Effect of Aerosol Inhalation Combined With a Vibration Expectoration Machine on Sputum Volume, IGF-1, α1-AT and PDGF-B in Patients With Chronic Obstructive Pulmonary Emphysema.

Objective: Aerosol inhalation is commonly used in the treatment of chronic obstructive pulmonary emphysema (COPE). This study aimed to evaluate the effectiveness of aerosol inhalation combined with a vibration expectoration machine on COPE. Methods: From June 2019 to June 2020, 110 patients receiving COPE treatment in Linyi Central Hospital in China were included in this randomized controlled trial. All patients were randomly assigned into one of two groups using the random number table. A total of 55 patients were given aerosol inhalation combined with the use of a vibration expectoration machine in the study group, and 55 patients were given aerosol inhalation alone in the control group. The general data, clinical efficacy arterial blood gas index, pulmonary function index and serum levels of insulin-like growth factor 1 (IGF-1), alpha 1 antitrypsin (α1-AT) and platelet-derived growth factor-B (PDGF-B) were compared. Results: There was no difference in baseline characteristics between the 2 groups (P > .05). After treatment, the clinical efficacy in the study group was significantly higher than in the control group (96.36% vs 81.82%, respectively; P = .023), daily sputum production in the study group was significantly higher compared with the control group (80.92 ± 10.29 vs 58.63 ± 9.02 ml, respectively; P < .001) and hospitalization time was significantly reduced in the study group (11.87 ± 1.76 vs 17.62 ± 1.92 days, respectively; P < .001). In addition, the respiratory rate was significantly lower in the study group (17.43 ± 1.61 vs 22.08 ± 3.25, respectively; P < .001). Partial pressure of oxygen (P[O2]) was significantly higher (76.29 ± 7.34 vs 66.81 ± 7.93 mmHg, respectively; P < .001) and partial pressure of carbon dioxide (P[CO2]) was significantly lower (34.82 ± 6.02 vs 39.83 ± 6.01 mmHg respectively; P < .001) in the study group compared with the control group. In addition, forced expiratory volume in the first second (FEV1) (1.79 ± 0.36 vs 1.66 ± 0.28 L, respectively), forced vital capacity (FVC) (2.58 ± 0.28 vs 2.42 ± 0.11 L, respectively), forced expiratory volume in the first second as a percentage of the predicted value (FEV1%pred) (65.32 ± 4.03 vs 59.83 ± 4.76 L, respectively) and maximal mid-expiratory flow (MMEF) (1.51 ± 0.27% vs 1.36 ± 0.12%, respectively) were all significantly increased after treatment in the study group compared with the control group (all P < .001). The IGF-1 (104.92 ± 11.27 vs 137.83 ± 11.02 ng/mL, respectively) and PDGF-B (124.39 ± 14.29 vs 249.93 ± 33.49 ng/L, respectively) were significantly reduced in the study group after treatment (all P < .001). The α1-AT (2.82 ± 0.38 vs 2.17 ± 0.22 g/L, respectively) were significantly increased after treatment in the study group compared with the control group. Conclusion: Aerosol inhalation combined with the use of a vibration expectoration machine is worthy of clinical application, and can effectively improve outcomes in patients with COPE.

A reactive center loop-based prediction platform to enhance the design of therapeutic SERPINs.

Serine proteases are essential for many physiological processes and require tight regulation by serine protease inhibitors (SERPINs). A disturbed SERPIN-protease balance may result in disease. The reactive center loop (RCL) contains an enzymatic cleavage site between the P1 through P1' residues that controls SERPIN specificity. This RCL can be modified to improve SERPIN function; however, a lack of insight into sequence-function relationships limits SERPIN development. This is complicated by more than 25 billion mutants needed to screen the entire P4 to P4' region. Here, we developed a platform to predict the effects of RCL mutagenesis by using α1-antitrypsin as a model SERPIN. We generated variants for each of the residues in P4 to P4' region, mutating them into each of the 20 naturally occurring amino acids. Subsequently, we profiled the reactivity of the resulting 160 variants against seven proteases involved in coagulation. These profiles formed the basis of an in silico prediction platform for SERPIN inhibitory behavior with combined P4 to P4' RCL mutations, which were validated experimentally. This prediction platform accurately predicted SERPIN behavior against five out of the seven screened proteases, one of which was activated protein C (APC). Using these findings, a next-generation APC-inhibiting α1-antitrypsin variant was designed (KMPR/RIRA; / indicates the cleavage site). This variant attenuates blood loss in an in vivo hemophilia A model at a lower dosage than the previously developed variant AIKR/KIPP because of improved potency and specificity. We propose that this SERPIN-based RCL mutagenesis approach improves our understanding of SERPIN behavior and will facilitate the design of therapeutic SERPINs.

