CN117659122A - Carbon monoxide release molecule-transmembrane peptide complex and preparation and application thereof - Google Patents

Abstract

The invention relates to a carbon monoxide releasing molecule-penetrating peptide complex CORM 401-R9 (CRs) and its preparation method and application. The adult lens does not have any blood supply, and oral and intravenous drugs are difficult to accumulate here, so the lens provides Medication modalities are limited to topical administration or intraocular injection, with topical administration being the simplest and least invasive route of delivering drugs to the lens. Due to the unique physiology and anatomy of the eye, drug delivery through the cornea is limited, so CORM‑401 has a low bioavailability of less than 5% when given alone. Oligoarginine R9 is a hydrophilic peptide containing 9 positively charged arginine residues and has strong biomembrane penetration ability. This complex breaks through the technical problems in the existing technology with regard to age-related cataract drug delivery obstacles and limited therapeutic effects. Its sequence is (CORM‑401)‑Arg‑Arg‑Arg‑Arg‑Arg‑Arg‑Arg‑Arg‑ Arg; the molecular weight of CORM 401-R9 is 1709kDa. The invention can be used to prepare medicines for preventing and treating age-related cataracts.

Description

A carbon monoxide releasing molecule-penetrating membrane peptide complex and its preparation and application

Technical field

The present invention relates to an ophthalmic drug, specifically to a carbon monoxide releasing molecule-penetrating peptide complex CORM 401-R9 (CRs) and its preparation method and application.

Background technique

Age-related cataract (ARC) is a disease in which the transparency of the lens decreases with age, affecting vision. It is the leading blinding eye disease in the world. Its etiology and pathogenesis are still not fully understood. Oxidative stress-mediated apoptosis of lens epithelial cells (LECs) is closely related to the occurrence and development of ARC. Hydrogen peroxide (H2O2) is the main oxidative substance in the lens and aqueous humor of patients with age-related cataract, and is also detected in the aqueous humor of patients. to significantly higher than normal H2O2 concentrations.

Currently the only feasible way to treat cataracts is surgery. Every year, more than 200,000 cataract patients worldwide undergo lens removal and intraocular lens implantation. However, the surgery has many limitations and potential complications, and also brings a heavy economic burden. Based on the proposed ARC formation mechanism, people have tried to use antioxidants to slow down the occurrence and development of cataracts. To date, compounds with antioxidant and free radical scavenging activities have shown great potential in relevant experimental studies.

CORM-401 is a newly discovered carbon monoxide-releasing molecule that has become the focus of research in recent years and plays an important role in reducing oxidative stress damage. But since the adult lens does not have any blood supply, oral and intravenous drugs are difficult to accumulate here, so the lens drug delivery method is limited to topical drug administration or intraocular injection, where topical drug administration is the simplest and smallest way to deliver drugs to the lens. Invasive pathways. Due to the unique physiology and anatomy of the eye, drug delivery through the cornea is limited, so CORM-401 has a low bioavailability of less than 5% when given alone. Oligoarginine R9 is a hydrophilic peptide containing 9 positively charged arginine residues and has strong biomembrane penetration ability.

