CN115025218A - Active combination of combined therapy for human immunodeficiency virus and its application - Google Patents

Abstract

The invention provides an active combination for treating human immunodeficiency virus in a combined way and application thereof. In particular, the use of an active ingredient combination comprising a first active ingredient bicinchonin and a second active ingredient neutralizing antibodies against human immunodeficiency virus is provided and for the preparation of a pharmaceutical composition or kit for the synergistic treatment and prevention of HIV. Aiconing and the neutralizing antibody of the human immunodeficiency virus can act synergistically on anti-HIV treatment, thereby inhibiting the proliferation of the HIV virus more remarkably.

Description

Active combination of combined therapy for human immunodeficiency virus and its application

technical field

The present invention relates to the technical field of anti-HIV drugs, more particularly to an active combination for combined treatment of human immunodeficiency virus and its application.

Background technique

Aikening (Aibovirtai for Injection, ABT for short) is the first long-acting injection anti-AIDS drug independently developed by Frontier Bio, and it is a national class 1 new drug. On May 23, 2018, the State Drug Administration issued the new drug certificate and drug registration approval for alprenavir and alprenavir for injection, which were approved for use in combination with other antiviral drugs, and the treatment has received antiviral treatment And HIV-1 adult infected people who still have viral replication. Aibovirtide has a new molecular mechanism of action. It is an HIV-1 fusion inhibitor. It acts on the HIV-1 outer membrane glycoprotein gp41 to prevent the virus from entering cells. In vitro tests have shown that it is effective against epidemic strains and drug-resistant viruses in Europe, America and China. It has broad-spectrum and high-efficiency activity. Consisting of 34 amino acids and a modified chemical group, ebavitaxel rapidly forms a 1:1 covalent conjugate with albumin in vivo, thereby extending the half-life to 12 days. Clinical Phase I-III trials have shown good safety and efficacy.

Human immunodeficiency virus neutralizing antibodies are a class of antibodies isolated from HIV-infected individuals by biotechnology. This type of antibody can recognize the HIV virus, inhibit the replication of the virus in the body by directly neutralizing the HIV virus strain, and reduce the level of HIV-1 virus in the body. The antibody can also stimulate other immune components in the body to jointly kill the virus and improve immunity. and remove the virus from the body.

In the prior art, the neutralizing antibody against human immunodeficiency virus, and Aikonin still have many deficiencies, such as the deficiency of insufficient virus coverage, and cannot be well used for the treatment and prevention of HIV virus. Therefore, there is an urgent need in the art to develop a more effective method for treating or preventing human immunodeficiency virus.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide an active combination for combined treatment of human immunodeficiency virus and its application, so as to solve the problem of insufficient coverage of HIV virus by using Aikonin alone or by using neutralizing antibody against human immunodeficiency virus (HIV neutralizing antibody). It provides a broad spectrum of anti-HIV, and provides a more effective option for HIV treatment and prevention.

In a first aspect of the present invention, there is provided an active ingredient combination, the active ingredient combination comprising a therapeutically effective amount of Aiconine and a human immunodeficiency virus neutralizing antibody, wherein the human immunodeficiency virus neutralizing antibody Included are the heavy chain shown in SEQ ID NO: 1 and the light chain shown in SEQ ID NO: 2, or long-acting mutants thereof.

In another preferred embodiment, the mass ratio of Aikenine to human immunodeficiency virus neutralizing antibody is 1:200 to 50:1, preferably 1:100 to 20:1, more preferably 1:20 to 10:1.

In another preferred embodiment, the administration concentration of Aikenine is selected from 0.1-1000mg/ml; preferably, 0.5-500mg/ml; more preferably, 2-320mg/ml; and the human immunodeficiency The administration concentration of virus neutralizing antibody is 0.5-5000 mg/ml; preferably, 1-2000 mg/ml; more preferably, 5-1000 mg/ml.

In another preferred example, the ratio (nmol/mg) of Aikenine to human immunodeficiency virus neutralizing antibody is 0.2-4000nmol/mg, preferably 1-2000nmol/mg, more preferably 5-1000nmol/mg /mg or 10-500nmol/mg.

In another preferred embodiment, the active ingredient combination further includes additional therapeutic drugs.

In another preferred embodiment, the additional therapeutic drug is an antiviral agent, an anti-infective drug or a combination thereof.

In another preferred embodiment, the active ingredient combination is used for the treatment and/or prevention of mammals or administered to mammals, more preferably, the mammals are rodents (such as mice, rats) or humans .

In another preferred embodiment, the long-acting mutant refers to the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 optionally subjected to addition, deletion, modification and/or substitution of at least one amino acid, and derived sequences that retain binding affinity to human immunodeficiency virus.

In the second aspect of the present invention, there is provided a medicine box, the medicine box comprises:

(a) a first preparation, the first preparation contains Aconine and a pharmaceutically acceptable carrier;

(b) a second formulation comprising human immunodeficiency virus neutralizing antibodies and a pharmaceutically acceptable carrier;

(c) Instructions describing a method of treating and/or preventing HIV in combination with human immunodeficiency virus neutralizing antibodies.

In another preferred embodiment, the first preparation and the second preparation are independent of each other.

In another preferred embodiment, the first preparation and the second preparation are freeze-dried preparations or liquid preparations.

In another preferred embodiment, the first preparation and the second preparation are injections.

In another preferred embodiment, the first formulation is administered before, during or after or mixed with the second formulation.

In another preferred embodiment, the human immunodeficiency virus neutralizing antibody includes an antibody with a heavy chain shown in SEQ ID NO: 1 and a light chain shown in SEQ ID NO: 2, or a long-acting mutant thereof .

In a third aspect of the present invention, a pharmaceutical composition is provided, the pharmaceutical composition comprising the above-mentioned combination of active ingredients and a pharmaceutically acceptable carrier.

In a fourth aspect of the present invention, there is provided a use of an active ingredient combination in the preparation of a pharmaceutical composition or kit for treating and/or preventing HIV, the active ingredient combination comprising a first active ingredient Aconine and The second active ingredient is human immunodeficiency virus neutralizing antibody.

In another preferred embodiment, the mass ratio of Aikenine to human immunodeficiency virus neutralizing antibody is 1:200 to 50:1, preferably 1:100 to 20:1, more preferably 1:20 to 10:1.

In another preferred embodiment, the administration concentration of Aikenine is selected from 0.1-1000mg/ml; preferably, 0.5-500mg/ml; more preferably, 2-320mg/ml; and the human immunodeficiency The administration concentration of virus neutralizing antibody is 0.5-5000 mg/ml; preferably, 1-2000 mg/ml; more preferably, 5-1000 mg/ml.

