WO1998051319A1

WO1998051319A1 - Antiviral compositions for the stimulation of the immune system and for other therapeutic and prophylactic applications

Info

Publication number: WO1998051319A1
Application number: PCT/CA1998/000445
Authority: WO
Inventors: Mohamed Ben Amar; Richard Morisset; Parviz Ghadirian; Fernando St-Hilaire
Original assignee: Lafond St-Hilaire, Gilberte
Priority date: 1997-05-09
Filing date: 1998-05-06
Publication date: 1998-11-19
Also published as: CA2205070A1; AU7328398A

Abstract

A composition which includes a mixture of Angelica oil and Red Cedar oil. This composition is an effective antiviral agent and a stimulant of the immune system. It may be used to treat infectious and/or immune diseases as well as neoplasic pathologies. In particular, it may be used as an anti-HIV and immunomodulatory agent. It may also be used to develop vaccines for the different pathologies mentioned above.

Description

ANTIVIRAL COMPOSITIONS FOR THE SΗMULATION OF THE IMMUNE SYSTEM AND FOR OTHER THERAPEUTIC AND PROPHYLACTIC APPLICATIONS

FIELD OF THE INVENTION

The invention relates to a pharmaceutical composition which may be used as an anti-HIV and immunomodulatory agent, and to a method of using the same. More particularly the composition of the invention significantly stimulates the immune system and particularly results in the increase of the CD8⁺ cells number. The composition of the invention includes a synergetic association of two essential oils, viz Angelica oil and Red Cedar oil.

BACKGROUND OF THE INVENTION

The oil of Angelica is a volatile oil extracted from root or fruit of Angelica archangelica

L, Umbelliferae. The oil of Angelica is obtained from distillation of roots or fruits of Angelica archangelica. This oil is a yellow liquid having a poignant odour and a sweet and sour flavour. Its physicochemical characteristics are as follows:

Oil of Roots Oil of fruits

Density at 25°C 0.850 to 0.880 0.853 to 0.876

Refractive index at 20°C 1.4735 to 1.4870 1.4800 to 1.4880

Specific optical rotation at 25°C inactive or no more 4° to 16° than 46° pH < 7 ≤ 3

In the following description it will be understood that the expression "oil of Angelica" stands for and is equivalent to the oil of the Roots of Angelica.

The major constituent (more than 25%) of the oil of Angelica is phellandrene. The oil also comprises umbelliferone (also known as 7-hydroxycoumarin); furo [3,2-g] coumarin (also known as psoralen), α-pinene; osthole; bergaptene; limonene; caryophyllene and linalol. The oil of Angelica is used for the manufacture of liquors. It has been used in the past as an antibacterial, fungicidal, antitussive, expectorant, stimulant, antiflatulent, antispasmodic, anti-inflammatory, sudorific, emmenagogue, diuretic, anti-rheumatic and sedative agent.

Therefore, in phytotherapy, the oil of Angelica has been used to treat a wide variety of disorders like general exhaustion, anaemia, flue, coughing, tuberculosis, chronic bronchitis, indigestion, digestive atonia, gastric acidity, aerophagy, stomachal and intestinal spasms, enteritis, hepatic disorders, migraine, faintness, impotence, menstrual disorders, leucorrhea, chlorosis, rachitis, scun y, etc.

The oil of Red Cedar is a volatile oil extracted from the Red Cedar tree Juniperus virginiana L_. Cupressaceae of the cypress family which is mainly found in North America. The oil of Red Cedar is also known as the oil of Cedar Wood and is extracted by distillation of Red Cedar wood. This oil is a viscous liquid almost colourless or yellowish which presents a characteristic Cedar odour. Its density at 25°C ranges from 0.938 to 0.953 and its refractive index at 20°C ranges from 1.5020 to 1.5100. Its major constituents are cedrene (a liquid sesquiterpene), cedral (a sesquiterpene alcohol also called cedrol) and longipinene. It also contains bergaptene.

The oil of Red Cedar is used in perfumery or as an insect repellant. In phytotherapy, the oil of Cedar Wood has been used as an anti-infectious agent or a stimulant.

