WO2006008115A1 - Diterpenic labdans as immunostimulants for treating infectious diseases - Google Patents

Abstract

The present invention provides the use of a pharmaceutical composition as an immunostimulating agent for improving the immunological response against diseases produced by different pathogens, the composition comprising diterpenic labdans, from Andrographis paniculata, in a defined dose and a pharmaceutically acceptable carrier.

Description

Description

DISTERPENIC LABDANS AS IM MUNOSTIMULANTS FOR TREATING INFECTIOUS DISEASES

Background art

[0001] Evidence is available for demonstrating the immunostimulating action of andrographolides, based in the lymphocytes proliferation and the IL-2 production increase. Even, andrographolides are capable of improving the alpha tumoral necrosis factor production and the expression of the CD marker, resulting in an enhancement of the lymphocytes cytotoxic activity against cancer cells. This results suggest that the andrographolide compound is a pharmacophore with potential anticancer and immunomodulating activities ["Andrographolide, a potential cancer therapeutic agent isolated from Andrographis paniculata", Rajagopal S, Kumar RA, Deevi DS, Satyanarayana C, Rajagopalan R. Discovery Research, Dr. Reddy's Laboratories, Miyapur, Hyderabad, lndia-500050. J. Exp. Ther. Oncol. 2003 May-Jun;3(3):147-58].

[0002] Other studies are available, wherein the anticancer and immunomodulating activity has been assessed for the methanolic extract of Andrographis paniculata, in human cancer and in immunological cells. The results of said studies indicate that the dichloromethane fraction of the methanolic extract retains the active compounds which contribute for both the anticancer and the immunostimuiating activities. The dichloromethane fraction inhibits in an important way the HT-29 cells proliferation (colon cancer) and increases the proliferation of human peripheral blood lymphocytes (HPBL) at low concentrations ["Anticancer and immunostimulatory compounds from Andrographis paniculata". Kumar RA, Sridevi K, Kumar NV, Nanduri S, Rajagopal S. Discovery Research, Dr. Reddy's Laboratories, Miyapur, Hyderabad 500050, India. J Ethnofarmacol. 2004 Jun; 92(2-3):291-5].

[0003] Puri et al. found that using an ethanolic and purified extract of andrographolides which have been isolated from Andrographis paniculata (Acanthaceae) the stimulation of antibodies and the response to the delayed type hypersensibility (DTH) against ovine red blood cells (SRBC) in mouse was induced in an important way. Also, the preparations from the plant, were able to stimulating the non-specific immune response of animals, which has been measured in terms of the macrophages migration index (MMI) during the phagocytosis of E. α?//marked with ¹⁴C-leucyne and over the spleen lymphocytes proliferation. Nevertheless, the stimulation of both responses, the one specific for antigen and the one non specific, was lower when using andrographolide regarding the ethanolic extract, which suggests that other substances differing from andrographolide are comprised within the extract, wherein said substances can contribute for said immunostimulation ["Immunostimulant agents from Andrographis paniculata". Puri A, Saxena R, Saxena RP, Saxena KC, Srivastava V, Tandon JS. Division of Biochemistry, Central Drug Research Institute, Lucknow, India. J. Nat. Prod. 1993 Jul;56(7):995- 9]-

[0004] Due to the wide use of this herb, a number of chemical substances have been identified as secondary metabolites, additionally, some reports have been published revealing diterpenoid compounds which have biologic activity. The hepatoprotective activity of these diterpenoids has been shown (Kapil et al. 1993), as well as the immunostimulating activity (Puri et al. 1993), the choleretic (Shukla et al. 1992), as inductors of the cell differentiation (Matsuda et al. 1994) and as enzymatic inductors (Choudhury et al. 1987) also have been shown those which inhibit the enzymatic activity (Choudhury et al. 1987). The methanolic extract of Andrographis paniculata is comprised of about 11 % of diterpenoids (Handa and Sharma, 1990b).

[0005] Considering the current state of the art, the use of a composition comprising diterpenic labdanes, from Andrographis paniculata, constitutes an invention intended to stimulate the TH1 type immunologic response, without causing the undesirable side effects.

[0006] Cell-Mediated Immunity

[0007] Cell-mediated immunity (CMI) was once thought to be mediated solely by T lymphocytes; however, it is now clear that it is mediated by a variety of cell types, cell factors, or both. Virus-infected or virally transformed cells activate strong cell-mediated immune responses. For some viral infections, cell-mediated immune reactions may be more important than antibody in early termination of viral infection and prevention of dissemination within the host. Recent evidence shows that cell mediated immunity functions at the body surfaces, as well as internally. Cell- mediated immune responses to viral infections involve T lymphocytes, ADCC, macrophages, natural killer (NK) cells, lymphokines, and monokines (Figures 2 and 3).

[0008] Cytotoxic effector cells that can destroy virus-infected cells include cytotoxic T cells, natural killer cells, and activated macrophages. Cytotoxic T lymphocytes can recognize and destroy virus-infected cells, and this recognition is virus specific and major histocompatibility complex (MHC) restricted. Activated macrophages and natural killer cells can also recognize virus-infected cells, but this is not virus specific or major histocompatibility complex restricted.

[0009] T Lymphocytes

[0010] T lymphocytes are thymus-derived lymphocytes and play a central role in the generation and regulation of the immune response to protein antigens. T cells originate from bone marrow stem cells that develop into T precursor cells that migrate to the thymus where they multiply and differentiate. The thymus production of T cells is very high in childhood and declines thereafter. Because mature T cells are long lived and recirculate, they comprise about 70-80% of lymphocytes in blood and lymph, and they are responsible for much of the immunologic memory.

[0011] Maturation

[0012] The maturation of T cells takes place in the thymus and is characterized by a sequential appearance of certain cell surface molecules. Among the first surface molecules to appear are CD3, T cell receptors (TcR), which are α/β positive; CD4; and CD8. Thus immature thymocytes are CD3⁺CD4⁺CD8⁺. Cortical T cells lose either CD4 or CD8 molecules to become CD3⁺TcR⁺CD4⁺ or CD3⁺TcR⁺CD8⁺. Mature T cells migrate to the medulla of the thymus from where they exit into the systemic circulation. [0013] The TcR recognizes protein antigen determinants that are presented by

MHC molecules. In addition, TcR are physically associated with CD3. This association with CD3 is required for transmembrane signaling that culminates in T cell activation.

[0014] Role of MHC in T Cell Development

[0015] One major function of MHC molecules is to present antigens to T cells.

Lymphocytes in the thymus are exposed to various endogenous proteins, particularly the products of MHC. Some T cells that have specificity towards self MHC molecules are eliminated (negative selection), while other T cells become "educated" to recognize foreign antigenic peptides that are associated with MHC (positive selection). Thus, antigen recognition by T cells becomes "MHC restricted," that is, the mature T cell recognizes its specific antigen only if that antigen is presented by the correct MHC molecule.

[0016] Two kinds of MHC genes, class I and class II, are involved in the development of T cells. In the course of selective adaptation, T cells learn to recognize foreign antigens in association with protein products of either MHC class I or Il genes. MHC class l-restricted T cells express CD8 molecules that bind to the invariant portion of MHC class I, whereas MHC class I l-restricted T cells express the CD4 molecule that binds to MHC class Il molecule. Thus, mature T lymphocytes leaving the thymus are either CD4⁺ or CD8⁺ (single positive) and express CD3 and TcR.

[0017] T-CeII Subpopulations

[0018] Both CD4 and CD8 molecules participate in T cell activation. CD4⁺ T cells are principally regulatory cells, which control the functions of other lymphocytes. Based on the lymphokines they produce, CD4⁺ Th cells are divided into two subsets, namely Th1 cells that promote cellular immunity, and Th2 cells that help antibody production. CD8⁺ T cells are cytotoxic/suppressor cells which participate in cell-mediated immunity against viruses, fungi, bacteria, and against certain tumors and play a role in immune regulation.