In another preferred example, the ratio (nmol/mg) of Aikenine to human immunodeficiency virus neutralizing antibody is 0.2-4000nmol/mg, preferably 1-2000nmol/mg, more preferably 5-1000nmol/mg /mg or 10-500nmol/mg.

In another preferred embodiment, the pharmaceutical composition or kit is used to treat and/or prevent mammals, more preferably, the mammals are rodents (eg, mice, rats) or humans.

In another preferred embodiment, the HIV comprises any one or a combination of HIV strains selected from the following group:

92US159, 99KE_KNH1088, 92BR025, 931N101, 02ET14, 92UG001, 00KENKU3006, 93BR020, G3, 96TH NI1149, 01CM_0002BBY, SF162, ADA, CNE1, CNE15, CNE17, BJOX25.01, BJOX27 and BJOX41 strains (synergy).

In another preferred embodiment, the HIV comprises any one or a combination of HIV strains selected from the following group: 92UG037, 00KE_KER2008, 91US001, 91US004, 96TH_NP1538, JR-CSF, 98US_MSC5016, 02ET288, 00UG_D26830M4, 93BR019, 93BR029, JV1083, 90TH_CM235, 97TH-NP 1525, 91DJ263, 02CM_0014BBY, BCF01, BCF02, BCF03 and CNE23 strains (additive effect).

In the fifth aspect of the present invention, there is provided a method for treating and preventing HIV, comprising the steps of: administering the combination of active ingredients described in the second aspect of the present invention and/or the combination of the active ingredients described in the third aspect to a subject (eg, a human) in need thereof Pharmaceutical compositions for the treatment and/or prevention of HIV.

In another preferred embodiment, the administration of Aconine is performed before, during and/or after or mixed with the human immunodeficiency virus neutralizing antibody administered to the subject.

In another preferred embodiment, the human immunodeficiency virus neutralizing antibody includes the heavy chain shown in SEQ ID NO: 1 and the light chain shown in SEQ ID NO: 2, or a long-acting mutant thereof.

In another preferred embodiment, the subject includes human and non-human mammals.

In another preferred embodiment, the subject is an HIV patient.

In another preferred embodiment, the HIV is selected from any one of the following HIV strains or a combination thereof:

92US159, 99KE_KNH1088, 92BR025, 931N101, 02ET14, 92UG001, 00KENKU3006, 93BR020, G3, 96TH NI1149, 01CM_0002BBY, SF162, ADA, CNE1, CNE15, CNE17, BJOX25.01, BJOX27 and BJOX41 strains.

In another preferred embodiment, the HIV is selected from any one of the following HIV strains or a combination thereof:

92UG037、00KE_KER2008、91US001、91US004、96TH_NP1538、JR-CSF、98US_MSC5016、02ET288、00UG_D26830M4、93BR019、93BR029、JV1083、90TH_CM235、97TH-NP 1525、91DJ263、02CM_0014BBY、BCF01、BCF02、BCF03和CNE23病毒株。

In a sixth aspect of the present invention, a kit for detecting HIV is provided, the kit comprising:

(1) a first container, the first container contains Aikenine;

(2) a second container containing HIV neutralizing antibodies; and

(3) Reagents for accompanying diagnosis of HIV virus.

In another preferred embodiment, the HIV comprises any one or a combination of HIV strains selected from the following group:

92US159, 99KE_KNH1088, 92BR025, 931N101, 02ET14, 92UG001, 00KENKU3006, 93BR020, G3, 96TH NI1149, 01CM_0002BBY, SF162, ADA, CNE1, CNE15, CNE17, BJOX25.01, BJOX27 and BJOX41 strains;

The HIV comprises any one or a combination of HIV strains selected from the group 92UG037, 00KE_KER2008, 91US001, 91US004, 96TH_NP1538, JR-CSF, 98US_MSC5016, 02ET288, 00UG_D26830M4, 93BR019, 93BR029, JV1083, 90TH_CM-235, 97TH NP 1525, 91DJ263, 02CM_0014BBY, BCF01, BCF02, BCF03, CNE23.

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (eg, the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, it is not repeated here.

Description of drawings

Figure 1 shows a schematic diagram of sample addition in the neutralization test;

Figure 2 shows the neutralizing activity of the HIV-neutralizing antibody of the present invention to HIV-1 epidemic strains in China;

Figure 3 shows the neutralizing activity of ABT against HIV-1 epidemic strains in China;

Fig. 4 shows the synergistic test result of HIV neutralizing antibody of the present invention and ABT on CNE1;

Fig. 5 shows the synergistic test result of HIV neutralizing antibody of the present invention and ABT on CNE15;

Fig. 6 shows the synergistic test result of HIV neutralizing antibody of the present invention and ABT to CNE17;

Fig. 7 shows the synergistic test result of HIV neutralizing antibody of the present invention and ABT on CNE19;

Fig. 8 shows the synergistic test result of HIV neutralizing antibody of the present invention and ABT on CNE23;

Fig. 9 shows the synergistic test result of HIV neutralizing antibody of the present invention and ABT showing 01 to BJOX25;

Figure 10 shows the synergistic test results of the HIV neutralizing antibody of the present invention and ABT on BJOX27;

Figure 11 shows the synergistic test results of the HIV neutralizing antibodies of the present invention and ABT on BJOX41;

Figure 12 shows gene taxonomy, catalog number and tropism information for HIV-1 isolates from the NIH AIDS Reagent Program.

Detailed ways

After extensive and in-depth research, the present inventors have discovered, for the first time, that Aikenine and human immunodeficiency virus neutralizing antibodies have a good synergistic effect in the treatment of viruses (especially HIV). The combined use of Aikenine and human immunodeficiency virus neutralizing antibody can effectively inhibit the growth of HIV virus, and its effect is far better than the additive effect of the two. On this basis, the present invention has been completed.

Preferably, the HIV virus includes viruses CNE1, CNE15, CNE17, BJOX25.01, BJOX27 and BJOX41 from China's circulating strains, and HIV strains 92US159, 99KE_KNH1088, 92BR025, 931N101, 02ET14, 92U G00 1 from NIH AIDS, 00KENKU3006, 93BR020, G3, 96TH NI1149, 01CM_0002BBY, SF162, ADA strains.

the term

As used herein, the terms "containing" or "including (including)" can be open, semi-closed, and closed. In other words, the term also includes "consisting essentially of," or "consisting of."

As used herein, the term "first active ingredient" refers to ecconine. It is to be understood that this term includes derivatives that have the same function as iconine.

As used herein, the term "second active ingredient" refers to human immunodeficiency virus neutralizing antibodies and long-acting mutants.