Hypothesis: Alpha-1-antitrypsin is a promising treatment option for COVID-19.

No definitive treatment for COVID-19 exists although promising results have been reported with remdesivir and glucocorticoids. Short of a truly effective preventive or curative vaccine against SARS-CoV-2, it is becoming increasingly clear that multiple pathophysiologic processes seen with COVID-19 as well as SARS-CoV-2 itself should be targeted. Because alpha-1-antitrypsin (AAT) embraces a panoply of biologic activities that may antagonize several pathophysiologic mechanisms induced by SARS-CoV-2, we hypothesize that this naturally occurring molecule is a promising agent to ameliorate COVID-19. We posit at least seven different mechanisms by which AAT may alleviate COVID-19. First, AAT is a serine protease inhibitor (SERPIN) shown to inhibit TMPRSS-2, the host serine protease that cleaves the spike protein of SARS-CoV-2, a necessary preparatory step for the virus to bind its cell surface receptor ACE2 to gain intracellular entry. Second, AAT has anti-viral activity against other RNA viruses HIV and influenza as well as induces autophagy, a known host effector mechanism against MERS-CoV, a related coronavirus that causes the Middle East Respiratory Syndrome. Third, AAT has potent anti-inflammatory properties, in part through inhibiting both nuclear factor-kappa B (NFκB) activation and ADAM17 (also known as tumor necrosis factor-alpha converting enzyme), and thus may dampen the hyper-inflammatory response of COVID-19. Fourth, AAT inhibits neutrophil elastase, a serine protease that helps recruit potentially injurious neutrophils and implicated in acute lung injury. AAT inhibition of ADAM17 also prevents shedding of ACE2 and hence may preserve ACE2 inhibition of bradykinin, reducing the ability of bradykinin to cause a capillary leak in COVID-19. Fifth, AAT inhibits thrombin, and venous thromboembolism and in situ microthrombi and macrothrombi are increasingly implicated in COVID-19. Sixth, AAT inhibition of elastase can antagonize the formation of neutrophil extracellular traps (NETs), a complex extracellular structure comprised of neutrophil-derived DNA, histones, and proteases, and implicated in the immunothrombosis of COVID-19; indeed, AAT has been shown to change the shape and adherence of non-COVID-19-related NETs. Seventh, AAT inhibition of endothelial cell apoptosis may limit the endothelial injury linked to severe COVID-19-associated acute lung injury, multi-organ dysfunction, and pre-eclampsia-like syndrome seen in gravid women. Furthermore, because both NETs formation and the presence of anti-phospholipid antibodies are increased in both COVID-19 and non-COVID pre-eclampsia, it suggests a similar vascular pathogenesis in both disorders. As a final point, AAT has an excellent safety profile when administered to patients with AAT deficiency and is dosed intravenously once weekly but also comes in an inhaled preparation. Thus, AAT is an appealing drug candidate to treat COVID-19 and should be studied.

Alpha 1-antitrypsin for treating ventilator-associated lung injury in acute respiratory distress syndrome rats.

Purpose: Mechanical ventilation (MV) is an essential life support tool for patients with acute respiratory distress syndrome (ARDS). However, MV for ARDS can result in ventilator-induced lung injury (VILI). This study aimed to assess whether alpha 1-antitrypsin (AAT) can reduce VILI in ARDS rats. Materials and Methods: Rats were randomly divided into five groups: the sham (S) group, MV (V) group, lipopolysaccharide (LPS) (L) group, MV/LPS (VL) group and MV/AAT (VA) group. Rats in the S group were anesthetized. The rats in the L group received LPS but not ventilation, the rats in the V group received only MV, and the rats in the VL and VA groups received LPS and MV. Additionally, the rats in the VA group were treated with AAT, and the other rats were injected with saline. The PaO2/FiO2 ratio and the wet/dry weight were assessed. The total protein and neutrophil elastase concentrations and the neutrophil and macrophage counts in bronchoalveolar lavage fluid (BALF) were evaluated. Proinflammatory factors in BALF and ICAM-1 and MIP-2 in serum were also tested. Furthermore, the oxidative stress response was detected, and histological injury and apoptosis were evaluated. Results: All the rats in the V, L and VL groups had significant lung injury, with the VL group exhibiting the most severe injury. Compared with the findings in the VL group, AAT significantly upregulated the PaO2/FiO2 ratio but decreased the wet/dry weight ratio and protein levels in BALF. AAT also reduced proinflammatory cytokine levels and inflammatory cell counts in BALF. Lung tissue injury and cell apoptosis were mitigated by AAT. Conclusions: AAT ameliorated VILI in ARDS rats. The protection conferred by AAT may be associated with the anti-inflammatory, antioxidative stress response and anti-apoptotic effects of AAT.