[0019] T Helper (Th) Cells [0020] These cells are involved in the regulation of both T cell and B cell- mediated immune responses. IgG, IgA, and IgE antibody responses against T-dependent antigens require Th2 cells. Th2 cells aid antigen- activated B cells to proliferate and differentiate into antibody-producing plasma cells. They recognize foreign antigens complexed with MHC class Il molecules on antigen-presenting cells (B cells, macrophages, dendritic cells and Langerhans cells).

[0021] Antigens are presented to Th2 cells in two ways. In the first, the antigen is taken up and processed by accessory cells, such as macrophages or B cells, that present the Ag/MHC complex to Th2 cells. Activated T cells then produce lymphokines that recruit and activate B cells to produce antibodies. Unlike phagocytic cells, B cells bind the antigen by specific antibodies, then they internalize and process the protein, and express a fragment of it bound to MHC class Il molecules on the cell surface. Antigen-specific Th2 cells that bind the Ag/MHC complex on the antigen- presenting cells become activated and produce helper factors for B cells. Furthermore, macrophages may process and present antigens without MHC product to B cells or, in the case of complex polysaccharides, the antigen may be presented directly to B cells without the aid of other cells. Which pathway is used depends on the nature of the antigen.

[0022] Helper T cells (Th 1) also aid effector T lymphocytes (vide infra) in cell- mediated immunity. This process occurs according to the pathway depicted, except that the recipients of the helper factor are effector T cells.

[0023] B and T cells require different cytokines for growth and differentiation. The pattern of the production of those particular factors define whether the cells are Th1 or Th2. For example, Th1 cells produce IFN-γ, a cytokine that activates macrophages. Those activated macrophages in turn participate in delayed hypersensitivity, a major aspect of cell-mediated immunity. In contrast, Th2 cells produce cytokines such as IL-4 and IL-10, which activate certain phases of antibody production and inhibit the genesis of delayed hypersensitivity.

[0024] The factors that determine whether a proliferating CD4 T cell will differentiate into a TH1 or a TH2 cell are not fully understood. The cytokines elicited by infectious agents (principally IFN-γ, IL-12, and IL-4), the co-stimulators used to drive the response, and the nature of the peptide:MHC ligand all have an effect. In particular, because the decision to differentiate into TH1 versus TH2 cells occurs early in the immune response, the cytokines produced in response to pathogens by cells of the innate immune system play an important part in shaping the adaptive response.

[0025] The consequences of inducing TH1 or TH2 cells are profound: the selective production of TH1 cells leads to cell-mediated immunity, whereas the production of predominantly TH2 cells provides humoral immunity. An example is the difference this can make to the outcome of infection is seen in leprosy, a disease caused by infection with Mycobacterium leprae. M. leprae, like M. tuberculosis, grows in macrophage vesicles, and effective host defense requires macrophage activation by TH1 cells. In patients with tuberculoid leprosy, in which TH1 cells are preferentially induced, few live bacteria are found, little antibody is produced, and, although skin and peripheral nerves are damaged by the inflammatory responses associated with macrophage activation, the disease progresses slowly and the patient usually survives. However, when TH2 cells are preferentially induced, the main response is humoral, the antibodies produced cannot reach the intracellular bacteria, and the patients develop lepromatous leprosy, in which M. leprae grows abundantly in macrophages, causing gross tissue destruction that is eventually fatal

[0026] Much evidence indicates that T lymphocytes are important in recovery from viral infections. Of the many functional subsets of T cells, those that express specific cytotoxic activity against virus-infected or transformed cells have aroused the most interest.

[0027] Cytotoxic T lymphocytes

[0028] The generation of virus-specific cytotoxic T lymphocytes (CTLs) is believed to be important in preventing viral multiplication. Presumably, the T lymphocytes prevent virus multiplication by destroying infected cells before mature, infectious virus particles can be assembled. This hypothesis assumes that viral antigens appear on the plasma membrane before the release of virus progeny, a view that is substantiated by studies of many, but not all, infections.

[0029] Exposure to a virus-infected cell can cause the antigen-specific T lymphocytes to differentiate into cytotoxic effector T cells, which can lyse virus infected or virally transformed cells. These cytotoxic T cells are specific not only for the viral antigen but also for self major histocompatibility antigens and will lyse virus-infected cells only if these cells also express the correct major histocompatibility complex (MHC) gene product.

[0030] Activation of cytotoxic and other T lymphocytes may be one of the earliest manifestations of an immune response. T-cell effector functions occur as early as 3 to 4 days after initiation of a viral infection. However, T-cell responses often decrease rapidly, within 5 to 10 days of elimination of the virus (although virus-specific memory T cells persist for long periods). In contrast, antibodies usually become measurable later in the viral infection (after 7 days) and persist at high levels for much longer (years).

[0031] Helper T cells may be as important as cytotoxic T cells in the immune response to virus infections. Helper T cells are required for the generation of cytotoxic T cells and for optimal antibody production. In addition, helper T cells, and cytotoxic T cells produce a number of important soluble factors (lymphokines) that can recruit and influence other cellular components of the immune and inflammatory responses.

[0032] Animal studies indicate that impairing the T-cell defenses enhances infections by herpes simplex virus, poxviruses, and Sindbis virus and enhances the development of tumors induced by polyomavirus. Since the host retains some resistance to infections, T lymphocytes probably are not the sole defense against these viruses. Impairment of T lymphocytes also hinders T cell-dependent antibody production. In humans, T-cell impairment is associated mainly with more frequent and severe poxvirus and herpesvirus infections. Nevertheless, these infections still do not develop in most individuals with T-cell deficiencies, even though the prevalence of herpesviruses (and many other viruses) is great.

[0033] Macrophages [0034] Macrophages are important in both specific and nonspecific responses to viral infections (e.g., herpesvirus infections). Factors that modify macrophage activity can influence the outcome of an infection. Moreover, since macrophages are central to the induction of T and B lymphocyte responses, any effect on macrophages will influence B and T cells.

[0035] Macrophages confer protection against viruses through either an intrinsic or an extrinsic process. In the former, virions are disposed of within macrophages acting either as phagocytes or as non permissive host cells. In the latter case, macrophages retard or ablate virus multiplication in neighboring cells by destroying virus-infected cells or by producing soluble factors (interferons) that act on these cells. Phagocytosis of some viruses by macrophages decreases virus levels in body fluids (as during viremia) and thereby impedes virus spread. These effects are produced only if the virus is destroyed or contained by macrophages. If a virus replicates in macrophages, the infected macrophages may aid in transmission of the virus to other body cells. The permissiveness of macrophages for virus replication may depend on the age and genetic constitution of the host and on the specific condition of the macrophages.

[0036] Macrophage activation mediated either by products of infection (viral and cellular) or by soluble factors produced by T cells (e.g., gamma interferon) often enhance phagocytosis and the elimination of free virus particles. Another important effector mechanism of activated macrophages is their ability to recognize and destroy virus-infected and virus-transformed cells. In addition, activated macrophages participate in virus inhibition by producing cytokines (interferon, etc.) and mediating ADCC (Antibody- Dependent Cell-Mediated Cytotoxicity).

[0037] Natural Killer Cells

[0038] Natural killer (NK) cells exhibit cytotoxic activity against a number of tumor cell lines, particularly against virus-infected or virus-transformed cells (Figure 2). Natural killer or natural killer-like cells, which have been found in almost every mammalian species examined and even in some invertebrates, are identified as large granular lymphocytes that possess Fc receptors. They can mediate ADCC activity; their nonspecific cytotoxic activity is increased by interferon and interleukin-2 (IL-2); and they can produce a number of different cytokines including interferon when stimulated with virus or virus-infected cells.

[0039] Although natural killer cells display cytotoxic activity against virus-infected or transformed cells, they show little or no cytotoxic activity against normal cells. Unlike that of cytotoxic T lymphocytes, natural killer cell killing is not human leukocyte antigen (HLA) restricted, and natural killer cells do not exhibit conventional immunologic specificity. There is evidence that natural killer cells play an important defensive role in virus infections in humans and animals. Their importance is believed to be due to their ability to produce cytokines and to kill virus-infected cells.