To the inventor's knowledge, it has never been reported so far that the above mentioned oil of Red Cedar has any beneficial effect on the immune system.

At present, considerable efforts are being made towards finding compounds capable of fighting retroviruses, particularly the human immunodeficiency virus (HIV). Nucleoside analogs and protease inhibitors are common antiretroviral agents targeted against the HIV. As HIV disease is characterized by progressive deterioration of immune functions, attention has been focused on the restoration of immunity. The following investigators have demonstrated the strong anti-HIV properties of CD8⁺ lymphocytes in vitro.

The teams of Klatzmann, Vilmer, Brun-Vezinet and Montagnier (Paris hospitals and the Pasteur Institute) observed that when CD8⁺ lymphocytes were cocultivated with cord blood lymphocytes in the presence of HIV-1 (formerly called LAV), no reverse transcriptase activity was detectable¹. They further noted that when serum samples from different HIV⁺ subjects were tested, a positive specific staining was observed in the CD4⁺ but never in the CD8⁺ fraction.

The team of Jay Levy (University of California) demonstrated that CD8⁺ lymphocytes are able to suppress HIV replication in peripheral blood mononuclear cells (dose- dependent effect) . They suggested that an unidentified soluble factor was involved in part in this antiviral response.³'⁴

Baier et al (Paul-Ehrlich Institute) reported that interleukin-16, secreted from activated CD8⁺ cells, suppresses HIV and SIV replication via an unknown mechanism.

The teams of Robert Gallo (Institute of Human Virology at the University of Maryland) and Paolo Lusso (San Raffaele Scientific Institute in Milan) illustrated that 3 cytokines namely RANTES, MIP-1α and MIP-1 β, isolated from CD8⁺ lymphocytes, reduced the production of a broad range of HIV-1 , HIV-2 and SIV strains growing in laboratory cultures.^ They also found that the 3 factors had to be combined to obtain optimal inhibition.

Furthermore, CD8⁺ lymphocytes are the major antiviral and anti-cancer cells of the immune system^. The following observations support their role in other pathologies. Interferons and other cytokines (also called chemokines) are produced by the CD8⁺ lymphocytes and have been efficient in the treatment of different types of cancers. Several researchers have identified specific viruses responsible of different types of cancers, the first one being the HTLV (human T leukemia virus) involved in leukemia (Robert Gallo, 1979) and one of the latest ones being a herpes virus involved in

Kaposi Sarcoma (1995).

The team of Riddell and Greenberg (Fred Hutchinson Cancer Research Center in Seattle) restored immunity in immunodeficient humans by the transfer of CD8⁺ lymphocytes isolated from bone marrow donors .

The team of Mizoguchi and Ochoa (National Cancer Institute in Maryland) found that in different types of mice tumors and in 7 of 12 human cancer patients, their T lymphocytes, particularly the CD8⁺ lymphocytes, were depressed. They also demonstrated that when the T lymphocytes are no longer in contact with the cancer cells, they return to their normal state. They suggest that cancerous tumors release a substance that alters the structure of a key molecule on the surface of the T cells of the immune system which disrupts their recognition and response to foreign antigens (alteration of T cell receptor signal) and leads to suppression of immune responses¹".

The team of Gaur (Stanford University in California) demonstrated that rats which were vaccinated with CD8⁺ lymphocytes did not develop an experimental animal model of multiple sclerosis. In contrast, the absence of the CD8⁺ cells did not protect

17 the animals against the experimental disease ' ' .

The team of Allison (Department of Molecular and Cell Biology and Cancer Research

Laboratory, University of California at Berkeley) showed that expression of the costimulatory ligand B7 on melanoma cells induced the rejection of a murine melanoma in vivo. This rejection was mediated by CD8⁺ T cells; CD4⁺ T-cells were not required. These results suggest that B7 expression renders tumor cells capable of effective antigen presentation, leading to their eradication in vivo '⁸.

The team of Erard (Ciba-Geigy laboratories in Basel) illustrated that CD8⁺ lymphocytes, after stimulation of their ligand B7, are capable of rejecting melanoma tumors in mice .