Reasons for choosing CORM-401: Although many CORMs containing different types of metal centers or organic molecules are currently known, most CORMs still lack some necessary properties for clinically applied drugs. Ideal CORMs need to meet many properties: 1. Effective therapeutic effect and low toxicity; 2. Appropriate absorption, distribution, metabolism and excretion properties; 3. Good water solubility and stability; 4. Good biocompatibility. CORM-1 is only soluble in organic solvents and releases CO slowly under a cold light source. Its half-life is <1 min and has now been eliminated; CORM-2 (soluble in organic solvents) and CORM-3 (water-soluble) are metal carbonyl groups. compounds, with ruthenium (Ru) as their metal center; CORM-A1 is a water-soluble borane carbonate that spontaneously releases CO in solution in a pH-dependent manner. Despite their different half-lives, these CORMs release only one mole of CO per mole. CORMs of metal carbonyl compounds have more favorable pharmacological effects than CORM-A1, but CORM-2 must release CO under the stimulation of physical and chemical conditions, which is not suitable for the actual environment of the body. The metal ruthenium center of CORM-2 is phased with glycine. The combination results in CORM-3, which has better water solubility, but ruthenium-based CORMs are more toxic than CO gas and CORM-401. In terms of toxicity, manganese has low toxicity and is one of the trace elements necessary for the body. It can constitute enzymes or coenzymes with important physiological functions in organisms (such as Mn-SOD). The water solubility of CORM-401 is greatly increased compared with previous CORMs. The introduction of -CH2CO2H group makes CORM-401 soluble in phosphate buffer saline (PBS), improving its biocompatibility. The ability to generate CO is higher than that of commercially available CORM-3 three times. CORM-401 is a CO-releasing molecule that is sensitive to oxidants. It can accelerate the release of CO under the action of oxidants and reduce the production of ROS induced by H2O2. In terms of stability, CORM-2 and CORM-3 release CO very quickly (<5 minutes), while CORM-401 releases CO continuously (>50 minutes). Regarding the storage stability of CORM stock solution, only CORM-401 remains stable within 7 days of storage. investigated the impact of CORMs on the respiratory chain using high-resolution respiration measurements and extracellular flux techniques. Interference of CORM-2 and CORM-3 in oxygen measurements occurs because even in the absence of cells in the culture medium Rapid oxygen consumption was also detected, while CORM-401 did not interfere with oxygen measurements, producing the most reliable CO-specific results for typical CO-targeted modulation. In terms of therapeutic effect, some studies have found that under conditions of oxidative stress induced by H2O2, CORM-401 is superior to others in protecting intestinal epithelial cells from oxidative stress and apoptosis induced by high concentrations of exogenous H2O2. CO-RMs. The ability of CORM-401 to release multiple CO molecules is therapeutically important because administration of lower doses of the compound is sufficient to deliver pharmacologically relevant amounts of CO to tissues, inducing greater intracellular CO accumulation. And the interaction with biologically relevant oxidants (such as H2O2) increases the CO released by CORM-401. As the concentration of oxidants increases, the rate of CO released by CORM-401 accelerates. In the stored stock solution, CORM-401 releases CO in a reversible reaction, which also explains its stability. Under the action of oxidants, such as H2O2, H2O2 will coordinate with the 16-electron Mn(CO)3(S2CNR2) after CORM-401 (Mn(CO)4(S2CNR2)) loses the first CO, and quickly Its oxidation prevents reverse reaction with CO and may immediately release the remaining 3 COs, thereby achieving targeted modulation of CO.

Reason for choosing R9: Based on physical and chemical properties, membrane-penetrating peptides (CPPs) can be divided into three major categories: cationic, amphipathic and hydrophobic. Cationic membrane-penetrating peptides are the main research objects in current research. They usually contain more than 5 positively charged amino acids. The more basic amino acids they contain, the stronger their penetrating ability. Positively charged agents can interact with negatively charged ocular components, such as epithelial cell membranes and external mucins, to increase drug persistence on the ocular surface and subsequently mediate drug absorption. The arginine residue is highlighted as the “magic residue” that has been shown to internalize efficiently and penetrate the plasma membrane most efficiently. The transduction capacity of oligoarginine peptides increases with the number of consecutive arginine residues and the concentration of the peptide. As the length of the peptide increases, the cytotoxicity of the peptide becomes proportionally more pronounced. Oligoarginine The minimum number of arginine residues required for acid internalization is 9, which represents an optimal outcome between cellular internalization efficacy, low cytotoxicity, and low production cost. In addition, R9 is also a typical hydrophilic peptide, which contains multiple positively charged arginine residues, which allows it to form hydrogen bonds with water molecules and dissolve well in aqueous solutions such as PBS.

