed whether pharmacokinetics (PK) could elucidate the pulmonary fate of orally inhaled drug products (OIDPs). Three fluticasone propionate (FP) dry powder inhaler (DPI) formulations (A-4.5, B-3.8, and C-3.7), differing only in type and composition of lactose fines, exhibited median mass aerodynamic diameter (MMAD) of 4.5 µm (A-4.5), 3.8 µm (B-3.8), and 3.7 µm (C-3.7) and varied in dissolution rates (A-4.5 slower than B-3.8 and C-3.7). In vitro total lung dose (TLDin vitro) was determined as the average dose passing through three anatomical mouth-throat (MT) models and yielded dose normalization factors (DNF) for each DPI formulation X (DNFx = TLDin vitro,x/TLDin vitro,A-4.5). The DNF was 1.00 for A-4.5, 1.32 for B-3.8, and 1.21 for C-3.7. Systemic PK after inhalation of 500 µg FP was assessed in a randomized, double-blind, four-way crossover study in 24 healthy volunteers. Peak concentrations (Cmax) of A-4.5 relative to those of B-3.8 or C-3.7 lacked bioequivalence without or with dose normalization. The area under the curve (AUC0-Inf) was bio-IN-equivalent before dose normalization and bioequivalent after dose normalization. Thus, PK could detect differences in pulmonary available dose (AUC0-Inf) and residence time (dose-normalized Cmax). The differences in dose-normalized Cmax could not be explained by differences in in vitro dissolution. This might suggest that Cmax differences may indicate differences in regional lung deposition. Overall this study supports the use of PK studies to provide relevant information on the pulmonary performance characteristics (i.e., available dose, residence time, and regional lung deposition).

Immunomodulatory Therapeutic Proteins in COVID-19: Current Clinical Development and Clinical Pharmacology Considerations.

The coronavirus disease 2019 (COVID-19) pandemic caused by infection with SARS-CoV-2 has led to more than 600 000 deaths worldwide. Patients with severe disease often experience acute respiratory distress characterized by upregulation of multiple cytokines. Immunomodulatory biological therapies are being evaluated in clinical trials for the management of the systemic inflammatory response and pulmonary complications in patients with advanced stages of COVID-19. In this review, we summarize the clinical pharmacology considerations in the development of immunomodulatory therapeutic proteins for mitigating the heightened inflammatory response identified in COVID-19.

Clinical Pharmacology Considerations for Developing Small-Molecule Treatments for COVID-19.

Numerous drugs are being investigated for the treatment of COVID-19, including antivirals and therapies targeting complications related to COVID-19. The clinical presentation of COVID-19 varies from mild fever, cough, and dyspnea in the early stages of disease to severe complications such as acute respiratory distress syndrome, systemic hyperinflammation, and sepsis. A thorough understanding of the disease pathogenesis and the disease complications is essential to developing effective therapies to treat this potentially life-threatening disease. This review offers key clinical pharmacology considerations involved in the development of small molecules for the treatment of COVID-19. They are based on the major observed disease complications that impact drug absorption, distribution, metabolism, and elimination. We also address considerations regarding potential drug interactions, alternative routes and methods of administration, and dosing in patients on hemodialysis.

A Comprehensive Updated Review on SARS-CoV-2 and COVID-19.

This literature review aims to provide a comprehensive current summary of the pathogenesis, clinical features, disease course, host immune responses, and current investigational antiviral and immunomodulatory pharmacotherapies to facilitate the development of future therapies and measures for prevention and control.

Development of an Aerosol Dose Collection Apparatus for In Vitro Dissolution Measurements of Orally Inhaled Drug Products.

