CN117603299A - Complexes and prodrugs containing tetravalent platinum and preparation methods and applications thereof - Google Patents

Abstract

The present disclosure relates to the field of biomedicine technology, specifically, to a complex containing tetravalent platinum, a prodrug and a preparation method and application thereof. The complexes of the present disclosure comprise a tetravalent platinum moiety and an oligopeptide as an axial ligand chemically linked to the tetravalent platinum moiety, the oligopeptide being a dipeptide Xaa1-Xaa4, a tripeptide Xaa1-Xaa2-Xaa4, or a tetrapeptide Xaa1-Xaa2-Xaa3-Xaa4, where Xaa1, Xaa2 and Xaa3 each represent any natural amino acid, and Xaa4 represents tryptophan, phenylalanine, histidine, tyrosine or arginine.

Description

Complex containing tetravalent platinum, prodrug and preparation method and application thereof

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to Chinese Patent Application No. 202211005108.0 and No. 202211006334.0 filed on August 22, 2022, the disclosures of which are incorporated herein by reference in their entirety for all purposes.

Technical Field

The present disclosure relates to the field of biomedicine technology, and in particular to a tetravalent platinum-containing complex, a prodrug assembled from the precursor, and a preparation method and application of the complex and the prodrug, such as a peptide-based platinum self-assembled nanoprodrug and a preparation method and application thereof.

Background Art

Divalent platinum drugs (such as cisplatin, carboplatin, and oxaliplatin) have strong broad-spectrum anticancer activity, mainly through destroying the DNA of tumor cells, inhibiting replication, and inducing apoptosis. They have been widely used in the clinical treatment of various malignant tumors. However, this type of drug has the physical and chemical characteristics of low solubility and poor stability, and lacks selectivity for tumor tissues. It is very easy to cause serious side effects such as nephrotoxicity, neurotoxicity, and bone marrow suppression, and is affected by innate and acquired drug resistance, which seriously limits its clinical efficacy and application.

In recent years, drug precursor technology has provided new solutions to improve the physical and chemical properties of platinum drugs and reduce toxic side effects. Compared with the divalent platinum complex Pt(II), the tetravalent platinum precursor complex Pt(IV) formed by adding two axial ligands has lower reactivity and toxic side effects. After entering tumor cells, it can shed the axial ligand under the action of intracellular reducing mediators such as ascorbic acid, methionine, and glutathione and transform into a cytotoxic Pt(II) structure, thereby producing a cell-killing effect. However, platinum small molecule precursor drugs also have limitations in the human body, such as rapid metabolism, poor accumulation in lesion sites, and easy development of drug resistance.

Nano drug delivery systems constructed based on various carrier materials show great potential in the application value and development prospects in achieving effective in vivo transport of chemotherapeutic drugs. They can carry drugs to overcome physiological and pathological barriers, achieve effective enrichment of tumor tissues and cells, and complete continuous, controllable and targeted delivery, thereby achieving the purpose of enhancing therapeutic effects and reducing adverse reactions. However, platinum small molecules are metal drugs that are poorly soluble in both oil and water, have weak affinity for most drug carriers, and have limited effective loading capacity. Therefore, it is necessary to structurally modify existing platinum drugs and develop multifunctional platinum prodrugs with one or more of the following: high anti-tumor activity, low toxicity and side effects, strong selectivity for tumor tissues, and controllable release, so as to improve the clinical application potential of such drugs.

Summary of the invention

In a first aspect of the present disclosure, a complex is provided, comprising a tetravalent platinum portion and an oligopeptide as an axial ligand chemically linked to the tetravalent platinum portion, wherein the oligopeptide may be a dipeptide Xaa1-Xaa4, a tripeptide Xaa1-Xaa2-Xaa4 or a tetrapeptide Xaa1-Xaa2-Xaa3-Xaa4, wherein Xaa1, Xaa2 and Xaa3 each represent any natural amino acid, and Xaa4 represents tryptophan, phenylalanine, histidine, tyrosine or arginine.

In some embodiments, the tetravalent platinum portion in the complex is represented by the following structural formula (a):

Wherein R ₁ , R ₂ , R ₃ , R ₄ and R ₅ can be independently selected from: Cl, Br, I, OH, NH ₃ ; R ₁ and R ₂ can form a ring with Pt, R ₃ and R ₄ can form a ring with Pt, and when forming a ring, R ₁ and R ₂ or R ₃ and R ₄ can be selected in combination from

In some embodiments, the tetravalent platinum moiety in the complex is represented by the following structural formulas (a-1) to (a-6):

In some embodiments, Xaa1 can represent glutamic acid, aspartic acid, tryptophan, proline, tyrosine or phenylalanine. In other embodiments, Xaa1 in a dipeptide can represent glutamic acid, aspartic acid, tryptophan or tyrosine. In still other embodiments, Xaa1 in a tripeptide can represent aspartic acid, tryptophan, proline, phenylalanine or glutamic acid. In still other embodiments, Xaa1 in a tetrapeptide can represent glutamic acid, tryptophan or aspartic acid.

In some embodiments, Xaa2 can represent tryptophan, glycine, alanine, valine, leucine, phenylalanine, proline, serine, threonine, cysteine, tyrosine, methionine, asparagine, glutamine, aspartic acid, glutamic acid, histidine or arginine. In other embodiments, Xaa2 in the tripeptide can represent tryptophan, glycine, alanine, valine, leucine, phenylalanine, proline, serine, threonine, cysteine, tyrosine, methionine, asparagine, glutamine, aspartic acid, glutamic acid or histidine. In yet other embodiments, Xaa2 in the tetrapeptide can represent tryptophan, glycine, valine, leucine, glutamic acid, phenylalanine, proline, aspartic acid, threonine, histidine, tyrosine or arginine.

In some embodiments, Xaa3 can represent tryptophan, glycine, leucine, phenylalanine, proline, histidine, aspartic acid, lysine, tyrosine or arginine. In some other embodiments, two or three of Xaa2, Xaa3 and Xaa4 in the tetrapeptide are the same. In some other embodiments, Xaa2 or Xaa3, when present, is the same as Xaa4. In this embodiment, for the tripeptide, Xaa2 and Xaa4 can both be tryptophan, and for the tetrapeptide, Xaa3 and Xaa4 can both be tryptophan.

In some embodiments, the tetravalent platinum moiety is chemically linked to any one of Xaa1 to Xaa3, for example, to Xaa1, Xaa2 or Xaa3, preferably, the tetravalent platinum moiety is chemically linked to Xaa1. In other embodiments, the oligopeptide is represented by Xaa1-Xaa2-Xaa4, wherein Xaa1 may represent aspartic acid, tryptophan, proline, phenylalanine or glutamic acid, Xaa2 represents histidine, phenylalanine, tryptophan or tyrosine, and Xaa4 represents tryptophan. In yet other embodiments, the oligopeptide is represented by Xaa1-Xaa2-Xaa4, Xaa1 may represent aspartic acid, tryptophan, proline, phenylalanine or glutamic acid, and Xaa2 and Xaa4 both represent tryptophan.

In some embodiments, the oligopeptide in the complex can be selected from the compounds shown in Table 1 below:

Table 1

In some embodiments, the complex is represented by formula (I):

Wherein, R ₁ , R ₂ , R ₃ , R ₄ and R ₅ can be independently selected from: Cl, Br, I, OH, NH ₃ ; R ₁ and R ₂ can form a ring with Pt, R ₃ and R ₄ can form a ring with Pt, and when forming a ring, R ₁ and R ₂ or R ₃ and R ₄ can be selected in combination from

In a more specific embodiment, the complex can be selected from any of the following:

In this embodiment, the complex can be selected from any of the following:

In a second aspect of the present disclosure, a peptide-based platinum prodrug is provided, comprising a tetravalent platinum complex having an axial ligand containing a tryptophan dipeptide. This embodiment provides a peptide-based platinum self-assembling nanoprodrug, characterized in that the axial ligand contains a tetravalent platinum complex containing a tryptophan dipeptide.