[0040] Lymphokines and Monokines

[0041] Soluble factors from T lymphocytes (lymphokines) and macrophages

(monokines) regulate the degree and duration of the immune responses generated by T lymphocytes, B lymphocytes, and macrophages. Interleukin-2 and gamma interferon are two such important factors produced by activated T cells, and interleukin-l is produced by macrophages. All three of these factors are essential for the full differentiation and proliferation of cytotoxic T cells. The two interleukins are also important for antibody production by B lymphocytes.

[0042] Macrophages and T lymphocytes also produce several other factors that act in both the immune and the inflammatory responses. Gamma interferon can activate macrophages to become cytotoxic toward virus- infected cells and can increase the level of phagocytosis and degradation. Lymphotoxins produced by T cells also may participate in the destruction of virus-infected cells. Virus can stimulate alpha interferon production from macrophages; this enhances natural killer cell function and inhibits virus multiplication in neighboring cells.

[0043] Intracellular microbial pathogens

[0044] Intracellular pathogens are responsible for an important amount of mortality worldwide. The list of intracellular infectious agents that have had a significant impact on global health and economy includes viral pathogens (responsible for AIDS, hepatitis, influenza, Newcastle disease virus), protozoan parasites (causative agents of Chagas disease, malaria, leishmaniasis, eimeria), and bacterial pathogens (including the agents responsible for tuberculosis, chlamydia, leprosy, listeria, brucella, Bartonella spp, Piscirickettsia salmon is).

[0045] The understanding of the molecular and cellular basis of intracellular pathogenesis is limited, and this lack of knowledge in many instances has severely reduced the ability to develop effective means of combating or preventing infection.

[0046] Intracellular microbial pathogens cause a plethora of diseases that pose a huge public health challenge. Efficacious prophylactic vaccines are needed to protect the population from this myriad of infectious diseases. Contemporary approaches to vaccine design are guided by the immuno- biological paradigm that extracellular pathogens are controlled principally by humoral immunity, involving specific antibodies, whereas host protection against intracellular pathogens requires effectors of cell- mediated immunity. However, this distinct T-helper (Th) type 1 and 2 paradigm of host defense has encountered a major challenge due to the reality that most antigens or vaccines induce mixed immune responses comprising of both humoral and CMI effectors. Besides, the true functional independence of antibodies and T-cells under in vivo physiologic conditions is uncertain. Recent findings have revealed that antibodies exert a significant immuno-regulatory effect on T-cell immunity. Thus, a robust and protective T-cell memory response against microbial pathogens such as Chlamydia and Mycobacteria require an effective primary humoral immune response characterized by specific antibody isotypes whose role is to modulate Th1 activation via Fc receptors (FcR) by facilitating a rapid uptake, processing and presentation of pathogen-derived antigens for an enhanced T-cell response.

Disclosure of the invention

[0047] The main objective of the instant invention is to provide the use of an immunostimulant, for improving the immunological response for diseases produced by different pathogens, using a composition comprising diterpenic labdanes, from Andrographis paniculata. [0048] One of the main objectives of immunopharmacology and biopharmacy, is the continuous search of therapeutic solutions for improving the immunological response in living beings.

[0049] The immunostimulating drugs action, which can be used for treating infections, immunodeficiency stages or cancer, have been scarcely studied. Among the main problems of the current available immunostimulants, are comprised the widespread or systemic effects, or their scarce efficacy. Those disclosed in the literature, by instance include Levamizol which has been clinically mentioned as a coadjuvant, with fluorouracil, after removing a colon cancer. However, its use has been related with the appearance of agranulocytosis, sometimes lethal. Other immunostimulant, thalidomide, which use, due to its teratogenic activity, is restricted, has been indicated for the treatment of patients with leprosus nodous erythema, since apparently it is able of diminishing the TNF alpha circulating levels, being able of increasing them in HIV seropositive subjects. It has been used as an immunostimulant agent Calmette-Guerfn Bacillus (BCG), from a strain of Mycobacterium bovis, for the treatment of bladder tumors and carcinoma, however its use has caused hypersensibility reactions, shock, shivers, fever, generally unwear and disease by immunologic complexes.

[0050] On the other hand, the recombinant cytokines can be mentioned, existing two types, those alpha, beta and gamma interferons, which are useful for treating viral infections, or for treating tumors and chronic granulomatous diseases, and the IL-2. Interferons are responsible of the reactions as cold, fever, shivers, migraine, myalgia, and gastrointestinal disorders. Recombinant IL-2 has been used for treating metastasis renal cells carcinomas and melanoma in adults. This interleukyne has caused great cardiovascular disorders as a consequence from the leakage syndrome or capillary effusion wherein a vascular tone loss is found, as well as occur an outflow of plasmatic proteins and liquid through the extravascular space. Whereby, hypotension, a decrease of the blood flow and dead can arise. Finally, within the biological therapeutic arsenal, the active (vaccines) or passive (immunoglobulins) immunizations can be found. [0051] All the above, clearly states the lack of drugs for inducing the immunostimulation and the need of increasing the therapeutic arsenal with new drugs.

Brief description of the drawings [0052] Figures 1 A and 1 B: show the effect of andrographolide over the cytokines production in mouse lymphocytes stimulated with Concanavalin A. [0053] Figures 2C and 2D: Exhibits the effect of andrographolide on the thymidine incorporation in mouse and healthy volunteers lymphocytes. n=4, mean ± E. E. [0054] Figures 3A y 3B: Show the effect of andrographolide on CON-A and LPS- induced thymidine incorporation. n=4, mean ± E. E. [0055] Figures 4A, 4B, 4C, 4D: Show the effect of andrographolide over the Th1 cytokines production (IFN-gamma, IL-2) and the one induced by Con-A. n=4, mean ± E. E. [0056] Figures 5A and 5B: Show the effect of andrographolide over the mRNA for

INF-gamma expression in mouse lymphocytes incubated in vitro during 24 hrs. n=4, mean ± E.E. [0057] Figures 6A and 6B: Show the effect of andrographolide over the Th2 cytokines production (IL-4) and the one induced by Con-A. n=4, mean ±

E.E. [0058] Figure 7. Shows the effect of the oral administration of the composition during 6 days over the in vitro Interferon gamma production by T lymphocytes. n=4, mean ± E.E. [0059] Figure 8. Shows the effect of the oral administration of the composition during 6 days over the in i//frp³H-thymidine incorporation on T lymphocytes, in mice immunized with the vaccine RB51. n=4, mean ± E.E. [0060] Figure 9. Shows the effect of andrographolide over the blastic transformation of animals immunized with different concentrations of proteins from Brucella abortus. n=4, mean ± E.E. [0061] Figure 10A. Shows the effect of andrographolide over the IFN-gamma production on animals immunized with different concentrations of proteins from Brucella abortus. n=4, mean ± E.E. [0062] Figure 10B. Shows the effect of andrographolide on the IL-2 production on animals immunized with different concentrations of proteins from Brucella abortus. n=4, mean ± E. E. [0063] Figure 11. Shows the effect of andrographolide on the production of IL-4 of animals immunized with different concentrations of proteins from Brucella abortus. n=4, mean ± E. E. [0064] Figure 12. Shows the effect of the andrographolide administration on the elimination (clearance) of the Ochrobactrum a bacteria. [0065] Figure 13. Effect of andrographolide over the stimulation of the NFKB route. The effect on the luciferase activity induced by andrographolide, the expression of IκBα y RT-PCR and genarray, are showed. Best mode for carrying out the invention [0066] It can be observed on the assays carried out in the instant invention

(Figures 1A and 1 B) that in all the used andrographolide concentrations, it is significantly inhibited the production of IL-2 in those cells stimulated with

Concanavalin A. [0067] At the 0,05 and 0,5 nm concentrations (Figures 2A and 2B), the used andrographolides are able of stimulating in a significant way the in vitro incorporation of tritiated thymidine in mouse lymphocytes and in lymphocytes from healthy volunteers, which indicates a higher cellular activity. [0068] Additionally, it can be observed that the immunostimulanting effect of andrographolide (Figures 3A and 3B) does not affect the response induced by Con-A and LPS, this means that no an increase of the immuno specific stimuli is obtained. [0069] Other important aspect, is that the andrographolide stimulates the production of the IFN-gamma and e IL-2 cytokines and no an increase to non specific immuno stimuli is observed, such as can be observed from

Figures 4A, 4B, 4C, 4D. [0070] Partly, the increase of the IFN-gamma production occurs due to a higher expression of the mARN, which can be deduced from observing figures 5A and 5B. [0071] On the other hand, the IL-4 production does not seem stimulated by an andrographolide effect, as well as neither it affects the response induced by CON-A, no modification of the immunological non specific stimuli effects can be observed (see figure 6).