The aim of the study was to develop a robust and standardized in vitro dissolution methodology for orally inhaled drug products (OIDPs). An aerosol dose collection (ADC) system was designed to uniformly deposit the whole impactor stage mass (ISM) over a large filter area for dissolution testing. All dissolution tests were performed under sink conditions in a sodium phosphate buffered saline solution containing 0.2%w/w sodium dodecyl sulphate. An adapted USP Apparatus V, Paddle over Disk (POD), was used throughout the study. The dissolution characteristics of the ISM dose of a commercial metered-dose inhaler (MDI) and a range of dry powder inhaler (DPI) formulations containing inhaled corticosteroids were tested. The uniform distribution of the validated ISM dose considerably reduced drug loading effects on the dissolution profiles for both MDI and DPI formulations. The improvement in the robustness and discriminatory capability of the technique enabled characterization of dissolution rate differences between inhaler platforms and between different DPI product strengths containing fluticasone propionate. A good correlation between in vivo mean absorption time and in vitro dissolution half-life was found for a range of the inhaled corticosteroids. The ADC system and the reproducible in vitro POD dissolution measurements provided a quantitative-based approach for measuring the relationship between the influence of device and the dispersion characteristics on the aerosol dissolution of low solubility compounds. The in vitro dissolution method could potentially be applied as a dissolution methodology for compendial, quality control release testing, and during development of both branded orally inhaled drug products and their generic counterparts.

Drug Interaction Between Febuxostat and Thiopurine Antimetabolites: A Review of the FDA Adverse Event Reporting System and Medical Literature.

BACKGROUND: There is a known drug interaction (DI) between xanthine oxidase (XO) inhibitors and the thiopurine immunosuppressants, azathioprine (AZA) and mercaptopurine (6-MP). Xanthine oxidase inhibition increases concentrations of AZA and 6-MP active metabolites, possibly resulting in myelosuppression. When allopurinol is used with AZA or 6-MP, dose reduction of AZA or 6-MP is recommended. Febuxostat is a newer XO inhibitor approved for the treatment of gout. OBJECTIVE: To determine the clinical impact of the febuxostat-thiopurine DI. DESIGN AND SETTING: Case series derived from the U.S. Food and Drug Administration Adverse Event Reporting System (FAERS) and published medical literature. PATIENTS: Nineteen patients who received concomitant febuxostat and either AZA or 6-MP. MEASUREMENTS: Laboratory and clinical data. RESULTS: Nineteen cases reporting myelosuppressive events were identified in patients receiving febuxostat with AZA or 6-MP. Eighteen cases were treated with the combination of AZA and febuxostat. A median of 1.6 months elapsed from initiation of the drug combination until discovery of the event. Sixteen cases required hospitalization; 15 reported administration of blood products. Thirteen cases reported resolution of the event with discontinuation of both drugs, two reported discontinuation of the thiopurine only, and one reported discontinuation of febuxostat only. LIMITATIONS: Thiopurine monotherapy may cause myelosuppression. Complications of immunosuppression that may contribute to the real-world morbidity and mortality associated with the febuxostat-thiopurine DI were not examined. Finally, FAERS data are limited by the voluntary nature of reporting. CONCLUSION: Current febuxostat labeling contraindicates concomitant administration of febuxostat with either AZA or 6-MP. This case series demonstrates that the DI can result in clinically significant adverse events and is supportive of current febuxostat labeling.

Scientific Considerations for the Review and Approval of First Generic Mometasone Furoate Nasal Suspension Spray in the United States from the Bioequivalence Perspective.

In 2016, the US Food and Drug Administration (FDA) approved the first Abbreviated New Drug Application for Mometasone Furoate Nasal Suspension Spray. To establish the bioequivalence of this generic nasal suspension spray with the reference listed drug product (RLD), Nasonex®, a "weight-of-evidence" approach was utilized by the applicant that included formulation and device similarities, equivalent in vitro performance, equivalent systemic exposure, and equivalent local delivery. In addition to these testing for comprehensive evaluation of the drug product, FDA also considered supportive data generated by a novel in vitro method, Morphologically-Directed Raman Spectroscopy (MDRS), to characterize the particle size distribution (PSD) of active pharmaceutical ingredient (API) in the drug product. In this case, MDRS data eliminated the need for a comparative clinical endpoint bioequivalence study. The approval of the first generic Mometasone Furoate Nasal Suspension Spray is precedent-setting and paves a new pathway to establish bioequivalence for generic nasal suspension sprays. This approval also exemplifies FDA's commitment to advance regulatory science for evaluation of generic drug products.

