EP0724445A1

EP0724445A1 - The use of inositoltrisphosphate for the preparing of medicaments

Info

Publication number: EP0724445A1
Application number: EP94929713A
Authority: EP
Inventors: Matti Siren
Original assignee: Perstorp AB
Current assignee: Perstorp AB
Priority date: 1993-10-08
Filing date: 1994-10-06
Publication date: 1996-08-07
Also published as: JPH09503519A; SE9303289D0; SE501793C2; SE9303289L; AU7866894A; WO1995010285A1

Abstract

The present invention relates to the use of inositoltrisphosphate (IP3) for the preparing of a medicament effective against retroviral diseases.

Description

THE USE OF INOSITOLTRISPHOSPHATE FOR THE PREPARING OF MEDICAMENTS

The present invention relates to the use of at least one isomer inositoltrisphosphate (IP_) for the preparing of a medicament effective against retroviral diseases.

Acquired immunodeficiency syndrome (AIDS) is a serious immunodeficiency disease induced by human immunodeficiency virus (HIV), type 1 and type 2 (HIV-1 and HIV-2) . HIV, which is the aetiological agent for AIDS is a nononcogenic, cytopathic retrovirus of the lentiviruε subfamily. Retro¬ viruses have their genetic material in the form of RNA, ribonucleic acid, instead of DNA, deoxyribonucleic acid. In order to turn their RNA into DNA, retroviruses have a special enzyme called reverse transcriptase.

The HIV-virus, 100 nm in diameter is covered by an envelope, and contains a surface glycoprotein as well as an internal cylindrical core. The envelope is formed by phospholipids and glycoproteins and the core contains the genome and several enzymes.

HIV infects human cells primarily by binding to CD4 receptors on the surface of susceptible cells. This binding is mediated by the oligomeric envelope glycoprotein (gp) of HIV and the receptor on the target cell surface followed by the fusion between the viral envelope and the plasma membrane. The post- binding events which lead to membrane fusion are poorly understood but presumably include a conformational change in the envelope protein which exposes the hydrophopic amino terminus of a gp 41 envelope protein in the fusion reaction. The binding of the HIV-1 envelope glycoprotein, gp 120, to the cellular receptor is the first step in HIV infection. However, there is also evidence for CD4 independent mechanism of infection. There are reports of HIV infection in a number of CD4-negative cells in vitro (Harouse, J.M. et al., J. Virol, 63, 2527, 1989, Zachar, V.B. et al. J. Virol, 65, 2102, 1991) .

These data show that the expression of CD4 alone is not absolute obligatory and not sufficient to support HIV infection and implies that there are other molecules required for infection.

The patophysiological basis of the profound and irreversible immune depression following the infection is obscure.

AIDS was first recognized in 1981 in young, homosexual men from the U.S. with opportunistic infections, Kaposi's sarcoma, and primary CNS lymphoma. Although unusual cases of systemic malignant lymphoma were also recognised at that time, statistically significant increases were not apparent until 1985. Victims also suffered from other opportunistic infections, caused by microorganisms that are ubiquitous but ordinarily not able to cause disease. Indeed, the infections and cancers seen in AIDS patients were previously known only in people born with certain defects in their immune system.

Since the disease was first recognised the number of cases have risen swiftly. According to the World Health Organisation the increase of AIDS will result in up to 40 million people infected with HIV by the year 2000.

Recently the HIV infection has been classified into three distinct stages: the acute phase, lasting weeks, the chronic phase, lasting years, and the final phase of crisis (generally referred to as AIDS) lasting months to years.

AIDS is an unique disease. No other known infectious disease causes comparable harm by directly attacking the human immune system.

Once in the human body the virus attacks the cells that usually defend the body against infectious diseases. These cells include monocytes, macrophages and dendritic cells, so called antigen presenting cells (APC) . Furthermore HIV can remain hidden in cells latent for months or years. A third difficulty is that HIV is extraordinary variable in its genetic make-up.

In 1985, zidovudine (AZT) was found to have in vitro activity against the human immunodeficiency virus, HIV. The in vitro and clinical activity of zidovudine is not disputed, but there is considerable debate when to initiate treatment (J.G. Bartlett,: New Engl. J. of Med., 329, 351, 1993; Cooper D.A. et al.: New Engl. J. of Med., 329, 297, 1993).

