HK1096850B - Compositions useful for the treatment of pathologies responding to the activation of ppar-gamma receptor - Google Patents

Description

Composition for treating pathologies responding to the activation of the PPAR-gamma receptor

The present invention relates to the use of spirolaxine (spirolaxine) in the treatment of such pathologies responding to the activation of a PPAR gamma receptor (peroxisome proliferator-activated receptor), such as insulin-resistant diabetes mellitus type 2.

The present invention also relates to a pharmaceutical composition comprising spirolain and all-trans retinoic acid as active ingredients for treating pathologies responding to activation of PPAR γ receptors, such as acute hematological malignancies.

PPAR γ is a member of the nuclear receptor superfamily, heterodimerizes with the Retinoid X Receptor (RXR) and acts as a transcriptional regulator of genes linked to glucose and lipid metabolism (Diabetes47 (4): 5071998, 4, month 4, 14).

Diabetes is a syndrome caused by the interaction of genetic and environmental factors; it is characterized by insulin secretion and other metabolic disorders as well as vascular abnormalities, i.e., elevated blood glucose levels, accelerated nonspecific atherosclerosis, neuropathy, and thickening of the capillary base layer resulting from renal and retinal degeneration.

According to modern classification, diabetes is largely divided into two categories:

1-insulin dependent diabetes mellitus (also known as type 1 diabetes); patients with this type of diabetes mellitus are completely dependent on insulin to prevent ketoacidosis and death patients with type 1 diabetes mellitus exhibit reduced insulin secretion in terms of endogenous insulin secretion.

2-non-insulin dependent diabetes mellitus (also known as type 2 diabetes); patients with this type of diabetes do not require insulin to live: they can decide whether to use it to control the symptoms of diabetes. With respect to endogenous insulin secretion, patients with type 2 diabetes can be further divided into two categories. In the first category, insulin levels are normal or below normal; in the second category, insulin values are higher than normal and patients exhibit insulin resistance.

As described above, PPAR γ also acts as a transcriptional regulator of genes involved in lipid and glucose metabolism.

Insulin sensitizers, ligands for PPAR γ, which are useful in the treatment of diabetes, are already known.

Thiazolidinedione derivatives are described, for example, as substances that are useful in treating patients with type 2 insulin resistant diabetes; it has anti-diabetic activity due to its high binding affinity to the receptor in vivo (Nippon Rinsho February 2000; 58 (2): 401-4).

Similarly, PPAR γ has high levels of expression in some leukemic cell lines, which cannot be differentiated so that accumulation occurs at the least mature level later (Jan, bxp.4, 281-99, 1995).

Acute hematologic malignancies are blood cancers, which are growing progressively in developed countries.

There are increasing pollutants in the air that mutate the human gene bank. These mutations are often responsible for solid cancers and hematologic malignancies.

As mentioned above, acute hematological malignancies are characterized by an inability of lymphoid or myeloid lineage cells to differentiate, which allows subsequent accumulation at the least mature level.

These lesions can usually be treated with drugs that either destroy the tumor cells or induce their terminal differentiation (differentiation therapy).

PPAR γ ligands with antiproliferative activity are described in European Journal of Cell Biology 67, 379-85-1995 and Burpean Journal of Cell Biology 77, 214-19-1998, at month 11, which show strong pro-differentiation synergy on different myeloid leukemia Cell lines when used in combination with retinoids.

In fact, PPAR γ determines an increase in the activity of the activated receptor complex and the simultaneous activation of both receptors, by heterodimerization with the RXT retinoid receptor (Cell volume 93, 241-52, 4 months 1998).

Combination therapy with retinoic acid and PPAR γ ligands provides therapeutic advantages for the treatment of pathologies characterized by insufficient cell differentiation, such as acute hematological malignancies.

Spirolaxine is a known compound; this substance is described as a metabolite of the fungus Sporotrichum laxum in Phytochemistry (1990) Vol 29, No 2, p 613-616. The anti-tumor activity of spirolein is described in Japanese patent application JP 08177033 the experimental model described in this application relates to an in vitro test for inhibition of tumor line proliferation which shows that proliferation is significantly inhibited by direct cytoxicity of the tested tumor lines.

