COMPOSITIONS INCLUDING ALAMANDINE PEPTIDES AND METHODS FOR TREATMENT OF PULMONARY DISEASE USING ALAMANDINE PEPTIDES

Field of the Invention

The invention is generally related to the field of medicine and pulmonary pathology, particularly to peptide therapeutics for lung pathologies, and most particularly to therapies using alamandine (ALA) peptides (Ala-Arg-Val-Tyr-Ile-His-Pro; SEQ ID NO:l) for treatment of pulmonary disease, such as pulmonary fibrosis.

Background

The essential role of the renin-angiotensin system (RAS) in regulating blood pressure and electrolyte balance is well known (1, 2 Reference List A). Further, involvement of the classical axis of the RAS angiotensin II (AngII)/ATl receptor in the pathogenesis of of pulmonary, renal, and cardiovascular disease has been reported. Research has demonstrated that higher levels of renin and Angll are associated with poor survival (3, reference list A).

Considering that lungs are an important site for the RAS cascade (4, reference list A), it is surprising that RAS has not been more extensively explored in the pathophysiology of pulmonary disease. However, a new challenge has recently stimulated the scienctific community. It was demostrated that Angll could be converted to form a new peptide; alamandine (Ala-Arg-Val-Tyr-Ile-His-Pro; SEQ ID NO:l) (22, reference list B). Alamandine can be formed by action of angiotensin-converting enzyme 2 (ACE2) on the octapeptide angiotensin A or by the decarboxylation of the N-terminal amino acid of Ang-(l-7), Asp, into Ala. The active alamandine peptide has vasodilating, antiproliferative, and anti-fibrotic properties. Thus, binding to MrgD receptor, alamandine probably has an important role antagonizing the actions of Angll/ ATI axis. The schematic illustration of the role of alamandine is shown in FIG. 1 (6, reference list A). Additionally, unlike Angll and Ang-(l-7), which play their effects through interaction with different receptors (ATI and Mas receptors, respectively), alamandine can, but not always, play systemic hypotensive actions and reduces cardiac fibrosis via MrgD (5, reference list A).

Considering these findings, the instant inventors hypothesized that alamandine could be involved in both pulmonary fibrosis and autonomic nervous system modulation. In beginning to explore this hypothesis, the instant inventors collected blood from patients having idiopathic pulmonary fibrosis and demonstrated that plasmatic alamandine was reduced in these patients. Sipriani TS, dos Santos RAS, Rigatto K. The Renin-Angiotensin System: Alamandine is reduced in patients with Idiopathic Pulmonary Fibrosis . J Cardiol Cardiovasc Med. 2019; 4:210-215 (30, reference list B).

Since alamandine has been shown to be decreased in fibrosis, it would be a great advantage to patients having chronic disease, if alamandine could be used to inhibit progression of fibrosis.

Summary of the Invention

The compositions and methods described herein provide this advantage as the peptide alamandine (ALA) offers the potential for promising new treatments for pulmonary disease, particularly pulmonary fibrosis. Further, the experiments described herein show that the increased oxidative stress caused by Angiotensin II can be decreased by alamandine.

Pulmonary fibrosis (PF) is characterized by an accelerated decline in pulmonary function and has limited treatment options. Alamandine (ALA) is a recently described protective peptide of the renin-angiotensin system (RAS) with essential tasks in several conditions. The instant inventors previously demonstrated that ALA is reduced by 365% in the plasma of patients having idiopathic PF and, thus, it is plausible to believe that stimulation of this peptide could represent an important therapeutic target. In this sense, the studies described herein investigate the effects of ALA in an experimental model of PF.

In these studies, bleomycin (BLM) was administrated in Wistar rats and these fibrotic animals were treated with ALA for 14 days. Body weight, histology, respiratory and hemodynamic parameters were analyzed to study the effects of ALA. ALA treatment attenuated the development of fibrosis (PO.OOOl), reduced respiratory system elastance (P<0.0001) and preserved weight gain (P<0.0001) in fibrotic animals without affecting the autonomic control of blood pressure and heart rate.

The data from this study demonstrate the potential of alamandine to alleviate pulmonary fibrosis and improve respiratory system mechanics in vivo.

In a general aspect, the invention provides a new treatment modality for conditions causing fibrosis, including, but not limited to, lung and/or respiratory disease.

In a basic aspect, the invention provides compositions including an antifibrotic peptide.

The antifibrotic peptide can be, but is not limited to, alamandine (ALA) heptapeptide having an amino acid sequence of Ala-Arg-Val-Tyr-Ile-His-Pro (SEQ ID NO:l). These compositions can be used in the treatment of any condition causing fibrosis. The preferred subject for receiving this treatment is a human. But the subject recipient is not limited and can be any animal that would benefit from treatment.

In another basic aspect, the invention provides a composition including the alamandine (ALA) peptide. In an aspect, the invention provides a composition including the alamandine (ALA) heptapeptide having an amino acid sequence of Ala-Arg-Val-Tyr-Ile-His-Pro (SEQ ID NO:l).

In an aspect, the invention provides a composition for treatment of pulmonary disease comprising an alamandine (ALA) peptide.

In an aspect, the invention provides compositions and methods for the treatment of pulmonary disease, particularly, but not limited to, pulmonary fibrosis and/or idiopathic pulmonary fibrosis. The pulmonary disease to be treated is characterized by at least one of inflammation, autonomic nervous system impairment, pulmonary fibrosis, and formation of fibrotic scar tissue in lungs.

In an embodiment, the invention provides a pharmaceutical composition for treatment of pulmonary disease comprising a therapeutically effective dosage of an alamandine (ALA) peptide in a pharmaceutical carrier. The “pharmaceutical carrier” can be any inactive and non toxic agent useful for preparation of medications. The phrase “therapeutically-effective dosage” or “therapeutically-effective amount” refers to the amount of a composition required to achieve the desired function; for example, attenuation of development of pulmonary fibrosis or attenuation or reduction in decline of lung/respiratory function. The pharmaceutical composition can further include a therapeutically effective dosage of an agonist of an MrgD receptor or a therapeutically effective dosage of an antifibrotic agent. The pharmaceutical composition can be formulated for a specific situation of a patient or subject, for example the pharmaceutical composition can be formulated for oral, buccal, nasal, rectal, parenteral, intra-periactivityal, intradermal, transdermal, subcutaneous, or intra-tracheal administration.