[0072] On figure 7, it is shown that the composition of e the instant invention when administered in vivo is able of increasing the in vitro production of IFN-gamma in T lymphocytes.

[0073] The specific immune response to intracellular pathogens (vaccine RB-51) is increased at the indicated andrographolide concentrations, such as can be observed on figure 8. Likewise, when observing figure 9, it is found that the specific immune response for intracellular pathogens (protein from Brucella abortus) is enhanced by the effect of andrographolide administered at the indicated concentrations.

[0074] During the experimental development, it has also been found, of the instant invention that the use of andrographolide is able of stimulating the production of INF-gamma in animals immunized with protein from Brucella abortus (see figure 10A). As well as it is able of stimulating the production of IL-2 in animals immunized with protein from Brucella abortus (see figure 10 B). Nevertheless, the use of andrographolide did not produce any effect on the IL-4 production in animals immunized with protein from Brucella abortus (see figure 11 ).

[0075] Other important effect regarding the use of said andrographolides compounds, is that the ability of bacteria disposal is improved per example, Ochrobactrum a., such as it can be observed in figure 12.

[0076] The NfKb mediator is stimulated by the use of andrographolide, which explains the increase of the INF-gamma and IL-2 cytokines and as well as over other genes such as IkappaBα (see figure 13).

[0077] In the instant patent application it is described the use of a composition which comprises an standardized extract of Andrographis paniculata, as the immunostimulating agent. Which allows to improve the immune response of the living beings.

[0078] Preferably, the composition is useful for enhancing the immune response on mammals, including the human and the production animals, being used as a medicament, or, being administered with the normal diet of said animals.

[0079] The term "production animals" represents all those animals selected from the group formed by: porks, ovines, bovines, birds, poultry, equines, camels, fishes and others.

[0080] Andrographis paniculata (Nees), is a medicinal plant which belongs to the Acantaceae family from Asia, native from India, Malaysia, China and Korea. In these countries it has been widely used due to the benefic effects of the same, as the fresh plant, the dried plant and its components for treating different diseases, as common cold, hepatic conditions, diabetes, etc.

[0081] At the literature suggested doses, the inventors of the instant application have found no stimulation, however it has been found a marked inhibition of the T lymphocytes response for the Th1 cytokines production (IFNγ and IL-2), see figure 1.

[0082] It is shown, on in vitro experiments carried out in mouse lymphocytes that at concentrations between 0.05 and 5 nM the andrographolide increases the ³H-thymidine incorporation in said cells (Figure 2A). This phenomenon is also shown in human lymphocytes (Figure 2B). Despite the increase of the thymidine incorporation, the andrographolide was not able of promoting the non specific response lipopolysaccharides induced-immune response (LPS, Figure 3B) and by mitogens (Concanavalin A, Figure 3A). These same doses were able of significantly increasing the INF-gamma and IL-2 production (Figures 4A), as well as the increase of the mARN for these cytokines (Figures 5A and 5B). However, an increase of the immune response induced by LPS and mitogens was not observed. On the other hand, the immune response due to TH2 cytokines as IL4 was not modified by an andrographolide effect (Figures 6A and 6B/ In experiments carried out with the composition of the instant invention in mice orally administered with 0.3 mg/kg it was observed an increase of the basal production of IFN-gamma (Figure 1). In other experiments, it was observed, with immunized mice using a vaccine against Brucella abortus (RB51) which develops a type TH1 specific immunity, that the composition of the instant invention at an orally administered dose of 0.3 mg/kg can increase the blastic transformation significantly regarding the control. This suggests, that the composition of the instant invention increases the ability of an immunologic response against vaccines, which stimulate the specific immune response through the TH1 cells (Figures 8A and 8B). In animals treated with the composition of the instant invention, reinforces the fact that the iFN-gamma and IL-2 is increased in animals vaccinated with protein from Brucella abortus (Figure 9 and 10) and does not modify the immune response through the Th2 lymphocytes, as IL4 (Figure 11) and IL5. I support to the specific effect of the composition of the instant invention is the absence of an increase of antibodies against the vaccine. Moreover, the composition of the instant invention helps the disposal of bacteria (Figure 12). The mechanism for explaining the increase of the expression and synthesis of cytokines is associated to an increase of the transcription factors NFKB and NFAT (Figure 13).

[0083] DESCRIPTION OF THE ANDROGRAPHOLIDES COMPOSITION [0084] The composition used in the present application, comprises a mixture of diterpenic labdanes, obtained from an extract of Andrographis paniculata dried extract, having the following general formulae: [0085]

Table 1

[0086] Which main features are: [0087]

Table 2

[0088] A representative mixture of this composition, is comprised by the following pharmaceutical formulation:

[0089]

Table 3

[0090] Said formulation is acceptable for manufacturing medicines which can be administered with a pharmaceutically acceptable carrier, i.e. a tablet form, which have been administered in the following doses:

[0091] a) 0.015-0.07 mg Andrographolide/kg per day, [0092] b) 0.002 - 0.01 mg 14-Deoxiandrographolide/kg per day, [0093] c) 0.0003 - 0.0017 mg Neoandrographolide/ kg per day. [0094] Therefore, the composition as well as its pharmaceutical formulation, particularly when administered in the tablet form and in the above indicated doses, provide a medicine that can be used as immunostimulating agents. [0095] The pharmaceutical composition that can be manufactured with the herein disclosed composition, specially according to the revealed formulation, can correspond to enteral, parenteral, dermal, ocular, nasal, otic, rectal, vaginal, urethral, bucal, pharyngeal-tracheal-bronchial pharmaceutical forms.

[0096] METHOD OF OBTAINING AND ANALYSIS OF ANDROGRAPHIS PANICULA TA RAW MATERIAL

[0097]

Table 4

[0098] The green leaves, stems and higher parts, organically cultivated under supervision of the inventor including the seeds are sun dried. All foreign materials are manually removed and the raw material is cut into 1-1.5 cm size pieces, which are stored in a ventilated area. Routine analysis is carried out in order to asses the identity: macro and microscopic analysis, organoleptic parameters and TLC analysis (thin layer chromatography) is performed according to European Pharmacopoeia.

[0099] METHOD FOR OBTAINING THE ANDROGRAPHIS PANICULATA DRIED EXTRACT

[0100] The extraction of A. paniculata is performed by continuous percolation of the grinded dried plant (aerial part) with hydroethanolic solutions (ethanol 70%/water).

[0101] The duly analyzed drug material is grinded to suitable particle size in a knife-hammer mill (0.8 cm²). The grinded material is charged into stainless steel percolators and the extraction solution is added at a temperature of 5O⁰C. The percolation time is of approximately 6 days (6x24 hours) in two extraction cycles. The percolate is collected in stainless steel tanks until the percolation is completed. The percolate is transferred directly to an evaporation unit in order to eliminate the solvent and most of the water. Evaporation is performed in a LUWA thin-film evaporator at 140-158⁰F (60-70⁰C) and 0.65-0.85 bar vacuum. The evaporation process is performed in 3-4 cycles, where the extract is kept under mixing, 4 times during 30 minutes per day. When the spissum extract has the right content of water, the following analysis are made: ash content, HCL-ash, loss on drying, pH value, TLC identity and HPLC (high performance liquid chromatography), analysis for Andrographolide, 14-Deoxiandrographolide and Neoandrographolide. Then the spissum extract is transferred to the drying unit. Before drying, the final dried extract is mixed with a sufficient amount of maltodextrin used as a carrier and spray dried at an inner temperature of 175-195°C. The dried extract is packaged in plastic bags in fiber drums for subsequent analysis.

