ITMI20011833A1 - AGENTS REGULATING VASCULAR PERMEABILITY - Google Patents

Description

Description of the patent for industrial invention entitled:

"AGENTS THAT REGULATE VASCULAR PERMEABILITY"

The present invention relates to the use of chromogranin A or its fragments for the treatment of pathologies related to excessive vascular permeability ("vascular leakage").

Chromogranin A (CgA) is an acidic protein of 439 amino acids, with a low isoelectric point, characterized by various post-translational modifications, such as glycosylation, sulfation, phosphorylation, and is contained in the secretory granules of many endocrine and neuroendocrine tissues (Winkler, H . et al., Neuroscience 49, 497-528 (1992), and Rosa, P. et al., J. Endocr. Invest. 17, 207-225 (1994)). Elevated levels of circulating CgA have been found in the blood of patients suffering from endocrine and neuroendocrine tumors, renal insufficiency and heart failure.

Its biological function is not yet fully understood. It is believed that it is involved in the packing of hormones inside the secretory granules and that it constitutes a precursor of biologically active peptides with endocrine, paracrine and autocrine functions. It has been shown that the fragments of CgA corresponding to amino acids 1-76 and 1-113, named respectively vasostatin I and Π, are able to inhibit the vascular tension of isolated segments of blood vessels. Furthermore, the fragment corresponding to residues 1-78 can induce adhesion of fibroblasts and smooth muscle cells on solid supports, suggesting a role for CgA in the modulation of cell adhesion.

By investigating the effect of CgA on the vascular epithelium, it was surprisingly found that the protein and the peptides derived from it have a marked effect of reducing vascular permeability induced by substances that cause an alteration of the barrier function of the endothelium, and that they are involved in various pathologies related to increased vascular permeability and edema. It is known that substances such as TNF-α, thrombin, oxidants, bradykinin, histamine and others, in endothelial cells cause modifications at the level of the cytoskeleton and cell adhesion mechanisms. It has now been found that CgA and its fragments are effective in reducing the vascular permeability induced by these active substances on the endothelium, in particular TNF-α and thrombin.

The study was conducted in in vitro and in vivo models. A mouse assay based on the measurement of labeled albumin accumulated in the extra-vascular hepatic compartment following administration of the substance capable of inducing increase in vascular permeability was used as an in vivo model; a study was also performed in a mouse model of TNF-induced lethal inflammatory response. A monolayer of human umbilical vein endothelial cells (HUVECs) in a double chamber permeability assay was used as an in vitro model.

A significant reduction in induced vascular permeability was observed both with the whole CgA protein and with fragments of the same, in particular with the fragments corresponding to residues 1-115, 1-78 and 7-57. Structure-activity studies indicate that region 7-57 contains the active site. The disulfide bridge (Cys-Cys) present in this region does not seem necessary for the interaction with endothelial cells, since the synthetic peptide CgA7-57-SetM, in which the two Cys residues are reduced and alkylated, shows the same activity of the unmodified product. Previous works show that the 47-57 region contains the active site for the adhesion of fibroblasts to solid supports (Ratti et al J. Biol. Chem. 275 (2000), 29257-29263). The importance of region 47-57 for the biological activity of CgA is evidenced by the high degree of homology found between different animal species, compared to other regions of the same molecule.

Several evidences suggest that the mechanism underlying the permeability inhibition effects observed with CgA or with its active fragments, consists in the interaction of these with a component of the endothelial cell membrane and in the subsequent inhibition of the reorganization of the cytoskeleton. Previous observations indicate that agents capable of blocking actin depolymerization also inhibit the permeability induced by other agonists (Lum, H. et al., Am J. Physiol. 267 (1994), 223-241). Without in any way binding the effects found with the use of CgA or its fragments to a particular mechanism of action, it is likely that these molecules exert a protective activity of the vessels by preventing the change in shape and contraction of endothelial cells. , regardless of the particular stimulus capable of inducing the increase in vascular permeability.

Therefore, the object of the invention is the use of the CgA protein or one of its active fragments for the preparation of a medicament useful for the treatment of diseases in which there is an increase in vascular permeability. The term "active fragment" means a portion of the CgA molecule that has an activity of protection of the endothelium against the increase in permeability, the portion containing the amino acid sequence 47-57 of CgA being preferred (sequence numbers in according to Koneki et al. J. Biol. Chem. 262, 17026-17030, 1993), more preferably the sequence 7-57. The preferred fragments are contained in the region comprised between residues 1-115, preferably between residues 1-78 of CgA.