In WO 9605204, spirolain is described as a compound for the treatment of gastroduodenal diseases caused by helicobacter pylori.

In Japanese patent application JP 94-82785, spirolein is described as a lipid-lowering compound having anti-cholesterolemia activity.

Methods for the preparation of spirolein are described in Phytochemistry (1990) volume 29, phase 2, pages 613-.

Retinoic acid is also a known compound. The toxicology and teratogenicity of this compound is disclosed in J.Kamm in J.Am.Acad.Dermatol.6,652 (1982). C.D.Robertson et al describe the synthesis of this compound in J.Am.chem.Soc.77, 4111 (1955).

The use of spirolain alone or in combination with all-trans retinoic acid as a substance for the treatment of these pathologies responding to the activation of the PPAR γ receptor has never been described.

Retinoic acid is a substance used for the treatment of Acute Promyelocytic Leukemia (APL), particularly hematological malignancies, because it stimulates the differentiation of the tumor cell clone promyelocytic (differentiation therapy).

APL exhibits less pronounced leukocytosis, anemia, and thrombocytopenia compared to other types of leukemia, and also exhibits a lower percentage of remission and higher mortality when treated with conventional chemotherapy.

APL is characterized by an abnormal translocation that involves the long arms of chromosomes 15 and 17 [ translocations (15; 17) ] involving the retinoic acid receptor alpha gene (Cin. Lab. Sci.2000 Spring; 13 (2): 106-16).

Oral administration of ATRA achieves complete remission in most patients with APL, however, treatment with ATRA may in some cases result in so-called "retinoic acid syndrome". This syndrome is characterized by a rapid and progressive increase in the white blood cell count of the treated patient, which can be treated with other chemotherapies.

Furthermore, since tumor cells become resistant to this compound during treatment with ATRA, late-stage remission therapy is required.

Despite recent efforts, there is still a need for new compounds which can be used alone or in combination for the treatment of these pathologies responding to the activation of the PPAR γ receptor.

It has been found that spirolain of formula (I) is a substance which can be used for the preparation of a medicament for the treatment of these pathologies which respond to the activation of the PPAR γ receptor.

An object of the present invention is the use of spirolain of formula (I) for the preparation of a medicament for the treatment of pathologies responding to the activation of PPAR γ receptors, wherein said pathologies responding to the activation of such receptors are insulin-resistant diabetes mellitus type 2.

Another object of the present invention is a pharmaceutical composition for treating these pathologies responding to the activation of the PPAR γ receptor, comprising as active ingredients spirolain of formula (I) and all-trans retinoic acid of formula (II).

Another object of the invention is the combination of spirolain of formula (I) with all-trans retinoic acid of formula (II).

Another object of the present invention is a pharmaceutical composition comprising as active ingredients spirolain of formula (I) and all-trans retinoic acid of formula (II) together with at least one excipient and/or carrier.

Another object of the present invention is the use of spirolain of formula (I) in combination with all-trans retinoic acid of formula (II) for the preparation of a medicament for the treatment of these pathologies responding to the activation of PPAR γ receptors, wherein said pathologies responding to the activation of PPAR γ are acute hematological malignancies, comprising: lymphocytic leukemia, myeloid leukemia, monocytic leukemia, and megakaryocytic leukemia.

Another object of the present invention is the use of spirolein of formula (I) in combination with all-trans retinoic acid of formula (II) for the preparation of a medicament for the treatment of acute promyelocytic leukemia.

Furthermore, the use of therapeutic regimens in which multiple antineoplastic agents are administered simultaneously or sequentially is well known in the medical arts.

The necessity of administering multiple antineoplastic agents in a treatment regimen can be justified by the fact that, through action at different metabolic levels, it contributes in some cases to complete remission of the cancer, while in other cases it contributes to making the patient being treated live longer and/or improving their quality of life. The combination of the invention may be used together with one or more known antineoplastic agents for the treatment of acute hematological malignancies.