Effects of Formulation Variables on Lung Dosimetry of Albuterol Sulfate Suspension and Beclomethasone Dipropionate Solution Metered Dose Inhalers.

The performance of pressurized metered dose inhalers (MDIs) is affected by formulation and device variables that impact delivered dose, aerodynamic particle size distribution, and consequently lung deposition and therapeutic effect. Specific formulation variables of relevance to two commercially available products-Proventil® HFA [albuterol sulfate (AS) suspension] and Qvar® [beclomethasone dipropionate (BDP) solution]-were evaluated to determine their influence on key performance attributes measured experimentally with in vitro cascade impaction studies. These commercial MDIs, utilized as model systems, provided mid-points for a design of experiments (DoE) plan to manufacture multiple suspension and solution MDI formulations. The experimental results were utilized as input variables in a computational dosimetry model to predict the effects of MDI formulation variables on lung deposition. For the BDP solution DoE MDIs, increased concentrations of surfactant oleic acid (0-2% w/w) increased lung deposition from 24 to 46%, whereas changes in concentration of the cosolvent ethanol (7-9% w/w) had no effect on lung deposition. For the AS suspension DoE MDIs, changes in oleic acid concentration (0.005-0.25% w/w) did not have significant effects on lung deposition, whereas lung deposition decreased from 48 to 26% as ethanol concentration increased from 2 to 20% w/w, and changes in micronized drug volumetric median particle size distribution (X50, 1.4-2.5 µm) increased deposition in the tracheobronchial airways from 5 to 11%. A direct correlation was observed between fine particle fraction and predicted lung deposition. These results demonstrate the value of using dosimetry models to further explore relationships between performance variables and lung deposition.

In Vitro Assessment of Nasal Insufflation of Comminuted Drug Products Designed as Abuse Deterrent Using the Vertical Diffusion Cell.

In vitro evaluation of abuse deterrent formulations (ADFs) is a challenge since real abuse situations are variable and ADF technology is evolving. Specifically, an assessment of an ADF to deter nasal insufflation would be valuable. In this study, a vertical diffusion cell (VDC) was used to evaluate polyethylene oxide (PEO)-based tablets manipulated by three different forces. The commercially available products Oxycontin®, an ADF, Opana®, and metoprolol tartrate tablet formulations made in our laboratory were studied. Particle size distribution and percent recovery of manipulated tablets were measured. Grinding produced the lowest recovery and the smallest particle size distribution. Drug release was examined using a VDC by placing the dry comminuted particles on an enclosed wetted cellulose membrane. Dispensing dry particles on a VDC is atypical but includes some key features associated with an abuse situation where once the particles are snorted, the moisture in the nasal mucosa activates hydration and swelling of the polymers in the formulation, retarding drug release. Drug release from OxyContin®, Opana®, and metoprolol tablets were analyzed for the cutting, grinding, and milling modes of abuse. The analysis showed that in most cases, the mode of abuse produced different particle sizes with different release rates. Statistically different release rates were observed for metoprolol tablets made with different molecular weight PEO and with different porosities. These results indicate that within detection limits, the VDC can be used to quantitate release differences due to various modes of abuse used in this study.

Predicting Pulmonary Pharmacokinetics from In Vitro Properties of Dry Powder Inhalers.

PURPOSE: The ability of two semi-mechanistic simulation approaches to predict the systemic pharmacokinetics (PK) of inhaled corticosteroids (ICSs) delivered via dry powder inhalers (DPIs) was assessed for mometasone furoate, budesonide and fluticasone propionate. METHODS: Both approaches derived the total lung doses and the central to peripheral lung deposition ratios from clinically relevant cascade impactor studies, but differed in the way the pulmonary absorption rate was derived. In approach 1, the rate of in vivo drug dissolution/absorption was predicted for the included ICSs from in vitro aerodynamic particle size distribution and in vitro drug solubility estimates measured in an in vivo predictive dissolution medium. Approach 2 derived a first order absorption rate from the mean dissolution time (MDT), determined for the test formulations in an in vitro Transwell® based dissolution system. RESULTS: Approach 1 suggested PK profiles which agreed well with the published pharmacokinetic profiles. Similarly, within approach 2, input parameters for the pulmonary absorption rate constant derived from dissolution rate experiments were able to reasonably predict the pharmacokinetic profiles published in literature. CONCLUSION: Approach 1 utilizes more complex strategies for predicting the dissolution/absorption process without providing a significant advantage over approach 2 with regard to accuracy of in vivo predictions.