In another aspect, the invention provides a method for treating pulmonary disease in a subject in need thereof. The subject can be, but is not limited to, a human or animal that would benefit from the treatment. Generally, the method includes steps for providing a composition including an alamandine (ALA) peptide and administering the composition to the subject. The pulmonary disease can be, but is not limited to, idiopathic pulmonary fibrosis (IPF), asthma, chronic bronchitis, pulmonary hypertension, emphysema, chronic obstructive pulmonary disease (COPD), or COVID-19.

In another aspect of the inventive methods, rather than providing an alamandine (ALA) peptide, an angiotensin-converting enzyme 2 (ACE2) peptide/protein and an angiotensin A peptide/protein are provided in the composition administered. The ACE2 will act on the angiotensin A to form alamandine in situ or in vivo in the subject.

The treatment or treating of pulmonary disease can include, but is not limited to, attenuating development of pulmonary fibrosis, attenuating decline in lung or respiratory function, attenuating pathologic changes in pulmonary architecture or lung tissue structure, decreasing collagen or fibronectin deposition in the lungs or in lung tissue, or improving lung or respiratory function. Some non-limiting parameters of lung and thoracic chamber (rib cage) or respiratory function measured are elastance (E), compliance (C), central airway resistance (R _n ), tissue resistance (G), tissue elastance (H), and resistance in lungs.

In yet another aspect, any of the above-described antifibrotic peptides or combination of peptides, such as alamandine (ALA) heptapeptide having an amino acid sequence of Ala-Arg- Val-Tyr-Ile-His-Pro (SEQ ID NO:l) or a combination of ACE2 and angiotensin A peptides/proteins, can be used in the manufacture of any of the above-described compositions and pharmaceutical compositions for use in any of the above-described methods.

Other objectives and advantages of this invention will become apparent from the following description, wherein are set forth, by way of example, certain embodiments of this invention.

Brief Description of the Drawings

A more complete understanding of the present invention may be obtained by references to the accompanying drawings when considered in conjunction with the subsequent detailed description. The embodiments illustrated in the drawings are intended only to exemplify the invention and should not be construed as limiting the invention to the illustrated embodiments.

FIG. 1 is a schematic illustration of the physiological role of alamandine (ALA) peptide (6, Reference List A).

FIGS. 2A-C show protein expression of ATI and Mas receptors in lung tissue from 12 patients with Idiopathic Pulmonary Fibrosis (IPF). FIG. 2A shows western blots of ATI and Mas receptors in the lung tissue of IPF patients and control subjects. Protein normalization with b-actin. FIG. 2B is a graph showing ATI receptor protein quantification in lung tissue of patients with IPF and controls (p<0.06). FIG. 2C is a graph showing Mas receptor protein quantification in lung tissue of patients with IPF and controls (p<0.046).

FIGS. 3A-D show, in the same patients, a series of dispersion graphs of the correlation between functional data (spirometry values; FEV 1% and FVC%) and the Mas and ATI receptors expression (n=12); FEV1= forced expiratory volume in the first second; FVC= forced vital capacity.

FIGS. 4A-B show, in an experimental model of PF, the protective effect of alamandine on the development of pulmonary fibrosis. FIG. 4A is a graph showing degree of fibrosis and collagen deposition in the lungs (F=33.10) according to Ashcroft modified score. FIG. 4B is a graph showing body weight variation (%) analyzed by two-way analysis of variance (ANOVA) followed by Tukey multiple comparison post-test. FIGS. 5A-D show effect of alamandine on the development of pulmonary fibrosis. These figures show representative images of effects at 2 weeks after alamandine treatment on histological findings and collagen deposition in the lungs. Hematoxylin and eosin (HE) and Masson trichrome (MT) staining; 40x. Tissue changes were analyzed by one-way ANOVA followed by Tukey multiple comparison post-test. All data represent mean±SEM; n=7; P<0.05 was considered statistically significant. *P<0.05; ****P<0.0001. FIG. 5A is CO; FIG. 5B is ALA; FIG. 5C is BLM; and FIG. 5D is BLM+ALA.

FIGS. 6A-C are graphs showing lung mechanics on day 14. FIG. 6 A shows Elastance=elastic rigidity, F=11.28; FIG. 6B shows Compliance=ease of extension, F=20.50; and FIG. 6C shows Resistance=level of constriction (cicatrization process (fibrosis) that makes lung expansion difficult, F=5.31. Control=animals that received only saline; ALA=saline intratracheally and alamandine in the osmotic minipumps; BLM=bleomycin intratracheally and saline in the osmotic minipumps; BLM+ALA=bleomycin intratracheally and alamandine in the osmotic minipumps. One-way ANOVA followed by Tukey multiple comparison test was used. Data represent mean±SEM; n=6-7; PO.05 was considered statistically significant. *p<0.05;

**p<0.01; ***p=0.0001; ****p<0.0001.

FIGS. 7A-C are graphs showing association between Ashcroft Score and pulmonary elastance. FIG. 7A shows BLM and BLM+ALA groups (r=0.7143); FIG. 7B shows BLM group (r=0.8640); and FIG. 7C shows BLM+ALA group (r=0.1347). BLM=bleomycin intratracheally and saline in the osmotic minipumps; BLM+ALA=bleomycin intratracheally and alamandine in the osmotic minipumps. (n=6). *=BLM group °=BLM+ALA group.

FIGS. 8A-B show anti-oxidative effects of alamandine measured in the lung tissue. FIG.

8A is a graph showing glutathione content and FIG. 8B is a graph showing intracellular reactive species production. *P<0.05; ***P=0.0001, ****P<0.0001. #P<0.05 vs ALA. DCF=2,7- diclorodihydrofluorescein; CO=Control; ALA=rats treated with alamandine; BLM= rats with lung fibrosis induced by bleomycin; BLM+ALA= BLM group+alamandine treatment. Data are expressed as mean ± SEM. Oxidative stress results were obtained from the same animals as all experimental data.

FIG. 9 is a graph showing a comparison between advanced oxidation protein products (AOPP) in lung tissue. **P<0.005. CO=Control; ALA=rats treated with alamandine; BLM= rats with lung fibrosis induced by bleomycin; BLM+ALA= BLM group+alamandine treatment. Data are expressed as mean ± SEM.

FIG. 10 is a graph showing the potential of alamandine in total antioxidant capacity in lung tissue. *P<0.05; **P<0.005 and ****P<0.0001. CO=Control; ALA=rats treated with alamandine; BLM= rats with lung fibrosis induced by bleomycin; BLM+ALA= BLM group+alamandine treatment. Data are expressed as mean ± SEM.

FIGS. 11 A-C are graphs showing the effect of alamandine on development of pulmonary fibrosis (F=8.265 for collagen area quantification in the lungs).