The CgA protein can be prepared by purification starting from a tissue or cell extract, or produced with recombinant DNA techniques in eukaryotic or prokaryotic cells (see Gasparri et al J. Biol. Chem 272, 20835-20843, 1997, and Corti et al. Eur. J. Biochem. 248, 692-699, 1997, incorporated herein by reference; for recombinant DNA techniques, see also Sambrook et al., Molecular Cloning. A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, New York (1982)). The fragments can be prepared by synthesis (see e.g. Stewart and Young, Solid Phase Peptide Synthesis, 2nd ed., Pierce Chemical Co. (1984)), by recombinant DNA techniques or by fragmentation or enzymatic digestion of the natural protein. In addition, the amino acid residues present in the natural protein or its fragments can be modified in order to improve the pharmacokinetic properties of the molecules intended for use in therapy, while keeping their specific biological activity unchanged. For example, single residues can be substituted with other residues of L- or D-amino acids, or they can be chemically modified by glycosylation, binding of groups with variable polarity or lipophilia, amidation or esterification of carboxyl groups, conjugation with other molecules or peptides.

The pathologies that can benefit from treatment with CgA or with its fragments are those in which symptoms or etiopathogenesis present an increased vascular permeability (vascular leakage), i.e. the leakage of liquids, piasmatic proteins and in some cases cells from the blood vessels to the surrounding tissues. Without limiting the invention in any way, among the diseases affected by an increase in vascular permeability, shock, acute and chronic inflammatory diseases, edema and associated tissue damage are cited in particular.

According to a preferred embodiment, chromogranin or fragments derived therefrom are used to reduce the increase in vascular permeability induced by TNF-oc, which is associated with various pathological conditions such as endotoxic shock, adult respiratory distress syndrome (SDRA ), systemic inflammatory response syndrome (SIRS), heart failure, rheumatoid arthritis and multiple sclerosis.

For use in therapy, CgA and its fragments can be formulated with pharmaceutically acceptable carriers and excipients, such as buffers, stabilizers, dissolving agents, preservatives, dispersants, surfactants, disintegrants, lubricants, thickeners, filling agents, dyes and others. The pharmaceutical compositions can be administered orally, intravenously, subcutaneously, intramuscularly, transdermally, inhaled, sublingually, topically. The principles and methods for the preparation of pharmaceutical compositions are known to those skilled in the art and are described for example in Remington: The Science and Practice of Pharmacy, Lippincott, Williams and Wilkins Eds, Dee. 2000.

The dosage of CgA or its fragments will vary according to the specific pathology, its severity and the general condition of the patient. In general, an amount of active ingredient ranging from 0.03 to 3 mg / kg, preferably 0.3 mg / kg, can be administered one or more times a day.

In support of the invention described here, the experimental results and techniques used are shown below.

METHODS

Cell cultures and reagents

HUVEC cells were isolated from human umbilical veins by collagenase treatment as described (39) and cultured in 1% gelatin-coated bottles (Falcon, Becton-Dikinson, Bedford, MA) containing 199 endotoxin-free medium (Sigma, S. Louis, MO), 20% heat-inactive fetal calf serum (Hylcone, Logan, UT), 1% bovine retinal-derived growth factor, 90 pg / mL heparin, 100 IU / mL penicillin, 100 pg / mL streptomycin (Biochrom, Berlin, Germany) (complete medium, CM). All experiments were performed with HUVEC cells in step 2 - 4. Mouse monoclonal anti-CgA mAb 5A8 (IgG1) was described previously (20). Human TNF (5.45 x 107) ± 3.1 units / mg and mouse TNF (1.2 ± 0.14) x 108 units / mg were prepared by recombinant DNA techniques as described (40).

Production of natural CgA and N-terminal fragments of recombinant CgA.

Natural human CgA was purified from pheochromocytoma tissue extracts (heat stable fraction) by immunoaffinity chromatography, essentially as described above (41), using a column bearing anti-CgA mAb 5A8 antibody. The SDS-PAGE of the final product showed two bands of 70 and 60 kDa. Various recombinant and synthetic fragments have been prepared to study the structure-activity relationships of CgA. Each fragment is indicated here with its relative position in the sequence (42). Recombinant CgAj.78 and CgA1-115 were obtained by expression in E. coli (15) and purified by reverse-phase HPLC using a SOURCE 15 RPC column (Pharmacia Upjiohn, Ussala, Sweden) followed by gelfiltration chromatography on Sephacryl S column -200 HR as described above (20). SDS-PAGE of CgA ^ g and CgAM15 showed that both products were homogeneous under reducing and non-reducing conditions. The molecular masses of CgA1-78 and CgA1-115, measured by electrospray mass spectrometry, were 9069.7 and 13247.4 Da, respectively (predicted 9069.3 and 13247.5 Da). The endotoxin content was 0.008 - 0.016 units / pg with Limulus Amoebocyte Lysate (LAL) Pyrotest (Difco Laboratories, Detroit, MI). Peptide 7 - 57 with reduced tanks and alkylated with N-ethylmaleimide (CgA7-57 ^ EtM) was prepared as described (20). The molecular mass of CgA7-57-sEtM was 6038 Da (predicted 6039 Da).