The team of Papadopoulos (Memorial Sloan-Kettering Cancer Centre in New York) showed that infusions of CD8⁺ lymphocytes to 5 patients suffering from lymphoma associated with Epstein-Barr virus led to the complete remission of the tumors in all the 5 subjects within 14 to 30 days of treatment²⁰.

The team of Steven Rosenberg (National Cancer Institute in Maryland) demonstrated that interleukin-2 has strong anti-cancer effects in several patients with advanced metastatic melanoma or renal cell cancer. Interleukin-2 has no direct antitumour activity and is produced by CD8⁺ and CD4⁺ lymphocytes. Its antitumour effectiveness is mediated by the stimulation of the host immune system and shows that immunotherapy can beat different forms of cancer²¹.

Williams suggests that progress made in the understanding of the mechanisms of action of the immune system will lead to effective immunotherapies for cancer²².

In view of these observations, it has been suggested that CD8⁺ lymphocytes play a significant role in the control of HIV infection' ^"1 and might be a key factor for long- term survival.4,11-14

It is also suggested in the above findings and literature that the CD8⁺ cells play an important role in other immune diseases of the immune system such as different forms of cancer, different forms of leukemia, multiple sclerosis and different immunodeficient pathologies. SUMMARY OF THE INVENTION

It has been found that a mixture of Angelica oil and Red Cedar oil, properly combined and mixed in a specific way, is an effective stimulant of the immune system and may be used to treat human immune diseases.

Therefore, it is a first object of the present invention to provide a composition which contains a mixture of Angelica oil and Red Cedar oil properly and advantageously combined with an acceptable pharmaceutical carrier.

Another object of the invention is to provide a method and a pharmaceutical composition to stimulate and activate the immune system of a patient, more particularly the T-cells and even more particularly the CD8⁺ cells.

Yet another object of the invention is to provide a pharmaceutical composition showing anti-HIV and immunomodulatory effects.

Still another object of the invention is to provide a pharmaceutical composition which can control HIV infections and related complications or disorders.

Still another object of the invention is to provide a method to control HIV infections and particularly to stimulate CD8⁺ lymphocytes in HIV infected patients.

Still another object of the invention is to provide a method and a pharmaceutical composition which presents the above mentioned advantages while showing no toxicity nor side-effects at therapeutic doses, and which is well tolerated.

Still another object of the invention is a method of medical and pharmacological treatment which consists in administrating to a patient an effective amount of the composition to treat an immune disease and/or to stimulate the immune system and/or to prevent the deterioration of the immune system. As can be appreciated, the composition of the present invention is used for treating a wide range of immune diseases such as HIV/AIDS, different forms of cancer, different forms of leukemia, multiple sclerosis and different immunodeficient pathologies.

These and other objects, advantages and features of the present invention will become more apparent to those skilled in the art upon reading the detailed description of the invention set forth below.

DETAILED DESCRIPTION OF THE INVENTION

The pharmaceutical composition according to the present invention is primarily a mixture of Angelica oil extracted from Angelica archangelica L, Umbelliferae and Red Cedar oil extracted from Juniperus virginiana L, Cupressaceae. The oil mixture is combined with an acceptable and suitable pharmaceutical carrier.

The composition according to the invention may be administered in different forms. To do so it can be formulated as an enteral, parental or topical preparation.

Hence, it is obvious that the quality and quantity of the oil mixture and the pharmaceutical carrier will vary depending on the form of the composition. The inventors consider that when the composition is applied in one of the above mentioned alternate forms, an increased concentration would be desirable.

According to a preferred embodiment of the invention, an oral preparation of oil of Angelica and oil of Red Cedar has been investigated in vitro and in vivo.

Hereinbelow is an example of a composition (referred to as SH-1^* and/or Stimulin SH-1* composition) according to the present invention and its effects on HIV- infected individuals.

Trade-marks EXAMPLE 1

PREPARATION SH-1* AND/OR STIMULIN SH-1* COMPOSITION The preferred oral composition which has been used in these studies was prepared by adding the amount of distilled or purified water (7) to the constituents (1 ) to (6) mentioned hereinbelow.

