CN1901914B

CN1901914B - Pharmaceutical composition of chloroquine and protease inhibitors, and its uses

Info

Publication number: CN1901914B
Application number: CN2004800048772A
Authority: CN
Inventors: 安德里亚·萨瓦里诺
Original assignee: Jarrow Formulas Inc
Current assignee: Jarrow Formulas Inc
Priority date: 2003-02-21
Filing date: 2004-02-20
Publication date: 2011-06-08
Anticipated expiration: 2024-02-20
Also published as: ZA200506667B; CN1901914A

Abstract

The present invention relates to a drug combination capable of conferring therapeutic benefits in the treatment of both AIDS and malaria. In particular, it relates to a drug combination including at least one quinolinic antimalarial compound such as chloroquine or hydroxychloroquine, and at least one inhibitor of the Human Immunodeficiency Virus (HIV) protease enzyme. This drug combination is capable of inhibiting the replication of both HIV and Plasmodium sp. It also relates to the direct antimalarial effects of the HIV PIs.

Description

Comprise the pharmaceutical composition and the application thereof of the combination of quinoline antimalarial compound and protease inhibitor

The application requires the temporary patent application No.60/449 that submitted on February 21st, 2003, the temporary patent application No.60/471 that on May 16th, 517 and 2003 submitted to, 038 priority, the content of these two pieces of provisional application at this with its hereby incorporated by reference.

Invention field

The present invention relates in the treatment of acquired immune deficiency syndrome (AIDS) (AIDS) and malaria, can bring the pharmaceutical composition of treatment benefit.Particularly, the present invention relates to comprise can HIV inhibiting (HIV) and the chloroquine that duplicates of plasmodium or the pharmaceutical composition of hydroxychloroquine and hiv protease inhibitor.The invention still further relates to the direct antimalarial effect of hiv protease inhibitor.

Background of invention

Acquired immune deficiency syndrome (AIDS) (AIDS) and malaria are one of the most serious infectious disease of affects humans, cause the nearly 5,000,000 people's death in the whole world every year.In undeveloped country, the appreciable impact of these diseases is that disease is accompanied by originally miserable finance and living condition.The national load of several resource poverties does not play effective treatment, and these treatments might prevent the death of numerous disease.Difficulty itself part of not only having treated AIDS but also having treated malaria is because the Drug resistance of both cause of diseases causes, that is, human immunodeficiency virus (HIV) and protozoacide belong to Plasmodium, becomes when medicine weapon extreme is limited and exaggerates.In the high resource impoverished nation of several HIV serum prevalence rates, the major obstacle of using high activity antiretroviral therapy (HAART) to run into is its complexity expensive and prescription.Recently, on humanitarian consideration, the anti-HIV medicine is to provide to the popular high LDCs (Least-developed countries) of HIV serum at a low price.Yet problem is solved far away.Compare with antiretroviral agent, the cost of antimalarial is lower, in any case this may increase the weight of the expenditure of these impoverished nations.First line treatment malaria medicine that chloroquine (CQ) is recommended for a long time as The World Health Organization (WHO), remain in Africa and to afford most and the antimalarial of the most extensive employing is selected, but, occur drug-fast plasmodium strain continuously and make that in Africa the large areas administration in Latin America and Southeast Asia is invalid.

Because great majority are subjected to the area of the seriously ill wound of AIDS also to show endemicity malaria (and individuality is a coinfection usually), therefore exploitation will be useful at these two kinds of effective treatments of disease.

In this respect, CQ may be particularly useful because its outer anti-HIV-1 of display body duplicate and anti-some kinds of relevant chance activation of microorganism of AIDS-.It also fully proves when the individuality that is used for non-responsiveness and is the malaria drug prophylaxis and is long-term safety when being used for the treatment of rheumatism.Although do not obtain the information that influences in the body of CQ to virus quantity, its hydroxy analogs, hydroxychloroquine (HCQ) have proved anti-HIV-1 activity in the body.The anti-HIV activity of CQ gives the credit to the communicable infringement of the virion that cell produced of treated with medicaments.Though the invention is not restricted to any concrete mechanism, it is believed that this inhibitory action mechanism behind is because the glycosylated inhibition of gp120.This hypothesis has obtained the formation that following result's support: CQ has damaged serious glycosylation epi-position 2G12, and described epi-position is positioned at gp120 envelope protein surface, and is most important to viral infectivity.These effects show that by being different from the mechanism of used antiretroviral drugs, CQ suppresses virus replication, test and this new mechanism has caused CQ and antiretroviral agent to be combined in the clinical trial.

The more detailed information of CQ anti-HIV effect can find in following two pieces of articles, incorporates in the present patent application at this in full with it:

Savarino A, Gennero L, Chen HC, Serrano D, Malavasi F, BoelaertJR, the anti-HIV effect of Sperber K. chloroquine: suppress mechanism and activity profile (Anti-HIV effectsof chloroquine:mechanisms of inhibition and spectrum of activity).AIDS2001?Nov?23；15(17)：2221-9。

Savarino A, Gennero L, Sperber K, anti-HIV-1 activity of Boelaert JR. chloroquine (The anti-HIV-1 activity of chloroquine).J?Clin?Virol?2001?Feb；20(3)：131-5。

Known CQ with other anti-HIV medicines, such as ddI, when hydroxyurea and AZT combined administration, can bring into play additive effect.But just know the effect of combined administration CQ and hiv protease inhibitor (PIs) fully up to the present invention.In the therapy of the local lesion of malaria extensive administration PI base, this interaction can provide following phenomenon in view of in the future: 1) CQ/HCQ and PIs are that the integration after-stage of testing in the people suppresses unique medicine that HIV duplicates; 2) effect of CQ and PIs all causes damaging the infectiousness of newborn virion; 3) CQ and PIs are the substrates of important cells surface drug transport protein, and suppress this transport protein at varying level, described transport protein is P-glycoprotein (P-gp) and multi-drug resistance associated protein (MRP), they belong to ATP-binding cassette family, and regulate the intracellular concentration of antiretroviral drugs.Meaningfully, nearest data show CQ can increase the outflow of the P-gp-that applied by PIs and MRP-mediation at CD4 ⁺Inhibition level in the lymphocyte (Savarino etc., JAIDS 2004, wait to publish).