As used herein, the term "long-acting mutant" refers to an amino acid sequence set forth in SEQ ID NO: 1 or 2 that has optionally undergone addition, deletion, modification and/or substitution of at least one amino acid and is capable of retaining immunity to humans Derivative sequences deficient in viral binding affinity. Further, the long-acting mutant of the present invention refers to at most 10, preferably at most 8, more preferably at most 5, and optimally at most 3 amino acids compared with the amino acid sequence of the antibody of the present invention. Polypeptides formed by substitution of similar or similar amino acids. These long-acting mutants are best generated by amino acid substitutions according to Table A.

Table A

initial residue representative substitution Preferred substitution Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln; His; Lys; Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn Glu(E) Asp Asp Gly(G) Pro; Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe Leu Leu(L) Ile; Val; Met; Ala; Phe Ile Lys(K) Arg; Gln; Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Leu; Val; Ile; Ala; Tyr Leu Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Ala Leu

As used herein, the terms "combination of active ingredients of the present invention", "combination of substances of the present invention", "combination of pharmaceutical ingredients of the present invention" are used interchangeably and refer to the combination of the first and second active ingredients described above.

As used herein, the term "pharmaceutical composition of the present invention" refers to a composition comprising or containing a first active ingredient (or a first formulation containing the first active ingredient) and a second active ingredient (or a formulation containing the second active ingredient). second formulation), wherein the first formulation and the second formulation may be the same or different formulations. Furthermore, the first formulation and the second formulation may be the same formulation, or may be separate formulations.

The HIV-neutralizing antibody of the present invention can specifically bind to the CD4 binding site of HIV-1 outer membrane protein gp120. The HIV neutralizing antibody of the present invention is composed of 1318 amino acid residues, has a molecular weight of 146280 Daltons, and has a half-life of 17.6 days in a healthy human body. Preclinical and clinical results show that the antibody not only has efficient and broad virus neutralization ability, but also can activate the immune response and delay the time of virus rebound after drug withdrawal. In vitro experiments showed that the combination of Aikenine and this antibody has synergistic or additive antiviral activity.

Both Aikoning and this antibody have a unique new mechanism of action. The two act on different envelope glycoproteins of HIV, respectively blocking their entry into cells. The combined use is expected to cover a wider range of virus strains, enhance and prolong Antiviral effect and reduce the generation of drug resistance; the two have the common characteristics of long half-life and intravenous administration. When used together, they can form a complete new drug formula for full injection and long-acting anti-AIDS. The frequency of administration is expected to be reduced to weekly or biweekly. , once a month or a few months to change the treatment mode of daily medication.

As used herein, "human immunodeficiency virus neutralizing antibody of the present invention", "HIV neutralizing antibody of the present invention", and "human immunodeficiency virus HIV neutralizing antibody of the present invention" are used interchangeably and refer to the following The heavy chain, light chain sequence of human immunodeficiency virus neutralizing antibody, which includes heavy chain (SEQ ID NO: 1) and light chain (SEQ ID NO: 2):

Heavy chain (SEQ ID NO: 1):

QVQLLQSGAAVTKPGASVRVSCEASGYNIRDYFIHWWRQAPGQGLQWVGWINPKTGQPNNPRQFQGRVSLTRHASWDFDTFSFYMDLKALRSDDTAVYFCARQRSDYWDFDVWGSGTQVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Light chain (SEQ ID NO: 2):

DIQMTQSPSSLSASVGDTVTITCQANGYLNWYQQRRGKAPKLLIYDGSKLERGVPSRFSGRRWGQEYNLTINNLQPEDIATYFCQVYEFVVPGTRLDLKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC;

As used herein, "aconine," "exavitax," and "ABT" are used interchangeably and all refer to compounds having the structures shown below. The structure of Aikonin in the present invention is as follows:

Expression vector

As used herein, the term "expression vector" refers to a vector capable of transferring a polynucleotide sequence of interest to a target cell. Such vectors can self-replicate or integrate into host cell chromosomes (host cells include, for example, prokaryotic cells, yeast, animal cells, plant cells, insect cells, animal individuals, and plant individuals, etc.), and can be used in polynuclear cells suitable for the present invention. The site of nucleotide transcription contains a promoter. Expression vectors may contain structural genes and promoters that regulate their expression, in addition to various regulatory elements that function in the host cell. It is well known in the art that the type of expression vector for a living organism (eg, animal) and the type of regulatory elements used can vary depending on the type of host cell used.

The viral vector that can be used in the present invention is not particularly limited, and can be any viral vector that can utilize the characteristics of viruses to transmit their genomes to bring genetic material into other cells for infection. Can occur in whole in vivo or in cell culture. Including lentiviral vector, adenoviral vector, herpes virus vector, poxvirus vector. In the present invention, a preferred expression vector is a lentiviral vector.

Active Ingredient Combinations, Pharmaceutical Compositions, and Modes of Administration

The present invention also provides an active ingredient combination that can be used for synergistic treatment of HIV, which can be used to inhibit HIV virus replication. The active ingredient combination of the present invention comprises: an effective amount of Aconine, and an effective amount of human immunodeficiency virus neutralizing antibody.

The present invention also provides a pharmaceutical composition comprising the active ingredient combination and a pharmaceutically acceptable carrier.

Unless otherwise specified, the "combination of active ingredients" herein may be admixed or separately formulated into iconic and neutralizing antibodies, used sequentially or simultaneously. The "pharmaceutical composition" may be formulated as a liquid preparation. The preparation mainly includes an effective amount of Aconine and an effective amount of neutralizing antibody against human immunodeficiency virus, which can be mixed or configured into Aconine and a neutralizing antibody, which can be used separately or simultaneously.

In general, the econine of the present invention, or the human immunodeficiency virus neutralizing antibody, can be formulated in a non-toxic, inert and pharmaceutically acceptable carrier medium, wherein the pH is usually about 5-9, preferably, pH is about 6-8.

As used herein, "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent, including various excipients and diluents.

The term refers to pharmaceutical carriers which are not themselves essential active ingredients and which are not unduly toxic after administration. Suitable carriers are well known to those of ordinary skill in the art. Pharmaceutically acceptable carriers in the compositions may contain liquids such as water, saline, buffers. In addition, auxiliary substances such as fillers, lubricants, glidants, wetting or emulsifying agents, pH buffering substances and the like may also be present in these carriers. The carrier may also contain cell transfection reagents.

As used herein, the term "effective amount" or "effective dose" refers to an amount that is functional or active in humans and/or animals and/or cells and which is acceptable to humans and/or animals.

As used herein, a "pharmaceutically acceptable" ingredient is one that is suitable for use in humans and/or mammals without undue adverse side effects (eg, toxicity, irritation, and allergy), ie, a substance with a reasonable benefit/risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent, including various excipients and diluents. Such carriers include, but are not limited to, saline, buffers, dextrose, water, glycerol, polysorbates, ethanol, and combinations thereof. Usually, the pharmaceutical preparation should match the mode of administration, and the pharmaceutical composition of the present invention can be prepared in the form of injection, for example, by using normal saline or an aqueous solution containing glucose and other adjuvants by conventional methods. The pharmaceutical compositions are preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount. The pharmaceutical preparation of the present invention can also be made into a sustained-release preparation.