Influence of Formulation Factors on the Aerosol Performance of Suspension and Solution Metered Dose Inhalers: A Systematic Approach.

Metered dose inhalers (MDIs) are complex drug-device combination products widely used to treat pulmonary disorders. The efficacy, driven by aerosol performance of the products, depends on a multitude of factors including, but not limited to, the physicochemical properties of drug and nature and amount of excipient(s). Under the quality by design (QbD) paradigm, systematic investigations are necessary to understand how changes in critical quality attributes (CQAs) of formulation, device, and manufacturing process influence key product performance parameters, such as delivered dose (DD) and fine particle dose (FPD). The purpose of this work is to provide a better understanding of the effects of different levels of excipients and drug particle size distribution on the aerosol performance of MDI products, while using two fundamentally different MDI products as relevant model systems, Proventil® HFA (albuterol sulfate suspension) and Qvar® (beclomethasone dipropionate solution). These MDI products, as model systems, provided mid-points around which a design of experiments (DOE), consisting of 22 suspension and 9 solution MDI formulations, were defined and manufactured. The DOE included formulations factors with varying ethanol (2 to 20% w/w and 7 to 9% w/w for the suspension and solution, respectively) and oleic acid concentrations (0.005 to 0.25% w/w and 0 to 2% w/w for the suspension and solution, respectively) and drug volumetric median particle size distribution (PSD D50, 1.4 to 2.5 µm for the suspension). The MDI formulations were analyzed using compendial methods to elucidate the effect of these formulation variables (ethanol, oleic acid, and PSD D50) on DD and FPD. The outcomes of this study allowed defining design spaces for the formulation factors, such that DD and FPD would remain within specific pre-defined requirements. The systematic approach utilized in this work can contribute as a QbD tool to evaluate the extent to which the formulation factors govern the aerosol performance of MDI drug products, helping to design MDI formulations with desired product performance parameters.

Patient Focus and Regulatory Considerations for Inhalation Device Design: Report from the 2015 IPAC-RS/ISAM Workshop.

This article reports on discussions at the 2015 workshop cosponsored by the International Pharmaceutical Aerosol Consortium on Regulation and Science (IPAC-RS) and the International Society for Aerosols in Medicine (ISAM), entitled "Regulatory and Patient Considerations for Inhalation Device Design, Development and Use." Key topics addressed at the workshop and presented here include patient-focused device design for orally inhaled products (OIPs), instructions for use (IFU), human factors, regulatory considerations in the United States and Europe, development of generic inhalers, quality-by-design, and change management of OIP devices. Workshop participants also identified several areas for further consideration and emphasized the need for increased focus on the patient to create therapeutic products (inclusive of device design, IFU, education, training) that support adherence with an individual patient's treatment regimen. Advances in patient-centric product development will require engagement and collaboration by industry, regulators, patients, physicians, and other stakeholders. The article includes summaries of presented talks as well as of panel and audience discussions.

Regulatory Considerations for Approval of Generic Inhalation Drug Products in the US, EU, Brazil, China, and India.