AZT can have serious side effects such as anaemia, and in people using the drug, HIV frequently mutates to produce strains that are unaffected by it.

Other nucleoside analogues are on trial but these will probably only be of limited usage as the mode of action including side effects ressemble those of zidovudine.

The function of AZT is to block the action of the enzyme reverse transcriptase of HIV which stops the virus from replicating in the cells.

Retroviruses vary at a notariously high rate resulting in rapid appearance of HIV-1 strains which are resistant against drugs and antibodies given to the patient. In addition several other stages in the HIV replicate cycle have been envisioned as targets for therapeutic intervention. One such target is the HIV protease which is essential for the assenbly of fully infections HIV particles.

In the past years an intensive search to find effective antiviral therapies has taken place. Active agents that have been discovered during the last years include carbovir (Vince, R. et al: Biochem. Biophys. Res. Commun. 156, 1046, 1988) , and a class of oxathin benzoic acid esters and derivates (Schultz, RJ, et al.: Proc. Am. Assoc. Cancer Res. 33, 409, 1990) .

While searching for a new drug which would prevent the spreading of HIV viruses, the following conditions should be fulfilled:

-It should have no or few side effects, or at least these side effects should be minimal and tolerable

-It should have high therapeutic indices -It should prevent the prolification of the HIV virus by preventing it from penetrating those cells in which the virus reproduces, or preventing it from reproducing inside these cells.

-The half-life of the drug should be long enough to allow and administration once or twice daily.

-The compound should be easily identified in the blood in order to establish individual dosage.

-The drug should effect the target cell in the same form as it is administered and not only as a result of metabolic activity.

-The drug should be teratologically safe.

According to the present invention it has surprisingly become possible to use at least one isomer of inositoltrisphosphate

(IP_) , for the preparing of a medicament effective against retroviral diseases.

In preferred embodiments of the invention the medicament is intended to be used for preventing, alleviating and combatting acquired immunodeficiency syndrome (AIDS) and AIDS-related diseases. The medicament can also be used against other conditions caused by retroviruses.

The medicament exerts significant inhibitory effects against retroviral infections without side-effects which is very beneficial for the patient. Furthermore the medicament is also intended to be used against viral diseases caused by other enveloped viruses.

For example the medicament is intended to be used against viral diseases caused by cytomegalovirus and different types of herpes virus.

From the European Patent No 179439 a pharmaceutical compo¬ sition comprising as a pharmaceutically active ingredient at least one isomer of inositoltrisphosphate is known. In said patent the effect of this pharmaceutical composition is shown for different areas, such as platelet aggregation.

The production of IP_ and the isolation of the different isomers thereof are disclosed in the US patent No 4.777.134. The IP_ isomers can also be produced by synthetic methods, chemically or enzymatically, starting with e.g. inositol and a phosphorus source. Furthermore, microbiological production methods including hybrid DNA-techniques of IP are also suitable.

The structure of IP_ and the different isomers thereof are disclosed in the US patent No 4.735.936 and the US patent No. 4.797.390.

It is suitable that the medicament used according to the invention exists in unit dosage form. Tablets, granules or capsules are suitable administration forms for such unit dosage. Furthermore, tablets and granules can easily be surface treated such as to provide an enteric coating to prevent an uncontrolled hydrolysis in the stomach and to bring about a desired absorption in the intestine. Other suitable administration forms are slow release and trans- dermal administration. A usual pharmaceutically acceptable additive, excipient and/or carrier can be included in the medicament. The tablets or granules can also contain a disintegrant which causes the tablets or the granules, respectively, to disintegrate easily in the intestine. In certain cases, especially in acute situations, it is preferable to use the unit dosage in the form of a solution for intravenous administration. In other situations suspensions comprising the compound can be preferably used as administration form.

The medicament can also consist as such of IP solely without any additive, excipient or carrier.

If desired, the medicament can be free of other inositol phosphates, IP^{, ιp} ₂' ^IP ₄' ^IP ₅ ^{and IP} ₆* ^Accordingly, the mixture of IP isomers can have a purity of 90-100 %, such as 93-100 % or preferably 95-100 %.

Alternatively, the medicament can consist of or comprise one or more specific IP isomers, each present in substantially pure form. Thus, the different isomers can be isolated from each other in substantially pure form, which means that they have a purity of 80-100 %, such as 82-100 % or 85-100 %, preferably 90-100 %. Since the isomers can be produced in pure form they can be mixed in any proportion, of course.