The inhibitory action of PIs pair cell surface drug transport protein makes the combination of CQ and PI especially can be used for treating malaria.

The transportation of medicine on cell surface participated in plasmodium drug resistance by hypothesis.This theory has obtained the support of several evidences.

At first, the glycoprotein of Plasmodium falciparum (P.Falciparum), promptly Pf-MDR has the homology of height with people P-gp, and be relevant to the CQ-resistance on some approach.Ward SA, definite evidence of the effect of Bray PG.Pfmdr 1 in Plasmodium falciparum quinoline resistance (Definitive proof for a role of pfmdr 1 in quinoline resistance inPlasmodium falciparum) .Drug Resist Updat 2000 Apr; 3 (2): 80-81.

Secondly, external CQ-resistance characteristic ground is replied by verapamil, and verapamil is the known inhibitor of ATP-binding cassette in people's cell.Sidhu AB, Verdier-Pinard D, the chloroquine resistance in Plasmodium falciparum (Plasmodium falciparum) malarial parasite (Chloroquine resistance in Plasmodium falciparum malariaparasites conferred by pfcrt mutations) .Science 2002 Oct 4 that Fidock DA. gives by the pfcrt sudden change; 298 (5591): 210-3.

The 3rd, by the erythrocyte of CQ-resistance Plasmodium falciparum (P.Falciparum) strain parasitism than by CQ pond in the more limited born of the same parents of the accumulation of CQ-sensitive strain parasitism.Plasmodium falciparum (P.Falciparum) strain reduces the cumulative ability strictness of CQ and is relevant to so-called CQ resistance transhipment (CRT) proteic gene of coding (Pf-crt) sudden change in erythrocyte.Plasmodium falciparum (P.Falciparum) CRT get involved these phenomenons really cutter system do not illustrated as yet.Meaningfully, these sudden changes are present in and most suddenly change in the open-air separated strain of geographic CQ resistance from the world, and are not present in the separated strain of CQ-sensitivity.Sidhu AB, Verdier-Pinard D, the chloroquine resistance in Plasmodium falciparum (Plasmodium falciparum) malarial parasite (Chloroquine resistance in Plasmodium falciparum malariaparasites conferred by pfcrt mutations) .Science 2002 Oct 4 that Fidock DA. gives by the pfcrt sudden change; 298 (5591): 210-3.

Have that not only to suppress HIV but also suppress the compositions of plasmodial CQ and PI and utilize the said composition treatment will be useful.

Summary of the invention

The present invention relates in the treatment of AIDS and malaria, can bring the pharmaceutical composition of treatment benefit.Particularly, the present invention relates to comprise that the hiv protease inhibitor adds that CQ or HCQ or another have the pharmaceutical composition of the antimalarial of similar characteristic.Since this pharmaceutical composition can suppress that HIV duplicates and can suppress plasmodium and duplicate.The invention still further relates to the direct antimalarial effect of HIV PIs.

The compositions that present patent application is protected has proved unexpectedly than using single medicament separately and has given more persistently not only to suppress HIV but also suppress plasmodial ability that promptly, CQ can strengthen the antiretroviral activity of PI, and PI can strengthen the antimalarial active of CQ.

Therefore, PI adds that the compositions of CQ can be used for suppressing the purpose that HIV duplicates, and is used to suppress the purpose of growth of malaria parasites, or is used to suppress the purpose of these two kinds of virulence factors.

From clinical angle, PI adds that perhaps the compositions of CQ/HCQ can treat AIDS and malaria.Therefore, it can be used for treating the individuality that infection has HIV, and be subjected to the individual of malaria invasion and attack or be in the individuality that contacts in the malaria risk, or the people of HIV/ malaria coinfection.

Equally, the compositions of CQ and PIs can be used to recover HIV and Plasmodium falciparum (P.Falciparum) Drug resistance separated strain respectively to the sensitivity of PIs and CQ.

In another embodiment, the present invention relates to the inherent malaria effect of PIs.The inventor finds that PIs is used for the treatment of HIV clinically and has direct antimalarial effect.These direct effects with treat attainable concentration external be observable (referring to EXAMPLE III).

Although the present invention does not relate to any specific mechanism, the bioinformatic analysis prompting, the target of PIs may be plasmepsin II, it is the plasmepsins family member, the potential target of new antimalarial.

Accompanying drawing describes in detail

Fig. 1 and 2 illustrates and utilizes corresponding sequence alignment between three dimensional structure double exposure that VAST algorithm (can derive from the NCBI network address, http://www.ncbi.nlm.nih.gov) obtains and Plasmodium falciparum (P.Falciparum) plasmepsin II and the HIV-1 protease.The three dimensional structure of plasmepsin II derives from albumen database (PDB; Http:// rcsb.org/pdb/).Shown in HIV-1 protease for by submitting to plasmepsin II structure to ncbi database to be used for one of many retroviral Protease sequences that the close thing of index structure searches out.Therefore, illustrating of it only is an example, is not to be intended to limit the scope of the invention by any way.

Fig. 1 illustrates two kinds of different images of the three dimensional structure double exposure of Plasmodium falciparum (P.Falciparum) plasmepsin II and HIV-1 protease comparison domain.Significantly comparison and corresponding to the residue in two Journal of Molecular Catalysis sites by labelling (a ...) expression.The lower plasmepsinII zone of comparison level is by cross hairs labelling (* * * *) expression.The similar area of hiv protease is by dotted line (---) expression.PI indinavir (Indinavir) (IDV) molecular structure in the complex of HIV-1 protease also shows with dark shadow region.

Fig. 2 shows the sequence alignment corresponding to three-dimensional comparison shown in Figure 1.Labelling strictness shown in about the residue is corresponding to labelling among Fig. 1, and explanation in last joint.Without any the zone of correspondence markings corresponding to the domain (not shown in figure 1) of comparison not.