[0102] IDENTITY METHOD OF DITERPENIC LABDANS

[0103] To 1 g herbal extract, 20 ml of methanol is added, shaken for about 1 hour and the methanol is decanted through a filter. The residue is shaken with 20 ml methanol, filtered and mixed with the first extract (making 40 ml of test solution).

[0104] As reference solutions are use Andrographolide, 14-Deoxiandrographo)ide and Neoandrographolide, dissolved in methanol. 20-30 ml test solution is applied to a TLC-plate (silica gel GF254 as coating substance) and developed over a path of 15 cm using a mixture of 77 volumes of ethyl acetate, 15 volumes of methanol and 8 volumes of water (77:15:8). Subsequently, the plate is allowed to dry in air and examined under UV (254 nM). The few dark spots of the chromatogram correspond to Andrographolide at a Rf: 0.65-07; 14-Deoxiandrographolide Rf: 0.75-0.8 and Neoandrographolide, Rf: 0.60-0.65.

[0105] HPLC METHOD FOR THE QUANTIFICATION OF DITERPENIC LABDANES

[0106] The three compounds are extracted with acetone (4:1) and then analyzed by HPLC using a reverse phase RP-C18 licrospher column (4x125mm). The mobile phase consists of acetonitrile 26% and phosphoric acid 0.5%, at a rate of 1.1 ml/min, and is detected at 228 nm according to Burgos et al.; 1999, Acta Hort. (ISHS) 501 :83-86.

[0107] The Andrographis paniculata dried extract is standardized to a minimum of 30% of total Andrographolides, which comprises 24,6% of andrographolide, 4.8% w/w of 14-Deoxyandrographolide, and 0.6% w/w of Neoandrographolide, for subsequently being produced as various pharmaceutical forms.

[0108] EXAMPLES

[0109] The following examples are intended to be illustrative, in no case they are intended to restrict the invention: [0110] EXAMPLE No. 1 [0111] Dried Andrographis paniculata NΘΘS, extract obtainment, dried herb 100, 0 mg.

[0112] Dried extract (Total andrographolides 30%) [0113]

Table 5

Other ingredients: mg

Potato starch 15.0

Talc 106.9

Gelatin 11.5

Magnesium stereate 5.6

Shellac 3.5

Anhydrous. Silicon dioxide 2.0

Polyethylene glycol 0.7

Tablet weight 450.0

[0114] Administrating route.

[0115] This pharmaceutical composition can be administered as enteral, parenteral, dermal, ocular, nasal, otic, rectal, vaginal, urethral and bucal pharmaceutical forms.

[0116] Additionally, the composition of the instant invention has a low toxicity, and does not exhibit harmful side effects.

[0117] Considering the current "state of the art" in science, the use of said composition cannot be deduced by a person skill in the art, wherein said composition is intended to work as an immunostimulating agent, useful for improving the immunologic response of a subject, per instance, when exposed to various antigens from viral, bacterial or protozoan pathogens, for preventing or treating those diseases caused by said agents, without provoking side effects, such as it occurs with other substances that are generally used for the same purpose of the instant invention. [0118] DESCRIPTION OF THE ANDROGRAPHOLIDES COMPOSITION [0119] The composition used in the present application, comprises a mixture of diterpenic labdanes, obtained from an extract of Andrographis panicu/ata dried extract, having the following general formulae:

[0120]

Table 6

[0121] A representative extract of the present composition, is constituted by a mixture of the following Andrographolides: Andrographolide, 14- Deoxyandrographolide, and Neoandrographolide. Wherein said individual components are contained approximately 20 to 40% w/w of Andrographolide, about 3 to 6% w/w of 14-Deoxyandrographolide, and about 0.2 to 0.8% w/w of Neoandrographolide in the dried extract. Preferably these compounds are contained from about 25 to 35% w/w of Andrographolide, from about 4.5 to 5.5% w/w of 14-deoxyandrographolide, and approximately 0.4 to 0.8% w/w of Neoandrographolide in the final extract.

[0122] In a more preferred embodiment, the novel extract comprises: [0123]

Table 7

[0124] Said formulation is acceptable for manufacturing medicines which can be administered with a pharmaceutically acceptable carrier, i.e. a tablet form, administered in a dose comprising approximately 0.015 to 0.07 mg/kg BW/day of the andrographolides mixture.a) 0.015-0.07 mg Andrographolide/kg per day

[0125] b) 0.002 - 0.01 mg 14-Deoxiandrographolide/kg per day [0126] c) 0.0003 - 0.0017 mg Neoandrographolide/ kg per day. [0127] Without affecting other formulation and administration embodiments, those herein disclosed, contribute efficiently and effectively for improving the immunologic response in the treatment of diseases caused by viral, bacterial and protozoan intracellular pathogen agents.

[0128] Said viral agents are selected from the group consisting of: the humane immunodeficiency virus, hepatitis virus, influenza virus, syncytial respiratory virus, Newcastle disease virus.

[0129] Said protozoan pathogen agents are selected from the group consisting of Tripanosoma cruzi, Plasmodium falciparum, Leishmania, Coccidium.

[0130] Said bacterial pathogen agents are selected from the group consisting of: Mycohacteriaceae, Chlamydia, Mycobacterium leprae, Listeria monocytogenes, Brucellae, Bartonella spp, and Piscirickettsia salmonis.

[0131] Therefore, both the composition and its pharmaceutical formulation particularly when administered in the tablet form and in the above indicated doses, provide a medicine useful as an immunostimulating agent, which final effect will be the one of improving the Th1 type immunologic response when occurs an infectious disease caused by a viral, bacterial or protozoan pathogen agent.

[0132] The pharmaceutical composition prepared with the composition of the instant invention, specifically according to the revealed formulation, may correspond to enteral, parenteral, dermal, ocular, nasal, otic, rectal, vaginal, urethral, bucal, and pharyngeal-tracheal-bronchial pharmaceutical forms.

[0133] METHOD OF OBTAINING AND ANALYSIS OF ANDROGRAPHIS PANICULA TA RAW MATERIAL

[0134]

Table 8

Active ingredient: Andrographis paniculata Nees {Burm. f.) Family: Acantaceae

Used part: herba

[0135] The green leaves, stems and higher parts, organically cultivated under supervision of the inventor, including the seeds are sun dried. All foreign materials are manually removed and the raw material is cut into 1-1.5 cm size pieces, which are stored in a ventilated area. Routine analysis is carried out in order to asses the identity, which consists of: macro and microscopic analysis, organoleptic parameters and TLC analysis (thin layer chromatography) are performed according to European Pharmacopoeia.

[0136] METHOD FOR OBTAINING THE ANDROGRAPHIS PANICULA TA DRIED EXTRACT

[0137] The extraction of A paniculata is performed by continuous percolation of the grinded dried plant (aerial part) using a polar solvent in part 1.

[0138] The duly analyzed drug material is grinded to suitable particle size in a knife-hammer mill (0.8 cm²). The grinded material is charged into stainless steel percolators and the extraction solution is added at a temperature of 50⁰C. The percolation time is of approximately 6 days (6x24 hours) in two extraction cycles. The percolate is collected in stainless steel tanks until the percolation is completed. The percolate is transferred directly to an evaporation unit in order to remove the solvent and most of the water. Evaporation is performed in a LUWA thin-film evaporator at 140-158⁰F (60-70⁰C) and 0.65-0.85 bar vacuum. The evaporation process is performed in 3-4 cycles, wherein the extract is kept under mixing, 4 times during 30 minutes per day. When the spissum extract has the right content of water, the following analysis are made: ash content, HCL-ash, loss on drying, pH value, TLC and HPLC (high performance liquid chromatography) identity, analysis for Andrographolide, 14- Deoxiandrographolide and Neoandrographolide. Then the spissum extract is transferred to the drying unit. Before drying, the final dried extract is packaged in plastic bags in fiber drums for subsequent analysis.