FIG. 12 shows representative images of effects (similar effects are shown in FIGS. 5A-D) at two weeks after alamandine treatment, on histological findings and collagen deposition in the lungs. Hematoxylin and eosin (HE) and Masson’s tri chrome (TM) staining. Magnification at 400x. CO=animals that received only saline; ALA=saline intratracheally and alamandine in the osmotic minipumps; BLM=bleomycin intratracheally and saline in the osmotic minipumps; BLM+ALA=bleomycin intratracheally and alamandine in the osmotic minipumps. Arrows: alveolar septa; asterisks: fibrous bands or fibrous masses; arrowhead: inflammatory cells. Tissue changes were analyzed by one-way ANOVA, followed by Tukey multiple comparison post-test. All data represent mean±SEM; n=7-9. PO.05 was considered statistically significant. *P<0.05; ***p<0 ooi; ****P<0.0001.

Detailed Description of the Invention

For the purpose of promoting an understanding of the principles of the invention, reference will now be made to embodiments illustrated herein and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended. Any alterations and further modification in the described compositions, peptides, pharmaceutical compositions, treatments, and methods along with any further application of the principles of the invention as described herein, are contemplated as would normally occur to one skilled in the art to which the invention relates.

The phrase "at least one of," when used with a list of items, means that different combinations of one or more of the listed items can be used, and only one item in the list can be needed. For example, "at least one of: A, B, and C" includes any of the following combinations: A, B, and C; A and B; A and C; B and C; A alone; B alone; and C alone. Any examples provided are non-limited examples.

For the Detailed Description of the Invention: Reference List B is referenced in the Introduction and Example 1; Reference List C is referenced in Example 2; and Reference List D is referenced in Example 3.

Introduction to Experimental Examples

Fibrosis can occur in tissues that are exposed to potentially noxious chemical compounds, such as drugs, and/or biological agents, like viruses. In general, diseases that cause fibrosis have a strong impact on organ function, compromising the patient's quality of life and, frequently, evolving to death. In the case of pulmonary fibrosis, in addition to causing a significant loss of quality of life due to the increase in lung elastance and respiratory failure, the outcome is not different. ^1-6

Currently, the respiratory disease (COVID-19) resulting from infection with new coronavirus (2019-nCoV/SARS-CoV-2) has caused a pandemic and puts the medical and scientific communities on alert. In China, the first country to suffer from the outbreak of COVID-19, despite the fact that many infected patients have had a quick recovery, there were thousands of deaths ^7-9 . The clinical manifestations of patients who survived and those who died are similar ¹⁰ . Respiratory failure can be considered a strong predictor of death, being responsible alone for 53% of deaths in a study with 68 fatal cases of this disease ¹¹ . Severe respiratory discomfort, and organ failure, is also seen in other respiratory diseases such as idiopathic pulmonary fibrosis, asthma, chronic bronchitis, and represents significant increases in morbidity and mortality in these patients.

According to Wan et al. (2020) ¹² , in humans, the pathogenic coronavirus can bind to the angiotensin converting enzyme 2 (ACE2), a specific target in pulmonary, renal, intestinal, and blood vessel epithelial cells. Moreover, it is not new that the ACE2 diverts the route of the renin angiotensin system (RAS) to induce antifibrotic and anti-inflammatory effects ¹³ , which are highly desirable in all diseases that cause fibrosis. Thus, with inactivation of ACE2, there is a reduction in angiotensin (1-7) and alamandine (ALA) formation, the protective arm for lung tissue ^14-23 .

Alamandine (ALA) is a heptapeptide generated in the ACE2 axis which, although recently discovered ²² , is well-established as providing protective action in several tissues ^24-29 . Therefore, it is not a surprise that the ACE2 inactivation, and consequently the reduction of ALA, favor the development of pulmonary fibrosis. This conclusion is confirmed by the instant inventors’ previous experiments showing that, compared to a control group, patients with idiopathic pulmonary fibrosis have ALA plasma concentration three times or approximately four times lower. ³⁰

In this context, it is reasonable to believe that the exogenous administration of alamandine can attenuate the development of pulmonary fibrosis and, consequently, can also reduce the decline in lung function. Therefore, one aim of the instant inventors was to evaluate the protective effect of alamandine on the development of pulmonary fibrosis in an experimental model that is proved substantially to have pulmonary fibrosis.

In rodents, the intratracheal single injection of bleomycin (BLM) is commonly used to provoke pulmonary fibrosis to study its pathogenesis and therapeutic options ^31-35 . Discovered in 1966, BLM is an antibiotic isolated from the fungus Streptomyces verticillus that has chemotherapeutic properties. It leads to pulmonary changes, such as inflammatory and fibrotic reactions, similar to those reported in patients with idiopathic pulmonary fibrosis. ^33-38

Thus, in the absence of an experimental model for COVID-19, the bleomycin-induced pulmonary fibrosis model can be considered an alternative for the pre-clinical respiratory pathology studies. Thus, the instant inventors’ objective is not only to demonstrate that alamandine can prevent the spread of the disease in the lung but also to find new alternatives that might, in the future, treat patients who will suffer for the rest of their lives with the consequences of pulmonary fibrosis due to known or unknown causes.

Example 1: Assessment of Alamandine in Pulmonary Fibrosis

Data resulting from this study shows that the lungs of patients having fibrosis expressed more ATI and less Mas receptors than controls. FIGS. 2A-C and FIGS. 3A-D show association of receptor expression with functional parameters in patients and show that the two axes (Angll/ATl and Ang-(l-7)/alamandine/Mas/MrgD axes) of RAS are unbalanced.

MATERIALS AND METHODS Animals

Male Wistar rats (5 weeks old) were randomly assigned to the experimental groups. They were housed in a room with temperature controlled (25±2°C) in a 12: 12h dark/light circadian rhythm. All rats had access to standard diet and water ad libitum.

The experimental protocol was approved by Ethics Research Committee of the Universidade Federal de Ciencias da Safide de Porto Alegre (protocol 17/207). All procedures of the experiments were in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health ³⁹ .

Bleomycin (BLM)-induced pulmonary fibrosis and alamandine treatment

Animals received saline (0.9%) or bleomycin (BLM; 2.5 mg/kg - Bonar, Ache) intratracheally. In addition, according to the group, all rats were treated subcutaneously with saline (0.9%) or alamandine (50 pg/kg/day - Sigma Aldrich, St. Louis, MO, USA diluted in 0.2mL of saline) using osmotic minipumps (ALZET®, model 2004).