Fig. 3 shows CQ and the HIV PIs combined effect to virus replication.In brief, MT-4 cell or elementary peripheral blood lymphocytes (PBMC) are respectively with laboratory strains or primary separation strain inoculation.Then, the cell of HIV infection and the IDV and/or the CQ incubation of selected concentration.

Fig. 3 A is presented at IDV and exists down, and the influence that CQ duplicates in the MT-4 cell HIV-1 IIIB wherein shows the reduction (meansigma methods ± S.E.M. of HIV-1 p24 output in the presence of CQ+IDV; Three experiments).In this case, cell infects with low infection multiplicity (0.01), is intended to disclose better the IDV/CQ combination and HIV-1 is duplicated has any favorable influence.

The branch inhibition concentration that the isobologram proof CQ+IDV combination anti-HIV-1 IIIB of Fig. 3 B duplicates.The branch inhibition concentration of CQ+PI combination shows can suppress virus replication 90% (EC ₉₀).According to nonlinear regression model (NLRM), calculate the curve of best fit data point.This figure is illustrated in the line of estimating under the situation of threshold values between simple addition effect (connecting 1.0FIC on x and the y axle) and inferior collaborative and the actual synergy (that is the line that, connects the 0.5FIC value).

Isoboles shown in Fig. 3 C proof CQ+ saquinavir (saquinavir) (SQV) and (RTV) the anti-HIV-1 IIIB branch inhibition concentration of duplicating of CQ+ ritonavir (ritonavir).As among the pro-width of cloth figure, this figure demonstration causes EC ₉₀The FIC of CQ+PI.The line and the curve that present obtain as described in the previous paragraph.

Fig. 3 D is illustrated in and has or lack under the CQ increased response to primary separation strain (VI829, HIV-1 CladeC).In this figure and following figure, virus replication is represented with the percentage ratio of untreated control, so that compare the effect of IDV under existence and shortage CQ (1 μ M) easily.CQ-handles and the difference that is untreated between the culture is conspicuous from the regression line, described regression line best match data point and at the EC of IDV ₅₀In cause the difference (labelling in the drawings) of nearly 1Log.

Fig. 3 E illustrates by CQ and partly recovers the reaction from the IDV in the PAVIA12 multi-drug resistance separated strain of HIV-1 Clade B.

Fig. 3 F illustrates by CQ and partly recovers reaction to the IDV of the separated strain that belongs to HIV-1 Clade A.This separated strain (UG3) is similar to some PI resistance viruses, and the virus replication peak appears under the PI intermediate concentration.From this figure, CQ obviously impels the displacement of the inductive virus replication peak value of IDV-to the minimum nM concentration of PI.The peak amplitude is low most probably owing to the direct anti-HIV effect of antimalarial in the presence of CQ.

Fig. 4 illustrates hiv protease inhibitor RTV and the IDV influence to laboratory plasmodium strain (3D7) and open-air separated strain (Ibginovia).The result represents with the percentage ratio of control value.

Fig. 5 A illustrates the influence of HIV-1 protease inhibitor ritonavir combination CQ to the open-air separated strain of CQ-resistance Plasmodium falciparum (P.Falciparum) (Ibginovia).In order to set forth typical consequence, described in embodiment part material and method, when the reaction by plasmodium lactic acid dehydrogenase (LDH) mediation finished, tables of data was shown as meansigma methods ± S.D. optical density (O.D.).O.D. value is directly proportional with Plasmodium falciparum (P.Falciparum) cell survival rate.

Fig. 5 B illustrates the influence of IDV (5 μ M) combination CQ to CQ-resistance Plasmodium falciparum (P.Falciparum) strain (W2).The result represents with the percentage ratio of control value.

Fig. 5 C illustrates the influence of IDV (5 μ M) combination CQ to Plasmodium falciparum (P.Falciparum) strain (3D7) of CQ-sensitivity.The result represents with the percentage ratio of control value.

Fig. 6 has antimalarial effect in the mice of Bai Shi plasmodium (Plasmodium berghei) for RTV in infection.

Fig. 6 A is the influence that RTV (50mg/kg) grows in the Balb/c mice to Bai Shi plasmodium (P.Berghei).The result is expressed as meansigma methods ± S.D. that difference is followed up a case by regular visits to the Red blood corpuscle percentage ratio of natural law parasitism.

Fig. 6 B is RTV (50mg/kg) has Bai Shi plasmodium mice survival rate to infection influence.The result is expressed as Kaplan Meyer curve, and with the difference of P value reporting survival rate.

Fig. 6 C is the influence that RTV (150mg/kg) grows in the Balb/c mice to the Bai Shi plasmodium.The result is expressed as meansigma methods ± S.D. that difference is followed up a case by regular visits to the Red blood corpuscle percentage ratio of natural law parasitism.

Fig. 6 D is RTV (150mg/kg) has Bai Shi plasmodium mice survival rate to infection influence.The result is expressed as Kaplan Meyer curve, and with the difference of P value reporting survival rate.

Detailed Description Of The Invention

The present invention relates to effectively in the world two kinds of Infectious Diseases, the i.e. pharmaceutical composition of AIDS and malaria virulence factor. Particularly, the present invention relates to comprise and to suppress that HIV copies and can suppress again the hiv protease inhibitor that plasmodium copies adds antimalarial, such as the pharmaceutical composition of CQ or HCQ.

In the present patent application claimed composition can than single component alone administration give more lasting inhibition to HIV and plasmodium, that is, CQ can strengthen the antimalarial active of PI, and PI can strengthen the antimalarial active of CQ.

Because above-mentioned composition is to all visible result for the treatment of of HIV and plasmodium, composition can be used for suppressing the purpose that HIV copies, and is used for suppressing the purpose of growth of malaria parasites, or is used for not only suppressing the purpose that HIV copied but also suppressed growth of malaria parasites.