In some embodiments, the structure of the self-assembled nanoprodrug is shown in Formula I:

Wherein, R ₁ , R ₂ , R ₃ , R ₄ and R ₅ can be independently selected from: Cl, Br, I, OH, NH ₃ ; R ₁ and R ₂ can form a ring with Pt, R ₃ and R ₄ can form a ring with Pt, and when forming a ring, R ₁ and R ₂ or R ₃ and R ₄ can be selected in combination from

In other embodiments, the self-assembled nanoprodrug has a structure shown in any one of Formulas II-XIII;

In some embodiments, the prodrug can self-assemble into a nanostructure with a particle size of 10-1000 nm, preferably 20-200 nm, in an aqueous medium.

In some embodiments, the pH of the aqueous medium in which the prodrug is capable of self-assembly is 7.0-7.8.

In some embodiments, the aqueous medium in which the prodrug can self-assemble is preferably purified water, water for injection, 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) buffer, tris (hydroxymethylaminomethane) (Tris) buffer or PBS buffer.

In other embodiments, the preparation method of the peptide-based platinum prodrug comprises the following steps: oxidizing the divalent platinum complex Pt(II) with chlorosuccinimide to a monochloromonohydroxy tetravalent platinum complex Pt(IV), and then condensing it with an oligopeptide under the catalysis of benzotriazoletetramethyltetrafluoroboric acid (TBTU) and triethylamine to obtain the peptide-based platinum prodrug.

In some embodiments, the method for preparing a self-assembled nano-prodrug includes: dissolving a peptide-based platinum prodrug in a first solvent to obtain an organic phase A; mixing the organic phase A with an aqueous phase B to obtain a mixed solution C; and removing the first solvent from the mixed solution C to obtain a monodisperse nano-prodrug.

In some embodiments, the first solvent is one or a mixture of at least two of acetone, ethanol, methanol, tetrahydrofuran, acetonitrile, dimethyl sulfoxide and N,N-dimethylformamide; preferably, the first solvent is acetone and/or ethanol; aqueous phase B is purified water, water for injection, 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) buffer, tris(hydroxymethylaminomethane) (Tris) buffer or PBS buffer; preferably, the aqueous phase B is water for injection or HEPES buffer; preferably, the pH value of the aqueous phase B is 7.0-7.8.

In some embodiments, the volume ratio of aqueous phase B and organic phase A when mixed is 1-100: 1, preferably 1-40: 1. In other embodiments, the method for removing the solvent from the mixed solution C is reduced pressure evaporation, high-speed centrifugation, dialysis or ultrafiltration; preferably, the reduced pressure evaporation method is used to remove the solvent from the mixed solution C.

In some embodiments, the use of peptide-based platinum self-assembled nano-prodrugs in the preparation of anti-tumor drugs is provided. The anti-tumor drugs can be drugs for lung cancer, breast cancer, gastric cancer, esophageal cancer, adrenocortical carcinoma, skin squamous cell carcinoma, head and neck cancer, thyroid cancer, liver cancer, pancreatic cancer, bile duct cancer, colorectal cancer, ovarian cancer, cervical cancer, endometrial cancer, vaginal squamous cell carcinoma, testicular cancer, prostate cancer, bladder cancer, urothelial carcinoma, melanoma, osteosarcoma, malignant lymphoma, neuroblastoma, cisplatin-acquired lung cancer, cisplatin-acquired resistant ovarian cancer, cisplatin-acquired resistant breast cancer, cisplatin-acquired resistant cervical cancer, cisplatin-acquired resistant gastric cancer, and cisplatin-acquired resistant colorectal cancer.

In a third aspect of the present disclosure, a self-assembly is provided, which is self-assembled from the complex described above or the peptide-based platinum prodrug described above. In some embodiments, the self-assembly is self-assembled from the complex described above or the peptide-based platinum prodrug described above in an aqueous medium. In other embodiments, the self-assembly has a particle size of 10 nm to 10000 nm. In yet other embodiments, the self-assembly has a particle size of 20 nm to 200 nm.

In a fourth aspect of the present disclosure, a method for preparing the complex described above is provided, comprising: oxidizing divalent platinum Pt(II) to hydroxy tetravalent platinum Pt(IV), and then condensing with an oligopeptide in the presence of a catalyst to obtain the complex. In some embodiments, the oxidant may be chlorosuccinimide. In other embodiments, the catalyst may be TBTU, triethylamine, or a combination thereof.

In a fifth aspect of the present disclosure, a method for preparing the self-assembly described above is provided, comprising dissolving the complex described above in a polar solvent to obtain an organic phase; mixing the organic phase with an aqueous phase to obtain a solution; and removing the polar solvent from the solution to obtain the self-assembly.

In some embodiments, the polar solvent can be one or a mixture of at least two of acetone, ethanol, methanol, tetrahydrofuran, acetonitrile, dimethyl sulfoxide and N,N-dimethylformamide. In a preferred embodiment, the polar solvent is acetone and/or ethanol. In some embodiments, the aqueous phase can be a combination of one or more of purified water, water for injection, HEPES buffer, Tris buffer or PBS buffer. In a preferred embodiment, the aqueous phase is water for injection or HEPES buffer. In some embodiments, the pH value of the aqueous phase is 7.0-7.8.

In some embodiments, the volume ratio of the aqueous phase to the organic phase is 1-100: 1. In other embodiments, the volume ratio of the aqueous phase to the organic phase is 1-40: 1. In some embodiments, the method for removing the solvent in the solution is reduced pressure evaporation, high-speed centrifugation, dialysis or ultrafiltration. In other embodiments, the method for removing the solvent in the solution is reduced pressure evaporation.

In a sixth aspect of the present disclosure, some embodiments provide a pharmaceutical composition comprising the complex, peptidyl platinum prodrug or self-assembly described above. In other embodiments, the pharmaceutical composition comprises a therapeutically effective amount of the complex, peptidyl platinum prodrug or self-assembly described above. In yet other embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

In the seventh aspect of the present disclosure, some embodiments provide the above-described complex, peptide-based platinum prodrug or self-assembly for treating tumors. Other embodiments provide the use of the above-described complex, peptide-based platinum prodrug or self-assembly in the preparation of anti-tumor drugs. Still other embodiments provide a method for treating tumors, the method comprising administering a therapeutically effective amount of the above-described complex, peptide-based platinum prodrug or self-assembly to a subject in need thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings are included to provide a further understanding of the present disclosure and are incorporated in and constitute a part of the present disclosure, and thus the accompanying drawings together with the related description serve to explain the concepts of the present disclosure. In the drawings:

FIG1 is a transmission electron microscope morphology photograph of the nanoprodrug prepared in Example 5;

FIG2 is a particle size distribution diagram of the nanoprodrug prepared in Example 5;

FIG3 is a particle size distribution diagram of the nanoprodrug prepared in Example 6;

FIG4 is a particle size distribution diagram of the nanoprodrug prepared in Example 7;

FIG5 is a release curve of the active ingredient of the nano prodrug prepared in Example 5;

FIG6 is a graph showing the particle size distribution of the nanoprodrug prepared in Example 5;

FIG7 is an in vitro toxicity curve of the nanoprodrug and cisplatin prepared in Example 5 based on human ovarian cancer (SKOV3);

FIG8 is an in vitro toxicity curve of the nano-prodrug and cisplatin prepared in Example 5 based on a cisplatin-resistant human ovarian cancer strain (SKOV3/DDP);

FIG9 is an in vivo antitumor efficacy evaluation of the nano-prodrug and cisplatin prepared in Example 5;

FIG10 is an in vivo antitumor efficacy evaluation of the nanoprodrug and cisplatin prepared in Example 5;

FIG11 is a pharmacokinetic curve of the nanoprodrug and cisplatin prepared in Example 5;

FIG12 shows the tissue distribution of the nanoprodrug and cisplatin prepared in Example 5;

FIG13 is a diagram showing the body weight changes of the in vivo safety evaluation of the nanoprodrugs prepared in Example 5;

FIG. 14 is H&E staining of tissue sections for in vivo safety evaluation of the nanoprodrug prepared in Example 5.