The medicament can consist of IP , wherein said IP- is provided by at least one of IP_fi/ IP or IP and a degradative substance such as an enzyme suitable to form IP-. It is in most cases suitable that the IP -isomer or isomers used for the preparing of the medicament according to the invention are present in salt form in order not to affect the mineral balance negatively. The salt should preferably consist of a sodium, potassium, calcium zinc or magnesium salt or a mixture of two or more of these salts.

For the above mentioned reasons it is also an advantage if the medicament contains a surplus or an extra addition of at least one pharmaceutically acceptable salt of calcium, zinc or magnesium with a mineral acid or organic acid. This is especially valuable for elderly persons who are often deficient in these minerals.

For administration to human patients appropriate dosages can routinely be determined by those skilled in this art by extension of the results obtained in animals at various dosages. The preferred dosage for humans falls within the range of 0.1 to 1000 g, especially 0.1-200 mg IP_/day/kg body weight.

In animal experiments, no toxic effects were seen after ad¬ ministration of very high doses of IP_, 160 g/kg body weight by intraperitoneal injection to mice.

The medicament usually contains 0.01-1.5 g, such as 0.05-1.3 g or preferably 0.1-1 g of IP₃ per unit dosage.

The composition used according to the present invention contains at least one, sometimes two or more of the following substances, which correspond to the essential IP_-isomer or isomer mentioned above: D-myo-inositol-l,2,6-trisphosphate of the formula

where X is hydrogen, at least one univalent, divalent or multivalent cation, or a mixture thereof, n is the number of ions, and z is the charge of the respective ion;

Myo-inositol-l,2,3-trisphosphate of the formula

where X, n and z have the above mentioned meaning; L-myo-inositol-l,3,4-trisphosphate of the formula

where X, n and z have the above mentioned meaning.

In each of the above formulas n ranges between 6 to 1 inclusive and z ranges from 1 to 6 inclusive. Preferably, n is between 3 to 6 inclusive and z is 3, 2 or 1. Of the above isomers D-myo-inositol-1,2,6-trisphosphate is preferred.

Other inositol trisphosphate isomers that may be utilized in the present invention as the active IP- ingredient in the composition have the structural formula

(I)

One group of inositol trisphosphate compound is defined by the structural formula (I) where three of R., R , R₅, R , R and R are hydroxyl and the remaining three are phosphate and R , R , R , R , R and R are hydrogen.

Another group of inositol trisphosphates is defined by the structural formula (I) where three of Rl. , R_j, R_co_l R„/, R_Qy and

R_1? are hydroxyl and the remaining three are phosphate and ϊ₂, R₄, R₅, R₈, ₁₀ and R_1:L are hydrogen.

Still another group of inositol trisphosphates is defined by the structural formula (I) where three of R 1. , R3_, RD_R, Ro₀, R.It and R.„ are hydroxyl and the remaining three are phosphate and R , R , R , R , R and R are hydrogen.

Yet another group of inositol trisphosphates is defined by the structural formula (I) where three of R , R , R,., R , R_ and R are hydroxyl and the remaining three are phosphate and R , R , R , R , R and R are hydrogen.

Still yet another group of inositol trisphosphates is defined by the structural formula (I) where three of R 1. , RJ_, R_co , Ro₀,

R- and R are hydroxyl and the remaining three are phosphate and R , R , R , R , R and R . are hydrogen.

Even still another group of inositol trisphosphates is defined by the structural formula (I) where three of R , R , R_fi, R , R and R are hydroxyl and the remaining three are phosphate and R , R , R₅, R , R_g and R . are hydrogen.

Even yet another group of inositol trisphosphates is defined by the structural formula (I) where three of R , R , R , R , R. _ and R.. are hydroxyl and the remaining three are phosphate and R , R., R_g, R_?, R and R are hydrogen.

Finally, another group of inositol trisphosphates is defined by the structural formula (I) where three of R , R , R , R , R_g and R.. are hydroxyl and the remaining three are phosphate and R , R., R , R_g, R and R are hydrogen.