Judging from the structural formula of the complex (CORM-401)-R9, the site where CORM-401 releases CO and the arginine residues of R9 that provide penetration ability are distributed at both poles of the molecule, and their combination will not interfere with each other. The ability of CORM-401 to release CO and the ability of R9 to penetrate cells. Secondly, they are covalently bonded. In addition to allowing R9 to more stably carry CORM-401 through the cornea to the target location, the structure is relatively simple, the molecular weight is small, and the preparation is simpler and more economical in cost. Both CORM-401 and R9 are soluble in PBS. The complex is also soluble in PBS solution and has good biocompatibility. However, water-soluble drugs are affected by the lipophilic epithelium during the process of penetrating the cornea, entering the aqueous humor, and contacting the lens. Cells and endothelial cells. Their combination also has the effect of "1+1>2". Although R9 is helpful in penetrating the corneal barrier, it does not have a targeting function itself, while CORM-401 has an oxidation response release mechanism and can be reversibly released in the stored stock solution. CO is released in a reaction manner, and a large amount of CO can be quickly released in response to oxidants, especially H2O2.

Contents of the invention

The present invention is to solve the technical problems in the prior art of drug delivery obstacles and limited therapeutic effects for age-related cataracts, and to provide a carbon monoxide releasing molecule-penetrating peptide complex CORM 401-R9 (CRs) with good antioxidant effect. ) and its preparation methods and applications.

To this end, the present invention provides a carbon monoxide releasing molecule-penetrating peptide complex CORM 401-R9 (CRs), whose sequence is (CORM-401)-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg- Arg; the molecular weight of CORM401-R9 (CRS) is 1709kDa.

The invention has the following beneficial effects:

CORM401-R9 (CRS) provided in the present invention can safely and controllably release and increase the CO content in lens epithelial cells, significantly reduce intracellular ROS accumulation, enhance antioxidant enzyme activity, and reduce oxidative stress damage; at the same time, it can significantly Overcome the challenges of anterior segment drug delivery barriers, including static barriers (corneal epithelium, corneal stroma and blood-aqueous humor barrier), dynamic barriers (conjunctival blood flow, lymphatic flow and tear drainage) and metabolic barriers, penetrating the cornea into the aqueous humor and Lens epithelial cells as drug candidates for topical ocular delivery.

Description of drawings

Figure 1 evaluates the lens epithelial cell penetration and toxicity of CORM-401 combined with different oligoarginines (R5-R12). A live cell analysis method was used to quantitatively analyze the uptake and cytotoxicity of oligoarginine by cells using a fluorescence microplate reader. Figure 1A shows that the lens epithelial cell penetration ability of arginine increases with the increase in the number of arginine residues and drug concentration, and Figure 1B shows that the CORM 401-oligoarginine complex was treated for 3 hours at a concentration of 15 μM. Under the conditions, the survival rate of lens epithelial cells decreases with the increase in the number of arginine residues. Based on the performance of cell penetration ability and toxicity, R9 was selected for drug design.

Figure 2 is a reaction formula for the preparation of the carbon monoxide releasing molecule-penetrating peptide complex CORM 401-R9 (CRs) in the present invention;

Figure 3A, Figure 3B, and Figure 3C are respectively the freeze-dried finished product, mass spectrometry analysis, and HPLC analysis of the carbon monoxide releasing molecule-penetrating peptide complex CORM 401-R9 (CRs) in the present invention.

Figure 4A, Figure 4B, and Figure 4C respectively show the CCK8 experimental results of the carbon monoxide releasing molecule-penetrating peptide complex CORM 401-R9 (CRs) in the present invention. Figure 4A shows that it was observed that CORM 401 and the compound CORM 401-R9 (CRs) alone treated lens epithelial cells for 3 hours with higher cell survival rates, and the drugs had no obvious cytotoxicity; Figures 4B and 4C respectively show CORM under 500μMH2O2 injury treatment The cell survival rate of lens epithelial cells treated with 401 and CORM 401-R9 (CRs) for 3 hours and 24 hours. The drugs can significantly reduce the damage of H2O2 to cells.