DETAILED DESCRIPTION

Exemplary embodiments will now be described more fully below; however, they may be implemented in different forms and should not be construed as limited to the manner shown herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the exemplary embodiments to those skilled in the art.

In addition, in this document, the technical features described in relation to a certain embodiment may be applied to another embodiment, or may be combined with the technical features of another embodiment, as long as such application or combination does not violate the teachings given in this document.

The term "prodrug" is intended to mean a substance that can be converted into a biologically active agent under physiological conditions or by solvolysis. A prodrug may be inactive when administered to a patient in need thereof, but is converted into an active agent in vivo. Prodrugs are generally rapidly converted in vivo, for example by hydrolysis in the blood.

The term "subject" can include humans and domestic animals such as laboratory animals and household pets (eg, cats, dogs, pigs, cows, sheep, goats, horses, rabbits), and non-domesticated animals such as wild animals and the like.

As used herein, a "therapeutically effective amount" refers to an amount that, when administered to a mammal, preferably a human, is sufficient to achieve treatment of a tumor in the mammal as defined below. What constitutes a "therapeutically effective amount" varies depending on the substance being administered, the disease state and its severity, the mode of administration, and the age of the mammal to be treated, but can be routinely determined by a person of ordinary skill in the art based on his or her own knowledge and the present disclosure.

As used herein, "pharmaceutically acceptable excipients" refer to additives other than the main drug in pharmaceutical preparations that are approved by the national drug administration agency for use in humans or livestock, and may also be called excipients. For example, binders, fillers, disintegrants, lubricants in tablets; matrix parts in semisolid preparations; solvents, preservatives, antioxidants, flavoring agents, aromatics, cosolvents, emulsifiers, solubilizers, osmotic pressure regulators, colorants, etc. in liquid preparations.

As used herein, "treating" or "treatment" encompasses the treatment of a tumor in a mammal, preferably a human, having a tumor, and includes: (i) preventing the occurrence of a tumor in a mammal, especially when such mammal is susceptible to a tumor but has not yet been diagnosed with a tumor; (ii) inhibiting a tumor, i.e., arresting its development; (iii) remitting a tumor, i.e., causing regression of a tumor; or (iv) alleviating symptoms caused by a tumor.

In this article, complex, peptide-based platinum prodrug, peptide-based platinum self-assembled nanoprodrug, nano self-assembled prodrug can be used interchangeably unless explicitly stated. For example, the explicitly stated "tetravalent platinum complex" means the tetravalent platinum part represented by the following structural formula (a), and thus cannot be interchanged with peptide-based platinum prodrugs, etc., because the two refer to different substances.

Herein, self-assembly, nanoprodrug, self-assembled nanoprodrug, nanoprodrug and nano self-assembled prodrug can be used interchangeably in certain contexts as long as they refer to the same substance.

In this article, natural amino acid refers to 20 kinds of amino acids existing in nature, including alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine. Unless explicitly stated, when referring to these amino acids, L-type and D-type may be included.

The present disclosure provides a complex comprising a tetravalent platinum moiety represented by the following structural formula (a) and an oligopeptide as an axial ligand chemically linked to the tetravalent platinum moiety,

Wherein R ₁ , R ₂ , R ₃ , R ₄ and R ₅ can be independently selected from: Cl, Br, I, OH, NH ₃ ; R ₁ and R ₂ can form a ring with Pt, R ₃ and R ₄ can form a ring with Pt, and when forming a ring, R ₁ and R ₂ or R ₃ and R ₄ can be selected in combination from and Here, those skilled in the art can reasonably select R ₁ , R ₂ , R ₃ , R ₄ and R ₅ according to the coordination principle of tetravalent platinum.

For example, R ₁ , R ₂ , R ₃ , R ₄ and R ₅ may be independently Cl, Br, I, OH or NH ₃ , or two of R ₁ , R ₂ , R ₃ and R ₄ may be combined with each other to form

The tetravalent platinum moiety of the present disclosure is represented by the following structural formulas (a-1) to (a-6):

The oligopeptide of the present disclosure may be a dipeptide Xaa1-Xaa4, a tripeptide Xaa1-Xaa2-Xaa4 or a tetrapeptide Xaa1-Xaa2-Xaa3-Xaa4. The tetravalent platinum moiety may be chemically linked to various parts of the oligopeptide, for example, a divalent platinum complex may be oxidized to a monochloromonohydroxy tetravalent platinum moiety, and the hydroxyl group of the tetravalent platinum moiety may be condensed with an amino acid to form a connection.

In the above oligopeptide, Xaa4 represents tryptophan, phenylalanine, histidine, tyrosine or arginine, preferably tryptophan, phenylalanine, histidine or tyrosine. These amino acids can drive the platinum part to self-assemble in an aqueous medium to form a stable monodisperse nanostructure through π-π stacking.

Typically, the tetravalent platinum moiety is chemically linked to Xaa1, wherein Xaa1 can be glutamic acid, aspartic acid, tryptophan, proline, tyrosine or phenylalanine. Of course, the tetravalent platinum moiety can also be chemically linked to Xaa2 or Xaa3, as long as their positions do not hinder π-π stacking.

In the present disclosure, when the oligopeptide is a dipeptide, Xaa1 may be glutamic acid, aspartic acid, tryptophan or tyrosine, and Xaa4 may be tryptophan, phenylalanine, histidine, tyrosine or arginine.

When the oligopeptide is a tripeptide, Xaa1 may be aspartic acid, tryptophan, proline, phenylalanine or glutamic acid, Xaa2 may be tryptophan, glycine, alanine, valine, leucine, phenylalanine, proline, serine, threonine, cysteine, tyrosine, methionine, asparagine, glutamine, aspartic acid, glutamic acid or histidine, and Xaa4 may be tryptophan, phenylalanine, histidine or tyrosine. For example, Xaa1 may represent aspartic acid, tryptophan, proline, phenylalanine or glutamic acid, Xaa2 represents histidine, phenylalanine, tryptophan or tyrosine, and Xaa4 represents tryptophan. For another example, Xaa1 may represent aspartic acid, tryptophan, proline, phenylalanine or glutamic acid, and Xaa2 and Xaa4 both represent tryptophan.

When the oligopeptide is a tetrapeptide, Xaa1 can be glutamic acid, tryptophan or aspartic acid, Xaa2 can be tryptophan, glycine, valine, leucine, glutamic acid, phenylalanine, proline, aspartic acid, threonine, histidine, tyrosine or arginine, Xaa3 can be tryptophan, glycine, leucine, phenylalanine, proline, histidine, aspartic acid, lysine, tyrosine or arginine, and Xaa4 can be tryptophan, phenylalanine, histidine or tyrosine.

In the tripeptide, Xaa2 may be the same as Xaa4, or Xaa1 may be the same as Xaa4, preferably, Xaa2 and Xaa4 may both be tryptophan. In the tetrapeptide, two or three of Xaa2, Xaa3 and Xaa4 may be the same, for example, Xaa2 and Xaa3 are the same, or Xaa3 and Xaa4 are the same, preferably, Xaa3 and Xaa4 may both be tryptophan.

The complexes of the present disclosure may be selected from any of the following:

In this embodiment, the complex can be selected from any of the following:

For the above compounds, the oligopeptide (eg, tryptophan dipeptide) motif in the prodrug molecular structure can provide an aromatic indole ring as an assembly motif, which drives the platinum complex to self-assemble in an aqueous medium to form a stable monodisperse nanostructure through π-π stacking.

The preparation method of peptide-based platinum prodrugs is characterized by comprising the following steps: oxidizing a divalent platinum complex Pt(II) with chlorosuccinimide to a monochloromonohydroxy tetravalent platinum complex Pt(IV), and then condensing it with an oligopeptide containing a tryptophan dipeptide motif under the catalysis of TBTU and triethylamine to obtain a peptide-based platinum prodrug.