[0139] METHOD OF PREPARATION OF ANDROGRAPHIS PANICULA TA 30% EXTRACT:

[0140] Cut and sieved leaves/stem of andrographis paniculata are collected from farms under direct control of the inventors. The aerial parts are analyzed for identity as previously described and then taken for extraction.

[0141] The aerial parts are extracted in a Stainless steel extraction unit under vacuum with a low polarity solvent (A), such as N-Hexane or Chloroform. Following successive extractions, the solvent is removed and the marc is treated with a second solvent having higher polarity (B) such as Pet Ether 40:60 or Ethyl acetate, after a single extraction, the solvent is removed and the marc is treated with a solvent (C) having greater polarity such as ethanol or water.

[0142] The third solvent is recovered and evaporated leaving behind a mass with 30-40% moisture, the marc is treated with a solvent having low polarity as previously described, the mass is then filtered and dried under vacuum till there is less than 5% moisture. The granules are ground to a fine powder having not less than 30% labdane diterpenes calculated as andrographolides.

[0143] DETAILS OF EXTRACTION:

[0144] Step 1 : Finely cut leaves/stem of Andrographis paniculata are loaded in an S S reactor with between 3-5 times w/w of solvent A.

[0145] Step 2: The herb is extracted for between 4-6 hours repeatedly and the solvent removed.

[0146] Step 3: The marc is then treated with a Solvent B having higher polarity than Solvent A and extracted once for 3-5 hours.

[0147] Step 4: The solvent B is removed and the marc is then extracted with Solvent C having higher polarity than Solvents A or B.

[0148] Step 5: The Solvent C is circulated through the marc for 3-5 hours and removed under vacuum to an S S evaporator and the marc is again extracted with Solvent C, this process is repeated 3-4 times.

[0149] Step 6: The solvent C is recovered from all the washings and the resulting mass pooled together.

[0150] Step 7: The mass obtained from Steps 4-7 is then treated with Solvents A or B and the residue is dried under vacuum at a temperature not exceeding 60 C.

[0151] Step 8: The dried mass that is obtained from Step 7 is powdered using a GMP Grinder having stainless steel meshing between 100-200 ASTM.

[0152] Step 9: The powder obtained from Step 8 is sieved through an auto siever and directly filled into sterilized PP bags ready to be sealed. [0153] The powder obtained as described above is analyzed as per the protocols described in this document having 25 to 35% w/w of Andrographoiide, from about 4.5 to 5.5% w/w of 14-deoxyandrographolide, and approximately 0.4 to 0.8% w/w of Neoandrographolide in the final extract.

[0154] IDENTITY OF AN D ROG RAP H I S PANICULATA - TLC

[0155] Test solution: To 1 g herbal extract, 20 ml of methanol is added, shaken for about 1 hour and the methanol is decanted through a filter. The residue is shaken with 20 ml methanol, filtered and mixed with the first extract (making 40 ml of test solution).

[0156] Reference solutions:

[0157] 1 Andrographoiide (A), 14-Deoxiandrographolide (DA) and Neoandrographolide (NA), dissolved in methanol.

[0158] 2. Reference-extract treated in the same way as the test-extract.

[0159] 20-30 ml test solution is applied to a TLC-plate (silica gel GF254 as coating substance) and developed over a path of 15 cm using a mixture of 77 volumes of ethyl acetate, 15 volumes of methanol and 8 volumes of water (77:15:8). Subsequently, the plate is allowed to dry in air and examined under UV (254nM). The few dark spots of the chromatogram correspond to Andrographoiide at a Rf. 0.65-07; 14-Deoxiandrographolide R_f: 0.75-0.8 and Neoandrographolide, R_f: 0.60-0.65.

[0160] HPLC METHOD FOR THE QUANTIFICATION OF DITERPENIC LABDANES

[0161] The three compounds are extracted with acetone (4:1) and then analyzed by HPLC using a reverse phase RP-C18 licrospher column (4x125 mm). The mobile phase consists of acetonitrile 26% and phosphoric acid 0.5%, at a rate of 1.1 ml/min, and is detected at 228 nm according to Burgos et al.; 1999, Acta Hort. (ISHS) 501:83-86.

[0162] The Andrographis particulate dried extract is standardized to a minimum of 30% of total Andrographolides, which comprises approximately 20 to 40% w/w of andrographoiide, 3 to 6% w/w of 14-Deoxyandrographolide, and 0.2 to 0.8% w/w of Neoandrographolide.

[0163] The composition according to the present invention has not been previously disclosed in the current "state of the art" in science and there are no antecedents about the use of the same in order to solve the described methodological problems concerning the use of the composition as an immunostimulating agent.

[0164] The pharmaceutical compositions of this invention may be administered orally or parenterally, and the parenteral administration comprises intravenous injection, subcutaneous injection, intramuscular injection and intraarticular injection.

[0165] The correct dosage of the pharmaceutical compositions of the invention will vary depending on the particular formulation, the mode of application, age, body weight and gender of the patient, diet, disease status of the patient, complementary drugs and adverse reactions. It is understood that the ordinary skilled physician will readily be able to determine and prescribe a correct dosage of this pharmaceutical compositions. Preferably, the daily dosage of this pharmaceutical compositions ranges from 1- 6.5 mg of the andrographolides mixture per kg body weight.

[0166] According to the conventional techniques known to those skilled in the art, the pharmaceutical compositions of this invention can be formulated with a pharmaceutical acceptable carrier, such as a unit dosage form. Non- limiting examples of the formulations include, but not limited to, a sterile solution, a solution, a suspension or an emulsion, an extract, an elixir, a powder, a granule, a tablet, a capsule, a liniment, a lotion and an ointment.

[0167] The present invention also embraces the pharmaceutical compositions containing the andrographolide, 14-deoxiandrographolide and Neoandrographolide labdanes compounds in combination with pharmaceutically acceptable carriers normally employed in preparing such compositions.

[0168] In the pharmaceutical compositions of this invention, the pharmaceutically acceptable carrier may be any conventional one described for pharmaceutical formulations, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, Hydroxypropylmethylcellulose (HPMC), methylhydroxy benzoate, propylhydroxy benzoate, talc, stearic acid, magnesium and mineral oil, but not limited thereto. Additionally, the pharmaceutical compositions of the present invention may contain any of a wetting agent, sweetening agent, emulsifying agent, suspending agent, preservatives, flavors, perfumes, lubricating agent, or mixtures of these substances.

[0169] Typically, the pharmaceutical compositions contains from 20-40%, preferably from 25 to 35%, and most preferably 30% w/w of andrographolide, 14-Deoxiandrographolide and Neoandrographolide Labdanes of the mixture, and the pharmaceutically acceptable carriers.

[0170] The pharmaceutical composition of the present invention can be administered to mammals in need thereof, via oral route administered singly or as a divided dose.

[0171] Thus, for oral administration, the compounds can be combined with a suitable solid carrier to form capsules, tablets, powders. Additionally, the pharmaceutical compositions may contain other components such as flavour agents, sweeteners, excipients and the like.

[0172] Additionally, the present invention provides a method for treating patients with the composition containing the andrographolides mixtures which comprises: intravenous administering of the solution and orally administering the tablets comprising the composition of the present invention to patients in need thereof. The preferred dosage of the injection solution formulation is about 60 to 210 mg/day, most preferably, 60-80 mg/day, of the composition per day in one, two or three injections.

[0173] The present formulation in the injectable solution form comprises 8-16 mg approximately of the composition per ml. When administering to patients, the composition is preferably diluted to about 1 :5 to 1 :10 volume of 0.9% saline solution.

[0174] The following examples are illustrative, but do not limit the scope of the present invention. Reasonable variations, such as those occurring to a reasonable artisan, can be made herein without departing from the scope of the present invention.