Figure 5A and Figure 5B are respectively the reactive oxygen species detection experimental results of the carbon monoxide releasing molecule-penetrating peptide complex CORM 401-R9 (CRs) in the present invention; Figure 5A is the fluorescence microplate reader after 1 hour of 600 μM H2O2 injury stimulation. The reactive oxygen species fluorescence intensity of lens epithelial cells treated with CORM401 and the compound CORM 401-R9 (CRs); Figure 5B is a schematic diagram of the fluorescence intensity observed under a fluorescence microscope under the same stimulation. The drug can significantly reduce the level of intracellular reactive oxygen species under H2O2 treatment, thereby alleviating the oxidative stress state of cells.

Figure 6 is the H2O2 level detection experimental results of the carbon monoxide releasing molecule-penetrating peptide complex CORM 401-R9 (CRs) in the present invention; after 600 μM H2O2 injury stimulation for 2 hours, the CORM 401 and the complex CORM 401 measured by the microplate reader -H2O2 levels in lens epithelial cells treated with R9(CRs). Drugs can significantly reduce intracellular H2O2 levels under H2O2 treatment, and the compound CORM 401-R9 (CRs) in the present invention can reduce H2O2 levels to normal levels.

Figure 7 shows the cell survival rate of lens epithelial cells treated with 600 μH2O2 for 3 hours with different concentrations of CORM 401-R9 (CRs) in the present invention. CORM 401-R9 (CRs) in a certain concentration range can significantly reduce the damage of H2O2 to cells. , among which the protective effect of 15μM-25μM concentration is better and there is no statistical difference between the groups.

The carbon monoxide releasing molecule-penetrating peptide complex CORM 401-R9 (CRs) prepared in the present invention can be used to prevent and treat age-related cataracts.

However, the above are only specific embodiments of the present invention, but they cannot limit the scope of the present invention. Therefore, the replacement of equivalent components, or equivalent changes and modifications made in accordance with the patent protection scope of the present invention, should be It still falls within the scope of the claims of the present invention.

Claims (3)

1. A carbon monoxide release molecule-transmembrane peptide complex CORM401-R9 (CRs) is characterized in that the carbon monoxide release molecule-transmembrane peptide complex is CORM-401 (C) ₈ H ₈ MnNO ₆ S ₂ ) Oligo-arginine R9 (C) ₅₄ H ₁₁₀ N ₃₆ O ₁₀ ) Is prepared by dehydration and carboxylation; the complex sequence is (CORM-401) -Arg-Arg-Arg-Arg-Arg-Arg and has a molecular weight of 1709kDa.

2. The method for preparing carbon monoxide releasing molecule-penetrating peptide complex CORM401-R9 (CRs) as defined in claim 1, wherein the reaction formula is shown in figure 2 of the accompanying drawings, and the synthesis method is Fmoc solid-phase synthesis method, and comprises the following steps:

synthetic raw materials and related reagents:

1) Fmoc-Arg (pbf) -OH, CORM-401 as a protected amino acid raw material

2) Condensation reagent

HBTU，DIEA

3) Solvents and solvents

DMF, DCM, methanol, acetonitrile

4) Resin

Chlorotrityl chloride resin with substitution degree of 1.1mmol/g

5) Deprotection reagent

Piperidine compounds

6) Detection reagent:

phenol reagent, pyridine reagent, ninhydrin reagent

7) Reagent for cutting

TFA, TIS, EDT, anhydrous diethyl ether

8) Nitrogen gas

9) Precision electronic balance

Instrument apparatus:

1) Twelve-channel semiautomatic polypeptide synthesizer

2) High performance liquid chromatograph

3. Freeze dryer

4) Centrifuge with a centrifugal separator

The synthesis process comprises the following steps:

swelling of resin

The chlorotrityl chloride resin was placed in a reaction tube, DMF (15 ml/g) was added, and shaking was performed for 60min.