The prepared peptide-based platinum prodrug self-assembles into a nanostructure; the prodrug can self-assemble into a nanostructure with a particle size of 10-1000 nm, preferably 20-200 nm in an aqueous medium. The pH of the aqueous medium for the self-assembly of the prodrug is 7.0-7.8. The aqueous medium for the self-assembly of the prodrug can be purified water, water for injection, HEPES buffer, Tris buffer or PBS buffer. The self-assembly method is as follows:

Dissolving the prepared peptide-based platinum prodrug in a first solvent to obtain an organic phase A; the first solvent is one or a mixture of at least two of acetone, ethanol, methanol, tetrahydrofuran, acetonitrile, dimethyl sulfoxide and N,N-dimethylformamide; preferably, the first solvent is acetone and/or ethanol;

The organic phase A is mixed with the aqueous phase B to obtain a mixed solution C; the aqueous phase B is purified water, water for injection, HEPES buffer, Tris buffer or PBS buffer; preferably, the aqueous phase B is water for injection or HEPES buffer; preferably, the pH value of the aqueous phase B is adjusted to 7.0-7.8; when the aqueous phase B is mixed with the organic phase A, the volume ratio is 1-100:1, preferably 1-40:1.

Removing the first solvent in the mixed solution C, wherein the method for removing the solvent in the mixed solution C is reduced pressure evaporation, high-speed centrifugation, dialysis or ultrafiltration; preferably, the reduced pressure evaporation method is used to remove the solvent in the mixed solution C, and a monodispersed nano-prodrug is obtained after removing the solvent;

Peptide-based platinum self-assembled nano-prodrugs can be used to prepare anti-tumor drugs. Nano-shaped drugs have good stability, which can avoid the premature release of active divalent platinum complexes in the blood circulation, thereby reducing toxic side effects, and promote the selective accumulation of platinum drugs in tumor tissues and the controlled release in tumor cells, thereby improving the inhibitory effect on the growth of drug-resistant tumors. The degradation products of the assembly units in the prodrugs are all endogenous amino acids in the human body, and their application in tumor treatment can significantly reduce the toxic side effects of platinum drugs.

The preparation method of the complex of the present disclosure may include oxidizing divalent platinum Pt(II) to hydroxy tetravalent platinum Pt(IV), and then condensing with an oligopeptide in the presence of a catalyst to obtain the complex. As shown above, the oxidant may be chlorosuccinimide, etc., and the catalyst may be TBTU, triethylamine, or a combination thereof, preferably a combination of the two.

The method for forming the self-assembly of the present disclosure may include dissolving the complex in a polar solvent to obtain an organic phase; mixing the organic phase with an aqueous phase to obtain a solution; removing the polar solvent in the solution to obtain the self-assembly. In this regard, the polar solvent may be one or a mixture of at least two of acetone, ethanol, methanol, tetrahydrofuran, acetonitrile, dimethyl sulfoxide and N,N-dimethylformamide, preferably acetone and/or ethanol. The aqueous phase may be a combination of one or more of purified water, water for injection, HEPES buffer, Tris buffer or PBS buffer, preferably water for injection or HEPES buffer. The pH value of the aqueous phase is 7.0-7.8. In the method, the volume ratio of the aqueous phase to the organic phase may be 1-100:1, preferably 1-40:1. In the method, the removal method may be reduced pressure evaporation, high-speed centrifugation, dialysis or ultrafiltration, preferably reduced pressure evaporation.

The self-assemblies of the present disclosure can be used to prepare anti-tumor drugs suitable for, for example, treating the following diseases: lung cancer, breast cancer, gastric cancer, esophageal cancer, adrenocortical carcinoma, skin squamous cell carcinoma, head and neck cancer, thyroid cancer, liver cancer, pancreatic cancer, bile duct cancer, colorectal cancer, ovarian cancer, cervical cancer, endometrial cancer, vaginal squamous cell carcinoma, testicular cancer, prostate cancer, bladder cancer, urothelial carcinoma, melanoma, osteosarcoma, malignant lymphoma, neuroblastoma, cisplatin-acquired resistance to lung cancer, cisplatin-acquired resistance to ovarian cancer, cisplatin-acquired resistance to breast cancer, cisplatin-acquired resistance to cervical cancer, cisplatin-acquired resistance to gastric cancer, and cisplatin-acquired resistance to colorectal cancer.

The pharmaceutical composition of the present disclosure may comprise the complex, prodrug or self-assembly described above, such as a therapeutically effective amount of the complex, prodrug or self-assembly, and a pharmaceutically acceptable excipient. Typical routes of the pharmaceutical composition of the present disclosure include, but are not limited to, oral, topical, inhalation, parenteral, intranasal, intraocular, intramuscular, subcutaneous, intravenous administration. The pharmaceutical composition of the present disclosure may be manufactured using methods well known in the art, such as conventional mixing, dissolution, granulation, sugar-coated pill making, grinding, emulsification, freeze drying, etc.

The present disclosure may involve one or more of the following advantages and effects:

The complexes, peptide-based self-assembled nano-prodrugs and self-assemblies described above are responsive to the tumor microenvironment and can promote the selective accumulation of platinum drugs in tumor tissues and the controlled release in tumor cells when used for tumor treatment, thereby improving the inhibitory effect on the growth of drug-resistant tumors.

The complexes, peptide-based self-assembled nano-prodrugs and self-assemblies described above can avoid the premature release of active divalent platinum complexes in the blood circulation, thereby reducing toxic side effects;

The complexes, peptide-based self-assembled nano-prodrugs and self-assemblies described above have good biocompatibility and safety. The assembly unit may contain one of the essential amino acids for the human body (such as tryptophan), participate in the protein synthesis and metabolic regulation of the body, and the degradation products are all endogenous substances of the human body. The application in tumor treatment can significantly reduce the toxic side effects of platinum drugs.

The above-mentioned complexes, peptide-based self-assembled nano-prodrugs and self-assemblies have mild preparation conditions and simple operations, and the production process does not involve harmful solvents, etc., and are suitable for industrial production.

Example

The following examples are provided to illustrate the present disclosure, wherein the experimental methods without specific operating steps are all carried out in accordance with the corresponding product instructions, and the instruments, reagents, and consumables used in the examples can all be purchased from commercial companies unless otherwise specified.

Abbreviations and structure:

H-Trp-OMe.HCl:

Cbz-Trp-OH:

Cbz-Glu(Obzl)-OH: H-Glu-Trp-Trp-OMe:

Example 1

A method for preparing a tetravalent peptide platinum having a structure as shown in formula (VIII) is provided, and the synthetic route is as follows:

(1) H-Trp-OMe.HCl (291 mg, 1 mmol) and N-methylmorpholine (145 μL, 1.3 mmol) were weighed and dissolved in 5 mL of dimethylformamide (DMF) to obtain mixture 1. Cbz-Trp-OH (338 mg, 1 mmol), 1-hydroxybenzotriazole (HOBt, 135 mg, 1 mmol) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 250 mg, 1.3 mmol) were weighed and dissolved in 20 mL of DMF, activated in an ice bath for 40 min, added to mixture 1, stirred for 2 h, and then naturally warmed to room temperature, and continued to stir and react for 12 h. After reduced pressure concentration, dialyzed in distilled water for 4 h, and freeze-dried to obtain Cbz-Trp-Trp-OMe.

Place Cbz-Trp-Trp-OMe in a 50 mL round-bottom flask and dissolve the peptide in 10 mL methanol. Add 150 mg of 5% Pd/C catalyst pre-suspended in 1 mL methanol, introduce hydrogen gas for 6 h under stirring, and filter. After the filtrate is concentrated under reduced pressure, dialyzed in distilled water for 4 h, and freeze-dried to obtain H-Trp-Trp-OMe.

H-Trp-Trp-OMe (243 mg, 0.6 mmol) and N-methylmorpholine (87 μL, 0.78 mmol) were weighed and dissolved in 5 mL DMF to obtain mixture 2. Cbz-Glu(Obzl)-OH (223 mg, 0.6 mmol), HOBt (81 mg, 0.6 mmol) and EDCI (150 mg, 0.78 mmol) were weighed and dissolved in 10 mL DMF, activated in an ice bath for 40 min, added to mixture 2, stirred for 2 h, and naturally warmed to room temperature, continued to stir and react for 12 h, dialyzed in distilled water for 4 h, and lyophilized to obtain Cbz-Glu(Obzl)-Trp-Trp-OMe.