This article describes regulatory approaches for approval of "generic" orally inhaled drug products (OIDPs) in the United States, European Union, Brazil, China and India. While registration of a generic OIDP in any given market may require some documentation of the formulation and device similarity to the "original" product as well as comparative testing of in vitro characteristics and in vivo performance, the specific documentation approaches, tests and acceptance criteria vary by the country. This divergence is due to several factors, including unique cultural, historical, legal and economic circumstances of each region; the diverse healthcare and regulatory systems; the different definitions of key terms such as "generic" and "reference" drug; the acknowledged absence of in vitro in vivo correlations for OIDPs; and the scientific and statistical issues related to OIDP testing (such as how best to account for the batch-to-batch variability of the Reference product, whether to use average bioequivalence or population bioequivalence in the statistical analysis of results, whether to use healthy volunteers or patients for pharmacokinetic studies, and which pharmacodynamic or clinical end-points should be used). As a result of this discrepancy, there are ample opportunities for the regulatory and scientific communities around the world to collaborate in developing more consistent, better aligned, science-based approaches. Moving in that direction will require both further research and further open discussion of the pros and cons of various approaches.

Effect of Device Design and Formulation on the In Vitro Comparability for Multi-Unit Dose Dry Powder Inhalers.

The focus of this investigation was to understand the design space to achieve comparable in vitro performance of two multi-unit dose dry powder inhalers (DPIs)­Flixotide® Accuhaler® (reference product) and MultiHaler® (test product). Flow field, pressure drop and particle trajectories within the test and reference DPI devices were modelled via computational fluid dynamics (CFD). Micronized fluticasone propionate (FP) was characterized to determine particle size distribution (PSD), specific surface area (SSA) and surface interfacial properties using cohesive-adhesive balance (CAB). CFD simulations suggested that the pressure drop and airflow velocity in the MultiHaler® were greater than Accuhaler®. Two modified test devices (MOD MH 1 and MOD MH 2) were manufactured with the introduction of by-pass channels in the airflow path, which achieved comparable specific resistance and airflow path between the test and reference devices. Assessment of reference product formulation in modified test devices suggested that MOD MH 2 achieved comparable in vitro performance to the reference product. CAB analysis suggested that adhesion of all FP batches to lactose was different, with batch D showing greatest and batch A least adhesion to lactose. Test DPI formulations were manufactured using four different batches of FP with milled or sieved lactose, and showed that batch A FP formulated with sieved lactose in MOD MH 2 device demonstrated the highest degree of similarity to the Accuhaler® in vitro deposition. Application of CFD modelling and material characterization of formulation raw materials enabled the modification of device and formulation critical material attributes to create an in vitro comparable device/formulation system to the reference product.

The influence of secondary processing on the structural relaxation dynamics of fluticasone propionate.

This study investigated the structural relaxation of micronized fluticasone propionate (FP) under different lagering conditions and its influence on aerodynamic particle size distribution (APSD) of binary and tertiary carrier-based dry powder inhaler (DPI) formulations. Micronized FP was lagered under low humidity (LH 25 C, 33% RH [relative humidity]), high humidity (HH 25°C, 75% RH) for 30, 60, and 90 days, respectively, and high temperature (HT 60°C, 44% RH) for 14 days. Physicochemical, surface interfacial properties via cohesive-adhesive balance (CAB) measurements and amorphous disorder levels of the FP samples were characterized. Particle size, surface area, and rugosity suggested minimal morphological changes of the lagered FP samples, with the exception of the 90-day HH (HH90) sample. HH90 FP samples appeared to undergo surface reconstruction with a reduction in surface rugosity. LH and HH lagering reduced the levels of amorphous content over 90-day exposure, which influenced the CAB measurements with lactose monohydrate and salmeterol xinafoate (SX). CAB analysis suggested that LH and HH lagering led to different interfacial interactions with lactose monohydrate but an increasing adhesive affinity with SX. HT lagering led to no detectable levels of the amorphous disorder, resulting in an increase in the adhesive interaction with lactose monohydrate. APSD analysis suggested that the fine particle mass of FP and SX was affected by the lagering of the FP. In conclusion, environmental conditions during the lagering of FP may have a profound effect on physicochemical and interfacial properties as well as product performance of binary and tertiary carrier-based DPI formulations.

Sulfated caffeic acid dehydropolymer attenuates elastase and cigarette smoke extract-induced emphysema in rats: sustained activity and a need of pulmonary delivery.