From Point of View of Clinical, PI adds antimalarial, can be used for treating AIDS and malaria such as the composition of CQ/HCQ. Therefore, described composition can be used for treating the individuality that infection has HIV, is subjected to the malaria invasion and attack or is in the individuality that contacts the malaria risk, or suffer from the people of HIV/ malaria coinfection. These the two kinds components that are used for composition can increase the inhibition level of drug susceptibility HIV, and the PI+CQ combination has recovered HIV and plasmodium falciparum (P.Falciparum) resistance to the action of a drug separated strain respectively to the sensitivity of PIs and CQ.

Treatment about HIV is important to note that, the effect of CQ combined protein enzyme inhibitor is worked in coordination with. When with the cell of PI combination medicine-feeding acute infection, CQ has reduced the HIV that produces to a certain degree and has suppressed necessary PI concentration [referring to embodiment 1].

In addition, following giving an example, CQ has partly recovered the sensitivity [referring to embodiment 1] in the PIs resistant strain to PIs.

Although the invention is not restricted to any specific mechanism, it is believed that and use P-gp and MRP blocking agent, such as CQ, can increase the intracellular concentration of PIs.

In one embodiment, the present invention gives therapeutic strategy, uses antimalarial altogether for thus the HIV positive individuals, makes the effective dose of PIs reduce such as CQ/HCQ or another quinoline medicament, and cost and possible toxicity reduce.Equally, CQ overcomes aspect the positive object of HIV that the ability of PIs resistance is experienced at medicine significant, and this positive object has developed the multiresistance to antiretroviral drugs, selects thereby have limited treatment.

Embodiment of the present invention also can be used for treating resistant malaria.In fact, the zone of several endemicity malaria in the world, Plasmodium falciparum (P.Falciparum) strain with multi-drug resistance phenotype just catches on, and use PI can recover the sensitivity to CQ.Therefore, a kind of like this practicality of medicine is estimated to save numerous life.In the third world area of cannot afford PIs at present, when PIs was used for treating on a large scale HIV and infects, the cost problem was expected in the near future to be solved to small part.Consider that AIDS and malaria often coexist as the same area, PIs might be than they present above-mentioned area and so hypothesis more economical these medicines that use effectively in the treatment malaria of more being usually used in.Equally, live in endemicity resistant malaria area also can prevent the malaria incident with the HIV-infected individuals of PI+CQ combination treatment generation.

And the described effect of PIs combination quinoline medicament can help medicine, such as CQ and first generation PIs (RTV, SVQ, resurrection IDV), the medicine that these medicines are doomed to be updated is originally in the near future replaced.

Generally speaking, the present invention relates to use to HIV and malaria drug composition effective.The embodiment of compositions comprises:

1) chloroquine (CQ) or hydroxychloroquine (HCQ) or another quinoline medicament, such as mefloquine (Mefloquine) (MQ) and quinine (quinine) (Q)

Combination has

2) one or more hiv protease inhibitor (PIs).

PIs comprises:

Indinavir (IDV), ritonavir (RTV), saquinavir (SQV), viracept see nelfinaivr (nelfinavir) (NFV), Lopinavir (lopinavir) is (LPV), RTV adds the combination of LPV, amprenavir (amprenavir) (APV), fosamprenavir (fosamprenavir) (FPV), tipranavir (tipranavir) is (TPV), atazanavir (atazanavir) (ATZ), TMC-114.

Antimalarial and PI compositions can be with nucleosidic inhibitors (NRTI) administrations altogether simultaneously of HIV reverse transcription.

NRTIs comprises:

Zidovudine (Zidovudine) (AZT or ZDV), lamivudine (lamivudine) (3TC), Abacavir (abacavir) (ABC), zalcitabine (zalcitabine) (ddC), remove hydroxyl machine glycosides (didanosine) (ddI), take charge of his furan pyridine (stavudine) (d4T), tenofovir (tenofovir) (TDF), emitricitabine (FTC), amdoxovir (DAPD).

The present invention is not subject in this respect, any suitable quinoline medicament, and PI and/or NRTI all can use.

Antimalarial and PI compositions also can with other antimalarials, or with antibiotic at concomitant infections, or with any medicine administration altogether simultaneously at coexistence or relevant disease.

The invention still further relates to the direct malaria effect of HIV PIs.Not only PIs can reply the CQ resistance, and PIs also possesses inherent malaria effect.These act directly on external with treat attainable concentration (referring to example II) and in the body of Mus malaria model (referring to EXAMPLE III) be observable.

The mechanism of the direct malaria effect of PIs is not set forth as yet.Yet interesting understanding is from following observed result: HIV-1 protease (that is, at these medicines of the target design) and plasmodium plasmepsins family member's protease are enjoyed significant sequence-and structure-similarity (Fig. 1 and Fig. 2).Similar to HIV-1 protease, plasmepsins is an aspartyl protease, and has basic role in the growth in the born of the same parents of Plasmodium falciparum (P.Falciparum).They participate in the first step of hemoglobin degrading, and described hemoglobin constitutes the main nutrient in stage in the parasitic erythrocyte.In view of the structural similarity between HIV-1 protease and the plasmepsins, might suppose that PIs passes through these enzyme infringement growth of malaria parasites of targeting.This hypothesis is kept by the following fact: the zone of maximum comparability is its catalytic site between two albumen, this site non-covalent PIs of being incorporated into and suppressed by PIs in HIV-1 protease.If this mechanism obtains experimental data and confirms that HIVPIs carries out safety test as first medicine in human body,, be designated as the potential target of the new antimalarial of exploitation recently by WHO to suppress the plasmepsins family member.Involve the continuous time that occurs in a criminal case at the Drug resistance plasmodium, the availability of Drug Storage that relates to the medicine of new target increases medicament selection.Other potential foundations of PI malaria effect are recently described by these medicines inductive downward modulation CD36 (receptor of Plasmodium falciparum (P.Falciparum)) in human red blood cell.Nathoo S, Serghides L, Kain KC.HIV-1 antiretroviral drugs pair cell adhere to and engulf the erythrocytic effect (Effect ofHIV-1 antiretroviral drugs on cytoadherence and phagocytic clearance ofPlasmodium falciparum-parasitised erythrocytes) of removing Plasmodium falciparum (Plasmodium falciparum)-parasitism.Lancet.2003?Sep?27；362(9389)：1039-41。

The explanation of above-mentioned mechanism is only for explanatory purpose: the present invention relates to the influence that PIs grows in the external and body to plasmodium, be not limited to any specific mechanism.