Preferably, the pharmaceutical composition of the present invention can be prepared in the form of a liquid preparation. The preparation mainly comprises an effective amount of Aconine, and an effective amount of human immunodeficiency virus neutralizing antibody; a buffer solution; a stabilizer; and a surfactant. Wherein, the buffer is an acidic buffer, and the acidic buffer can be a buffer of acetic acid, phosphoric acid, tartaric acid, ascorbic acid, malonic acid, maleic acid, fumaric acid, malic acid or oxalic acid; the stabilizer can be selected from One or more combinations of sodium chloride, amino acids, and polyols; the stabilizer may comprise polyols and/or sodium chloride, and the polyols are selected from sucrose, trehalose or sorbose; the The surfactant can be Tween 20 or Tween 80.

One or more other pharmaceutically acceptable carriers, excipients or stabilizers, such as those described in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980), may be included in the pharmaceutical compositions of the present invention, provided that They do not adversely affect the desired characteristics of the formulation. Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed, and include other co-solvents; antioxidants, including ascorbic acid and methionine; integrators, such as EDTA; metals complexes (eg, Zn-protein complexes); biodegradable polymers, such as polyesters; and/or salt-forming counterions.

The pharmaceutical composition of the present invention can be prepared by combining various components in a certain concentration by methods known in the art.

In addition, the active ingredient combinations of the present invention may also be used with other therapeutic agents such as antiviral or immunomodulatory agents.

When using a pharmaceutical composition, a safe and effective amount of a combination of active ingredients (including the first active ingredient (or a formulation thereof) and/or the second active ingredient (or a formulation thereof)) is administered to the mammal.

It should be understood that the effective amount of the first active ingredient (or its formulation) and/or the second active ingredient (or its formulation) in the active ingredient combination of the present invention may vary with the mode of administration and the severity of HIV, etc. . Preferably, selection of an effective amount can be determined by one of ordinary skill in the art based on a variety of factors (eg, through clinical trials). Such factors include, but are not limited to: pharmacokinetic parameters such as bioavailability, metabolism, half-life, etc.; severity of HIV, patient's weight, patient's immune status, route of administration, and the like.

Typically, for the first active ingredient, a safe and effective amount is usually at least about 10 ng/kg body weight, and in most cases no more than about 50 mg/kg body weight, preferably the dose is about 50 ng/kg body weight Body weight - about 10 mg/kg body weight. Of course, the specific dosage should also take into account the route of administration, the patient's health and other factors, which are all within the skill of the skilled physician.

For the second active ingredient, a safe and effective amount is usually at least about 10 ng/kg body weight, and in most cases no more than about 50 mg/kg body weight, preferably the dose is about 50 ng/kg body weight - about 10 mg/kg body weight. Of course, the specific dosage should also take into account the route of administration, the patient's health and other factors, which are all within the skill of the skilled physician.

Specifically, the active ingredient combination of the present invention includes the first active ingredient Aiconine and the second active ingredient human immunodeficiency virus neutralizing antibody.

In another preferred embodiment, the mass ratio of Aikenine to human immunodeficiency virus neutralizing antibody is 1:200 to 50:1, preferably 1:100 to 20:1, more preferably 1:20 to 10:1.

In another preferred embodiment, the administration concentration of Aikenine is selected from 0.1-1000mg/ml; preferably, 0.5-500mg/ml; more preferably, 2-320mg/ml; and the human immunodeficiency The administration concentration of virus neutralizing antibody is 0.5-5000 mg/ml; preferably, 1-2000 mg/ml; more preferably, 5-1000 mg/ml.

In another preferred example, the ratio (nmol/mg) of Aikenine to human immunodeficiency virus neutralizing antibody is 0.2-4000nmol/mg, preferably 1-2000nmol/mg, more preferably 5-1000nmol/mg /mg or 10-500nmol/mg.

The mode of administration of the pharmaceutical composition of the present invention is not particularly limited, and representative examples include (but are not limited to): intravenous injection, subcutaneous injection, intramuscular injection, and the like.

Pill box

The present invention provides a medicine box, the medicine box comprises:

Component (1): a preparation containing Aikenine;

Component (2): a preparation containing human immunodeficiency virus neutralizing antibodies;

Component (3): Instructions.

The formulations containing iconic include (but are not limited to): lyophilized formulations, liquid formulations or intravenous injections.

The preparations containing human immunodeficiency virus neutralizing antibodies include (but are not limited to): lyophilized preparations, liquid preparations, tablets, capsules, suppositories, or intravenous solutions.

In the present invention, protein preparations are usually freeze-dried preparations or injections.

Typically, the kit contains one or more (eg, at least two) unit dosage forms containing Aiconine and one or more (eg, at least two) unit dosage forms containing human immunodeficiency virus neutralizing antibodies; preferably Each place is 2-20.

As used herein, the term "unit dosage form" refers to the preparation of a composition into a dosage form required for a single administration for convenience of administration, including but not limited to various solids (eg, tablets), liquids, capsules, sustained-release preparations agent.

The instructions provided by the present invention can be described as follows: the using method of the kit is to use the unit dosage form containing iconine and the unit dosage form containing human immunodeficiency virus neutralizing antibody at the same time.

The medicine kit provided by the present invention is prepared by the following steps: placing the preparation containing econine and the preparation containing the neutralizing antibody of human immunodeficiency virus and the instructions together to form a medicine box.

Said preparation containing aconine preferably contains a unit dosage form of aconine, and said preparation containing a neutralizing antibody against human immunodeficiency virus preferably contains a unit dosage form of neutralizing antibody against human immunodeficiency virus.

In the step, preferably, at least one unit dosage form containing iconine and at least one unit dosage form containing human immunodeficiency virus neutralizing antibody, together with the instructions, are placed together to form a kit.

The main advantages of the present invention include

1. Aikonin and human immunodeficiency virus neutralizing antibodies can synergistically act on anti-HIV therapy, thereby inhibiting the proliferation of HIV virus more significantly.

2. Since ebavitaxel and HIV neutralizing antibodies act on gp41 and gp120 of viral glycoproteins in the entry stage of HIV virus entering human T cells, respectively, gp41 and gp120 constitute gp160. The first is that the gp120 part of HIV binds to human CD4, triggering binding to the coreceptor in the human body, resulting in a conformational change gp120 and gp41 dissociation, fully exposing gp41 and forming a trimeric transition state, and then fused to form a hexameric virus successfully into human T cells. The combined use of Aikenine acting on gp41 and HIV neutralizing antibody acting on gp120 is beneficial to improve the deficiency of insufficient virus coverage of the antibody or ABT; experiments show that the combination of Aikenine and the HIV neutralizing antibody of the present invention After use, it has synergistic and broad-spectrum antiviral activity against a variety of HIV strains, thus providing a new and more effective option for HIV treatment and/or prevention; and because of its synergistic and Broad-spectrum, more effective detection of HIV infection.