In vitro permeability assay.

The assay was conducted by measuring the flux of radiolabeled albumin through HUVEC monolayers, grown on gelatin-coated membranes in Transwell cell culture chambers (0.4 µm filters, Costar). The HUVECs (5 x 10 4 cells / well, in CM) were cultured for five days in the upper compartment of the Transwell. The culture medium was then replaced with solutions of human TNF (4 ng / ml) or thrombin (2 U / ml), in the absence or in the presence of CgA or the N-terminal fragments, in medium 199 containing 2 mM glutamine. , 100 IU / mL penicillin, 100 pg / mL streptomycin (200 μl / well). After 1 - 2 hours a mixture of 4 pCi / ml of bovine serum albumin labeled with 125I (NEN Boston, MA) and 1.5 mg / ml of human serum albumin (Farina Biagini SpA, Lucca, Italy), were added to the upper compartment (50 pl / well). The lower compartment was then filled with medium 199 containing 2 mM glutamine, 100 IU / ml penicillin, and 100 pg / ml streptomycin (600 / μl / well) and incubated for one hour with gentle shaking. At different times, samples (30 µl) were taken from the lower compartment and their activity measured using a γ-counter (Packard, Sterling, VA). Each experiment was conducted in triplicate and the results are expressed as mean ± SD.

In vivo permeability test.

The effect of mouse TNF on albumin-trypan blue complex permeability in liver vessels was evaluated in anesthetized male Balb / c mice (Charles River Italy, Calco, Italy) as described (14). Mouse TNF (2 ng) in 0.2 ml 0.9% sodium chloride was injected intraperitoneally. Other animals were treated with a mixture of TNF (2 ng) and CgA (1 pg), with or without mAb 5A8 (20 pg). After 30 minutes, the liver of each animal was perfused for 5 minutes with 4 ml of a solution containing 0.4% bovine serum albumin, 0.5% trypan blue, 0.85% sodium chloride via the inferior vena cava. . The perfuse was drained from the portal vein as described (43). Subsequently 5 ml of 0.9% sodium chloride was injected into the inferior vena cava to remove the dye present in the lumen of the vessels. The livers were dissected, homogenized with 3 ml of PBS and centrifuged. The absorbance at 540 nm of the sumatants was then measured using a Pye Unicam SP-550 spectrophotometer (Cambridge, United Kingdom).

Confocal microscopy.

The HUVECs (1 x 10 5 cells in CM) were grown on glass coverslips for 5 days and incubated for two hours with 4 ng / mL TNF alone, or with TNF in combination with CgA1-78 or CgA, in CM. The cells were then washed, fixed with 2% paraformaldehyde for 15 minutes, and stained with fluorescein isothiocyanate-conjugated goat anti-mouse immunoglobulin (Laboratories Zymed, Ine., South San Francisco, CA). The cells were then permeabilized with 0.01% Triton X-100 in PBS and incubated with phalloidin (Sigma) fluorescein tetraisocyanate to stain actin-F. The coverslips were fitted using 50% glycerol in PBS. Microscope analysis was conducted using a Bio-Rad MRC 1000 confocal scanning microscope (Biorad Laboratories, Milan, Italy) and fluorescence images were recorded on Kodak T-Max 100 film using a Focus Imagerecorder Plus (Focus Graphics , Foster City, CA).

TNF cytotoxicity assay.

The cytotoxic activity of TNF in the presence or absence of CgA was evaluated by standard cytolytic assays using mouse L-M fibroblasts (ATCC CCL1.2) as described (44).