Place Cbz-Glu(Obzl)-Trp-Trp-OMe in a 50 mL round-bottom flask and dissolve the peptide in 10 mL methanol. Add 180 mg of 5% Pd/C catalyst pre-suspended in 1.2 mL methanol, introduce hydrogen gas for 6 h under stirring, and filter. After the filtrate is concentrated under reduced pressure, dialyze in distilled water for 4 h, and freeze-dry to obtain H-Glu-Trp-Trp-OMe.

(2) Cisplatin (300 mg, 1 mmol) and N-chlorosuccinimide (NCS, 150 mg, 1.1 mmol) were suspended in 60 mL of distilled water and reacted at room temperature in the dark for 12 h. After filtering the reaction solution, the filtrate was concentrated under reduced pressure until a light yellow precipitate was precipitated. The precipitate was washed with acetone and ether in turn to obtain a tetravalent platinum intermediate product (yield 91.2%).

The tetravalent platinum intermediate (176 mg, 0.5 mmol), H-Glu-Trp-Trp-OMe (293 mg, 0.55 mmol), tetramethyluronium tetrafluoroborate (TBTU, 264 mg, 0.82 mmol), and triethylamine (84 mg, 0.82 mmol) were dissolved in dry dimethylformamide under nitrogen protection and reacted at room temperature in the dark for 24 h. 1M sodium hydroxide solution was added to adjust the pH to 12.8, and stirred at 15 ° C for 6 h; 1M hydrochloric acid solution was added to adjust the pH to 6.0; after vacuum concentration, it was added to 10 times the volume of ice ether for precipitation, centrifuged, and the precipitate was taken and cold ether was added. Repeat the above operation 5 times, and the product of structural formula (II) was obtained by vacuum drying. The mass spectrum and nuclear magnetic resonance data of the product are shown below.

ESI-MS[M+H]+:854.64

¹ HNMR (600MHz, DMSO-d6): δ10.22(s,1H),8.05(d,J＝6.7Hz,2H),7.62-7.58(m,2H),7.36(dd,J＝7.7,1.5Hz ,2H),7.21-7.10(m,4H),7.05(d,J＝6.7Hz,1H),6.98-6.91(m,2H),6.76(d,J＝6.7Hz,1H),4.55-4.47( m,2H),3.53(p,J＝5.6Hz,1H),3.26(dd,J＝14.5,6 .7Hz,1H),3.20(dd,J＝15.6,5.9Hz,1H),2.98(dd,J＝15.6,5.9Hz,1H),2.92(dd,J＝14.5,6.7Hz,1H),2.42( t,J＝7.5Hz,3H),2.37-2.28(m,1H),2.30-2.16(m,2H),1.96(p,J＝7.6Hz,2H),1.79(dtd,J＝15.3,8.4, 5.0Hz, 1H), 1.68 (d, J = 5.9Hz, 2H), 1.50 (s, 3H).

Example 2

A method for preparing a tetravalent peptide platinum having a structure as shown in formula (VIII) is provided, and the synthetic route is as follows:

(1) Weigh H-Trp-OMe.HCl (291 mg, 1 mmol) and N-methylmorpholine (145 μl, 1.3 mmol), dissolve in 5 mL DMF, and obtain mixture 1. Weigh Cbz-Trp-OH (338 mg, 1 mmol), HOBt (135 mg, 1 mmol), EDCI (250 mg, 1.3 mmol), dissolve in 20 mL DMF, and activate in an ice bath for 40 min; add ①, stir for 2 h, then naturally warm to room temperature, and continue stirring and reacting for 12 h. After reduced pressure concentration, dialyze in distilled water for 4 h, and freeze-dry to obtain Cbz-Trp-Trp-OMe.

Place Cbz-Trp-Trp-OMe in a 50 mL round-bottom flask and dissolve the peptide in 10 mL methanol. Add 150 mg of 5% Pd/C catalyst pre-suspended in 1 mL methanol, introduce hydrogen gas for 6 h under stirring, and filter. After the filtrate is concentrated under reduced pressure, dialyze in distilled water for 4 h, and freeze-dry to obtain Trp-Trp-OMe.

Trp-Trp-OMe (243 mg, 0.6 mmol) and N-methylmorpholine (87 μL, 0.78 mmol) were weighed and dissolved in 5 mL DMF to obtain mixture 2. Fmoc-Glu(Obzl)-OH (268 mg, 0.6 mmol), HOBt (81 mg, 0.6 mmol) and EDCI (150 mg, 0.78 mmol) were weighed and dissolved in 10 mL DMF, and activated in an ice bath for 40 min; mixture 2 was added, stirred for 2 h, and then naturally warmed to room temperature, and the reaction was continued with stirring for 12 h, dialyzed in distilled water for 4 h, and lyophilized to obtain Fmoc-Glu(Obzl)-Trp-Trp-OMe.

Place Fmoc-Glu(Obzl)-Trp-Trp-OMe in a 50 mL round-bottom flask and dissolve the peptide in 10 mL methanol. Add 180 mg of 5% Pd/C catalyst pre-suspended in 1.2 mL methanol, introduce hydrogen gas for 6 h under stirring, and filter. After the filtrate is concentrated under reduced pressure, dialyze in distilled water for 4 h, and freeze-dry to obtain Fmoc-Glu-Trp-Trp-OMe.

(2) Cisplatin (300 mg, 1 mmol) and NCS (150 mg, 1.1 mmol) were suspended in 60 mL of distilled water and reacted at room temperature in the dark for 12 h. The reaction solution was filtered and the filtrate was concentrated under reduced pressure until a light yellow precipitate was precipitated. The precipitate was washed with acetone and ether in turn to obtain a tetravalent platinum intermediate product.

The tetravalent platinum intermediate (176 mg, 0.5 mmol), Fmoc-Glu-Trp-Trp-OMe (415 mg, 0.55 mmol), TBTU (264 mg, 0.82 mmol), and triethylamine (84 mg, 0.82 mmol) were dissolved in dry dimethylformamide under nitrogen protection, and reacted at room temperature in the dark for 24 h; 1M sodium hydroxide solution was added to adjust the pH to 12.8, and stirred at 15°C for 6 h; 1M hydrochloric acid solution was added to adjust the pH to 6.0; after vacuum concentration, it was added to 10 times the amount of ice ether for precipitation, centrifuged, and the precipitate was added with cold ether and the above operation was repeated 5 times, and the product of structural formula (VIII) was obtained by vacuum drying. ESI-MS [M+H] +: 854.56

Example 3

A method for preparing a tetravalent peptide platinum having a structure as shown in formula (XIV) is provided, and the synthetic route is as follows:

Oxaliplatin (395 mg, 1 mmol) was reacted according to the synthesis method in Example 1 or Example 2 to obtain a product of structural formula (XIV) (yield 71%). The NMR data of the product are shown below.

¹ HNMR (600MHz, DMSO-d6): δ10.22(s,1H),8.05(d,J＝6.7Hz,2H),7.60(dd,J＝7.2,1.2Hz,2H),7.36(dd,J ＝7.7,1.5Hz,2H),7.21-7.10(m,4H),7.05(d,J＝6.7Hz,1H),6.98-6.91(m,2H),6.76(d,J＝6.7Hz,1H) ,4.55-4.47(m,2H),3.53(p,J＝5.5Hz,1H),3.26(dd,J＝14.5,6.7Hz,1H),3.2 0(dd,J＝15.6,5.9Hz,1H),2.98(dd,J＝15.6,5.9Hz,1H),2.92(dd,J＝14.5,6.7Hz,1H),2.56-2.48(m,2H) ,2.37-2.28(m,1H),2.30-2.16(m,2H),2.08-2.00(m,1H),1.84-1.66(m,7H),1.53-1.47(m,2H),1.38-1.30( m,1H),1.32-1.21(m,2H),1.15(dddd,J=12.4,8.4,5.9,3.9Hz,1H).