The present invention is further described below in conjunction with specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental method of unreceipted detailed conditions in the following examples, usually according to normal conditions such as people such as Sambrook, molecular cloning: conditions described in laboratory manual (New York:Cold Spring Harbor LaboratoryPress, 1989), or according to manufacturer's instructions. recommended conditions. Percentages and parts are by weight unless otherwise indicated.

Reagent and sample configuration

1) Ghost(3)X4/R5 cells, 293T cells, HIV-1 membrane protein expression plasmid, HIV-1 backbone plasmid pNL4-3.Luc.R-E- are all stored in the laboratory;

2) Human serum albumin, Albumin human, batch number: SLBS2806V, American Sigma company;

3) DMEM cell culture medium, 8117156, Gibco, USA;

4) Fetal bovine serum: FBS, batch number: 1856032, Gibco, USA;

5) 0.25% trypsin: Trypsin-EDTA 0.25%, batch number: G1509200, Macgene, USA;

6) Luciferase detection kit: Bright-Glo ^™ Luciferase Assay System, batch number: 173MB, Beijing Promage Company.

7) The HIV neutralizing antibody of the present invention (Lot#201307A) and Aibovitex (batch number 151201) were provided by Frontier Biopharmaceutical (Nanjing) Co., Ltd.

8) ABT-albumin preparation:

① Prepare 25% human serum albumin (3.79mM HSA) solution.

② Prepare 10mM (46.66mg/mL) Abovactide-dimethyl sulfoxide (DMSO) solution.

③ Prepare a 1 mM ebaviret-HSA (molecular ratio of about 1:3) solution.

④ in 37 ℃ water bath for 3 hours. Aliquot and store at 4°C.

9) Experimental equipment: microplate luminometer, Berthold Technologies, Germany.

10) Experimental data analysis software: Graphpad Prism 5.0, for processing, analyzing and neutralizing experimental data and drawing graphs.

general method

1. Packaging of HIV-1 pseudovirus

(1) 1×10 ⁷ 293T cells resuspended in DMEM medium with 10% FBS were seeded into a dish, and cultured overnight at 37° C., 5% CO ₂ . The bottom coverage of the monolayer of adherent cells should reach 80%-90% on the day of transfection.

(2) An appropriate amount of HIV-1 membrane protein expression plasmid and pNL4-3.Luc.R-E- were added to an EP tube containing serum-free DMEM medium. An appropriate amount of PEI was added to the above mixture, mixed well, and incubated at room temperature to form a transfection complex. Add the transfection complex to the culture dish of 293T cells and mix well.

(3) Incubate at 37°C, 5% CO ₂ for 6-8 hours, and the plasmids are transfected into cells. Carefully remove the medium containing the DNA-PEI transfection complex, add an appropriate amount of fresh 10% FBS DMEM medium, and continue the culture.

(4) After continuing the culture, collect the culture supernatant containing pseudovirus particles from the petri dish, centrifuge to remove cell debris and impurities, collect the supernatant, and store the aliquoted tube at -80°C.

2. Determination of pseudovirus titers with Ghost(3)X4/R5 cells

(1) 1×10 ⁴ Ghost(3)X4/R5 cells were seeded in each well of a 96-well plate, and incubated overnight (at least 15 hours).

(2) Dilute the virus with DMEM at a ratio of 1:2 and 1:10, respectively, add 100 μl of the diluted virus solution to each well of the 96-well plate inoculated with the cells, and incubate.

(3) After incubation, remove all the culture supernatants in the cell culture wells, then add an appropriate amount of PBS and luciferase detection reagent, and place in the dark for 5 minutes. After pipetting, transfer the above-mentioned cell lysis liquid to a 96-well white bottom plate, and measure the luciferase activity with a chemiluminometer.

3. Pseudovirus neutralization assay

(1) Add 300 μl of PBS to the surrounding wells of the 96-well plate (the surrounding wells are severely evaporated and are not used for testing). The schematic diagram of sample addition in the neutralization test is shown in Figure 1, and each sample was measured in 3 replicates.

HIV neutralizing antibody neutralization test of the present invention: add 100 μl of 10% FBS DMEM medium to each well of 60 wells in the middle, and the two leftmost columns are virus control (Virus Control, VC, only add virus and cells) and cell control ( CellControl, CC, cells only). Column CC was supplemented with 50 [mu]l medium (150 [mu]l total), and column 11 was supplemented with 39 [mu]l medium (139 [mu]l total). Among them, S1 and S2 are the wells of different concentrations in the sample loading column.

Neutralization test of peptide ebavitaxel: add 100 μl of 10% FBS DMEM medium to each well of column VC, add 150 μl of 10% FBS DMEM medium to each well of column CC, and add no medium to columns 4 to 11 until the polypeptide is diluted Join when done.

(2) Concentration gradient setting

HIV neutralizing antibody neutralization test of the present invention: add 11 μL of antibody (11 μg, 1 mg/ml) to each well in column 11, and the total volume is 150 μL. After mixing with a row gun, aspirate 50 μL to the 11th column, and then aspirate 50 μl and transfer it to the 10th column. Repeat this to the left for 3-fold dilution, and finally aspirate 50 μL from the 4th column and discard. The final antibody concentration from column 4 to column 11 was 0.022, 0.067, 0.201, 0.604, 1.811, 5.432, 16.296, 48.889 (μg/ml), respectively. The final volume of each well was 100 μl. Cell control and virus control wells were not involved in fold dilution.

Neutralization test of peptide albovirtide: The peptide ABT was diluted 8 times in DMEM medium containing 10% FBS, and the final concentrations of ABT-albumin (calculated as ABT) from 4 to 11 columns were 0.0233, 0.186, and 1.491, respectively. , 11.936, 95.486, 763.889, 6111.111, 48888.89 (nM). The concentration of each column was prepared separately, and then 100 μl of peptides were added in sequence, and the final volume of each well was 100 μl. Cell control and virus control wells were not involved in fold dilution.

(3) Add 50 μl of pseudovirus with a titration reading value of more than 100,000 to each well except the cell control well. Incubate the 96-well plate in a 37°C, 5% CO ₂ incubator to allow the antibody to fully bind to the virus particles, take out the 96-well plate, and add Ghost(3)X4/R5 cells to all 60 holes in the middle, and continue the culture. 48-72 hours.