The oils of Angelica and Red cedar are readily available on the market. The use of ethylic alcohol or ethanol in the present composition is optional. FD &C green No.3 and FD & C yellow No. 6 are common colourings used in sufficient quantities. Water is used as a solvent in the present oral preparation. It is worth mentioning that any solvent that is compatible with oils may be used. For example glycerin is a suitable solvent.

The resulting preparation is stirred and homogenated. This preparation will be referred to as SH-1* and/or Stimulin SH-1* hereinbelow.

The ratio of Oil of Red Cedar/ Oil of Angelica which was used in this preferred preparation was about 6/1 and the concentration of oils in the preparation was about 2%. However, the concentration and/or ratio of oils may vary in wide proportions. For example, oil concentrations up to 90% may be used with the obvious proviso * Trade-marks that in the posology, the toxicity level is not reached. The concentration of the oil mixture will vary with the severity of the disease to treat. For example, a lower concentration of the oil mixture will be used for treating moderate cases of immune- deficient diseases such as the preliminary stage of a cancer whereas a higher concentration of the oil mixture will be used to treat more severe cases of immune deficient diseases such as AIDS. As a matter of fact, someone skilled in the art will be able to discern the appropriate dosage or method of use without any undue experimentations.

The following study evaluates the effects of the above-mentioned preferred composition on the immune system of HIV-infected individuals with fewer than 500 CD4⁺ cells/mm³.

METHODS AND ANALYSIS OF THE SH-1* AND/OR STIMULIN SH-1^* EFFECTS

(I . Selection and follow-up of patients

Ten HIV-infected Caucasian men (identification of antibodies to HIV by ELISA and confirmation by WESTERN BLOT), aged between 31 and 47 (average age=37.3 years), were enrolled in the study after fulfilling all inclusion criteria. They had the following biological parameters:

- CD4⁺ lymphocytes <5OO cells/mm³;

- white cells ≥2000 cells/mm³; - hemoglobin ≥95 g/l;

- granulocytes ≥1000 cells/mm ,

- platelets ≥75000 cells/mm³;

- transaminases ≤150 lU/ml; and

- creatinine ≤ 1.5 x upper limit of normal. Subjects with evidence of severe cardiac, hepatic or renal illnesses, or who were suffering from serious nervous diseases other than those resulting from HIV infection, were excluded.

Eligible patients were not on any antiretroviral or immuno-modulatory agent for at least 30 days prior to treatment and had the following CD4⁺ lymphocyte counts at the onset of treatment: 422, 403, 386, 333, 251 , 210, 192, 189, 79 and 76 cells/mm³ (patients 1 to 10 respectively). After approval of the study by the Hospital's Ethics Committee, the volunteers gave their written informed consent to proceed in this study.

A complete evaluation of the clinical state of each patient was obtained through an exhaustive medical examination and laboratory tests before the study was initiated. Patients were closely monitored at the beginning, at the 15th and 30th days after starting treatment, then once every month till the 10^th month, and every 30 to 90 days subsequently. Lymphocyte and white cell counts were performed on an XL flow cytometer (Coulter Electronics, Hialeah, FL)

(II . Administration of SH-1* and/or Stimulin SH-1* The SH-1* and/or Stimulin SH-1* preparation of vegetal extracts of the present invention is administered orally as a syrup and a freshly-prepared solution of same is given every 30 days. The product is stored at room temperature. The SH-1^* and/or Stimulin SH-1* dosage was 5 ml bid for the first 2 months, 5 ml tid between the 3rd and 8th months, 15 ml bid from the 8th month to the 18th month, and 15 ml tid subsequently.

(Ill) Management of eventual toxic reactions

In the case of toxicity, SH-1* and/or Stimulin SH-1^* dosage must be reduced by half. If, after dosage reduction, the affected biological parameters do not return to initial values within 15 days, treatment must be interrupted until they do. SH-1* and/or

* Trade-marks Stimulin SH-1* must then be readministered at half the original dose. If more than one interruption of SH-1* and/or Stimulin SH-1* is required, the subject is eliminated from the study. Treatment must also be discontinued in the event of non-compliance, defined as failure to take 75% of the drug or absence from 2 consecutive medical consultations without valid reason.

(IV) Statistical analysis

Statistical comparisons were made on pretreatment, approximately 6 month and 12 month measures of the various immunologic parameters studied, using analysis of variance (ANOVA), followed by multiple comparisons based on the Tukey B method.