Example 4

A method for preparing a tetravalent peptide platinum having a structure as shown in formula (XV) is provided, and the synthetic route is as follows:

Carboplatin (371 mg, 1 mmol) was reacted according to the synthesis method in Example 1 or Example 2 to obtain a product of structural formula (XV) (yield 68%). The NMR data of the product are shown below.

¹ HNMR (600MHz, DMSO-d6): δ10.22(s,1H),8.05(d,J＝6.7Hz,2H),7.62-7.58(m,2H),7.36(dd,J＝7.7,1.5Hz ,2H),7.21-7.10(m,4H),7.05(d,J＝6.7Hz,1H),6.98-6.91(m,2H),6.76(d,J＝6.7Hz,1H),4.55-4.47( m,2H),3.53(p,J=5.6Hz,1H),3. 26(dd,J＝14.5,6.7Hz,1H),3.20(dd,J＝15.6,5.9Hz,1H),2.98(dd,J＝15.6,5.9Hz,1H),2.92(dd,J＝14.5, 6.7Hz,1H),2.42(t,J＝7.5Hz,3H),2.37-2.16(m,3H),1.96(p,J＝7.6Hz,2H),1.79(dtd,J＝15.3,8.4,5.0 Hz, 1H), 1.68 (d, J = 5.9Hz, 2H).

Example 5

4-Hydroxyethylpiperazineethanesulfonic acid buffer (HEPES, 1 M) was prepared as follows: 119.15 g HEPES was weighed and dissolved in 400 mL distilled water, 1 M NaOH aqueous solution was added to adjust the pH to 7.4, and then the volume was made up to 500 mL with distilled water and stored at 4°C.

The tetravalent peptide platinum (100 mg) prepared in Example 1 or Example 2 was dissolved in 1 mL of ethanol-acetone mixed solvent (1:1), and the resulting solution was injected into 100 mL of HEPES buffer (10 mM, pH 7.4). After the addition, stirring was continued for 30 min, and the solvent was removed by evaporation under reduced pressure to obtain a nanocolloid solution, which is a nano self-assembled prodrug (hereinafter sometimes referred to as a nano prodrug). The appearance of the obtained nano prodrug is a monodisperse sphere, as shown in FIG1 , and the particle size distribution is shown in FIG2 , the average particle size is 118 nm, and the PDI (polymer dispersibility index) is 0.12.

Example 6

The tetravalent peptide platinum (10 mg) obtained in Example 3 was self-assembled according to the method of Example 5. The particle size distribution of the obtained nano self-assembled prodrug is shown in Figure 3, with an average particle size of 132 nm and a PDI of 0.18.

Example 7

The tetravalent peptide platinum (10 mg) obtained in Example 4 was self-assembled according to the method of Example 5. The particle size distribution of the obtained nano prodrug is shown in FIG4 , with an average particle size of 121 nm and a PDI of 0.09.

Example 8

Environmentally responsive release behavior

The nano self-assembled prodrug obtained in Example 5 was dispersed in 20 mL of a buffer solution (10 mM, pH 7.4) containing different concentrations of reduced glutathione (GSH), 0.1 mL was sampled at specific time points, and high-speed centrifugation was performed at 20,000 rpm for 30 min. 20 uL of the supernatant was taken for HPLC determination of the drug concentration, and the release curve was drawn. The specific steps are as follows, and the results are shown in Figure 5.

The nanoprodrug is slowly released in a release medium without GSH, with a cumulative release rate of less than 20% in 48 hours; in a release medium containing a higher concentration of GSH (5mM), the release of the active ingredient is significantly accelerated, with a cumulative release rate of 80% in 48 hours. From the in vitro release results, it can be seen that the nanoprodrug has tumor microenvironment responsiveness and can quickly release the active ingredient under the action of the reducing environment in tumor cells.

(1) Determination method of cisplatin (CDDP) content:

Chromatographic conditions: Shim-Paok VP-ODS C18 (150×4.6 mm, particle size 5 μm); flow rate 1 mL/min;

Mobile phase: 0.9% sodium chloride; detection wavelength: 210 nm; column temperature: 30°C; injection volume: 20 μL.

(2) Standard curve drawing:

Accurately weigh cisplatin (10 mg) in a 10 mL volumetric flask, add 0.9% sodium chloride and dilute to the mark to obtain a concentrated cisplatin stock solution. Accurately measure the stock solution and prepare it into 2.5, 5, 10, 20, 40, 80, 100 μg/mL standard test solutions. Take the solution and inject it according to the above chromatographic conditions, record the peak area, and perform linear regression on the concentration C with the peak area A to obtain the regression equation: A = 24644C + 21689, r2 = 0.9993. Cisplatin has a good linear relationship in the range of 2.5-100 μg/mL.

Example 9

Serum stability

The nanometer self-assembled prodrug obtained in Example 5 was dispersed in a buffer solution (pH=7.4) containing 10% fetal bovine serum FBS, placed in a 37°C constant temperature shaker for shaking, and samples were taken at 0, 2, 4, 8, 12, 24, and 48 hours for particle size analysis. The results are shown in FIG6 , indicating that the particle size distribution of the nanoparticles obtained in Example 5 in 10% FBS did not change significantly within 24 hours, indicating that the nanoparticles obtained in Example 5 will have good stability in blood circulation.

Example 10

In vitro cytotoxicity

The CCK8 method was used to evaluate the in vitro cytotoxicity of the nano self-assembly prodrug prepared in Example 5. The specific method is as follows: tumor cells in the logarithmic growth phase were inoculated into a 96-well plate (5×10 ³ / well), and after the cells adhered to the wall, the culture medium was replaced with a nano self-assembly prodrug, and the cisplatin solution was used as the control group. After incubation at 37°C for 48 hours, 10 μL of CCK8 reagent was added to each well, and the OD value of each well was measured at a wavelength of 450 nm after incubation for 4 hours. The cell survival rate was calculated, and the cell survival rate was nonlinearly regressed against the concentration to obtain the half inhibitory concentration (IC50) of the drug on cell growth. The in vitro toxicity results of the prepared nano self-assembly prodrug on tumor cells are shown in Table 2, Figure 7 and Figure 8, where CDDP represents cisplatin and PDDP represents the nano self-assembly prodrug obtained in Example 5.

Table 2

From the experimental results, it can be seen that after incubation with human ovarian cancer SKOV3 cells for 48 hours, the half inhibition concentration of the self-assembled nano-prodrug is equivalent to that of cisplatin solution, indicating that the self-assembly of cisplatin into nano-prodrug does not affect its ability to inhibit the proliferation of tumor cells. In particular, after incubation with the platinum-resistant cell line SKOV3/DDP, the half inhibition concentration of the self-assembled nano-prodrug was significantly lower than that of the cisplatin solution, indicating that nano-prodrug has advantages in alleviating tumor resistance.

Embodiment 11

In vitro cytotoxicity.

The nano self-assembled prodrug prepared in Example 6 was used, and oxaliplatin solution was used as a control group, and the half-maximal inhibitory concentration (IC50) of human colon cancer HCT116 and its drug-resistant cell line HCT116/L-OHPD was tested according to the method in Example 10. The in vitro toxicity results of the prepared self-assembled nano prodrug on tumor cells are shown in Table 3.

Table 3

Embodiment 12:

In vitro cytotoxicity.

The nano self-assembled prodrug prepared in Example 7 was used, and carboplatin solution was used as a control group, and the half-maximal inhibitory concentration (IC50) of human ovarian cancer SKOV3 and its platinum-resistant cell line SKOV3/DDP was tested according to the method in Example 11. The in vitro toxicity results of the prepared self-assembled nano prodrug on tumor cells are shown in Table 4.