(4) Take out after 48-72 hours, discard the medium supernatant, add PBS, then add luciferase detection reagent, measure the luciferase activity, and calculate the percentage inhibition. Neutralization curves were fitted and _IC50s were calculated. The calculation formula of neutralization inhibition rate is: inhibition rate=[1-(mean value of luminescence intensity of sample group-mean value of blank control CC)/(mean value of luminescence intensity VC of negative group-mean value of blank control CC)]×100%.

4. HIV neutralizing antibody and ABT neutralization synergy test of the present invention

According to the IC ₅₀ values obtained by the HIV neutralizing antibody of the present invention and the ABT neutralization test alone, the highest concentrations of the HIV neutralizing antibody and ABT of the present invention were respectively set as 3 to ⁴ times their IC ₅₀ values, and a 3-fold dilution was carried out. , a total of 8 dilutions (the concentration of the 5th dilution is about the IC ₅₀ value), and each sample was measured in 3 wells. Using the test system described above, the neutralizing activity of the HIV-neutralizing antibody of the present invention administered in combination with ABT was evaluated. CompuSyn software (ComboSyn Inc., Paramus, NJ) was used to evaluate whether the HIV neutralizing antibody of the present invention and ABT combined possess synergy, additive or antagonism according to the median effect analysis method (Chou and Talalay). antiviral activity. The three main parameters used to evaluate the synergy test are fractional effects (FA), combined index (CI), logCI. In this experiment, FA refers to the neutralization percentage of the HIV neutralizing antibody, ABT, etc. of the present invention to the virus; CI is the combined index. Using the index, logCI is the log value of CI. When CI>1, that is, logCI>0, it indicates that the two drugs have antagonistic effects; when CI=1, that is, logCI=0, it indicates that the two drugs have additive effects; <1, i.e. logCI<0, indicates that the two drugs have synergistic effects.

Example 1 Neutralizing activity of HIV-neutralizing antibodies against HIV-1 epidemic strains in China

The test evaluated the neutralizing activity of the HIV-neutralizing antibody of the present invention to 22 HIV-1 pseudoviruses in the acute phase and chronic phase. VRC01 was used as a positive control, and its neutralizing activity was consistent with previous laboratory data, and the methodology was reliable.

The test results show that the HIV neutralizing antibody of the present invention can neutralize 77.2% of the circulating virus in China, the median IC ₅₀ and IC ₉₀ are 0.06 and 0.78 μg/ml, respectively, and the average IC ₅₀ and IC ₉₀ are 0.32±0.51 and 4.58 ±11.09 μg/ml. The HIV neutralizing antibody of the present invention cannot neutralize CNE6, CNE7, CNE15, CNE23 and BJOX41 strains, and its IC ₅₀ is >50 μg/ml. See Table 1 and Figure 2.

Table 1. The neutralizing activity of HIV-neutralizing antibodies of the present invention to HIV-1 epidemic strains in China

*:Except _IC50 or IC90>50μg/ml

Example 2 ABT neutralizing activity against HIV-1 Chinese epidemic strain pseudovirus

The neutralization activity of ABT against 22 HIV-1 pseudoviruses circulating in China in the acute phase and chronic phase was evaluated. VRC01 was used as a positive control, and the neutralizing activity was consistent with previous laboratory data. The test results showed that the median IC ₅₀ and IC ₉₀ of ABT against the circulating virus in China were 3.61 and 964.48 nM, respectively, and the average IC ₅₀ and IC ₉₀ were 71.41 ± 116.26 and 2101.42 ± 2258.81 nM, as shown in Table 2 and Figure 3.

The neutralization activity test is mainly used to evaluate the neutralization ability of monoclonal antibodies against HIV-1 virus. Aibovirtide is a synthetic polypeptide of 34 amino acids. The results of this test show that Aibovirtide has a certain ability to neutralize the HIV virus. However, the individual differences in the test were large, and the IC ₅₀ ranged from 0.00343 to 254.849nM.

Table 2. Neutralizing activity of ABT against HIV-1 epidemic strains in China

Example 3 HIV neutralizing antibody and ABT neutralization synergy test

The experiment evaluated the neutralizing activity of the HIV-neutralizing antibody of the present invention and ABT in combination with 8 Chinese epidemic strains of HIV-1 in acute phase and chronic phase.

As shown in Tables 3 and 4, the test shows that the combination of HIV neutralizing antibody and ABT of the present invention has synergistic anti-disease activity against 6 strains of viruses CNE1, CNE15, CNE17, BJOX25.01, BJOX27 and BJOX41; For additive effect; for CNE19 showed antagonism.

Table 3. HIV-neutralizing antibodies of the invention and ABT-neutralizing activity synergistically

Table 4. Results of neutralizing activity of HIV neutralizing antibodies of the present invention and ABT synergistic assay

(1) Synergistic effect on CNE1

Both the HIV-neutralizing antibody of the present invention and ABT have neutralizing activity on CNE1. After the HIV-neutralizing antibody of the present invention is used in combination with ABT, the neutralizing effect is significantly improved, and there is a synergistic relationship (Table 5, Figure 4).

Table 5. The neutralizing activity of HIV-neutralizing antibodies and ABTs of the present invention on CNE1

(2) Synergistic effect on CNE15

The HIV-neutralizing antibody of the present invention cannot neutralize CNE15, but the combined use of the HIV-neutralizing antibody of the present invention and ABT can improve the neutralizing activity of ABT on CNE15, and there is a synergistic effect (Table 6, Figure 5).

Table 6. The neutralizing activity of HIV-neutralizing antibodies and ABTs of the present invention on CNE15

(3) Synergistic effect on CNE17

Both the HIV neutralizing antibody and ABT of the present invention have neutralizing activity on CNE17, and the neutralizing activity of the two drugs in combination above the IC ₅₀ concentration increases, indicating that they have a synergistic effect at the therapeutic concentration. The neutralizing activity of the combination was reduced at lower concentrations, possibly antagonism (Table 7, Figure 6).

Table 7. The neutralizing activity of HIV-neutralizing antibodies and ABTs of the present invention on CNE17

(4) Antagonistic effect on CNE19

Both ABT and the HIV-neutralizing antibody of the present invention have neutralizing activity on CNE19, and the combined use shows no significant change in neutralizing activity. The combined use index is greater than 1, and there may be a certain antagonism (Table 8-9, Figure 7).

Table 8. HIV neutralizing antibody and ABT of the present invention neutralize synergistic test results for CNE19

Table 9. Neutralizing activity of HIV-neutralizing antibodies and ABTs of the present invention on CNE19

(5) Additive effect on CNE23

The HIV-neutralizing antibody of the present invention has no neutralizing activity against CNE23. When the HIV-neutralizing antibody of the present invention is used in combination with ABT, the neutralizing effect is similar to that of ABT alone, and there is an additive effect (Table 10, Figure 8).