As mentioned above, being used in combination of they and CQ consolidated in the direct malaria effect of PI.The direct malaria effect of PIs also shows, lives in the zone of endemicity malaria and the individuality that infects with the HIV-that the antiretroviral cocktail that comprises PI is treated, and prevents the generation of malaria incident to small part.Consider the expenditure that several impoverished nations are limited, prevent that the malaria incident from being favourable for treatment HIV.In fact, at Africa, inferior the Sahara (Sub-Saharian Africa), the zone of malaria endemic disease is arranged, the popular level of HIV serum can arrive at 30%.Because the people that HIV-infects are in high risk complicated malaria, can imagine that the direct malaria protozoon effect of PI can be saved large quantities of mankind and save financial resource.

The present invention will further be described by embodiment, and these embodiment are used to help those of ordinary skills to implement the present invention, are not intended to limit the scope of the invention by any way.

Embodiment

Material and method

Infection analysis

The HIV-1 that uses laboratory to be fit to _IIIBAnd HIV-2 _CBL/20Strain, primary separation strain HIV-1 _UG3(Clade A, R5) and HIV-1 _{VI 829}(Clade C R5), all derives from the inmature object of antiretroviral.These viruses fully are described in the paper of (the same) such as Savarino A..Also use HIV-1 _PAVIA12Separated strain, by Maurizia doctor Debiaggi, University of Pavia, the Italy present, Maurizia doctor Debiaggi has also carried out its gene type assay.HIV-1 _PAVIA12Separated strain belongs to Clade B and separates from the individuality of suffering from HAART depletion, and it has the genotype collection of illustrative plates (sudden change in the HIV-1 reverse transcription: 67N 69D 70R 74V 108I 181C 184V 219Q228R of multi-drug resistance to used antiretroviral in the current medical practice of all types; Sudden change in the HIV-1 protease: 10I 20R 36I 46L 54V 55R 63P 71V 82A90M).Utilize MT-2 cell (experiment strain) or the activatory peripheral blood lymphocytes of PHA-(PBMC) (primary separation strain), by 50% terminal point dilution method titration biology virus original seed.

In actute infection is analyzed, with the viral original seed of the cell of adequate types and infection multiplicity (MOI) nearly 0.1 37 ℃ of incubations 2 hours, unless otherwise prescribed.After three washings, cell 37 ℃ of incubations 7 days in fresh culture infect the back different intervals and collect acellular suspension and be used for ELISA and measure HIV-1 p24 (NEN Life Science Prod., Boston MA) or HIV-2 p27 (Coulter, Hialeah, FL) (26,33).Then after virus absorption, cell incubation under the accessible CQ concentration in the individual blood plasma of CQ treatment.

Selectivity index is calculated to be IC ₅₀/ EC ₅₀Ratio.Under the situation of infection of PBMCs, each donor is drawn the toxicity curve, with accurate estimation selectivity index.

CD4 ⁺CXCR4 ⁺MT-4T-cell line is used to estimate the influence of the strain that CQ and PIs be fit to the X4 laboratory, yet utilizes the primary separation strain, adopts by informed consent to obtain from healthy donors and with 2pg/ml phytohemagglutinin (PHA in analysis; Difco Laboratories, Detroit, MI) 3 days peripheral blood lymphocytes (PBMC) of stimulation.

The more details of the virusology program of relevant imitation can find in following article: Savarino A, Gennero L, Chen HC, Serrano D, Malavasi F, Boelaert JR, the anti-HIV effect inhibition mechanism and the activity profile of Sperber K. chloroquine: (Anti-HIV effects ofchloroquine:mechanisms of inhibition and spectrum of activity).AIDS2001 Nov 23; 15 (17): 2221-9, at this with its hereby incorporated by reference.

Toxicity assessment is analyzed

In the contrast of not infecting, cell survival rate and programmed death are by the determined trypan blue exclusion method of technology of the previous checking of the inventor, and mtt assay and iodate third ingot/annexin VFITC dyeing is analyzed.The more details of the virusology program of relevant imitation can find in following article: Andrea Savarino, Thea Bensi, Annalisa Chiocchetti, FlaviaBottarel, Riccardo Mesturini, Enza Ferrero, Liliana Calosso, Silvia Deaglio, Erika Ortolan, Stefano Butto, Aurelio Cafaro, Toshiaki Katada, BarbaraEnsoli, in the V3 of viral envelope glycoprotein gp120 ring, people CD38 disturbs HIV-1 to merge (Human CD38interferes with HIV-1 fusion through a sequence homologous to the V3loop of the viral envelope glycoprotein gp120) by sequence homology for Fabio Malavasi and Umberto Dianzani (2003).The FASEB JournalExpress Article 10.1096/fj.02-0512fje, at this with its hereby incorporated by reference.

The concertedness assessment

In order to measure concertedness, after virus was adsorbed onto on the cell, cell precipitation was resuspended in the culture medium of the CQ that contains various combination and IDV.Then, branch inhibition concentration (FIC) is calculated to be the medicine EC of medicine A ₉₀Medicine 2/EC with drug alone B ₉₀Combination.When FICs sum＜0.5, effect is considered as working in coordination with.

Parasite

Ibginovia is from the Istituto Superiore di Sanita, Rome, and the separated strain that Italy obtains is male to codon K76 in the pfcrt gene of giving the CQ resistance and A220 sudden change.This parasite is from Nigeria.All parasites are external to be maintained in RPMI 1640 culture medium, wherein adds people A type Red blood corpuscle (RBC) and 10% hot deactivation human serum.All cultures place the humidification incubator, and 37 ℃, 3%O ₂, 6%CO ₂, and 91%N ₂Gas control environment, and feed according to ready-made program.

The detection of parasitemia

Utilize the painted thin smear of Giemsa by visible light microscope and utilize dyestuff sulfo-benzo carboxylic purine to determine parasitemia by fluorescence microscope.