Experimental groups

The rats were randomly allocated (n=7/each): (1) Control: rats received sterile saline intratracheally and subcutaneously. (2) Alamandine (ALA): animals received sterile saline intratracheally and alamandine in the osmotic minipumps. (3) Bleomycin (BLM): animals received bleomycin intratracheally and saline in the osmotic minipumps. (4) Bleomycin and alamandine treatment (BLM+ALA): animals received bleomycin intratracheally and alamandine in the osmotic minipumps.

Protocol description

After being acclimated for one week, rats were anesthetized with ketamine (80 mg/kg) and xylazine (10 mg/kg) by intramuscular injection for BLM or saline intratracheal instillation. At this moment, the Alzet minipump containing saline or alamandine solution was introduced subcutaneously on the animal's back near the withers to deliver 0.25pl/hour (ALA=50pg; day or saline= 0.9%) for 15 days of the respective treatment. The health status and body weight of all animals were monitored daily.

Assessment of respiratory function

Fourteen days after treatment all animals received similar dose of ketamine and xylazine by intramuscular injection to be submitted to the respiratory mechanics and hemodynamic evaluations under anesthesia. The respiratory function was measured by invasive spirometry using a mechanical ventilator for small animals (flexiVent, SCIREQ, Montreal/Canada). Briefly, animals were tracheostomized to introduce a rigid-type cannula (2-mm ID) and positioned on a plane surgical table. The cannula was fixed to the trachea by a silk thread and then connected to the mechanical ventilator. After 5 minutes of animal adaptation, the experimental protocol for pulmonary function was started and parameters were determined: elastance (E), compliance (C), central airway resistance (Rn), tissue resistance (G), tissue elastance (H) and resistance in the lungs (R), as previously described ³³ · ⁴⁰ .

Hemodynamic analyzes

After respiratory function data collection, and still under anesthesia, a polyethylene catheter (PE-50) was inserted into the right carotid to record blood pressure (BP) for 10 minutes (sample rate=2000Hz/channel). The analogical signals were digitalized by a data-acquisition system (Windaq - AT/CODAS, Dataq 143 Instruments Inc., OH, USA). The BP data were analyzed by spectral analysis to assess the sympathovagal balance in the cardiovascular system. At the end of the experiment all animals were euthanized by intramuscular anesthetic overdose (240 mg/kg of ketamine and 30 mg/kg of xylazine) to collect the lungs.

Autonomic evaluation

The spectral analysis of autonomic modulation to the heart was performed observing randomly selected sequences of 200 to 250 beats of HR. The sequence was discarded when it presented non-stationary episodes and a new random selection was performed. ⁴¹ · ⁴² An autoregressive algorithm using the frequency domain analysis of HRV was performed on the PI interval sequences (tachogram). ⁴³ The power spectral density was calculated for each time series. The low frequency (LF, 0.25 to 0.75 Hz) spectral component represents predominantly sympathetic nervous system modulation and the high frequency (HF, 0.75 to 3.00 Hz) represents vagal participation. The components were expressed in absolute (LFa and HFa; ms ² ) and normalized (LFnu and HFnu; %) units. The spectral and symbolic analysis demonstrated the sympathetic and parasympathetic participation in the heart rate modulation. The ratio between the LFa and HFa (LF/HF) was considered the sympathovagal balance. In a reduced variability, linear methodologies have poor applicability (Montano et al., 1994). Therefore, the non-linear approach provides a new perspective to investigate neural control of the cardiovascular system (Casali et al., 2008; Porta et al., 2008). Symbolic analysis is a powerful validated tool (Guzzetti et al., 2005; Porta et al., 2007) that transforms a time series into short, three beat long patterns. The sequences are spread on six levels and all possible patterns are divided into four groups: i) no variations (0V, three symbols equal); ii) one variation (IV, two symbols equal and one different); iii) two like variations (2LV); and iv) two unlike variations (2UV) (Guzzetti et al., 2005).

Lung histonathology

Lung tissue was fixed in 10% phosphate-buffered formalin for 30 minutes at 20 mrrdHO pressure. The organ was immersed in this fixation solution for 72 hours, dehydrated, diaphanized, paraffin-embedded and embedded in paraffin. 5 pm thickness sections were obtained and stained with hematoxylin and eosin (H&E) and Masson's trichrome for histopathological analysis and quantitative analysis of fibrosis. For histopathological scoring of pulmonary fibrosis (modified Ashcroft score), the entire lung sections were reviewed and 20 fields of each blade were assessed using a 400x magnification ⁴⁴ . Each section, starting always from the same locus, was analyzed and a score ranging from 0 (normal lung) to 8 (total fibrosis) was assigned. The mean score of all fields was assumed as the fibrosis score of that lung section. Quantitative analysis of collagen fibers was performed using ImageJ software. 1.46r (RASBAND, 2012). In the chosen areas, the software calculates the average of color percentages by identifying the blue-stained collagen fibers in the tissue.

Statistical analysis

Data were tested for the normal distribution by Shapiro-Wilk test. Parametric data analysis was performed using the one-way or two-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison post-test. When data did not pass in the normality test, the Kruskal-Wallis was used follow by Dunn post hoc test. Correlations were identified through Pearson's correlation test. Data analysis was performed by GraphPad Prism software. Data are presented as mean±SEM and PO.05 was considered statistically significant. RESULTS

As expected, bleomycin (BLM) caused a reduction in body weight gain in the animals BLM groups when compared to the CO and alamandine (ALA) and groups during the protocol (P<0.0001) (FIG. 4B). A protective effect of alamandine was observed when comparing weight variation between groups at the end of the period (P=0.029) (FIG. 4A). All animals survived until the end of induction period (14 days). On the other hand, three animals in the BLM group did not survive after anesthesia on the day of analysis.

As described, H&E staining of lung tissues was performed for observation of histological changes (FIGS. 5A-D). Preservation of parenchyma was observed in the CO group (FIG. 5A), just with occasional infiltration of inflammatory cells. No difference existed between CO and ALA groups (FIGS.5 A and 5B).

As expected, this study showed the development of pulmonary fibrosis in BLM group and no changes on lung architecture was observed in the CO and ALA groups. BLM induced thickness of alveolar septa, destruction of part of bronchi structure, alveolar collapse, proliferation of fibroblast and infiltration of inflammation cells (FIG. 5C). Alamandine showed a significant decrease in Ashcroft fibrosis score when compared with the BLM group (p<0.05) (FIG. 5D).