Table 10. The neutralizing activity _IC50 of HIV neutralizing antibodies and ABTs of the present invention to CNE23

(6) Synergistic effect on BJOX25.01

The HIV-neutralizing antibody of the present invention has a weak neutralizing activity against BJOX25.01, but the combination of ABT and the HIV-neutralizing antibody of the present invention improves the neutralizing effect and has a synergistic effect (Table 11, Figure 9).

Table 11. Neutralizing activity of HIV neutralizing antibodies and ABTs of the present invention on BJOX25.01

(7) Synergistic effect on BJOX27

Both ABT and the HIV-neutralizing antibody of the present invention can neutralize and activate BJOX27. When the HIV-neutralizing antibody of the present invention is used in combination with ABT, the neutralizing effect of the single drug is improved, and there is a synergistic effect (Table 12, Figure 10). .

Table 12. Neutralizing activity of HIV-neutralizing antibodies and ABTs of the present invention on BJOX27

(8) Synergistic effect on BJOX41

The HIV-neutralizing antibody of the present invention cannot neutralize BJOX41, but the combined use of the HIV-neutralizing antibody of the present invention and ABT can enhance the neutralizing activity of ABT on BJOX41, showing a synergistic effect (Table 13, Figure 11).

Table 13. Neutralizing activity of HIV neutralizing antibodies and ABTs of the present invention on BJOX41

Example 4 Activity test of HIV neutralizing antibody and ABT on American HIV virus

Using a method similar to that in Examples 1-3, the neutralizing activity of the HIV-neutralizing antibodies of the present invention and ABT administered in combination against 32 HIV strains (Figure 12) from the NIH AIDS Reagent Program was evaluated.

Determination of combined effect:

Combination effects were determined using Chou and Talalay and CompuSyn software (CalcuSyn 2.0, BIOSOFT PO Box 1013, Great Shelford, Cambridge CB22 5WQ GB-United Kingdom, http://www.biosoft.com/w/calcusyn.htm). After subtracting the background signal control (cells only), the raw relative light unit data was expressed as a fraction of the maximum signal control (cells + virus) using the equation: 1-(experimental value/average virus control value). Combination index values were then calculated at ₅₀ % HIV inhibition (CI50). The combination index (CI) value represents the combined effect of two or more compounds. CI values <0.5 indicate strong compound combination synergy; >0.5 to 0.75 indicate synergy; ~0.76-1.25 indicate additivity; 1.26-1.5 indicate antagonism, and >1.5 indicate high antagonism. The dose reduction index (DRI) can also be calculated and indicates how much the concentration of compounds can be reduced to produce the same effect when acting in combination when acting alone (not in combination).

where CI = combination index, IC = inhibitory concentration, x = percent effect parameter, A = dose of drug A at time x, B = dose of drug B at time x, A+B = dose of combination drug at time x time dose.

where DRI = dose reduction index, IC = inhibitory concentration, x = percent effect parameter, A = dose of drug A at x, B = dose of drug B at x, A+B = combination drug at x dose at the time.

Experimental results: The results of combination index, inhibitory concentration value and dose reduction index at half HIV replication are shown in Table 14 below. Among them, CI value <0.5 indicates strong compound combination synergy; >0.5 to 0.75 indicates synergy; ~0.76-1.25 indicates additivity; 1.26-1.5 indicates antagonism, and >1.5 indicates high antagonism.

The results show that the HIV neutralizing antibody and ABT of the present invention have a synergistic effect on 92US159, 99KE_KNH1088, 92BR025, 931N101, 02ET14, 92UG001, 00KENKU3006, 93BR020, G3, 96TH NI1149, 01CM_0002BBY, SF162, ADA strains;对931N101、02ET14毒株具有强协同作用(CI ₅₀ <0.5)；对92UG037、00KE_KER2008、91US001、91US004、96TH_NP1538、JR-CSF、98US_MSC5016、02ET288、00UG_D26830M4、93BR019、93BR029、JV1083、90TH_CM235、97TH-NP1525、 91DJ263, 02CM_0014BBY, BCF01, BCF02, BCF03 showed additive effect.

Table 14. Summary of Combination Index, Inhibitory Concentration Values, and Dose Reduction Index at the maximal half of HIV replication

¹ ABT-HSA: 3BNC1 17 (μg/mL:μg/mL)

² -half maximal inhibitory combination index (C1 ₅₀ ) +/- standard deviation (SD), N=3

³ Interpretation of CI ₅₀ values: <0.5 means strong synergy; 0.5 to 0.75 means synergy; ~0.76-1.25 means additive; 1.26-1.5 means antagonistic; >1.5 means highly antagonistic (Syn=Synergistic, Add=Additive)

⁴ 2 isolates out of 3 experiments were insensitive to 3BNC117

⁵ cannot be calculated; it can be assumed that the combined effect is additive;

⁶ Taking into account the error (SD), DRI ~ 1.

It is worth noting that when the HIV neutralizing antibody of the present invention is used alone, it is not sensitive to HIV strains of 97TH-NP 1525, BCF01, BCF02, and BCF03, but after combined use with ABT, the neutralizing effect is improved, and the The effects of the strains are additive;

The HIV neutralizing antibody of the present invention has low activity on HIV virus strains of 92UG001 and 01CM_0002BBY when used alone, but can improve the neutralizing effect after being used in combination with ABT, and has a synergistic effect on the virus strains. The above results show that the combined use of the HIV neutralizing antibody of the present invention and ABT expands the antiviral broad-spectrum of the HIV neutralizing antibody of the present invention, and exhibits a synergistic effect on a large number of viruses.

in conclusion

The present invention evaluates the neutralizing activity of the HIV-neutralizing antibody and ABT of the present invention to 22 HIV-1 Chinese acute and chronic circulating pseudoviruses through experiments, and the results show that the HIV-neutralizing antibody of the present invention has a broad spectrum to Chinese circulating strains Neutralizing activity, can neutralize 77.2% of the virus. The median IC ₅₀ of the HIV neutralizing antibody of the present invention to 17 Chinese epidemic strains is 0.06 μg/ml, and the median IC ₉₀ is 0.78 μg/ml. The HIV neutralizing antibody of the present invention cannot neutralize CNE6 of B' subtype, BJOX41 of B subtype, CNE7 and CNE15 of B'C subtype, and CNE23 of CRF_08BC subtype, and its IC ₅₀ is >50 μg/ml. Aibovirtide has neutralizing activity against Chinese epidemic strains with a median IC ₅₀ of 3.61 nM and an average IC ₅₀ of 71.41±116.26 nM.

The combined use of antiviral activity in vitro shows that the HIV-neutralizing antibody and ABT of the present invention have synergistic anti-disease activity against 6 strains of viruses CNE1, CNE15, CNE17, BJOX25.01, BJOX27 and BJOX41; it has an additive effect on CNE23 virus; It has an antagonistic effect on CNE19.