Particular inositol trisphosphate compounds within the contemplation of the above formula include compounds having the structural formula (I) where

R„ and R are phosphate, R , R and R. are hydroxyl and

R„ R , R₆, R , R_g and R.- are hydrogen;

R, R and R are phosphate, R , R and R_ are hydroxyl and R₄, R₆, R_g, R₉ and R ₂ are hydrogen;

R and R are phosphate, R , R and R are hydroxyl and R₄, R₆, R₈, R_g and R₁₂ are hydrogen;

R, R₅ and R are phosphate, R , R and R are hydroxyl and R, R , R-, R , R_g and R₁₂ are hydrogen;

R„ and R are phosphate, R , - and R- are hydroxyl and R , R , R , R_g and R₁₂ are hydrogen;

R and R are phosphate, R , R and R are hydroxyl and

R, R., Rg, R , R_g and R-₂ are hydrogen;

R- and R are phosphate, R , R and R₁₂ are hydroxyl and

R, R₄, R₅, R₈, R₁₀ and R_χι are hydrogen; R , R_ and R are phosphate, R , R and R are hydroxyl and R . , R- , R_Q, R-_ and R are hydrogen;

R₅ and R_g are phosphate, R , R and R are hydroxyl and R₄, R_g, R„, R_g and R are hydrogen;

R, R₃ and R ₂ are phosphate, R , R and R are hydroxyl and R₄, R_g, R_, R_g and R are hydrogen;

R, R₃ and R₅ are phosphate, R_g, R_1Q and R are hydroxyl and

R, R₄, R_g, R_, R_g and R are hydrogen;

R, R₅ and R_g are phosphate, R₃, R and R are hydroxyl and

R, R₄, R_g,R_, R and R - are hydrogen;

R₅ and R_χ2 are phosphate, R₃, R and R are hydroxyl and R₄, R_g, R_, and R are hydrogen;

R„ R₃ and R are phosphate, R , R and are hydroxyl and R₄, R_, R_, R-₀ and R are hydrogen;

R, R_ and R are phosphate, R , R and R are hydroxyl and R₄, R₅, R_g, R and R. - are hydrogen;

R and R are phosphate, R , R and R are hydroxyl and R₃, R_g, R„, R and R₁₁ are hydrogen;

R. R₅ and R are phosphate, R , R and R are hydroxyl and

Rr R₄,R , R , R and R_χ2 are hydrogen;

R, R_ and R are phosphate, R , R and R are hydroxyl and R₄, R_g, R_, R_g and R are hydrogen; R^ R₃ and R₅ are phosphate, R_?, R_g and R _l are hydroxyl and

^R2 R₄, R₆, Rg, R₁₀ and R₁₂ are hydrogen;

^Rl R₃ and R₁₂ are phosphate, R , R and R are hydroxyl and ^R2 R₄, R_g, R_?, R_1Q and R₁₁ are hydrogen;

^Rl R₃ and R_g are phosphate, R₅, R_g and R are hydroxyl and ^R2 R₄, Rg, R_, R₁₀ and R. are hydrogen;

^R3 R₅ and R_χ2 are phosphate, R , R and R are hydroxyl and ^R2 R₄, R_g, R_, R and R- - are hydrogen;

^Rl R₅ and R_g are phosphate, R₃, R_g and R are hydroxyl and ^R2 R₄, Rg, R„, R. and R are hydrogen;

^Rl R₅ and R_χ2 are phosphate, R₃, R and R are hydroxyl and ^R2 R₄, R_g, R_, R₁₀ and R. are hydrogen;

^Rl R₃ and R_g are phosphate, R₅, R_g and R are hydroxyl and ^R2 R₄, R_g, R_, R₁₀ and R.. are hydrogen;

^R3 R₅ and R are phosphate, R^ R_g and R are hydroxyl and ^R2 R₄, R_g, R_, R₁₀ and R. are hydrogen;

^R5 R_g and R are phosphate, R , R and R are hydroxyl and ^R2 R₄, R_g, R„, R_1Q and R are hydrogen;

^Rl R_g and R are phosphate, R₃, R₅ and R are hydroxyl and ^R2 R₄, R_g, R_, R._ and R are hydrogen;

^Rl R_g and R are phosphate, R , R and R are hydroxyl and ^R2 R₄, R_g, R_, R₁₀ and R_l are hydrogen; R, R_g and R₁₂ are phosphate, R₁, R₃ and R_Q are hydroxyl and

R, R₄, R_g, R_?, R_1Q and R_χι are hydrogen;