FURIN Inhibition Reduces Vascular Remodeling and Atherosclerotic Lesion Progression in Mice.

Objective- Atherosclerotic coronary artery disease is the leading cause of death worldwide, and current treatment options are insufficient. Using systems-level network cluster analyses on a large coronary artery disease case-control cohort, we previously identified PCSK3 (proprotein convertase subtilisin/kexin family member 3; FURIN) as a member of several coronary artery disease-associated pathways. Thus, our objective is to determine the role of FURIN in atherosclerosis. Approach and Results- In vitro, FURIN inhibitor treatment resulted in reduced monocyte migration and reduced macrophage and vascular endothelial cell inflammatory and cytokine gene expression. In vivo, administration of an irreversible inhibitor of FURIN, α-1-PDX (α1-antitrypsin Portland), to hyperlipidemic Ldlr-/- mice resulted in lower atherosclerotic lesion area and a specific reduction in severe lesions. Significantly lower lesional macrophage and collagen area, as well as systemic inflammatory markers, were observed. MMP2 (matrix metallopeptidase 2), an effector of endothelial function and atherosclerotic lesion progression, and a FURIN substrate was significantly reduced in the aorta of inhibitor-treated mice. To determine FURIN's role in vascular endothelial function, we administered α-1-PDX to Apoe-/- mice harboring a wire injury in the common carotid artery. We observed significantly decreased carotid intimal thickness and lower plaque cellularity, smooth muscle cell, macrophage, and inflammatory marker content, suggesting protection against vascular remodeling. Overexpression of FURIN in this model resulted in a significant 67% increase in intimal plaque thickness, confirming that FURIN levels directly correlate with atherosclerosis. Conclusions- We show that systemic inhibition of FURIN in mice decreases vascular remodeling and atherosclerosis. FURIN-mediated modulation of MMP2 activity may contribute to the atheroprotection observed in these mice.

Inhaled Biologicals for the Treatment of Cystic Fibrosis.

BACKGROUND: Cystic Fibrosis (CF), one of the most frequent genetic diseases, is characterized by the production of viscous mucus in several organs. In the lungs, mucus clogs the airways and traps bacteria, leading to recurrent/resistant infections and lung damage. For cystic fibrosis patients, respiratory failure is still lethal in early adulthood since available treatments display incomplete efficacy. OBJECTIVE: The objective of this review is to extend the current knowledge in the field of available treatments for cystic fibrosis. A special focus has been given to inhaled peptide-based drugs. METHODS: The current review is based on recent and/or relevant literature and patents already available in various scientific databases, which include PubMed, PubMed Central, Patentscope and Science Direct. The information obtained through these diverse databases is compiled, critically interpreted and presented in the current study. An in-depth but not systematic approach to the specific research question has been adopted. RESULTS: Recently, peptides have been proposed as possible pharmacologic agents for the treatment of respiratory diseases. Of note, peptides are suitable to be administered by inhalation to maximize efficacy and reduce systemic side effects. Moreover, innovative delivery carriers have been developed for drug administration through inhalation, allowing not only protection against proteolysis, but also a prolonged and controlled release. CONCLUSION: Here, we summarize newly patented peptides that have been developed in the last few years and advanced technologies for inhaled drug delivery to treat cystic fibrosis.

Alpha-1-anti-trypsin-Fc fusion protein ameliorates gouty arthritis by reducing release and extracellular processing of IL-1ß and by the induction of endogenous IL-1Ra.