Table 4

Embodiment 13:

In vivo antitumor efficacy

150 μL of SKOV3/DDP tumor cell suspension (1×10 ⁷ ) was subcutaneously inoculated into the ventral subcutaneous tissue of Balb/c nude mice. When the tumor volume reached about 100-150 mm ³ , the tumor-bearing mice were randomly divided into three groups (5 mice in each group) and injected with 0.2 mL of normal saline, 0.2 mL of cisplatin solution and 0.2 mL of the nano self-assembled prodrug prepared in Example 5 (calculated as free cisplatin, dose 2 mg/kg) through the tail vein, respectively. The drugs were administered once every 2 days for 5 consecutive times. The long and short diameters of the tumors were measured every other day, and the tumor volume and tumor burden were calculated. The results are shown in Figures 9 and 10. In Figure 10, * indicates p<0.05, and ** indicates P<0.01.

During the treatment period, the tumor volume in the normal saline group continued to grow, and there was no inhibitory effect on tumor growth; the cisplatin solution group could not effectively inhibit tumor growth in tumor-bearing mice; the inhibitory effect of the self-assembled nanoprodrug group on tumor growth was significantly stronger than that of the cisplatin solution group.

Embodiment 14

Pharmacokinetic analysis

(1) Establishment of the determination method

Preparation of platinum standard stock solution (2.5 mg/L): Accurately measure 2.5 mL of platinum standard solution (1 mg/mL), and dilute to 10 mL with 0.2% nitric acid; accurately measure 100 μL, and dilute to 10 mL with 0.2% nitric acid.

Platinum standard working solution (200 μg/L): Accurately measure 0.8 mL of platinum standard stock solution and dilute to 10 mL with 0.2% nitric acid.

Analysis conditions: working wavelength 265.9 nm, bandwidth 0.2 nm, injection volume 20 μL.

Standard curve: good linear relationship in the range of 12.5-200μg/L.

(2) Sampling

SD rats (weight 200-250g) were randomly divided into 2 groups (n=5), fasted for 12h before formal administration, and had free access to water. Each group was injected with 3mg/kg (in terms of cisplatin content) of CDDP saline solution or the saline dilution of the nano prodrug (PDDP) prepared in Example 5 through the tail vein, and blood was collected from the orbit at 5min, 15min, 30min, 1h, 2h, 4h, 6h, 8h, 12h, 24h, and 48h after administration, mixed in an EP tube coated with sodium heparin, and centrifuged at 5000rpm for 10min to obtain plasma, which was stored in a -20℃ refrigerator.

Plasma sample processing and determination: Accurately measure 100 μL of plasma sample, add 2 mL of nitric acid-perchloric acid (9:1), and heat on a hot plate until the nitric acid is almost dry. The residue is dissolved with 0.2% nitric acid and fixed to 10 mL, measured by graphite furnace atomic absorption spectrophotometer, and substituted into the standard curve to calculate the blood drug concentration. The blood drug concentration-time curve is drawn with blood drug concentration as the ordinate and time as the abscissa. The measured Pt content is fitted by a two-compartment model using Kinetica software, and various pharmacokinetic parameters are calculated. The results are shown in Figure 11 and Table 5.

Table 5

The elimination half-life of the nanoprodrug prepared in Example 5 was 16.38 h, which was significantly higher than that of the cisplatin group (5.62 h), indicating that PDDP can significantly increase the blood circulation time of platinum drugs.

Embodiment 15

Organizational Distribution

150 μL of SKOV3 tumor cell suspension (1×10 ⁷ ) was subcutaneously inoculated into the ventral subcutaneous tissue of Balb/c nude mice. When the tumor volume reached about 150 mm ³ , the tumor-bearing mice were randomly divided into 2 groups (n=5) and injected with cisplatin (CDDP) saline solution and the nanoprodrug (PDDP) saline dilution prepared in Example 5 (calculated as free cisplatin, dose 5 mg/kg) through the tail vein. The mice were killed 12 hours after the injection, and the heart, liver, spleen, lung, kidney and tumor of different organs were taken out and weighed. The tissue samples were minced, added with 2 mL of nitric acid-perchloric acid (9:1), and placed on a hot plate and heated until the nitrolysis was almost dry. The residue was dissolved with 0.2% nitric acid and fixed to 10 mL, and the concentration was determined by graphite furnace atomic absorption spectrophotometer, and the Pt concentration was calculated by substituting into the standard curve.

The test results are shown in Figure 12. It can be seen that the nanoprodrug prepared in Example 5 has better tumor targeting ability, and the accumulation amount in the tumor site can reach 6512ng/g tissue, which is significantly better than the cisplatin group (1958ng/g tissue), verifying the targeted delivery efficiency of the nanoprodrug.

Example 16

In vivo safety evaluation

(1) Hemolytic test

Take a few milliliters of rabbit blood, put it into a conical flask containing glass beads and shake it for 10 minutes to remove fibrinogen to make defibrillated blood. Add about 10 times the amount of 0.9% sodium chloride solution, shake well, centrifuge at 1500rpm for 15 minutes, remove the supernatant, and wash the precipitated red blood cells with 0.9% sodium chloride solution 2-3 times according to the above method until the supernatant is no longer red. The obtained red blood cells are prepared into a 2% (V/V) suspension with 0.9% sodium chloride solution for test use.

Take 7 clean test tubes and number them. Tubes 1-5 are test tubes, tube 6 is a negative control tube, and tube 7 is a positive control tube. Add 2% red blood cell suspension, 0.9% sodium chloride solution or distilled water in sequence as shown in the table below. Add various solutions in the order of Table 6:

Table 6

Each group was set up in triplicate, vortexed and incubated at 37°C for 3 hours. The supernatant was centrifuged and the absorbance was measured at 540 nm. The results are shown in Table 7.

Hemolysis rate (%) = [(OD sample - OD negative) / (OD positive - OD negative)] x 100%.

Table 7

It can be seen from the above that the hemolysis rate of the nano prodrug (PDDP) prepared in Example 5 is less than 5% in the concentration range of 12.5 to 200 μg/mL.

(2) Weight changes

Healthy KM mice (5-7 weeks) weighing 20-23g were randomly divided into 3 groups (10 mice in each group) and injected with physiological saline, cisplatin physiological saline solution and the nano self-assembled prodrug (PDDP) physiological saline dilution prepared in Example 5 (calculated as free cisplatin, dose 2 mg/kg) through the tail vein, respectively. The mice were given once every 2 days for 5 consecutive times. After administration, they were fed routinely, the survival status of the mice was monitored daily, and the body weight was measured. The results are shown in Figure 13.

As can be seen from the results in Figure 13, the weight of mice in the cisplatin solution group decreased significantly after administration, and the weight loss was about 25% within 14 days, indicating that the cisplatin solution group had more serious systemic toxicity. The weight of mice in the nanoprodrug group increased slightly, similar to the weight change in the saline group. The mice were in good health and no deaths occurred.

(3) Histopathological examination

On the 14th day after administration, the mice were killed and the main organs (heart, liver, spleen, lung, and kidney) were collected and fixed with 4% paraformaldehyde for 24 hours. The samples were then embedded in paraffin and tissue sections were made and stained with H&E. The results are shown in FIG14 .

H&E staining was used to evaluate the tissue damage of PDDP to mice. From the liver tissue sections, the hepatocytes in the saline group were aggregated, evenly distributed, with clear membrane boundary structure, uniform eosinophilic staining, and round and clear nucleoli located in the center with lighter color. In the CDDP solution group, the hepatocytes were scattered with cavities, lighter staining, liver edema and degeneration, venous congestion and inflammation, which were consistent with the typical hepatotoxicity of cisplatin. The liver tissue in the PDDP nanoformulation group was more normal than that in the CDDP solution group. From the renal tissue sections, the glomeruli in the saline group were spherical, with vascular glomeruli in the center, quite round nucleoli, eosinophilic chromatin, and dark red staining. The degenerated glomeruli in the CDDP solution group had disordered structure, focal cell hyperplasia, and enlarged renal tubular epithelium. No obvious histological damage was observed in the renal tissue sections of the PDDP nanoformulation group.

It can be seen that the H&E staining results of the main organ tissue sections of mice showed that obvious liver and kidney damage was observed in the cisplatin solution group, while there were no obvious pathological changes in the organ tissue sections of the self-assembled nano-prodrug group and the normal saline group, indicating that the self-assembled nano-prodrug can significantly reduce the toxicity of the original drug, and its application in tumor treatment has a high in vivo safety.