The parasite lactic acid dehydrogenase is measured

This analysis is based on following principle: plasmodium lactic acid dehydrogenase (LDH) can utilize 3-acetylpyridine NAD (APAD) as coenzyme, and APAD changes into APADH in the lactic acid oxidizing process.The sample that all LDH determine passes through metric measurement at the 650nm place.Measure for these, the malaria sample of 10-50 μ l joins in the Mastat reagent, the optimization of C that this reagent detects for parasite LDH.Sample is made up of the malaria culture.Utilize 96 hole microtitration plates, all sample aliquot join the Malstat reagent of 0.2ml.Utilize multi-wavelength plate readout instrument, determine the formation of APADH at the 650nm place.Automatic measurement was carried out at interval with 30 seconds in each hole of testing for traces titer plate, and the individual data point storage, and drew with software program subsequently.The active spectrophotometer evaluation of LDH is promoted to Malstat reagent by adding NBT (0.24mM) and PES (0.033mM).Along with APADH forms, NBT reduces, and forms blue first (formazan) product, and naked eyes detect and measure at the 650nm place.This analysis is specific to parasite LDH, is not subjected to the influence of people's enzyme.The measurement of the lactic acid dehydrogenase activity of Makler MT and Hinrichs DJ (1993) Plasmodium falciparum (Plasmodium falciparum) is as assessment (the Measurement of lactate dehydrogenase activity of Plasmodiumfalciparum as an assessment of parasitem of parasitemia

Example I

Whether higher than the independent generation of IDV the purpose of this test be analyze to add CQ to IDV HIV inhibition level.The MT-4 cell that HIV-1IIIB-infects incubation under existence or shortage increase concentration C Q (1-6.25 μ M) is containing in the culture medium of 10nM IDV.Selected IDV concentration approaches the EC in the MT-4 cell that HIV-1 IIIB-infects ₅₀, as under this experiment condition, measuring (data not shown).Adding CQ and do not cause the significant difference of cell survival rate, is because the cell survival rate (data not shown) that special damage applies by CQ thereby get rid of observed difference.Infected back 5 days, the p24 level is than low (Fig. 3 A of the cell conditioned medium liquid of handling with IDV separately in the supernatant of the culture of handling with CQ+IDV; P＜0.05, repeated measure ANOVA).The effect of CQ is dose-dependent, and the anti-HIV-1 of this prompting CQ/IDV combination is renderd a service and reduced the collimation that CQ concentration increases.

In order to explore this phenomenon better, determine whether the work of different CQ/IDV combination is addition in order to the effect of estimating combination, collaborative or inferior collaborative.HIV-1 _IIIBThe cell of-infection is handled alone or in combination with the CQ or the IDV of multiple variable concentrations.Test is to estimate the concentration of each medicine in producing the different components that 90%HIV-1 duplicates inhibition.For each pharmaceutical composition, determine FIC.Utilize the analysis of isobolograms method to show, CQ during to the active FICs that acts on low CQ of the anti-HIV-1 of IDV be work in coordination with (corresponding to prevention malaria plasma concentration, promptly, 0.1-1 μ M, at intermediate concentration FICs (3.12-6.25 μ M) is inferior collaborative or addition, and when the FICs (10 μ M, irrelevant clinically concentration) of the highest CQ antagonism slightly.Similar action obtains (data not shown) with HIV-2 and utilizes CQ and RTV or SQV combination acquisition (Fig. 3 B).The result shows that the CQ concentration of finding is making up after-applied collaborative anti-HIV effect with PIs in malaria prevention process.Then, test CQ handles elementary HIV separated strain susceptible in the influence of IDV.For this purpose, exist or lack under the CQ of 1 μ M, PHA-stimulated peripheral mononuclear cells (PBMC) infects elementary HIV-1 separated strain, washing, and with IDV (0-1000nM) incubation that increases concentration.The result shows, in 5 days supernatant the level of p24 in the culture of handling with IDV+CQ than with coupling IDV concentration but do not have in the culture of CQ processing lower.Fig. 3 D shows the typical effect of CQ to the IDV-sensitivity separated strain that belongs to subtype C (VI 829).In this case, CQ has reduced EC ₅₀Nearly 1Log.And CQ has partly recovered the reaction (Fig. 3 E) in multiresistance HIV-1 separated strain to IDV.Then, tested the effect of IDV/CQ combination to the separated strain (UG3) that belongs to " West Africa " HIV-1 hypotype A.Infected back 5 days by the UG3 separated strain, present the typical peaks of p24 level in supernatant, similarly in the presence of similar PI concentration, increase from the described infectiousness of the PI-resistance particle of hypotype B with the cell of 100nM IDV incubation.Mammano F, Trouplin V, Zennou V, Clavel F. recalls the evolutionary path of 1 pair of protease inhibitor resistance of human immunodeficiency virus's type: medicine lack and in the presence of virus suit (Retracing the evolutionary pathways ofhuman immunodeficiency virus type 1 resistance to protease inhibitors:virus fitness in the absence and in the presence of drug).J?Virol.2000?Sep；74(18)：8524-31。Utilize the IDV of 1 μ M, as seen suppress effect on the contrary.The IDV that utilization begins from 100nM in the presence of CQ, recovers to be moved to the horizontal peak of p24 and the inhibition (Fig. 3 F) of minimum nM IDV concentration (far below those concentration that reached clinically).On the whole, these results meet the synergy of CQ to the IDV reaction.In fact, they can not be owing to being of unique addition effect, is minimum (data not shown) in the inhibitory action of the independent CQ of 1 μ M concentration.

These differences can not help the toxic action that applies by the CQ+IDV compositions, as the cell survival rate value of the PBMC that handles with 1 μ M CQ basically with lack at CQ under with those values identical (data not shown) of the cell of identical IDV concentration processing.