[0175] The pharmaceutical composition of the present invention is suitable for preparing in a typical scale for the pharmaceutical industry as well as for a smaller measure. Following conventional techniques of the pharmaceutical industry which involve wet granulation, dry granulation, direct compression, fluid bed granulation, when necessary, for tablet forms, as appropriate, to give the desired oral, or parenteral administration products.

[0176] The percentages indicated in the following examples are all indicated in a weight base.

[0177] EXAMPLES

[0178] Exemplary of a typical method for preparing a tablet containing the active agents is to first mix the agent with a binder such as gelatin, ethyl cellulose, or the like. Wherein the mixing is suitably carried out in a standard V-blender and usually under anhydrous conditions. Next, the fresh prepared mixture can be slugged through conventional tablet machines and the slugs fabricated into tablets. The freshly prepared tablets are coated, with suitable coatings including shellac, methylcellulose, camauba wax, styrene-maleic acid copolymers, and the like.

[0179] For oral administration, the compressed tablets containing from 30 mg up to 40 mg of the andrographolide mixture are manufactured according to the above disclosed methods of manufacturing techniques well known to the art and set forth in Remington's Pharmaceutical Science, Chapter 39, Mack Publishing Co., 1965.

[0180] The preferred pharmaceutical compositions of the present invention, correspond to formulations that are detailed in some of the following Examples.

[0181] EXAMPLE 1

[0182] Pharmaceutical composition for preparing a tablet of the present invention, using the andrographolide mixture contained in the dried extract obtained from the herb Andrographis paniculata Nees. [0183]

Table 9

Ingredients Per tablet mg.

Dried extract (andrographolides mixture) 15.0

Potato starch 168.8

Talc 106.9

Gelatin 11.5

Magnesium stereate 5.6

Hydroxypropylmethyl cellulose 3.5

Silicon dioxide, anhydrous 2.0

Polyethylene glycol 0.7

Carbonate, calcium (qsp.) 16.0

[0184] To formulate the tablet uniformly, the dried extract is blend

(Andrographolides mixture) active compound, potato starch, talc, gelatin, hydroxypropylmethylcellulose, silicon dioxide, anhydrous, polyethylene glycol, and calcium carbonate under dry conditions in a conventional V- blender until all the ingredients are uniformly mixed. The mixture is then passed through a standard light mesh screen, dried in an anhydrous atmosphere and then blended with magnesium stearate, and compressed into tablets, and coated with shellac. Other tablets containing from 116 to 162 mg, are prepared in a similar fashion. [0185] EXAMPLE 2

[0186] Pharmaceutical composition for preparing a capsule of the present invention, using the dried extract obtained from the herb Andrographis paniculata Nees. [0187]

Table 10

Ingredients Per tablet mg.

Dried extract, (andrographoiides mixture) 15.0

Potato starch 168.8

Talc 106.9

Gelatin 11.5

Magnesium stereate 5.6

Hydroxypropylmethyl cellulose 3.5

Silicon dioxide, anhydrous 2.0

Polyethylene glycol 0.7

Carbonate, calcium 16.0

[0188] The manufacture of capsules containing from 1 mg to 5 mg of the andrographolide mixture for oral use consists essentially of mixing the Dried Extract, (Andrographoiides mixture) with a carrier and enclosing the mixture in a polymeric sheath (capsule), usually gelatin or the like. The capsules can be in the art known soft form of a capsule made by enclosing the compound in intimate dispersion within an edible, compatible carrier, or the capsule can be a hard capsule consisting essentially of the novel compound mixed with a nontoxic solid such as talc, calcium stearate, calcium carbonate, or the like. Exemplary of a typical use for employing a capsule containing 1.0 mg to 5.0 mg for use as therapeutically indicated.

[0189] The dose administered, whether a single dose, multiple dose, or a daily dose, will of course, vary with the particular compound of the invention employed because of the varying potency of the compound, the chosen route of administration, the size of the patient and the nature of the disease condition. The administered dose corresponds to a general oral dose of 1.0 to 5.0 mg daily, with the oral dose of normally 2 mg three times per day; the usual intravenous dose of 2 to 6 mg, followed if indicated by 2 to 6 mg in a subsequent period, and the usual intramuscular dose of 2 to 6 mg every 24 hours, with 1 to 2 injections per day.

[0190] The pharmaceutical compositions of the instant invention, comprising the Dried Extract, (containing a Andrographolides mixture) are adaptable for the administration of their physiological expected effects from drug delivery systems, such as skin delivery systems, gastrointestinal drug delivery devices, and the like, wherein the delivery device is manufactured from naturally occurring and synthetic polymeric materials. Representative of materials acceptable for the fabrication of drug delivery systems containing the compounds for controlled drug administration include materials such as polyvinyl chloride, polyisoprene, polybutadiene, polyethylene, ethylene- vinyl acetate copolymers, polydimethylsiloxane, hydrophilic hydrogels of esters of acrylic and methacrylic acid, polyvinyl acetates, propylenevinyl acetate copolymers, and the like.

[0191] EXAMPLE 3

[0192] Shellac covered tablets containing the above indicated composition are prepared following conventional techniques of the pharmaceutical industry involving mixing, granulating, and compressing, when necessary, for tablet forms.

[0193] Specifically the composition of example 1 is thoroughly mixed with a sufficient amount of Andrographis paniculata dried extract. For the manufacture of tablets comprising Andrographolide, 14- Deoxiandrographolide and Neoandrographolide, the mixture is compressed in a direct form with the inactive ingredients mentioned in example N°1 , and subsequently covered with shellac, accordingly. [0194] EXAMPLE 4

[0195] Description of a compound from the composition of Andrographolides,

[0196] A representative composition of the present invention is a pharmaceutical formulation in tablets, which supplies the following mixture of compounds:

[0197]

Table 11

Andrographolide 24.6%,

14-Deoxyandrographolide 4.8%

Neoandrographolide 0.6%

[0198] For the subsequent manufacture of the different pharmaceutical forms, and applied in the following doses:

[0199] a) 1 - 5 mg Andrographolide/kg per day

[0200] b) 0.2 - 1 mg 14-Deoxiandrographolide/kg per day

[0201] c) 0,02 - 0.12 mg Neoandrographolide/ kg per day.

[0202] EXPERIMENTAL METHODS

[0203] For carrying out the instant invention the animals and materials were selected as indicated below and the following experiments were made:

[0204] Animals:

[0205] Mice from the strain "Rockefeller" from the animal care facilities of the lnstituto de lnmunologfa de Ia Universidad Austral de Chile were used, these were maintained at 2O⁰C, fed with a special concentrate for mice and water " ad libiturri' .

[0206] EXPERIMENTAL DESIGN No. 1

[0207] Immunization:

[0208] The animals were divided into tour groups each with tour subjects, with the same sex and age, and they were inoculated intraperitoneally with Andrographolide in concentrations of 0, 0.08, 0.8 and 8 nM (0; 0.224; 2.24 and 22.4 μg/kg of weight respectively), during six days. After that period, with the purpose of obtaining spleen lymphocytes, the mice were immunized with 1x10⁸ cells from Brucella abortus strain RB51 (i.p). In order to obtain lymphatic nodes lymphocytes, the mice were immunized with 2,5 μg of total protein from Brucella abortus RB51 in an emulsion form with Incomplete Freund coadjuvant (s.c.) in the underside of the foot cushions of the rear ends, using general anesthetics.

[0209] LYMPHOYD ORGANS OBTENTION:

[0210] The animals from the different groups were sacrificed six days after their immunization with Brucella RB51 , the sacrifice was carried out by an excessive ether inhalation. Their popliteal inguinal nodes and spleen were removed. The obtained organs were disintegrated on Petri plates containing complete sterile RPMI 1640 (Gibco). The obtained lymphocytes, were washed and taken up in 1 ml of RPMI 1640 for being quantified in a Neubauer chamber. Finally the lymphocytes suspension was adjusted to a concentration of 4x10⁶ cells per ml in complete RPMI.

[0211] LYMPHOID CELLS CULTURE FOR BLASTIC TRANSFORMATION.