Therefore, a further object of the present invention is also a pharmaceutical composition comprising the combination of spirolain of formula (I) and all-trans retinoic acid of formula (II) and one or more known antineoplastic agents for the treatment of acute malignant hematological pathologies. The above known antitumor agents include: an alkylating agent; a topoisomerase inhibitor; anti-tubulin drugs; intercalating agents (intercalants); an antimetabolite agent; natural products such as Vinca alkaloids (Vinca alcaloids), epipodophyllotoxins, antibiotics, enzymes, and taxanes (taxanes).

The following test data are reported to better illustrate the invention.

Example 1

Activation of PPAR γ by spirolein (ST 1397)

The ability of spirolein to bind to PPAR gamma receptors and the ability to determine the activation of these genes with PPAR-gamma (PPAR-gamma RE) response sequences was demonstrated by several Cell transfection experiments with PPAR-gamma RE-controlled plasmids expressing PPAR-gamma and information vectors encoding luciferase (Cell 68; 879-887; 1992; J.biol.chem.272; 25252-25259; 1997).

The activation of luciferase expression was demonstrated by transfection of NIH-3T3 murine fibroblasts with a PPAR-gamma plasmid and TK-PPAR-Reluc information vector; luciferase activity was measured after 24 hours of treatment with spirolein at a concentration of 40 nM.

The activity of spirorelin was compared with that of Troglitazone (TZD), a known compound used in the treatment of type 2 insulin-resistant diabetes, and tested at a concentration of 5. mu.M.

The results illustrated in figure 1 indicate that spirorelin is more effective than the above antidiabetic compound. In fact, the luciferase activity induced by the reference compound and mediated by the TK-PPAR-Reluc information carrier (which can be used as an index for receptor activation) was 5 times higher than that of the control, whereas the luciferase activity induced by the spirolein of the present invention was 7 times higher than that of the control.

Example 2

Effect of the combination of the invention on the differentiation of the human promyelocytic leukemia cell line (NB4)

The prophase differentiation activity of both spirodelaine (ST 1397) and ATRA, used alone and in combination, was evaluated in this experimental model.

It is well known that all-trans retinoic acid becomes effective at concentrations between 0.1 and 1 μ M; when growth is significantly halted, a peak in differentiation is generally observed within the third/fourth day of treatment.

NB4 cells at 25cm²The flasks were grown in 5ml RPMI1640 culture with 10% Fetal Calf Serum (FCS) at a density of about 100.000 cells/ml. One day later, with ATRA at 10^-7The concentration of M was either treated with ST1397 at doses of 0, 1, 0, 5 and 1 μ M, or with a combination of two compounds of equal volume. Then, the cells were cultured in an incubator for 2-3 days without replacing the culture medium.

At the end of the second or third day of treatment, cell to granulocyte differentiation was determined by NBT dye reduction and spectrophotometric analysis of the samples.

Retinoic acid was dissolved in the culture medium with a 1mM DMSO solution. Since this compound (DMSO) may also differentiate under certain assay conditions, control cultures were treated with equal volumes of DMSO.

To measure the differential differentiation, 500.000 cells were collected from each sample, centrifuged and resuspended in 1ml RPMI1640 culture with 10% serum, 1mg/ml Nitrobluetetrazolium (NBT) and 100 ngPMA. The resuspended cells were incubated at 37 ℃ for 20-60 minutes. At the end of the culture, the cells were centrifuged and the pellet thus obtained was resuspended in 1ml of PBS containing 10% Triton X100.

The sample was sonicated to dissolve it completely and then read with a spectrophotometer at a wavelength of 540 nm.

The results presented in figure 2 indicate that ST1397 is unable to induce differentiation in NB4 cells when administered alone. The differentiation effect of ATRA has been well known, but it was found that this effect was potentiated by the simultaneous administration of ST1397, which was ineffective when administered alone as described above.

Claims (3)

1. A composition of spirolain of formula (I) and all-trans retinoic acid of formula (II)

2. A pharmaceutical composition, wherein spirolain of formula (I) is used as an active ingredient in combination with all-trans retinoic acid of formula (II) and at least one excipient and/or carrier,

3. use of a composition according to claim 1 for the preparation of a medicament for the treatment of promyelocytic leukemia.