Example II

To CQ+PIs in conjunction with before experimentizing, estimated the effect of protease inhibitor when individually dosed erythrocyte to Plasmodium falciparum (P.Falciparum) parasitism.The erythrocyte of Plasmodium falciparum (P.Falciparum)-parasitism (beginning parasitemia 1%) was cultivated 48 hours with IDV and RTV concentration, and described concentration is arranged within the observable clinically scope of handling with these PIs of individuality.Then collect the sample aliquot of red blood cell suspension, and analyze Plasmodium falciparum (P.Falciparum) LDH activity.The result shows, RTV and IDV dose dependent ground suppress Plasmodium falciparum (P.Falciparum) growth (Fig. 4), Plasmodium falciparum (P.Falciparum) LDH activity measuring as cell survival rate analyzed in the CQ cultivation that the erythrocyte of Plasmodium falciparum (P.Falciparum)-parasitism increases with concentration under existence or shortage PI 48 hours then.The typical consequence that Fig. 5 A and Fig. 5 B obtain with combination PI and CQ.From these data, obviously RTV and IDV recover the reaction to CQ in CQ-resistance Plasmodium falciparum (P.Falciparum), when these PIs working concentrations are the suitableeest inhibition in Asia Plasmodium falciparum (P.Falciparum) growth own.In CQ-sensitivity Plasmodium falciparum (P.Falciparum) strain (3D7), IDV is more inapparent to the effect of CQ reaction, shown in Fig. 5 C.

For whether the effect of determining the CQ+PI compositions only is addition or collaborative, the inventor utilizes the method for FICs sum, has analyzed W2 (CQ-resistance) and 3D7 (the CQ-sensitivity) the strain reaction to IDV.These analysis reports to the value 05 (indication synergy) of W2 strain, and to 3D7 strain value＞0.5.Because cooperative effect is only at the CQ-resistant strain but not observe in the strain of CQ-sensitivity, conclusion is that IDV recovers the sensitivity to chloroquine.Therefore, the effect of CQ-resistant strain is not attributable simply to effect addition with IDV and CQ.

On the whole, these results show that PIs replys the CQ-resistance with the averapamil-sample loading mode.

EXAMPLE III

In order to estimate the effect of PI in the malaria artery model, inoculation has Bai Shi plasmodium (P.Berghei) in the Balb/c mouse peritoneum, be divided into three groups then: the 1) analogies of gastric inoculation phosphate buffer (PBS) processing, 2) gastric is used the ritonavir diluent (promptly under ritonavir (placebo) lacks, 47% alcoholic solution) handle, and 3) gastric is with 50 or the 150mg/kg processing.Through determining that ritonavir (handling 1) dose dependent ground has postponed the propagation of parasite in mice, handling 2 and 3 does not thus have similar effect (Fig. 6 A and 6C).Meaningfully, according to the Kaplan-Mayer curve shown in Fig. 6 B and the 6D, ritonavir has also increased the survival rate of handling mice in remarkable mode.

EXAMPLE IV

Utilization well known to a person skilled in the art technology administration malaria/PI compositions.Antimalarial/PI combination can be used by pharmaceutical composition, comprises appropriate excipients, diluent or carrier.The approach that administration antimalarial/PI compositions is recommended comprises oral, intramuscular, percutaneous, Sublingual, intravenous or subcutaneous methods.

The form of pharmaceutical composition can be tablet, sugar-coat, capsule, pill, solution, suspension, emulsion or any other the suitable form of transmitting pharmaceutical composition that be same as.The pharmaceutical composition of solid phase form can contain non-aqueous diluent or carrier, for example comprises filler, enriching substance, bonding agent, wetting agent, disintegrating agent, surfactant, absorption carrier, lubricant, or any other is to well known to a person skilled in the art suitable diluent or carrier.The pharmaceutical composition of liquid form comprises diluent, or carrier, and such as, water, ethanol, propylene glycol or any other are suitable for well known to a person skilled in the art diluent or carrier.For parenteral administration, solution and suspension should be aseptic, and if suitable, should be that blood is isoosmotic.

As used in this, term " treatment effective dose " expression antimalarial/PI compositions can suppress that HIV duplicates and/or plasmodial dosage.The treatment effective dose can be determined under the doctor instructs according to the standard medical principle.CQ and PI can any suitable form of medication, provide such as the acceptable salt of medicine.

In the prophylactic treatment malaria, the dosage of used PIs will depend on the selected PI of treatment, and whether PI uses separately or be used in combination with other antimalarials.Be used for the treatment of PI dosage range that malaria uses and be 1/2 to 2 times that is used for the treatment of typical doses that HIV uses.

For the acute malaria of treatment, the dosage of the PI that uses also depends on the selected PI of treatment, and whether PI uses separately and still be and the antimalarial combined administration.Be used for the treatment of PI dosage range that the polarity malaria used and be 1/2 to 3 times that is used for the treatment of typical doses that HIV uses.

For the malaria prevention, can use the combination of CQ and PI, wherein CQ comprises the gross weight of about 0.8% weight (for example utilizing, in amprenavir or the saquinavir combination) to the combination CQ/PI of about 15% weight (for example utilizing, in vitonavir/ Lopinavir 1: the 4w/w combination).Total CQ/PI combination that the amount of using CQ is less than 2% weight can allow to use once in a week CQ, and regularly uses the separately protease inhibitor of dosage form on the same day, such as per 12 hours or per 8 hours.Using protease inhibitor and being used to use the used dosage form foundation of protease inhibitor is to well known to a person skilled in the art method and dosage form.

Use of the total CQ/PI combination (depend on used protease inhibitor) of the amount of CQ, as mentioned above, can be undertaken by use two kinds of medicines in the single medicine dosage form greater than 2% to 10% weight.In high-caliber especially CQ resistance zone, the amount of the CQ that uses can increase the total amount until the CQ of about 33% weight and PI combination.CQ and PI also separately dosage form use in the patient, to realize the CQ and the PI of required dosage.

For treatment polarity malaria, CQ amount in the CQ/PI combination can increase to the CQ and the PI combination of about 75% weight.CQ and PI dosage form separately use to realize desired level in the patient.When CQ and PI are combined in the single dosage form, before the CQ/PI combination of using first dosage or use extra CQ separately with single dosage form simultaneously.The CQ that uses extra dose is to realize CQ load cell suitably.