[0212] The lymphocytes from the spleen and the lymph nodes were stimulated "in vJtro"W\Vn Concanavalin A (1 , 5 and 10 μg/ml), PHA (0, 4 and 40 μg/ml), total protein from Brucella abortus strain RB51 (0, 1 , 5 and 10 μg/ml) and thermally dead Brucella abortus strain RB51 (10⁶, 10⁷ and 10⁸ cells/ml). In 96 wells culture plates were loaded 100 μl of lymphoid cells plus 100 μl of the mitogen or the antigen, according to the experimental design. The plates were incubated in a oven at 37⁰C, 5% CO2 during 24 hours. Once finished that incubation period, to each well was added 0.5 μCi of tritiated Thymidine (Amersham) and it was incubated by 24 hours. Subsequently, the cells were harvested on a microfiber paper (Whatman), in a cell harvester (Multimash 2000, Dynatech). The microfiber paper was allowed to dry in the oven at 37⁰C and subsequently, each sample was trespassed to a vial adding each 3 ml of scintillation solution (Ecoscint, National Diagnostics). The determination of the tritiated thymidine incorporation to the cells, was carried out in a liquid scintillation counter (Tri-Carb 2100TR, Packard). The results were expressed as cpm.

[0213] EXPERIMENTAL DESIGN No. 2

[0214] Total IgG, IgGI and lgG2a determination

[0215] In a parallel way, the animal groups pretreated with Andrographolide (0, 0.08, 0.8 and 8 nM), during six days and immunized with 1x10⁸ cells from Brucella abortus strain RB51 (i.p) were bleeded through the caudal vein, each seven days and sacrificed at the 21 day. The collected blood was allowed to clot in an oven at 37⁰C. The extracted sera were centrifuged during 1 min at 1 ,000 x g and then they were stored at -2O⁰C until their use.

[0216] The determination of the different specific immunoglobulins for the

Brucella abortus antigen was carried out through non direct ELISA. In short, "low binding" ELISA plates were seeded with 0.5 μg of total protein from Brucella. It was incubated during the whole night at 4⁰C. Three washings were carried out, each with 0.05% Tween-20 in PBS pH 7.0. Then, the plate was blocked with 5% skim milk in PBS pH 7.0, during one hour at room temperature. It was washed and then, 100 μl of the sera corresponding to each group treated with Andrographolide were added, incubating during two hours at room temperature. After the washing were added 100 μl of the second anti-mouse antibody specific for each isotype peroxidase-conjugated (Santa Cruz) and it was incubated during one hour at room temperature. Alter the washing the reaction was revealed with 100 μl of TMB and H₂O₂ (Kit TMB Substrate Pierce, U.S.A.) and it was quenched with 50 μl of H2SO42M. The absorbance was determined at 450 nm in an ELISA microplate reader (ElxδOO, BioteK).

[0217] EXPERIMENTAL DESIGN No. 3:

[0218] IL-2, IL-4 and INF-γ determination:

[0219] The determination of cytokines was carried out in the supernatant of the mouse spleen cells culture, by using commercial kits for OptEIA Mouse IL- 2 Set, OptEIA Mouse IL-4 Set and OptEIA Mouse IFN-γ Set capture ELISA (PharMingen, U.S.A.). In 24 wells culture plates, the spleen cells were "in i//?ro"stimulated with Andrographolide (0, 0.08, 0.8 y 8 nM) in the presence of Concanavalin A (0, 1 , 3.3 and 10 μg/ml) and during 24 hours. Then, the supernatant is centrifuged for 1 minute at 1000 x g and maintained at -2O⁰C until its use. In short, "high binding" ELISA plates (Pierce, U.S.A.) were seeded or loaded with 100 μl of the first specific antibody for each cytokine. The plate was incubated at 4⁰C during the night. Three washings were carried out, each with 0,05 % Tween-20 in PBS pH 7,0. Subsequently, the plate was blocked with 5% skim milk in PBS pH 7,0, during one hour at room temperature. The plates were washed once more, and immediately added with 100 μl of the corresponding culture supernatant, and it was incubated for two hours at room temperature. Once it was washed, were added 100 μl of the second antibody conjugated with peroxidase and it was incubated during one hour at room temperature. Once it was washed, the reaction was revealed with 100 μl of TMB and H2O2 (Pierce, U.S.A.) and it was quenched with 50 μl of 2M H2SO4. The absorbance was read at 450 nm in an ELISA microplate reader (ElxβOO, BioteK). Additionally, a calibration curve was carried out for each cytokines determination, expressing the results in pg/ml units.

Claims

Claims

1. Use of a pharmaceutical composition which comprises a mixture of diterpenic labdanes containing from 20 to 40% w/w of Andrographolide, preferably 24.6% w/w of Andrographolide, from about 3 to 6% w/w of 14-Deoxiandrographolide, preferably 4.8% w/w of 14-Deoxiandrographolide and from about 0,2 to 0,8% w/w of Neoandrographolide, preferably 0,6% w/w of Neoandrographolide and a pharmaceutically acceptable carrier, CHARACTERIZED in that it is useful for preparing a medicament useful as an immunostimulating agent on the treatment and prevention of infectious diseases in a subject in need thereof.

2. The use according to claim 1 , CHARACTERIZED in that said medicament is useful as an immunostimulating agent of the Th1 response against disease produced by intracellular pathogens in animals.

3. The use according to claims 1 and 2, CHARACTERIZED in that the infectious disease is selected from the group of diseases caused by viral pathogen agents.

4. The use according to claims 1 and 2, CHARACTERIZED in that the infectious disease is selected from the group of diseases caused by bacterial pathogen agents.

5. The use according to claims 1 and 2, CHARACTERIZED in that Ia infectious disease is selected from the group of diseases caused by protozoan parasites.

6. The use according to claim 3, CHARACTERIZED in that the viral pathogen agent is the human immunodeficiency virus (HIV).

7. The use according to claim 3, CHARACTERIZED in that the viral pathogen agent is hepatitis virus.

8. The use according to claim 3, CHARACTERIZED in that the viral pathogen agent is influenza virus.

9. The use according to claim 3, CHARACTERIZED in that the viral pathogen agent is the syncytial respiratory virus.

10. The use according to claim 3, CHARACTERIZED in that the viral pathogen agent is the Newcastle disease virus.

11. The use according to claim 4, CHARACTERIZED in that the bacterial pathogen agent is selected from the Mycobacteriaceae family.

12. The use according to claim 4, CHARACTERIZED in that the bacterial pathogen agent is Chlamydia.

13. The use according to claim 4, CHARACTERIZED in that the bacterial pathogen agent is Mycobacterium leprae.

14. The use according to claim 4, CHARACTERIZED in that the bacterial pathogen agent is Listeria monocytogenes.

15. The use according to claim 4, CHARACTERIZED in that the bacterial pathogen agent is selected from the Brucellae family.

16. The use according to claim 15, CHARACTERIZED in that the bacterial pathogen agent is Brucella abortus.

17. The use according to claim 4, CHARACTERIZED in that the bacterial pathogen agent is Bartonella spp.

18. The use according to claim 4, CHARACTERIZED in that the bacterial pathogen agent is Piscirickettsia salmonis.

19. The use according to claim 5, CHARACTERIZED in that the protozoan pathogen agent is Trypanosoma cruzi.

20. The use according to claim 5, CHARACTERIZED in that the protozoan pathogen agent is Plasmodium falciparum.

21. The use according to claim 5, CHARACTERIZED in that the protozoan pathogen agent is Leishmania.

22. The use according to claim 5, CHARACTERIZED in that the protozoan pathogen agent is, Coccidium.

23. The use according to any of the preceding claims, CHARACTERIZED in that said subject is a mammal.

24. The use according to any of the preceding claims, CHARACTERIZED in that said subject is a human.

25. The use according to any of the preceding claims, CHARACTERIZED in that said subject a production animal.

26. The use according to claim 24, CHARACTERIZED in that said production animal is selected from the group formed by porks, ovines, bovines, birds, poultry, equines, camels, fishes and others.