RESULTS

CgA and its N-terminal fragments (CgA1-78 and CgA1-115) protect vessels against TNF-induced vascular permeability in mice

To evaluate whether CgA is able to inhibit TNF-induced vascular permeability, the effect of mouse TNF on albumin-trypan blue permeability in liver vessels of BALB / c mice was measured. This assay is based on the measurement of albumin-trypan blue passage within the extavascular compartment of the liver after treatment with TNF, as a measure of vascular permeability to macromolecules (14). Three separate experiments were conducted under different conditions (Fig. 1). As expected, TNF induced marked passage of dye into the liver parenchyma in the absence of inhibitor, while CgA alone had no effect on dye passage (Fig. La). The administration of 0.4 µg or 1 µg of natural CgA significantly inhibited the TNF-induced passage of dye (Fig. La). This effect was specific, given that the co-administration of the anti-CgA monoclonal antibody (mAb 5A8) but not the control antibody (mAb 19E12), partially neutralized the activity of CgA (Fig. La). Furthermore, injection of the recombinant CgA fragment CgAj1-87 or CgA1-115 (3 pg) inhibited the effect of TNF (Fig. 16) indicating that both CgA and its N-terminal fragments can inhibit induced permeability to macromolecules. from TNF in hepatic vessels.

CgA and its N-terminal fragments inhibit TNF-induced permeability in cultured HUVEC monolayers

The mechanism of action was then studied. To understand whether the effect of CgA on TNF-induced vascular permeability was related to the regulation of endothelial function, the activity of natural CgA and human TNF on the flux of radiolabeled albumin through confluent HUVEC monolayers was measured, using the in-chamber permeability assay. Double. Natural CgA (3 pg / mL) did not modify the transendothelial flux of albumin-125I when added alone (Fig. 2 a). Conversely TNF (4 ng / ml) significantly increased the permeability of HUVEC (Fig. 2 b). The effect of TNF was inhibited by CgA in a dose-dependent manner (Fig. 2 b). To evaluate the specificity of these effects, the activity of CgA was measured in the presence of mAb 5A8 anti-CgA. A mixture of 3 pg / mL CgA and 30 μ / mL 5A8 behaved like 0.3 pg / mL CgA (Fig. 2c), indicating that this antibody can block most of the CgA activity. Since mAb 5A8 is directed against the epitope located in the N-terminal domain of CgA (15), these results suggest that the N-terminal region is critical for activity.

To obtain more information on the localization of the active site of CgA, the effects of recombinant CgA1-115, CgA1-718 and the synthetic peptide CgA7-57-SEtM on TNF-induced permeability were measured in vitro. Both recombinant and synthetic fragments inhibited TNF-induced permeability (Fig. 3c and d), suggesting that region 7-57 contains the active site. However, dose-response experiments showed that natural CgA was at least ten times more potent (on molar basis) than recombinant CgA1-78 (Fig. 2b and Fig. 3).

Taken together, these results indicate that the N-terminal domain of CgA contains a site that acts on endothelial cell function in vitro and in vivo. These effects have been observed with HUVEC prepared from several different donors.

CgA does not block TNF-TNF receptor interactions and inhibits thrombin-induced permeability of HUVEC monolayers

It has previously been shown that the interaction of TNF with the TNF-p55 receptor is necessary and sufficient to cause an increase in vascular permeability in vivo and in HUVEC monolayer permeability in vitro (16). Then it was tested whether CgA could bind TNF or TNF-R and consequently inhibit TNF / TNF-R interactions. No binding of human TNF to CgA was observed, immobilized in microtiter plates, by ELISA, using anti-TNF polyclonal IgG as detection reagents (not shown). Furthermore, neither CgA nor CgA1-78 inhibited the cytolytic activity of human TNF (mouse TNF-p55 selective agonist) or mouse TNF (TNF-p55 and TNF-p75 receptor agonist) against mouse L-M cells in an assay. of standard cytotoxicity. Since the cytolytic activity of mouse TNF on these cells is dependent on p55 and p75 receptors (17) these results imply that CgA does not inhibit the binding of TNF to both membrane receptors.

To confirm this hypothesis and to evaluate whether CgA can also inhibit the activity of other inflammatory agents independent of the TNF / TNF receptor system, the effect of natural CgA on thrombin-induced HUVEC permeability was measured. As shown in Fig. 4, 3 pg / ml of CgA inhibited the increase in cell permeability caused by thrombin.

CgA and CgA1-78 inhibit the reorganization of the endothelial cytoskeleton

TNF-α and thrombin are known to promote changes in the size and shape of endothelial cells by rearranging the F-actin cytoskeleton and converting peripheral actin bundles into "stress fibers" (13). Since this phenomenon is believed to be related to increased endothelial permeability to macromolecules, the effect of CgA1-78 and CgA on TNF-induced cytoskeleton rearrangement in HUVEC was investigated by confocal microscopy. As expected, human TNF induced marked actin reorganization (Fig. 5 a and b). Adding 3 μg / ml of CgA1-78 to the cultures inhibited the reorganization of actin, observing peripheral bundles both in the presence and absence of TNF (Fig. 5 c and d). The inhibition was blocked by anti-CgA mAb 5A8, as indicated by the formation of "stress fìbers" in the presence of a mixture of antibody, CgA1-78 and TNF (Fig. 5é). Similarly, 3 μg / ml of CgA inhibited TNF-induced "stress fibers" formation (not shown). These results indicate that CgA1-78 and CgA can inhibit TNF-induced morphological changes associated with disruption of the barrier function of the endothelium.