Lung tissues also were stained by Masson’s tri chrome to evaluate the degree of interstitial fibrosis. Again, as expected, the CO group revealed normal lung tissue structure and slight collagen deposition in the alveolar septa. In contrast, the BLM showed severe collagen deposition, obliteration of interalveolar spaces, and disordered lung structure. The BLM- ALA group reduced the destruction to pulmonary interstitium, demonstrating again the protective role of alamandine. The IOD of the fibrosis area stained into blue reflected the degree of fibrosis and collagen deposition. As shown in Table 1, the IOD in animals that received the alamandine was significantly lower than BLM group.

Legend to Table 1: Spectral and symbolic analysis results. CO=control rats; ALA=rats treated only with alamandine; BLM=rats treated with bleomycin; BLM+ALA=rats treated with bleomycin+alamandine. HR=heart rate; bpm=beats per minute; ABP=average blood pressure. HRV=Heart rate variability; LF=Low and HF=High frequency component; a=absolute and nu=normalized units. A one-way analysis of variance (ANOVA) followed by Tukey multiple comparison post-test was used for ABP and HR evaluation. Kruskal-Wallis and the post hoc Dunn's multiple comparisons test was performed to detect differences in spectral and symbolic analysis. Data represent mean±SEM and a P<0.05 was considered statistically significant. Table 1: Hemodynamic data and Spectral and symbolic analysis results

Spectral Analysis

CO (n=7) ALA (n=7) BLM (n=7) BLM+ALA (n=7) P

ABP (mini I») 75±6 70±3 74±5 77±3 0.81

HR (bpm) 299±16 269±17 251±11 267±10 0.14

HRV (ms ² ) 9.18±2.26 9.58±2.81 9.43±4.28 12.69±6.68 0.90

LFa (ms ² ) 2.12±0.74 3.15±1.38 1.31±0.37 2.12±0.86 0.68

HFa (ms ² ) 5.50± 1.63 4.30±0.98 7.07±4.12 6.03±2.47 0.96

LFnu 0.32±0.09 0.32±0.07 0.28±0.08 0.30±0.07 0.96

HFnu 0.68±0.09 0.68±0.07 0.72±0.08 0.70±0.07 0.96

LF/HF ratio 0.83±0.38 0.62±0.19 0.56±0.21 0.50±0.16 0.97

Symbolic Analysis (%)

0V pattern 0.101±0.006 0.100±0.019 0.111±0.027 O. l l liO.OlO 0.99 IV pattern 0.370±0.010 0.372±0.021 0.361±0.017 0.392±0.010 0.40 2LV pattern 0.102±0.014 0.091±0.012 0.103±0.012 0.075±0.014 0.34 2UV pattern 0.418±0.020 0.434±0.031 0.424±0.035 0.413±0.020 0.94

Respiratory function

In addition, the results of this study showed that alamandine was able to improve respiratory function (FIGS. 7A-C).

Hemodynamic analysis

There was no difference in blood pressure between groups.

DISCUSSION These results demonstrated for the first time the protective effect of alamandine (ALA, a peptide exhibiting antifibrotic action) in attenuating pulmonary fibrosis and preserving the respiratory mechanic in an experimental model of pulmonary fibrosis induced by bleomycin (BLM). The data also showed a positive association between the respiratory mechanics impairment with the degree of fibrosis. In addition, the BLM administration significantly induced weight loss, one of the strongest indicators of BLM-induced injury, which was prevented by ALA treatment. The fibrotic lesions are characterized by activation of fibroblasts, excessive collagen deposition, thickening alveolar walls, and reduced lung compliance ⁴⁸ . Similarly to the findings in the BLM group, these structural changes result in progressive decline in lung function and ultimately respiratory failure ⁴⁹ .

In addition, recent studies also indicate that among the most frequent comorbidities of patients surviving the COVID-19, for example, are cardiovascular diseases and type 1 or type 2 diabetes mellitus ¹⁰ · ⁴⁵ . These data are a matter of concern for physicians and researchers because patients with hypertension and diabetes are generally treated with ACE and/or ATI receptor inhibitors, which results in an upregulation of ACE2 ⁵⁰ . Thus, when connecting to ECA2 for replication, COVID-19 infection could be facilitated, worsening pulmonary fibrosis ⁴⁶ probably also due to the reduction in ALA production.

In this study, it was shown that rats receiving bleomycin developed pulmonary fibrosis and had impaired lung function 2 weeks after its intratracheal instillation. ALA treatment alleviated the degree of fibrosis and subsequent collagen content. Other studies also indicated similar results, which confirm the ALA anti-fibrotic effects in liver ⁵¹ , kidney ⁵² and heart ^{52 54} .

Moreover, ALA has different effects on blood pressure regulation depending on where it is administered and on the pathophysiological condition of the body. It causes an increase in blood pressure and sympathetic participation when injected into the paraventricular nucleus of spontaneously hypertensive rats ⁵⁵ whereas; subcutaneous infusion of Alamandine attenuates hypertension and cardiac hypertrophy in those rats ⁵⁶ . It was also demonstrated by Wang et al (2019) that ALA attenuates cardiac fibrosis induced by long-term hypertension independently of blood pressure ⁵⁴ . The results presented herein also demonstrated that ALA attenuated lung fibrosis but did not change blood pressure, clearly indicating the versatility of ALA effects.

Additionally, these results demonstrated that the impairment in respiratory parameters, seen in the BLM group, was correlated with the degree of fibrosis and collagen deposition in the lung tissue. These findings agreed with the literature ⁵⁷ showing, in patients with IPF, that there is a decline in lung volumes, forced vital and carbon monoxide diffusion capacity. Despite the evidence in the literature about the involvement of RAS in lung fibrosis, there is no scientific paper showing the potential therapeutic action of the counter-regulatory axis of this system represented by ALA. The experiments described herein are the first to demonstrate the importance of ALA in attenuating the development of fibrosis in this model, and (these experiments) point to the possibility of use of it to treat pulmonary fibrosis. Probably, ALA may restore, counteract the Angll effect, and attenuate tissue injury, preventing functional decline.

To date, several studies have investigated the potential of another RAS peptide which antagonize Angll effects, the Ang 1-7 ^{58 60} . On the other hand, there are no reports in the literature investigating the action of ALA on the fibrotic process in the lungs. Collectively, pulmonary fibrosis having several causes, including the COVID-19, are diseases with high rates of mortality and morbidity worldwide that have no efficient treatment. In this study, using a well-established experimental model, the role of ALA to mitigate the damage caused by the fibrotic process was studied. These novel findings provide a strong physiological basis for further explorations of ALA as a therapy for patients.