The test in Example 4 evaluated the neutralizing activity of the HIV neutralizing antibodies and ABTs of the present invention against 32 HIV strains from the NIH AIDS Reagent Program, and showed that the neutralizing activity against HIV including A, B, C, D, F, G, BF , CRF01_AE, CRF02_AG, Group O and other HIV virus subtypes have broad-spectrum antiviral activity. The combined use of antiviral activity in vitro shows that the HIV neutralizing antibody and ABT of the present invention have synergistic or additive antiviral activity against the above 32 HIV strains.

This shows that the combination of ABT and HIV neutralizing antibodies is beneficial to improve the deficiency of insufficient viral coverage of antibodies or ABT, and has synergistic and broad-spectrum antiviral activity against a variety of HIV strains, so as to provide HIV treatment. And prevention provides new and more effective options.

All documents mentioned herein are incorporated by reference in this application as if each document were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

sequence listing

<110> Frontier Biopharmaceutical (Nanjing) Co., Ltd.

<120> The active combination of combined treatment of human immunodeficiency virus and its application

<130> P2022-1082

<150> 202110872475X

<151> 2021-07-30

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Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn

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Claims (10)

1. an active ingredient combination, it is characterized in that, described active ingredient combination comprises the Aikening of effective therapeutic dose and human immunodeficiency virus neutralizing antibody, wherein, described human immunodeficiency virus neutralizing antibody comprises as SEQ ID The heavy chain shown in NO: 1 and the light chain shown in SEQ ID NO: 2; or a long-acting mutant thereof. 2. active ingredient combination as claimed in claim 1 is characterized in that, the mass ratio of described Aikenine and human immunodeficiency virus neutralizing antibody is 1:200 to 50:1, preferably 1:100 to 20:1, more preferably 1:20 to 10:1. 3. The combination of active ingredients according to claim 1, characterized in that, the administration concentration of Aikenine is selected from 0.1-1000mg/ml; preferably, 0.5-500mg/ml; more preferably, 2- and the administration concentration of the human immunodeficiency virus neutralizing antibody is 0.5-5000 mg/ml; preferably, 1-2000 mg/ml; more preferably, 5-1000 mg/ml. 4. active ingredient combination as claimed in claim 1 is characterized in that, the ratio (nmol/mg) of described Aikenine and human immunodeficiency virus neutralizing antibody is 0.2-4000nmol/mg, preferably 1- 2000 nmol/mg, more preferably 5-1000 nmol/mg or 10-500 nmol/mg. 5. The active ingredient combination according to any one of claims 1 to 4, wherein the active ingredient combination further comprises additional therapeutic drugs (such as antiviral agents, anti-infective drugs or combinations thereof); preferably Preferably, the active ingredient combination is used for the treatment and/or prophylaxis of or administered to a mammal, more preferably the mammal is a rodent (eg, mouse, rat) or a human. 6. A medicine box, characterized in that, the medicine box comprises: (a) a first preparation, the first preparation contains Aconine and a pharmaceutically acceptable carrier; (b) a second formulation comprising human immunodeficiency virus neutralizing antibodies and a pharmaceutically acceptable carrier; (c) instructions describing a method of treating and/or preventing HIV in combination with Aiconine and human immunodeficiency virus neutralizing antibodies, and a method of treating and/or preventing HIV in combination with other antiviral drugs; Preferably, the kit further includes additional therapeutic agents (eg, antiviral agents, anti-infective agents, or combinations thereof). 7. A pharmaceutical composition comprising the active ingredient combination of any one of claims 1 to 5, further comprising a pharmaceutically acceptable carrier. 8. The application of an active ingredient combination in the preparation of a pharmaceutical composition or a kit for treating and/or preventing HIV, the active ingredient combination comprising the first active ingredient Aikenine and the second active ingredient human immunodeficiency virus neutralizing antibodies; Preferably, the human immunodeficiency virus neutralizing antibody is selected from antibodies comprising the heavy chain shown in SEQ ID NO: 1 and the light chain shown in SEQ ID NO: 2; or a long-acting mutant thereof; More preferably, the mass ratio of Aikenine to human immunodeficiency virus neutralizing antibody is 1:200 to 50:1, preferably 1:100 to 20:1, more preferably 1:20 to 10 :1; Further preferably, the administration concentration of said Aikenine is selected from 0.1-1000mg/ml; preferably, 0.5-500mg/ml; more preferably, 2-320mg/ml; and said human immunodeficiency virus neutralization The administration concentration of the antibody is 0.5-5000 mg/ml; preferably, 1-2000 mg/ml; more preferably, 5-1000 mg/ml; Still preferably, the ratio (nmol/mg) of described Aikenine to human immunodeficiency virus neutralizing antibody is 0.2-4000nmol/mg, preferably 1-2000nmol/mg, more preferably 5-1000nmol/mg or 10-500 nmol/mg. 9. The use of claim 8, wherein the HIV comprises any one or a combination of HIV strains selected from the group consisting of: 92US159, 99KE_KNH1088, 92BR025, 931N101, 02ET14, 92UG001, 00KENKU3006, 93BR020, G3, 96TH NI1149, 01CM_0002BBY, SF162, ADA, CNE1, CNE15, CNE17, BJOX25.01, BJOX27 and BJOX41 strains; The HIV includes any one or a combination of HIV strains selected from the group 92UG037, 00KE_KER2008, 91US001, 91US004, 96TH_NP1538, JR-CSF, 98US_MSC5016, 02ET288, 00UG_D26830M4, 93BR019, 93BR029, JV1083, 90TH_CM-235, 97TH NP 1525, 91DJ263, 02CM_0014BBY, BCF01, BCF02, BCF03, CNE23. 10. A kit for detecting HIV, wherein the kit comprises: (1) a first container, said first container containing iconic; and (2) a second container containing HIV neutralizing antibodies; (3) Reagents for accompanying diagnosis of HIV virus; Preferably, the HIV comprises any one or a combination of HIV strains selected from the group consisting of: 92US159, 99KE_KNH1088, 92BR025, 931N101, 02ET14, 92UG001, 00KENKU3006, 93BR020, G3, 96TH NI1149, 01CM_0002BBY, SF162, ADA, CNE1, CNE15, CNE17, BJOX25.01, BJOX27 and BJOX41 strains; The HIV includes any one or a combination of HIV strains selected from the group 92UG037, 00KE_KER2008, 91US001, 91US004, 96TH_NP1538, JR-CSF, 98US_MSC5016, 02ET288, 00UG_D26830M4, 93BR019, 93BR029, JV1083, 90TH_CM-235, 97TH NP 1525, 91DJ263, 02CM_0014BBY, BCF01, BCF02, BCF03, CNE23.

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