All analyses were interpreted at α=0.05 level.

(V. RESULTS

Five immunologic markers were used to assess the effects of SH-1* and/or Stimulin SH-1^* on the immune system: CD4⁺, CD8⁺ and CD3⁺ cells, total lymphocytes and white cells.

Table 1 illustrates the average variations of the group's parameters studied. As demonstrated by ANOVA, the CD4⁺ cell count increased between TO and T6 months but returned to baseline at T12 months (F=1.32; p=O.29). The group showed a significant elevation of CD8⁺ lymphocytes between TO and T6 and maintained this gain between T6 and T12 (F=4.90; p=0.02). For CD3⁺ lymphocytes, the group displayed a significant increase between TO and T6 and remained stable between T6 and T12 (F=7.30; p=0.016). Total lymphocyte count augmented significantly at TO-T6 and remained stable between T6 and T12 (F=10.6; p=0.006). Finally, no statistically significant variation was observed in the group between the 3 measures of white cells (F=l.08; p=0.36).

The long-term effects of SH-1^* and/or Stimulin SH-1^* on CD8⁺ lymphocytes are illustrated in Table 2. After 24 months of treatment, values were not available for

^* Trade-marks patients 7 and 10, as they voluntarily abandoned the project after 9 and 18 months respectively. Individually, 8 patients showed a significant increase (>10%) of CD8 cells after 6 months of therapy. After 12 months of treatment, this increase was still significant in 7 subjects, and after 24 months of SH-1* and/or Stimulin SH-1^* therapy, it remained significant in 5 patients.

Stimulation of CD8⁺ cells had clinical relevance: the 5 subjects (patients 2,4,5,6 and 8) whose CD8⁺ stimulation was sustained in time after 24 months of SH-1^* and/or Stimulin SH-1* therapy were the ones who showed the most clinical benefits: only a single new opportunistic infection (a herpes zoster episode) occurred among them during the first 2 years of treatment. In addition, during this period, there was only 1 death in the group: patient 7, who abandoned the project after 9 months of treatment, died 20 months after initiation of therapy. The drop of CD8⁺ cells in patient 3 at T=24 months was largely attributed to aggressive chemotherapy following the development of a lymphoma. Previous to this incidence, his CD8 count was 1705 cells/mm³ at T=21 months, which represented a significant increase compared to baseline. No side-effects were observed at therapeutic doses of SH- 1*and/or Stimulin SH-1* after 24 months of treatment.

TABLE 1 EVOLUTION OF IMMUNOLOGIC MARKERS DURING SH-1* AND/OR STIMULIN SH-1* THERAPY

^•Patient 7 a^bandoned the project after 9 months of treatment. His coun_t corresponds to T=10 months ' ^{Mis cou}nt

* Trede-ir.arks TABLE 2

EVOLUTION OF CD8 T LYMrøOCYTES*- DURING SH-1* AND/OR STIMULIN SH-1* THERAPY

c/>

CO CO

m

C/3

-_ rπ m

33 m r

* Trade-marks

* Values are expressed as number of CD8 cells/mm3

** Patient 7 abandoned the project after 9 months of treatment His count corresponds to T=10 months

SH-1* and/or Stimulin SH-1* is a potent stimulant of CD8⁺ lymphocytes. This study illustrates in HIV-infected individuals that the composition according to the present invention, SH-1* and/or Stimulin SH-1* is capable of stimulating in a significant (amplitude of increase) and sustained (24 months of therapy) way the production of CD8⁺ cells without manifesting side effects. CD8⁺ lymphocyte stimulation induced by SH-1^* and/or Stimulin SH-1* administration seems to play a key role in the control of HIV infection and in the clinical benefits observed in SH-1* and/or Stimulin SH-1* recipients.

In view of what is known about CD8⁺ cells, it is proposed the following hypotheses on their role in HIV disease. In the course of immune responses directed against HIV, stimulation of CD8⁺ lymphocyte production is a key factor in the control of HIV infection. In addition to their immunoregulatory properties, CD8+ cells seem to have a strong antiviral activity capable of partially neutralizing HIV through a virolytic effect (direct cytolysis or secretion of suppressive factors) and/or a virostatic action

(inhibition of viral replication).