Second, connect the first amino acid

The solvent was filtered off with suction through a sand core, 3-fold molar excess of Fmoc-Arg (pbf) -OH (C-terminal first amino acid) was added, followed by 10-fold molar excess of DIEA, and finally dissolved by adding DMF and shaking for 30min. Methanol seal head for 30min.

Three, deprotection

DMF was removed, 20% piperidine DMF solution (15 ml/g) was added for 5min, and 20% piperidine DMF solution (15 ml/g) was removed for 15min.

Fourth, detection

Pumping off piperidine solution, taking more than ten resin particles, washing with ethanol for three times, adding ninhydrin, KCN and phenol solution into the resin particles, heating the mixture at 105-110 ℃ for 5min, and turning deep blue into positive reaction.

Fifth, washing

DMF (10 ml/g) was twice, methanol (10 ml/g) was twice, and DMF (10 ml/g) was twice.

Sixth, condensation

3-fold molar excess Fmoc protected amino acid, 3-fold molar excess HBTU, 10-fold molar excess DIEA and finally DMF were added for dissolution and shaking for 45min.

Seventhly, detection

Taking more than ten pieces of resin, washing the resin with ethanol for three times, adding ninhydrin, pyridine and phenol solution into the resin, heating the resin at 105-110 ℃ for 5min, and taking colorless negative reaction.

Eighth step of washing

DMF (10 ml/g) was taken once, methanol (10 ml/g) was taken twice, and DMF (10 ml/g) was taken twice.

Nine. Synthesis of CORM401-R9 (CRs)

Repeating three to eight steps, sequentially connecting amino acids in the sequence from right to left, adding 3 times molar excess CORM-401,3 times molar excess HBTU, adding 10 times molar excess DIEA, adding DMF, dissolving, and oscillating for 45min.

Ten times of pumping

The resin was washed twice with DMF (10 ml/g), three times with DCM (10 ml/g) and four times with methanol (10 ml/g) and drained for 10min.

Eleven cutting

Cleavage (10/g) TFA95%; hydrating 2%; EDT 2%; TIS 1%, cutting time: 180min.

Twelve, blow-drying and washing

Drying the lysate with nitrogen as much as possible, precipitating diethyl ether, centrifuging to remove supernatant, washing precipitate with diethyl ether for six times, and volatilizing at normal temperature.

Thirteenth, purifying and preparing.

1) Taking a small amount of crude product, and dissolving the crude product by H2O/ACN.

2) And taking a small amount of sample, and analyzing and judging the peak time corresponding to the target peak on an HPLC analysis instrument.

3) Preparing a system by using C18 reverse phase chromatography, wherein the wave length is 220nm; flow Rate 15m/min; inj.Vol 20mL Column Temp:25 DEG C

Buffer A0.1% TFAin water; buffer B0.1%TFA in Acetonitrile; the target peak solution was collected.

4) A few target peak solutions were taken with a 1.5ml centrifuge tube for mass spectrometry and purity detection.

Fourteen freeze drying

And freeze-drying the qualified target peak solution to obtain a finished product, as shown in figure 3A.

Fifteen, authentication

A small amount of the final polypeptide was taken and subjected to molecular weight characterization by MS and purity characterization by HPLC analysis, respectively, as shown in fig. 3B and 3C.

Sixteen, packaging and preserving

Sealing and packaging the polypeptide powder, and preserving at-20deg.C.

3. Use of the carbon monoxide releasing molecule-transmembrane peptide complex CORM401-R9 (CRs) according to claim 1 for the preparation of a medicament for the prophylaxis and treatment of age-related cataracts.