The promising results from this study demonstrated the potential of ALA to protect respiratory function and attenuate changes in pulmonary architecture following BLM instillation. Knowing the complexity of the pathophysiology of fibrosis and the lack of an effective treatment for this disease, it is important to seek more efficient alternatives to solve the problem of thousands of people. As mentioned before, ALA is a peptide with antifibrotic effects on the liver, kidneys and cardiovascular system, therefore, it is not surprising that it also has this effect on the lungs, one of the main organs in the RAS activation.

Considering the high rate of deaths from respiratory failure ¹¹ , the use of ALA in combination with other antifibrotic agents may bring important and determinant advances in the clinical area. Although this study might represent a significant progress in the direction of increasing survival of patients with lung fibrosis, further research should be conducted to demonstrate the protective effect of ALA.

CONCLUSION

Collectively, these results indicate that much more important than decreasing the plasma concentration of Angll, necessary for hydro electrolytic balance and blood pressure maintenance, is to increase the peptide that counteracts the powerful negative effects of Angll without disturbing normal blood pressure.

Example 2: Oxidative Stress vs. Pulmonary Fibrosis-Protective Role of Alamandine

Reference List C is referenced in this example.

Results from this oxidative stress analysis came from the same methodology of experimental protocols used in other experiments described herein. Part of the lungs from the same animals were used to perform the oxidative stress analysis.

Oxidative stress is characterized by excessive production of reactive oxygen species (ROS) and/or insufficient of antioxidant defenses. This imbalance causes serious tissue damage and it is associated with a worsening of different conditions, such as pulmonary fibrosis (PF). ¹ · ²

The transforming growth factor beta (TGF-b) is the most potent profibrogenic cytokine involved in the mechanism of fibrosis. ^{3 5} This factor contributes to collagen deposition, ROS production and decreases glutathione (GSH) concentration ⁴ · ⁶ · ⁷ , decreasing neutralization of toxic oxidants, such as hydrogen peroxide (H2O2) in the respiratory system. ⁶

Fortunately, data from the literature also demonstrate that, in physiological conditions, the antioxidant defense is efficient to protect the organism against ROS. However, in diseases such as PF, oxidation may be overloaded ⁸ , inducing lesions in alveolar epithelial cells and contributing to PF progression. ⁶ · ⁹ · ¹⁰

It is very well established in the literature that oxidative stress occurs in different tissues of PF patients ² · ¹¹ · ¹² . In this context, the increase in ROS generated by NADPH oxidase (NOX4), a pulmonary constitutive enzyme, and the reduction in glutathione (GSH) and superoxide dismutase (SOD) participation support the concept that there is an imbalance between oxidant agents and the antioxidant capacity in the fibrotic disease.

In addition, regarding the renin-angiotensin system, it is not new that Angiotensin II (Ang II) induces oxidative damage through NOX4 stimulation, one of the main sources of ROS ^{13 14} also in the lung. Consequently, it is important to neutralize the Angll/ATl effects stimulating the counterbalance axis represented by alamandine (ALA) formation. Due to its potential to modulate the redox balance by inhibiting the oxidant action of Ang II, it was demonstrated that ALA reduces the hydrogen peroxide (H2O2) content and NOX4 levels, protecting the liver and the lungs. ^{14 15} Corroborating to this protection, Park et al. (2018) ¹⁶ have shown that ALA increases the expression of SOD, catalase (CAT) and heme oxygenase in ventricular tissue, important antioxidant components.

Therefore, aiming to measure oxidative stress in lung cells, the fluorescent dye, 2 T- dichlorodihydrofluorescein diacetate (DCF) has been used in the described method. For the first time, after 15 days of subcutaneous administration of ALA, it was found that there was a significant increase in the GSH content in the lungs of rats treated with bleomycin (P<0.0001 versus BLM group, FIG. 8A). ALA treatment also provoked a reduction in H2O2 (P<0.0001, FIG. 8B), both effects contributing to the decrease in oxidative stress provoked by bleomycin administration.

Several authors have drawn attention to the importance of increasing GSH content as an indicator of decreased oxidative stress. They also consider that a reduction in oxidative stress could be a promising form of treatment for idiopathic PF. ^{8 10 17}

This rational is based on the fact that the ROS causes cellular damage by several mechanisms such as protein peroxidation ^18-22 . As described by Witko-Sarsat et al. (1996) ²³ , the advanced oxidation protein products (AOPP) reflect tissue damage caused by oxidative stress. Additionally, according to Guilpain et al. (2011) ¹⁹ , compared to healthy controls, AOPP is increased in patients with PF. These authors found that there was a significant linear association between fibroblast proliferation and AOPP in those patients, corroborating the idea that oxidative stress participates in PF development.

In this study, it was found that in BLM rats the AOPP was higher and that ALA treatment reduced protein peroxidation. The BLM+ALA group was similar to the control after 15 days of ALA subcutaneous administration (FIG. 9).

In addition, another parameter that can be assessed is the total antioxidant potential to measure the ability to eliminate azinobis ethyl benzthiazoline sulfonic acid (ABTS) radicals. ^{10 17} As it is demonstrated in FIG.10, bleomycin decreased the total antioxidant capacity, indicating that the reduction in antioxidant defense is also present during development of fibrosis. On the other hand, these results demonstrated that ALA treatment reestablishes this capacity (P=0.03 versus BLM group). There is no significant difference between CO and BLM+ALA groups, while this difference is present between BLM+ALA and BLM group.

Although oxidative stress parameters are not markers of disease severity, they are directly involved in fibrotic pathogenesis. Thus, mitigating this imbalance could be essential to relieve lung injuries, reduce functional impairments and, consequently, improve the quality of life and the prognosis of PF patients.

Although Liu et al. (2021) ¹⁵ have investigated the role of ALA in H2O2 production in PF, the instant inventors are the first to demonstrate that ALA treatment restores the balance between oxidant versus antioxidant defenses in fibrotic lung tissue. In conclusion, they have shown, for the first time, that BLM rats had decreased antioxidant defense, probably due to the reduction in GSH concentrations, and increased H2O2 content and protein peroxidation in the pulmonary tissue. ALA treatment did restore those parameters and the total antioxidant capacity.

Example 3: Assessment of Alamandine (ALA) in Pulmonary Fibrosis and Respiratory Mechanics in Rodents

Reference List D is referenced in this example.

Pulmonary fibrosis (PF) is characterized by excessive extracellular matrix (ECM) deposition and frequently evolves to death. ¹ In PF patients, the elastance of the respiratory system is significantly increased ² , requiring greater respiratory work. Therefore, it is essential to find effective therapeutic strategies that facilitate lung expansion to reduce episodes of respiratory failure.