The antiviral activities of CD8⁺ lymphocytes are particularly important during the asymptomatic phase of HIV infection, when they seem to eliminate part of the virus present in peripheral blood (drop of the initial burst of plasma viraemia) and play the role of a protective haematologic barrier, confining the virus principally to the lymph nodes. This anti-HIV activity of CD8⁺ cells is durable and persists throughout the asymptomatic phase. CD8⁺ cells also seem to play a major but partial protective role against the destruction of CD4⁺ cells by HIV.

The development of opportunistic infections and progression to ARC and AIDS might be related in part to a long-term failure of the anti-HIV and immunostimulant effectiveness of CD8⁺ cells, which could result from one or several of the following phenomena: ^*Trade-marks ^■ the appearance of viral mutations and the emergence of highly pathogenic viral strains resistant to immune responses and particularly cytopathic to CD8⁺ and CD4⁺ lymphocytes;

^• the long-term failure of the antiviral properties of CD8⁺ lymphocytes, involving direct (loss of their lytic power to shut down HIV) and/or indirect mechanisms

(reduction or abolition of the secretion of cytokines which elicit anti-HIV properties);

^■ the decline of CD8⁺ cell capacity to engender an efficient immune response of cellular mediation; ^• the disruption of release by the immune system of chemical mediators necessary for the activity of CD8⁺ lymphocytes.

Furthermore, it is believed the drop in CD4⁺ cells is in part a consequence of a progressive decrease of the antiviral effectiveness of CD8⁺ lymphocytes and the corresponding decline of their protective role, leading ultimately to the disappearance of their benefits and to the final collapse of the immune system.

It therefore seems that the decrease in CD8⁺ cells causes a drop in CD4⁺ cells, while the inverse appears to be less predominant as an unlatching factor. However, CD4⁺ lymphocytes might also potentiate the antiviral effects of CD8⁺ lymphocytes, via the release of specific factors (interieukin 2 and/or other cytokines) which stimulate the functions of these CD8⁺ cells. Therefore, the sharp decrease in the number of CD4⁺ cells in the advanced or terminal stages of HIV disease could contribute to the disruption of responses related to cellular immunity and more particularly to the loss of beneficial effects of CD8⁺ lymphocytes. This suggests a close interdependence between CD4⁺ and CD8⁺ cells (cross-regulation CD4⁺ <=>

CD8⁺) and the existence of a threshold characterized by a critical number of CD8⁺ and CD4⁺ cells, below which the destruction and collapse of the immune system become irreversible. In addition, loss of anti-HIV activity by CD8⁺ lymphocytes may decrease their ability to contain HIV in ganglionary stocking sites, allowing release of the virus into the general circulation, which leads to the emergence of its pathogenicity. This hypothesis about CD8⁺ cells may also explain the early destruction of CD4⁺ cells during primary infection: at this stage, HIV would be able to multiply at a high rate in the presence of few activated lymphocytes, as it is only when a critical threshold has been reached that the immune system mobilizes CD8⁺ cells. Following mobilization, CD8⁺ lymphocytes might go from a passive state to a state of stimulation characterized by an anti-HIV capacity which could explain the inversion of the CD4⁺/CD8⁺ cell ratio. An increase in the number of CDS*- lymphocytes would then reflect an attempt by the immune system to control HIV, restricting the drop in CD4⁺ lymphocytes and the resultant organic damages.

Finally, the greater the impairment of the immune system (illustrated by a lower number of CD4⁺ lymphocytes), the more difficult it is to restore immunologic functions.

Hence, upon reading the proposed hypotheses of the role of CD8+ cells in HIV- infection, it is obvious that prolonging the asymptomatic phase is crucial for a better evolution of HIV diseases and other immune diseases. SH-1* and/or Stimulin SH-1^* administration in the early stages of HIV infection allows a more efficient neutralization of the AIDS virus and, more importantly, to stop the destruction of vital elements of the immune system.