BACKGROUND: Although emphysema destroys alveolar structures progressively and causes death eventually, no drug has been discovered to prevent, intervene, and/or resolve this life-threatening disease. We recently reported that sulfated caffeic acid dehydropolymer CDSO3 is a novel potent triple-action inhibitor of elastolysis, oxidation, and inflammation in vitro, and therefore, a potential anti-emphysema agent. However, the in vivo therapeutic potency, duration and mode of actions, and effective route remain to be demonstrated. METHODS: Emphysema was induced in rats with human sputum elastase (HSE) combined with cigarette smoke extract (CSE). CDSO3 at 5, 30, or 100 µg/kg was dosed to the lung or injected subcutaneously at 2, 6, or 24 h before or 1 or 24 h or 1 week after the HSE/CSE instillation. At 1 h or 48 h or on day 21-22 or day 28, lungs were examined for airway-to-blood injurious barrier damage; their elastolytic, oxidative, and inflammatory activities; lung luminal leukocytes infiltration; functional treadmill exercise endurance; and/or morphological airspace enlargement. RESULTS: CDSO3, when dosed to the lung at 30 or 100 µg/kg, but not via systemic subcutaneous injection, significantly (43-93 %) attenuated HSE/CSE-induced (1) barrier damage measured by luminal hemorrhage and protein leak; (2) elastolytic, oxidative, and inflammatory activities measured with elastase, reduced glutathione, and TNFα levels, respectively; (3) luminal neutrophil infiltration and tissue myeloperoxidase activity; (4) functional impairment of exercise endurance; and (5) airspace enlargement, in both preventive and interventional dosing protocols. Notably, the effects were shown to last for 24 h at the greater 100-µg/kg dose, and the 1-week-delayed administration was also capable of attenuating the development of emphysema. CONCLUSIONS: CDSO3 is a novel, potent, long-acting, nonpeptidic macromolecule that inhibits HSE/CSE-induced elastolysis, oxidation, and inflammation in the lung and thereby attenuates the development of emphysema in rats, in both preventive and interventional manners, when administered locally to the lung.

Novel low molecular weight lignins as potential anti-emphysema agents: In vitro triple inhibitory activity against elastase, oxidation and inflammation.

No molecule has been found to be effective against emphysema to date primarily because of its complex pathogenesis that involves elastolysis, oxidation and inflammation. We here describe novel unsulfated or sulfated low molecular weight lignins (LMWLs) chemo-enzymatically prepared from 4-hydroxycinnamic acids monomers, as the first potent triple-action inhibitors of neutrophil elastase, oxidation and inflammation. The inhibitory potencies of three different cinnamic acid-based LMWLs were determined in vitro using chromogenic substrate hydrolysis assays, radical scavenging and lung cellular oxidative biomarker reduced glutathione (rGSH) assays, and lung cellular inflammatory biomarker NFκB and IL-8 assays, respectively. Each LWML uniquely displayed triple-action inhibition, among which CDSO3, a sulfated caffeic acid-based LMWL, was most potent. The half-maximal anti-human neutrophil elastase (HNE) potency of CDSO3 was 0.43 µM. This high potency arose from lignin-like oligomerization, which was further potentiated by 6.6-fold due to sulfation. Mechanistically, this elastase inhibition was of mixed-type, time-dependent and more selective to positively charged elastases. The half-maximal anti-oxidative potency of CDSO3 was 3.52 µM, 4.8-fold potentiated from that of the monomer, caffeic acid (CA). In contrast, the half-maximal inhibitory potency to TNFα-induced inflammation was 5-10 µM, despite no activity with the monomer. More intriguingly, this anti-inflammatory activity was essentially identical with different stimuli, okadaic acid and hydrogen peroxide (H(2)O(2)), which implied that CDSO3 acts directly on inflammatory cascades within the cells. Overall, oligomerization and sulfation produced or significantly potentiated the activity, in comparison to the monomer. Thus, sulfated and unsulfated LMWLs are novel non-peptidic 2.8-4.1 kDa macromolecules that exhibit for the first time potent triple inhibitory activity against elastase, oxidation and inflammation, the three major pathogenic mechanisms known to cause emphysema.