CgA1-78 increases survival of C57BL6 mice at lethal doses of TNF and galactosamine

In order to verify the ability of CgA1-78 to protect animals from the lethal action of TNF, some C57BL6 mice were treated with 150 ng of mTNF and 36 mg of galactosamine, with or without pre-treatment with CgA1-78. (3 pg). In this model, TNF is able to cause a violent inflammatory response and fulminant hepatitis within 24 hours resulting from damage to the liver tissue. The results, reported in Table 1, indicate that CgA17-8 is able to increase the survival of mice. Animals treated with TNF and galactosamine, but not those pre-treated with CgA1-78, showed signs of distress a few hours after treatment (piloerection, difficulty breathing, numbness, poor reaction to stimuli).

a) Each compound was administered intraperitoneally (100 μΐ / mouse). The animals were treated with CgA ^ g three times: 1 h before mTNF and galactosamine administration, 5 min and 1 h after treatment. Galactosamine (Sigma) was diluted with 0.9% sodium chloride; mTNF and CgAi.78 were diluted with 0.9% sodium chloride containing 100 pg / ml of human serum albumin.

DESCRIPTION OF THE FIGURES

Figure 1. Effect of natural CgA (a) and N-terminal fragments of recombinant CgA (A) on TNF-induced permeability to albumin-biue trypan in mouse liver vessels. The animals were treated with the reagents indicated above each box, 30 minutes prior to liver perfusion with albumin-blue trypan as described in the Methods section. Three separate experiments were conducted. In "Experiment 1" (upper pane, a), and "Experiment 3" (b) each compound was injected sequentially. In "Experiment 2" (lower panel, a) the reagents were premixed and incubated 30 minutes before injection. The following doses of each reagent were injected intraperitoneally into the mouse: TNF (2ng), mAb 5A8 (20µg); mAb 19E12

Figure 2. Effect of CgA on human TNF-induced permeability in cultured HUVEC monolayers. The assay was conducted with untreated (a) or treated (b and c) cells with 4 ng / ml of human TNF. The effect of CgA and TNF was also measured in the presence of anti-CgA mAb 5A8 (30 pg / mL) (c). The Y axis reports albumin-125I present in the lower chamber of the Transwell system after one hour, expressed as% of the control (cells treated with TNF only). The dashed bars represent the basal permeability observed in the absence of TNF; the black bars represent the permeability induced by TNF. (*) PO, 05; (*) PO, 01 (cells treated with TNF vs CgA / TNF mixtures).

Figure 3. Effect of N-terminal CgA fragments on human TNF-induced permeability in cultured HUVEC monolayers. The assay was conducted with cells treated with culture medium (a) or containing medium only

in the absence (o) or in the presence (·) of 4 ng / ml of human TNF. The radioactivity present in the lower chamber of the Transwell systems was measured at the times indicated. Box /: Effect of different doses of CgA1-78 on cell permeability measured after two hours (see Fig. 2 for explanation of bars). Figure 4. Effect of CgA on thrombin-induced permeability of cultured HUVEC monolayers. Untreated cells (A), cells treated with 2 U / ml thrombin (o); cells treated with 2 U / ml of thrombin and 3 pg / ml CgA (·) ·

Claims (8)

CLAIMS 1. CgA or fragments thereof for use as a medicament. 2. CgA or fragments thereof for use according to claim 1 in the treatment of pathologies affected by an increase in vascular permeability. 3. CgA or fragments thereof according to claims 1-2, wherein said fragments contain the sequence 47-57 of the CgA protein. 4. CgA or fragments thereof according to claim 3, wherein said fragments contain the sequence 7-57 of the CgA protein. 5. CgA or fragments thereof according to claims 1-4, in which the pathologies affected by an increase in vascular permeability are acute and chronic inflammatory diseases, edema and associated tissue damage. 6. CgA or fragments thereof according to claims 1-3, wherein said increase in vascular permeability is induced by TNF-a. 7. Pharmaceutical composition containing CgA or its fragments as the active ingredient. Pharmaceutical composition according to claim 7, wherein the CgA fragments are as defined in claims 3 or 4.