This study seems to confirm that persistent high levels of activated CD8⁺ cells are a strong predictor of extended survival and a key factor for a long asymptomatic phase in HIV-infected subjects. The SH-1^* and/or Stimulin SH-1^* immunostimulant approach might also be useful for the development of a vaccine.

* Trade-marks In view of all these observations, SH-1* and/or Stimulin SH-1* may also be used to treat other infectious diseases and/or immune diseases and/or neoplasic diseases. It can also be used to prevent these diseases. It is therefore considered both as a therapeutic and/or a prophylactic agent for different types of diseases.

^*Trade-marks

REFERENCES

LKlatzmann D, Barre-Sinoussi F, Nugeyre MT et al. Selective tropism of lymphadenopathy associated virus (LAV) for helper-inducer T lymphocytes. Science 1984; 225: 59-63.

2.Walker CM, Moody DJ, Stites DP, et al. CD8+ lymphocytes can control HIV infection in vitro by suppressing virus replication. Science 1986; 234: 1563-1566.

3.Walker CM, Levy JA. A diffusible lymphokine produced by CD8⁺ T lymphocytes suppresses HIV replication. Immunology 1989; 66: 628-630.

4. Levy JA.Pathogenesis of human immunodeficiency virus infection. Microbiol Rev 1993; 57: 183-289.

δ.Baier M, Werner A, Banned N, et al. HIV suppression by interleukin-16. Nature 1995; 378: 563.

6.Cocchi F, DeVico AL, Garzino-Demo A, et al. Identification of RANTES, MlP-lα, and MlP-lβ as the major HIV-suppressive factors produced by CD8⁺ T cells. Science

1995; 270: 1811-1815.

7. Levy JA. Mysteries of HIV: challenges for therapy and prevention. Nature 1988; 333: 519-522.

8. Lang W, Perkins H, Anderson RE, et al. Patterns of T lymphocyte changes with human immunodeficiency virus infection: from seroconversion to the development of AIDS. J Acquir Immune Defic Syndr 1989; 2: 63-69.

9.Chen CH, Weinhold KJ, Bartlett JA, et al. CD8⁺ T lymphocyte-mediated inhibition of HIV-1 long terminal repeat transcription: a novel antiviral mechanism. Aids Res Hum Retroviruses 1993; 9: 1079-1086. 10.Rowland-Jones S, Sutton J, Ariyoshi K, et al. HlV-specific cytotoxic T-cells in HIV exposed but uninfected Gambian women. Nature Med 1995; 1 : 59-64.

11.Cao Y, Qin L, Zhang L, et al. Virologic and immunologic characterization of long- term survivors of human immunodeficiency virus type 1 infection. N Engl J Med 1995; 332: 201-208.

12. Ben Amar M. Les immunomodulateurs. In: Mise a jour de la situation du SIDA. Edited by R. Morisset and P. Ghadirian. Quebecor, Montreal, 1995; 387-408.

13. Levy JA. HIV in AIDS pathogenesis and long-term survival. 12th International Congress of Pharmacology. Montreal, Quebec, Canada, July 24- 29, 1994 (Summary PL.1 published in the Canadian Journal of Physiology and Pharmacology vol. 72, supplement 1. page 3, July 1994).

14.Montagnier L. Histoire d'une maladie. In: Des virus et des hommes. Editions Odile Jacob; Paris; 1994: 83-110

15.Riddell SR, Watanabe KS, Goodrich JM, et al. Restoration of viral immunity in immunodeficient humans by the adoptive transfer of T cell clones. Science 1992; 257: 238-240.

16. Mizoguchi H, O'Shea JJ, Longo DL, et al. Alterations in signal transduction molecules in T lymphocytes from tumor-bearing mice. Science 1992; 258: 1795-

1798.

17.Gaur A, Haspel R, Mayer JP, et al. Requirement for CD8⁺ cells in T cell receptor peptide-induced clonal unresponsiveness. Science 1993; 259: 91-93.

18.Townsend SE, Allison JP. Tumor rejection after direct costimulation of CD8⁺ T cells by B7- transfected melanoma cells. Science 1993; 259: 368-370. 19.Erard F, Wild MR, Garcia-Sanz JA, et al. Switch of CD8 ⁺ T cells to noncytolytic CD8⁺ CD4⁺ cells that make TH₂ cytokines and help B cells. Science 1993; 260: 1802-1805.