OBJECTIVES: In the present study, we generated a new protein, recombinant human alpha-1-anti-trypsin (AAT)-IgG1 Fc fusion protein (AAT-Fc), and evaluated its properties to suppress inflammation and interleukin (IL)-1ß in a mouse model of gouty arthritis. METHODS: A combination of monosodium urate (MSU) crystals and the fatty acid C16.0 (MSU/C16.0) was injected intra-articularly into the knee to induce gouty arthritis. Joint swelling, synovial cytokine production and histopathology were determined after 4 h. AAT-Fc was evaluated for inhibition of MSU/C16.0-induced IL-1ß release from human blood monocytes and for inhibition of extracellular IL-1ß precursor processing. RESULTS: AAT-Fc markedly suppressed MSU/C16.0-induced joint inflammation by 85-91% (p<0.001). Ex vivo production of IL-1ß and IL-6 from cultured synovia were similarly reduced (63% and 65%, respectively). The efficacy of 2.0 mg/kg AAT-Fc in reducing inflammation was comparable to 80 mg/kg of plasma-derived AAT. Injection of AAT-Fc into mice increased circulating levels of endogenous IL-1 receptor antagonist by fourfold. We also observed that joint swelling was reduced by 80%, cellular infiltration by 95% and synovial production of IL-1ß by 60% in transgenic mice expressing low levels of human AAT. In vitro, AAT-Fc reduced MSU/C16.0-induced release of IL-1ß from human blood monocytes and inhibited proteinase-3-mediated extracellular processing of the IL-1ß precursor into active IL-1ß. CONCLUSIONS: A single low dose of AAT-Fc is highly effective in reducing joint inflammation in this model of acute gouty arthritis. Considering the long-term safety of plasma-derived AAT use in humans, subcutaneous AAT-Fc emerges as a promising therapy for gout attacks.

Novel peptides as potential treatment of systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by a loss of immunologic tolerance, production of auto-antibodies, and inflammatory damage in multiple organs. We have tested the effect of anti-inflammatory peptide, a H2A histone fragment, termed IIIM1, on MRL/lpr mice, animal model of SLE. Oral administration of IIIM1 at early stage of disease caused reduction in proteinuria and serum anti-dsDNA antibodies. Starting the treatment at advanced stage of disease resulted in prolonged animal survival, decreased lymphadenosis and reduced levels of pathogenic or abnormal double negative CD4(-)CD8(-) cells and B220(+) cells in lymph nodes and spleen. We discovered that IIIM1 induces the production of an additional peptide, a fragment of alpha-1-antitrypsin, termed UBE. A relatively low dose (1 µg/kg) of UBE reduced proteinuria and hematuria in MRL/lpr mice. The beneficial effect of the peptide was corroborated by histological examination. Furthermore a significant reduction in serum IL17, IL12 and anti dsDNA antibodies was observed in the UBE-treated mice. Isolated CD4 cells incubated with the peptide showed a similar cytokine profile. Decreased levels of double negative CD4(-)CD8(-) and B220(+) cells were determined in lymph organs of UBE-treated animals. The beneficial effects of both UBE and IIIM1 suggest these peptides as potential drugs for SLE.

Combination of alpha-1 antitrypsin and doxycycline suppresses collagen-induced arthritis.

BACKGROUND: Rheumatoid arthritis (RA) is a complex disease characterized by autoimmune inflammation and joint destruction. Despite recent advances in RA treatment, current therapies require further improvement to overcome adverse events and ineffectiveness in some cases. By targeting different pathways/molecules using drug combinations, a better treatment can be obtained, whereas adverse events are reduced. In order to develop a new treatment option, the present study employs a gene therapy-based combination therapy using doxycycline and human alpha-1 antitrypsin (hAAT). METHODS: DBA/1 mice were immunized with type II collagen to induce arthritis. Four weeks before immunization, they received a doxycycline containing diet and a single injection of adeno-associated virus vector expressing hAAT under the control of a tetracycline-dependent promoter. Control groups received doxycycline alone or saline. Macroscopic arthritis development as well as histopathological changes in the joint were evaluated. In addition, the effects of hAAT and doxycycline on lipopolysaccharide (LPS)- or tumor necrosis factor-alpha-induced interleukin (IL)-6 production from mouse fibroblast cells were also determined. RESULTS: Combination therapy significantly reduced arthritis development and progression compared to the control group in respect to macroscopic as well as histopathological changes. Doxycycline and hAAT in combination also inhibited IL-6 expression from LPS-stimulated NIH/3T3 mouse fibroblast cells, indicating a contributing mechanism of arthritis inhibition. CONCLUSIONS: The results obtained in the present study indicate that a combination therapy using AAT and doxycycline holds promising potential as a new therapy for RA.