All documents cited in this disclosure, such as patents, patent application publications, patent applications, and non-patent publications, are incorporated herein by reference in their entirety.

It can be understood from the foregoing that although specific embodiments are described herein for illustrative purposes, various modifications or adjustments may be made without departing from the spirit and scope of the present disclosure. Therefore, the scope of the present disclosure should only be limited by the attached claims and their equivalents.

Claims (13)

1. A complex comprising a tetravalent platinum portion and an oligopeptide as an axial ligand chemically linked to the tetravalent platinum portion, wherein the oligopeptide is a dipeptide Xaa1-Xaa4, a tripeptide Xaa1-Xaa2-Xaa4 or a tetrapeptide Xaa1-Xaa2-Xaa3-Xaa4, wherein Xaa1, Xaa2 and Xaa3 each represent any natural amino acid, and Xaa4 represents tryptophan, phenylalanine, histidine, tyrosine or arginine. 2. The complex according to claim 1, wherein the tetravalent platinum portion is represented by the following structural formula (a): wherein R ₁ , R ₂ , R ₃ , R ₄ and R ₅ are independently selected from: Cl, Br, I, OH, NH ₃ ; or, R ₁ and R ₂ can form a ring with Pt, and R ₃ and R ₄ can form a ring with Pt, and when forming a ring, R ₁ and R ₂ or R ₃ and R ₄ are selected in combination from Optionally, the tetravalent platinum moiety is represented by the following structural formulas (a-1) to (a-6): 3. The complex according to claim 1 or 2, wherein Xaa1 represents glutamic acid, aspartic acid, tryptophan, proline, tyrosine or phenylalanine; optionally, in a dipeptide, Xaa1 represents glutamic acid, aspartic acid, tryptophan or tyrosine; optionally, in a tripeptide, Xaa1 represents aspartic acid, tryptophan, proline, phenylalanine or glutamic acid; optionally, in a tetrapeptide, Xaa1 represents glutamic acid, tryptophan or aspartic acid; Optionally, wherein Xaa2 represents tryptophan, glycine, alanine, valine, leucine, phenylalanine, proline, serine, threonine, cysteine, tyrosine, methionine, asparagine, glutamine, aspartic acid, glutamic acid, histidine or arginine; Optionally, in a tripeptide, Xaa2 represents tryptophan, glycine, alanine, valine, leucine, phenylalanine, proline, serine, threonine, cysteine, tyrosine, methionine, asparagine, glutamine, aspartic acid, glutamic acid or histidine; Optionally, in a tetrapeptide, Xaa2 represents tryptophan, glycine, valine, leucine, glutamic acid, phenylalanine, proline, aspartic acid, threonine, histidine, tyrosine or arginine; Optionally, wherein Xaa3 represents tryptophan, glycine, leucine, phenylalanine, proline, histidine, aspartic acid, lysine, tyrosine or arginine; Optionally, in the tetrapeptide two or three of Xaa2, Xaa3 and Xaa4 are identical; wherein Xaa2 or Xaa3, when present, is identical to Xaa4, preferably in the tripeptide, Xaa2 and Xaa4 are both tryptophan, and in the tetrapeptide, Xaa3 and Xaa4 are both tryptophan, Optionally, the tetravalent platinum moiety is chemically linked to any one of Xaa1 to Xaa3, preferably, the tetravalent platinum moiety is chemically linked to Xaa1, Optionally, the oligopeptide is represented by Xaa1-Xaa2-Xaa4, wherein Xaa1 represents aspartic acid, tryptophan, proline, phenylalanine or glutamic acid, Xaa2 represents histidine, phenylalanine, tryptophan or tyrosine, and Xaa4 represents tryptophan. Preferably, both Xaa2 and Xaa4 represent tryptophan. 4. The complex according to any one of claims 1 to 3, wherein the oligopeptide is selected from: 5. The complex according to any one of claims 1 to 4, wherein the complex is represented by formula (I): Wherein, R ₁ , R ₂ , R ₃ , R ₄ and R ₅ are independently selected from: Cl, Br, I, OH, NH ₃ ; or, R ₁ and R ₂ can form a ring with Pt, and R ₃ and R ₄ can form a ring with Pt, and when forming a ring, R ₁ and R ₂ or R ₃ and R ₄ are selected in combination from Optionally, the complex is selected from any one of the following: Optionally, selected from any of the following: 6. A peptide-based platinum prodrug comprising a tetravalent platinum complex having an axial ligand containing a tryptophan dipeptide. 7. A self-assembly, wherein the self-assembly is self-assembled by the complex according to any one of claims 1 to 5 or the peptide-based platinum prodrug according to claim 6. Optionally, the self-assembly is self-assembled by the complex according to any one of claims 1 to 5 or the peptide-based platinum prodrug according to claim 6 in an aqueous medium. Preferably, the pH of the aqueous medium is 7.0-7.8. Preferably, the aqueous medium is purified water, water for injection, HEPES buffer, Tris buffer or PBS buffer. Preferably, the self-assembly has a particle size of 10 nm to 10000 nm. More preferably, the self-assembly has a particle size of 20 nm to 200 nm. 8. A method for preparing the complex according to any one of claims 1 to 5, the method comprising: oxidizing divalent platinum Pt(II) to hydroxytetravalent platinum Pt(IV), and then condensing it with an oligopeptide in the presence of a catalyst to obtain the complex. 9. A method for preparing the peptide-based platinum prodrug according to claim 6, wherein a divalent platinum complex Pt(II) is oxidized with chlorosuccinimide to a hydroxy tetravalent platinum complex Pt(IV), and then condensed with an oligopeptide containing a tryptophan dipeptide motif under the catalysis of TBTU and triethylamine to obtain the peptide-based platinum prodrug. 10. A method for preparing the self-assembly according to claim 7, comprising dissolving the complex according to any one of claims 1 to 5 or the peptide-based platinum prodrug according to claim 6 in a polar solvent to obtain an organic phase; mixing the organic phase with an aqueous phase to obtain a solution; removing the polar solvent from the solution to obtain the self-assembly, Optionally, the polar solvent is one or a mixture of at least two of acetone, ethanol, methanol, tetrahydrofuran, acetonitrile, dimethyl sulfoxide and N,N-dimethylformamide; preferably, the polar solvent is acetone and/or ethanol; or The aqueous phase is a combination of one or more of purified water, water for injection, HEPES buffer, Tris buffer or PBS buffer; preferably, the aqueous phase is water for injection or HEPES buffer; more preferably, the pH value of the aqueous phase is 7.0-7.8; or The volume ratio of the aqueous phase to the organic phase when mixed is 1-100:1, preferably 1-40:1; or The method for removing the solvent in the solution is reduced pressure evaporation, high-speed centrifugation, dialysis or ultrafiltration; preferably, reduced pressure evaporation. 11. A pharmaceutical composition comprising the complex according to any one of claims 1 to 5, the peptide-based platinum prodrug according to claim 6 or the self-assembly according to claim 7, and a pharmaceutically acceptable excipient. 12. Use of the complex according to any one of claims 1 to 5, the peptide-based platinum prodrug according to claim 6, the self-assembly according to claim 7 or the pharmaceutical composition according to claim 11 in the preparation of an anti-tumor drug. 13. The use of claim 12, wherein the tumor is selected from lung cancer, breast cancer, gastric cancer, esophageal cancer, adrenocortical carcinoma, skin squamous cell carcinoma, head and neck cancer, thyroid cancer, liver cancer, pancreatic cancer, bile duct cancer, colorectal cancer, ovarian cancer, cervical cancer, endometrial cancer, vaginal squamous cell carcinoma, testicular cancer, prostate cancer, bladder cancer, urothelial carcinoma, melanoma, osteosarcoma, malignant lymphoma, neuroblastoma, cisplatin-acquired lung cancer, cisplatin-acquired resistant ovarian cancer, cisplatin-acquired resistant breast cancer, cisplatin-acquired resistant cervical cancer, cisplatin-acquired resistant gastric cancer, and cisplatin-acquired resistant colorectal cancer.