20. Papadopoulos EP, Ladanyi M, Emanuel D, et al. Infusions of donor leukocytes to treat Epstein-Barr virus-associated lymphoproliferative disorders after allogeneic bone marrow transplantation. N Engl J Med 1994; 330: 1185-1191.

21.Rosenberg SA Yang JC Topalian SL et al. Treatment of 283 consecutive patients with metastatic melanoma or renal cell cancer using high-dose bolus interleukin-2. JAMA 1994; 271 : 907-913.

22.Williams N. An immune boost to the war on cancer. Science 1996; 272: 28-30.

Claims

1. A pharmaceutical composition for effectively stimulating the immune system and treating and/or preventing different human immune diseases, said composition comprising a mixture of Angelica oil from Angelica archangelica L, Umbelliferae and

Red Cedar oil from Juniperus virginiana L, Cupressaceae, the mixture being combined with an acceptable pharmaceutical carrier.

2. A pharmaceutical composition according to claim 1 , characterized in that the pharmaceutical composition is in the form of an enteral, parenteral or topical preparation.

3. A pharmaceutical composition according to claim 2, characterized in that the composition is an enteral preparation.

4. A pharmaceutical composition according to claim 3, characterized in that the mixture of Angelica oil and Red Cedar oil is present in a concentration of about 2%.

5. A pharmaceutical composition according to claim 3 or 4, characterized in that the Red Cedar oil/Angelica oil are present at a ratio of about 6/1.

6. Use of a mixture of Angelica oil from Angelica archangelica L, Umbelliferae and Red Cedar oil from Juniperus virginiana L, Cupressaceae, for the manufacture of a pharmaceutical composition according to any one of claims 1 to 5, for stimulating and activating the immune system and/or preventing the deterioration of the immune system in a human.

7. Use according to claim 6, characterized in that the pharmaceutical composition stimulates the immune system including CD4⁺ lymphocytes, CD8⁺ lymphocytes, CD3⁺ lymphocytes, total lymphocytes, and white cells.

8. Use according to claim 7, characterized in that the pharmaceutical composition stimulates the CD8⁺ lymphocytes.

9. Use of a mixture of Angelica oil from Angelica archangelica L, Umbelliferae and Red Cedar oil from Juniperus virginiana L, Cupressaceae, for the manufacture of a pharmaceutical composition according to any one of claims 1 to 5, for controlling HIV infections in a human and for prolonging an asymptomatic phase of said human.

10. Use of a mixture of Angelica oil from Angelica archangelica L, Umbelliferae and Red Cedar oil from Juniperus virginiana L, Cupressaceae for the manufacture of a pharmaceutical composition according to any one of claims 1 to 5, for stimulating CD8+ lymphocytes in an HIV infected patient and in other immune diseases.

11. Use of a mixture of Angelica oil from Angelica archangelica L, Umbelliferae and Red Cedar oil from Juniperus virginiana L, Cupressaceae for the manufacture of a pharmaceutical composition according to any one of claims 1 to 5 as an antiviral, anti-HIV and/or immunostimulant and/or immunomodulatory agent.

12. Use of a mixture of Angelica oil from Angelica archangelica L, Umbelliferae and Red Cedar oil from Juniperus virginiana L, Cupressaceae for the manufacture of a pharmaceutical composition according to any one of claims 1 to 5 for treating an immune-deficient disease chosen from the group consisting of HIV, AIDS, any form of cancer, any form of leukemia, multiple sclerosis, any other immune diseases, hepatitis and inflammatory pathologies.

13. A method for preparing a pharmaceutical composition according to any one of claims 1 to 5, comprising the steps of a) mixing Red Cedar oil and Angelica oil, to obtain a first mixture; b) adding the pharmaceutical carrier to the first mixture to obtain a second mixture; and c) stirring and homogenizing the second mixture to obtain the requested composition.

14. A method for treating an immune disease and/or for stimulating the immune system and/or for preventing its deterioration, comprising the step of administering to a patient an effective amount of a pharmaceutical composition according to any one of claims 1 to 5.