AANA journal course. Update for nurse anesthetists--part 4--life in the balance: the role of serpins in disease genesis and prevention.

Complex biological systems are often shaped and maintained by opposing forces. A relevant biological example is the delicate balance between proteases and their inhibitors. Serine proteases contain a serine residue in the active site of the molecule that is essential to the activity of the enzyme. Protease inhibitors limit the activity of proteases in the body. As examples, aprotinin (Trasylol), a serine protease inhibitor, and aminocaproic acid (Amicar), a lysine protease inhibitor, are used to decrease the rate of fibrinolysis and have recently been the subject of considerable controversy in the literature regarding safety and efficacy. This AANA journal course reviews 2 common examples of protease inhibitor disorders, angioedema and a form of emphysema, that are of particular anesthetic relevance.

Anti-inflammatory approaches to the treatment of cystic fibrosis lung disease: past, present and future.

Inflammation has been increasingly recognized as a major factor in the pathogenesis of cystic fibrosis (CF) lung disease. The use of anti-inflammatory medications to slow pulmonary deterioration has been the focus of much research over the past two decades. Oral corticosteroids are effective, but are associated with significant adverse effects when used long-term. Inhaled corticosteroids are being studied as an alternative to systemic steroids. High-dose ibuprofen has also been shown to be of benefit in CF patients but has not been widely used. A variety of other non-specific anti-inflammatory agents, as well as antioxidants and antiproteinases, have been evaluated or are currently under investigation for use in CF. At present, the anti-inflammatory therapies used to treat CF lung disease are limited. There is hope that agents being evaluated in ongoing clinical trials will prove more effective than those already tested and that future research will provide additional anti-inflammatory therapies for CF airway disease.

Effect of recombinant human DNase on alpha1-proteinase inhibitor function: an experimental approach to the combined clinical use of rhDNase and alpha1-PI in CF patients.

Chronic inflammation in cystic fibrosis (CF) airways leads to high concentrations of deoxyribonucleic acid (DNA) and neutrophil elastase (NE). Both play a major role in CF lung pathophysiology and are aims of new therapeutic approaches: rhDNase degrades highly viscosic DNA and alpha1-proteinase inhibitor (alpha1-PI) inhibits NE activity and thereby pulmonary inflammation and hypersecretion. Given the reports on increased sputum NE concentrations upon rhDNase inhalation, there is a rationale for a combined rhDNase/alpha1-PI treatment. With the question of whether a combined therapy is feasible, we first investigated in vitro whether incubation of CF sputum with rhDNase changes proteolytic and secretagogue activity of sputum supernatants and its inhibition by alpha1-PI. Next, we studied whether incubation of alpha1-PI with rhDNase impairs the inhibitory effect of alpha1-PI on proteolytic activity of NE and the inhibitory effect of alpha1-PI on NE-induced secretion from a human mucoepidermoid cell line. Incubation of CF sputum with rhDNase led to a twofold increase in sputum NE activity. Correspondingly, the inhibitory effect of alpha1-PI on sputum NE activity and on secretion induced by these sputum samples was significantly reduced by rhDNase. Preincubation of alpha1-PI with rhDNase significantly reduced the inhibitory effect of alpha1-PI on purified NE activity and on NE-induced secretion. However, this effect was limited to alpha1-PI concentrations lower than those achievable after inhalation. Therefore, impairment of alpha1-PI function by rhDNase is not likely to be relevant in vivo, provided that a sufficient dosage of alpha1-PI is inhaled.

Recombinant antitrypsin Pittsburgh undergoes proteolytic cleavage during E. coli sepsis and fails to prevent the associated coagulopathy in a primate model.

During severe sepsis there is dramatic activation of both contact proteases and the coagulation pathway. These processes contribute to the development of shock and disseminated intravascular coagulation (DIC) respectively. The Pittsburgh mutant of antitrypsin (358Met-Arg) is a novel protease inhibitor with activity against both thrombin and the contact proteases and should therefore prove beneficial as a therapeutic agent in the management of septic shock. This hypothesis was supported by an earlier study in a pig model where recombinant antitrypsin Pittsburgh (rAT Pittsburgh) at a concentration of 1 microM alleviated some of the features of shock, but did not improve survival. In order to reduce the lethal effects of E. coli sepsis we postulated that a higher concentration of antitrypsin Pittsburgh would be necessary. To test this hypothesis we used rAT Pittsburgh in a primate model. This was chosen in preference to another species as E. coli sepsis in the primate has been well characterised and closely resembles the changes seen in man. Surprisingly this treatment did not alleviate the features of shock and unexpectedly appeared to exacerbate the associated coagulopathy. We propose two possible mechanisms for this unforeseen outcome. The first results from the broad spectrum of activity of antitrypsin Pittsburgh. As well as inhibiting thrombin and the contact proteases, the Pittsburgh mutant also inhibits activated protein C. Inhibition of the protein C system is known to exacerbate septic shock. Secondly, a significant quantity of inactive antitrypsin Pittsburgh, cleaved at the reactive centre, was detected in the plasma of the treated animals. Proteolytically altered serpins, including antitrypsin. have been shown to enhance the inflammatory process. Therefore the accumulation of cleaved rAT Pittsburgh might be expected to exacerbate septic shock.