PI is used for the treatment of the dosage scope of HIV for be used for the treatment of 1/4 to 1 of the common dosage of HIV under shortage CQ or other antimalarials.

In treatment HIV infected, the combination of CQ and PI made CQ comprise the gross weight of about 0.8% weight to the combination CQ/PI of about 33% weight.CQ/PI can single dosage form use.Perhaps, CQ and PI use CQ and PI to realize required relative quantity with dosage form separately.

Preferably, use CQ and PI and be used for the treatment of HIV at about 0.05 μ M to about 1 μ M, and be used for the clinical management malaria to about 6 μ M at about 0.005 μ M with the plasma concentration that realizes CQ.Preferably, use CQ and PI with the plasma concentration that realizes PI at about 500nm to about 30 μ M, be used for the clinical management malaria, and, be used for the treatment of HIV at about 10nM about 30nM extremely.Those of skill in the art will recognize that to the invention is not restricted in this respect, can use the CQ and the PI of any suitable treatment effective dose.

Claims

1. be used for the treatment of or prevent human immunodeficiency virus infection, malaria or the pharmaceutical composition of the two by suppressing HIV and plasmodium (plasmodium sp.), comprise the inhibitor of at least a quinoline antimalarial compound and at least a people's hiv protease or the combination of its officinal salt, wherein said quinoline antimalarial compound is selected from chloroquine, hydroxychloroquine, mefloquine, quinine and combination thereof, wherein said composition comprises the described quinoline antimalarial compound of 0.8% to 33% weight of the gross weight of the inhibitor that accounts for quinoline antimalarial compound and hiv protease or its officinal salt.

2. the compositions of claim 1, the inhibitor of wherein said hiv protease is selected from indinavir, ritonavir, saquinavir, viracept see nelfinaivr, Lopinavir, amprenavir, fosamprenavir, tipranavir, atazanavir, TMC-114 and combination thereof.

3. the compositions of claim 1, it further comprises the nucleosidic inhibitors of at least a HIV reverse transcription.

4. the compositions of claim 3, the nucleosidic inhibitors of wherein said HIV reverse transcription is selected from zidovudine, lamivudine, Abacavir, zalcitabine removes hydroxyl machine glycosides, takes charge of his furan pyridine, tenofovir, emitricitabine, amdoxovir and combination thereof.

5. the compositions of claim 1, wherein compositions comprises that a certain amount of described hiv protease inhibitor is enough to realize plasma concentration 10nM to 30 μ M.

6. the compositions of claim 5, wherein compositions comprises that a certain amount of described quinoline antimalarial compound is enough to realize plasma concentration 0.005 μ M to 6 μ M.

7. the compositions of claim 1, wherein compositions comprises that the dosage of ritonavir is that the 1mg/kg body weight is to the 150mg/kg body weight.

8. pharmaceutical kit, be used for infecting by suppressing HIV and plasmodium treatment or prevention and HIV, the physiological situation that malaria or both are correlated with, described test kit comprises a plurality of containers, wherein at least one described container contains at least a quinoline antimalarial compound or its officinal salt, and another described container contains at least a hiv protease inhibitor or its officinal salt, wherein said quinoline antimalarial compound is selected from chloroquine, hydroxychloroquine, mefloquine, quinine and combination thereof, wherein said composition comprises the described quinoline antimalarial compound of 0.8% to 33% weight of the gross weight of the inhibitor that accounts for quinoline antimalarial compound and hiv protease or its officinal salt.

9. the test kit of claim 8, the inhibitor of wherein said hiv protease is selected from indinavir, ritonavir, saquinavir, viracept see nelfinaivr, Lopinavir, amprenavir, fosamprenavir, tipranavir, atazanavir, TMC-114 and combination thereof.

10. the test kit of claim 8, wherein said pharmaceutical kit comprises that further at least one contains the container of the nucleosidic inhibitors of at least a HIV reverse transcription.

11. the test kit of claim 10, the nucleosidic inhibitors of wherein said HIV reverse transcription is selected from zidovudine, lamivudine, and Abacavir, zalcitabine removes hydroxyl machine glycosides, takes charge of his furan pyridine, tenofovir, emitricitabine, amdoxovir and combination thereof.

12. the test kit of claim 8, wherein the described hiv protease inhibitor of treatment effective dose is enough to realize plasma concentration 10nM to 30 μ M in the compositions.

13. the test kit of claim 12, wherein the described quinoline antimalarial of treatment effective dose is enough to realize plasma concentration 0.005 μ M to 6 μ M in the compositions.

14. pharmaceutical composition is used for by suppressing treatment of HIV and plasmodium or prevention people HIV infection in preparation, application in malaria or both medicines, it is characterized in that described compositions comprises: at least a quinoline antimalarial compound and at least a hiv protease inhibitor, or its officinal salt, wherein said quinoline antimalarial compound is selected from chloroquine, hydroxychloroquine, mefloquine, quinine and combination thereof, wherein said composition comprises the described quinoline antimalarial compound of 0.8% to 33% weight of the gross weight of the inhibitor that accounts for quinoline antimalarial compound and hiv protease or its officinal salt.

15. the application of claim 14, the inhibitor of wherein said hiv protease is selected from indinavir, ritonavir, saquinavir, viracept see nelfinaivr, Lopinavir, amprenavir), fosamprenavir, tipranavir, atazanavir, TMC-114 and combination thereof.

16. the application of claim 14, wherein compositions also comprises the nucleosidic inhibitors of at least a HIV reverse transcription.

17. the application of claim 16, wherein the nucleosidic inhibitors chemical compound of HIV reverse transcription is selected from zidovudine, lamivudine, and Abacavir, zalcitabine removes hydroxyl machine glycosides, takes charge of his furan pyridine, tenofovir, emitricitabine, amdoxovir and combination thereof.

18. the application of claim 14, wherein the described hiv protease inhibitor of treatment effective dose is enough to realize plasma concentration 10nM to 30 μ M in the compositions.

19. the application of claim 18, wherein the described quinoline antimalarial compound of treatment effective dose is enough to realize plasma concentration 0.005 μ M to 6 μ M in the compositions.