The participation of renin-angiotensin system (RAS) in PF has been well described. ^{3 5} The angiotensin-converting enzyme 2 (ACE2) has been shown to be an important counter-regulatory axis in several different conditions. ^{6 8} Alamandine (ALA), generated by ACE2 axis, although recently discovered ⁹ , is well-known due to its protech ve action in the cardiovascular system, i.e. vasodilation ⁹ and antifibrotic effects. ¹⁰ In addition, a previous study of the instant inventors showed that patients with idiopathic PF present 365% less ALA in plasma ¹¹ , probably indicates that the exogenous administration of this peptide may attenuate development of PF and, consequently, reduce the decline in lung function.

Therefore, since bleomycin (BLM) is the best characterized PF model ^{12 13} , this study aimed to evaluate the protective effect of ALA on the development of PF in BLM-induced rats.

MATERIALS AND METHODS Animals

All procedures followed the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health. ¹⁴ Ethics Research Committee of the Universidade Federal de Ciencias da Saride de Porto Alegre approved the study (protocol 17/207). Five-weeks old male Wistar rats were housed in a room (25±2°C) on a 12: 12h dark/light circadian rhythm with access to standard diet and water ad libitum.

Pulmonary fibrosis protocol

Rats were anesthetized with ketamine (80mg/kg) and xylazine (lOmg/kg) and BLM (2.5mg/kg, Bonar, Ache) or saline (0.9%) was administered by oropharyngeal aspiration (OA). On the same day, the mini-osmotic pumps (OM; Alzet 2004) containing saline or ALA (Sigma Aldrich, St. Louis, MO, USA) solution was introduced subcutaneously onto the animal’s back to deliver 0.25pl/hour (ALA; 50pg/kg or saline=0.9% a day) for 14 days, respectively. Health status of the rats and body weight were monitored daily.

Groups (N=7/per group): (1) CO: saline by OA and OM. (2) ALA: saline by OA and ALA (50pg/kg/day) in the OM. (3) BLM: BLM (2.5mg/kg) by OA and saline in the OM. (4) BLM+ALA: BLM (2.5mg/kg) by OA and ALA (50pg/kg/day) in the OM.

Histonathology

Lung was inflated and fixed in 10% phosphate-buffered formalin and 5pm thickness sections were stained with hematoxylin and eosin (HE) and Masson’s tri chrome (TM). 20 fields of each slide were examined at a magnification of 400x. The fibrosis classification was according to the modified Ashcroft score ¹⁵ . Quantitative analysis of stained collagen area was performed using Image Pro-Plus® 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA). Assessment of respiratory function

On day 14, animals were anesthetized with ketamine (80mg/kg) and xylazine (lOmg/kg), positioned on a plane surgical table and tracheostomized to introduce a rigid-type cannula (2- mm ID). The cannula was fixed to the trachea by a silk thread and connected to the mechanical ventilator. Respiratory function was analysed using a mechanical ventilator for small animals (FlexiVent, Scireq, Montreal, Canada). ¹⁶ Hemodynamic analyses and autonomic evaluation

After respiratory mechanics data collection, still under anesthesia, a polyethylene catheter (PE-50) was inserted into the right carotid to record arterial blood pressure (ABP) for 10 minutes (sample rate=2000 Hz/channel). The analogical signals were digitalized by a data acquisition system (Windaq-AT/CODAS, Dataq 143 Instruments Inc., OH, USA). The data were analyzed by spectral analysis to assess the sympathovagal balance in the cardiovascular system. ^{17 18} At the end of the experiment, animals were euthanized by intramuscular anaesthetic overdose (240mg/kg of ketamine and 30mg/kg of xylazine) for lungs collection.

Statistical analysis

The normal distribution was tested by the Shapiro-Wilk test. Parametric data analysis was performed using one-way or two-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test. For data with non-normal distribution, the Kruskal-Wallis test was used, followed by the Dunn post hoc test. The associations between data were demonstrated through Pearson’s correlation test. Data analysis was performed using GraphPad Prism 8 software and presented as mean±SEM. P<0.05 was considered statistically significant.

RESULTS

Alamandine attenuates weight loss of pulmonary fibrosis (PF)

From day 6, bleomycin (BLM) group gained less weight onwards when compared to the control (CO) and alamandine (ALA) groups (P<0.01). ALA treatment had a protective effect starting on day 8 (P<0.05). By day 14, PF rats gained significantly less body weight compared to the CO and ALA groups (P<0.0001). However, animals treated with ALA maintained a similar weight to healthy animals (P <0.0001, vs. BLM group; FIG. 4B).

Alamandine attenuates development of pulmonary fibrosis

The lung parenchyma was preserved in the CO and ALA groups (score=0) compared to the BLM group (score=3.6). ALA treatment had a potent effect (FIG. 4 A, FIGS. 11A-C) by attenuating fibrosis (BLM+ALA score=1.3; P<0.0001; F=33.10). Moreover, collagen deposition was confined to the regions around blood vessels and airways in the CO and ALA groups. As expected, there was considerable lungs interstitial collagen deposition in BLM group (P<0.05 vs. CO and ALA). ALA treatment significantly reduced collagen deposition compared to the BLM group (PO.OOl; F=8.285). Cellular and alveolar areas were similar among groups in FIG. 4A, FIGS. 11A-C. Images of lung histology are shown in FIG. 12. Alamandine treatment improves the respiratory mechanics in pulmonary fibrosis

There was significant increase in the dynamic elastance (Edyn) in the BLM group (7.27±1.6 cmThO/ml) compared to the CO (2.13±0.10 cmThO/ml) and ALA (1.92±0.08 cmLhO/ml) groups (FIG. 6A). BLM+ALA group demonstrated a significant attenuation in Edyn (2.22±0.18 cmEEO/ml) compared to the BLM group (PO.OOl; F=11.28; FIG. 6A).

As expected, the dynamic compliance was lower in the BLM group (0.18±0.04 cmFhO/ml) vs. CO (0.48±0.02 cmFhO/ml) and ALA (0.53±0.02 cmFhO/ml) groups. The results from the BLM+ALA (0.47+0.04 cmFhO/ml) demonstrated a protective role of ALA on respiratory mechanics (P0.0001 vs. BLM; F=20.50; FIG. 6B). Moreover, the BLM group showed significantly higher respiratory dynamic resistance (P<0.001 vs. CO; F=8.672; FIG. 6C), indicating the protective effect ALA in the BLM+ALA group (P<0.001) to prevent loss of tissue function.