Comparative study of protease inhibitors on coagulation abnormalities in canine pancreatitis.

Coagulation abnormalities induced by pancreatitis were studied in 36 dogs. The 12 dogs in group I underwent a duodenotomy alone. The six dogs in groups II, III, IV, and V had pancreatitis induced by bile injection (1 cc/kg) into the pancreatic duct. Twenty minutes after bile-induced pancreatitis, group III was given 1.0 mg/kg aprotinin (trasylol), group IV was given 10 mg/kg S-2441, a new synthetic protease inhibitor, and group V was given 0.5 mg/kg alpha 2-antitrypsin by intravenous infusion over 10 min. Blood was drawn for amylase, protime (PT), partial thromboplastin time (PTT), fibrinogen, and platelets, in addition to markers of hypercoagulation, fibrinopeptide A, and antithrombin III, and markers of fibrinolysis, B beta 15-42 immunoreactive peptide (IP), and alpha 2-antiplasmin at baseline, 1/2, 1, 3, 6, 24, 48, and 72 hr after duodenotomy or bile injection. There was no significant difference in PT, platelets, antithrombin III, and fibrinopeptide A among the five groups. With the induction of pancreatitis (group II), serum amylase was significantly elevated but fibrinogen only became elevated at 24 hr and PTT at 48 hr. The increase in B beta 15-42 IP seen 30 min after induction of pancreatitis and the decrease in alpha 2-antiplasmin were blunted by aprotinin, alpha 1-antitrypsin, and S-2441, but inhibition of the rise in amylase and B beta 15-42 IP only reached significance with S-2441 (P less than 0.05). Pancreatitis-induced fibrinolysis was inhibited by S-2441 suggesting that synthetic protease inhibitors may play a therapeutic role in pancreatitis.

Collagenase inhibitors: rationale for their use in treating corneal ulceration.

Tissue collagenases have been implicated in corneal ulceration in human corneal disease and in ulceration of the rabbit cornea that has served as a model system. Such enzymes from the rabbit and human cornea are inhibited by metal-binding agents of the EDTA type, by thiols, and by the human serum antiprotease alpha2-macroglobulin. Determination of the relative efficacies of collagenase inhibitors indicates that EDTA and Ca-EDTA are about one hundred times more effective on a molar basis than L-cysteine and its derivatives, N-acetyl-L-cysteine and D-penicillamine. The alpha2-macroglobulin on a molar basis, is superior as an inhibitor to the metal-binding agents and thiols. Although Ca may be a necessary cofactor of the corneal collagenases, such a requirement has not been established unequivocally. Inhibition and isotope studies do indicate a requirement for Zn. Thiols are thought to inhibit corneal collagenases by binding to or removing an intrinsic metal cofactor (Zn), and/or possibly by reducing one or more disulfide bonds. Inhibition by both EDTA-type agents and thiols is largely reversible by dialysis. The human alpha2-macroglobulin appears to inhibit corneal colleagenases irreversibly by forming tight complexes with them. Ca-EDTA, cysteine, and acetylcysteine, given as eyedrops, are able to prevent or retard ulceration in the alkali-burned rabbit cornea. They appear to have some efficacy in the prevention of corneal ulceration in humans. EDTA-type compounds are quite stable under routine storage, while acetylcysteine is more stable than cysteine. EDTA is quite toxic and should not be used as eye medication. Ca-EDTA has a low toxicity, and cysteine and acetylcysteine have even lower toxicity. It is not yet certain which inhibitor has the most favorable therapeutic index for clinical use, or is the optimal mode of drug delivery known. However, the collagenase inhibitors seem to have therapeutic promise in the prevention of corneal ulceration.