The correlation between the Ashcroft score and pulmonary elastance is shown in FIGS. 7A-C. There was a strong positive correlation between BLM and BLM+ALA groups (r=0.8452; R ² =0.7143; P=0.0005; FIG. 7A) probably due to the data from the BLM group alone (r=0.9295; R ² =0.8640; P=0.0073; FIG. 7B). Observing the correlation between those parameters in the BLM+ALA group, it is possible to verify that this association was weak (r=0.3670; R ² =0.1347; P=0.4742; FIG. 7C).

Hemodynamic analyses

Table 1 shows that there were no differences among the hemodynamic parameters. The ABP (P=0.8121) and heart rate (P=0.1432) were not different among groups. This result was confirmed by spectral analysis showing that the autonomic nervous system participation in the heart was also not different among groups. The sympathetic (LF) and parasympathetic components (HF) were similar in absolute and normalized units. These results were confirmed by the LF/HF ratio. In addition, the 0V, which represents the sympathetic participation to the heart through symbolic analysis, also indicates that there was no significant difference among groups.

DISCUSSION

The instant inventors have demonstrated for the first time the protective effect of alamandine (ALA) in attenuating pulmonary fibrosis (PF) and preserving respiratory mechanics. In addition, bleomycin (BLM) administration significantly induced weight loss, which was prevented by ALA treatment.

According to American Thoracic Society recommendations, induction by intratracheal BLM in male rats is the one that best mimics the disease in humans and is the most suitable for initial preclinical tests. ¹⁹ Kilic et al. (2014) ²⁰ showed that 2.5 mg/kg of BLM in Wistar rats causes histological changes compatible with PF. The inventive study also demonstrated that this dose is sufficient to study the effects of antifibrotic substances without causing lethal damage to animals. In addition, George, Wells and Jenkins (2020) ²¹ describes that studies for antifibrotic therapies using the BLM animal model of PF can be beneficial also to COVID-19. Even in patients recovered from COVID-19, the vims elimination does not preclude the development of progressive fibrosis. Thus, the promising results of ALA obtained in this study might encourage investigations of preventive fibrosis therapies after SARS-CoV-2 infection.

These findings agree with the literature showing that PF leads to a decrease in body weight ²² , a strong indication of health in animals. ²² While ALA treatment prevented the loss in body weight, the higher energy consumption to respiratory work probably explain this decrease in BLM group. This is true also for humans because treatment with nintedanib or pirfenidone normally provokes weight loss in patients ²³ , contributing to their poor prognosis. ²⁴

Furthermore, the antifibrotic effects of ALA have already been described in the cardiovascular system ²⁵ and, more recently, in the liver. ²⁶ Although there are strong indications of the protective effects of ALA, there are no reports in the literature investigating its action on the fibrotic process in the lung. To date, there are only studies suggesting the protective role of to ACE2 and angiotensin-(l-7) in PF ²⁷ and COVID-19. ⁸ · ²⁸ In this study, ALA alleviated the lung degree of fibrosis and collagen deposition. It is established in the literature that collagen is the primary determinant of overall lung tissue elasticity ²⁹ , therefore commits to functional capacity ³⁰ in the same proportion as the degree of fibrosis. ³¹ Thus, if ALA prevent the fibrosis, it is possible that ALA can also act by improving respiratory mechanics.

Furthermore, the inventive findings show that the degree of fibrosis was positively correlated with respiratory system elastance and that ALA treatment reduced this correlation. It is well-established that fibrosis increases the elastic recoil forces of the lung and therefore reduces lung compliance. Moreover, excess ECM alters ventilation/perfusion ratios in the lung, causing hypoxemia both at rest and with effort. ³² Consequently, these results indicate that treatment with ALA might overcome these mechanical changes and could be effective in reducing respiratory work also in IPF patients. Despite evidence in the literature regarding the involvement of the RAS in PF ³³ , this is the first study which demonstrates the protective effect of ALA in lungs, extending the knowledge about the potential of the ACE2 axis. ³⁴ In this sense, this data point to the possibility of using ALA to treat PF of varying etiology, mainly when involving ACE2 participation. As proposed by Wu (2020) ⁸ , the findings of the instant inventors indicate that the compensation of ACE2 function, with ALA administration, could be a promising alternative to treat the severe respiratory damage provoked by PF, as found in COVID-19. ²¹ · ²⁸ Studies have also indicated that ALA has different effects on arterial blood pressure (ABP) regulation, depending on where it is administered and/or the pathophysiological condition. It causes an increase in ABP and sympathetic participation when injected into the paraventricular nucleus of spontaneously hypertensive rats ³⁵ , whereas subcutaneous infusion of ALA attenuates hypertension. ³⁶ It was also demonstrated by Wang et al. (2019) ³⁷ that ALA attenuates cardiac fibrosis caused by long-term hypertension independently of ABP. Similar to the report by Wang et al. in 2019 ³⁷ , results of the instant inventors demonstrate that ALA attenuated lung fibrosis but did not change ABP, clearly indicating the versatility of ALA’s effects and confirming RAS pleiotropism.

As this disclosure demonstrates, alamandine (ALA) can represent an important strategy to improve idiopathic pulmonary fibrosis (IPF) patient quality of life. This histological and functional inventive study supports significant progress and may encourage further investigation into the mechanisms of ALA in PF.

All patents and publications mentioned in this specification are indicative of the levels of those skilled in the art to which the invention pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual publication were specifically and individually indicated to be incorporated by reference. It is to be understood that while a certain form of the invention is illustrated, it is not intended to be limited to the specific form or arrangement herein described and shown. It will be apparent to those skilled in the art that various changes may be made without departing from the scope of the invention and the invention is not to be considered limited to what is shown and described in the specification.

One skilled in the art will readily appreciate that the present invention is well adapted to carry out the objectives and obtain the ends and advantages mentioned, as well as those inherent therein. The compositions, peptides, pharmaceutical compositions, treatments, and methods described herein are presently representative of the preferred embodiments, are intended to be exemplary, and are not intended as limitations on the scope. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention. Although the invention has been described in connection with specific, preferred embodiments, it should be understood that the invention as ultimately claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the art are intended to be within the scope of the invention. References

Patents

1. WO 2016/161243 Al; Diapin Therapeutics LLC

2. WO 2018/204385 Al; Beth Israel Deaconess Medical Center, Inc.

3. US 2015/0313829 Al; Frezard et al.

4. US 9,974,825 B2; Souza dos Santos et al.

5. EP 2264048 A2; Universidade Federal de Minas Gerais

6. BR 102012 033605-7 A2; Universidade Federal de Minas Gerais

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