R, R_g and R are phosphate, R₃, R and R are hydroxyl and R₄, R_g, R_?, R_1Q and ₁ are hydrogen;

Rg and R_g are phosphate, R^ R₃ and R are hydroxyl and R₄, R_g, R_?, R₁₀ and R_±1 are hydrogen;

^R3 R₈ and R_g are phosphate, R , R₅ and R are hydroxyl and ^R2 R₄, R_g, R_?, R₁₀ and R_±1 are hydrogen;

R, R_g and R_χ2 are phosphate, R₁, R₅ and R_g are hydroxyl and

R, R₄, R_g, R₇, R _ and R. are hydrogen;

R. R_g and R₁₂ are phosphate, R_χ, R and R are hydroxyl and R₄, Rg, R_?, R _ and R are hydrogen; and

R_g and R are phosphate, R., R and R₅ are hydroxyl and

R. R₄, R_g, R_?, R - and R are hydrogen.

The above formula describes specific isomers of inositol trisphosphate where the inositol is selected from the group myoinositol, cisinositol, epiinositol, alloinositol, neoinositol, mucoinositol, chiroinositol and scylloinositol. The invention will be further explained in the following examples without limiting it thereto. Example l shows the manufacturing of a solution of IP for intravenous admini¬ stration and Example 2 and 3 demonstrate the effect of IP to inhibit infection induced by different HIV-cell lines. Example 4 illustrates the ability of IP to counteract HIV- infection induced by clinical isolates from HIV-patients. Example 1

Solution of the sodium salt of D-myo-inositol-1,2,6- trisphosphate (IP_) for injection.

0.5 g of the sodium salt of IP and 0.77 g sodium chloride were dissolved in 98.73 ml of water for injection to form a solution suitable for injection into a person or an animal.

Example 2

Inhibitory effect of the sodium salt of D-myo-inositol- 1,2,6-trisphosphate (IP₃) on HIV induced infection.

Five preparations of MT-4 cells (a human T-cell line; e

1 x 10 cells) were suspended in 500 μl growth medium comprising 10 % fetal calf serum and 20 μg/ml gentamicin and 100 IU/ml streptomycin. To four of the suspensions the sodium salt of D-myo-inositol-l,2,6-trisphosphate (IP₃) was added to final concentrations of 0.2, 2.0, 20 and 200 μg/ml respectively. The fifth suspension was used as a control with no additions of IP₃« 50 μl of HIV-3B (type code of HIV-virus) was added to the suspension followed by an incubation for 2 hours at 37°C. After extensive washing, the infected cells were resuspended in 5 ml growth medium containing appropriate amounts of IP in order to reach the final concentrations as mentioned above.

Quadriplicates of the suspensions were transferred to cell culture plates and were cultured for 4 days at 37°C. One measurement of the degree of infection is the expression of the antigen gp-120 on the cells. This HIV-antigen production was measured by an antibody-ELISA-technique and the results obtained for the different suspensions comprising IP were expressed relative to the suspension serving as a control.

The results were normalized and are given in the following table:

Concentration of IP₃ Percent infection (μg/ml)

0 100

0.2 55

2.0 55

20 18

200 0

The data demonstrate a significant inhibitory effect of IP against HIV-induced infection.

Example 3

Three preparation of MT-4 cells (1 x 10 cells) were added to 500 μl growth medium (consisting of fetal calf serum (10 % w/w) , gentamicin (20 μg/ml) and streptomycin (100 IU/ml) . The sodium salt of D-myo-inositol-1,2,6-trisphosphate (IP-) was added to two of the suspensions while the third suspension served as a control. The added amount of IP resulted in final concentra¬ tions of 125 μg/ml and 250 μg/ml respectively.

50 μl of HIV-MN (type code of HIV-virus) was added to each suspension followed by an incubation for 2 hours at 37°C. After washing the infected cells were resuspended in 5 ml growth medium containing the appropiate amounts of IP in order to reach the final concentrations as mentioned above.

Triplicates of the suspensions were transferred to cell culture plates and were cultured for 4 days at 37°C. The degree of infection was measured as the expression of the antigen gp-120 on the cells. The assay was performed using an antibody-ELISA-technique and the results obtained for the two suspensions comprising IP₃ were expressed relative to the suspension serving as a control.

The results were normalized and are given in the following table

Concentration of IP- Infection (%) (μg/ml)

0 100

125 70

250 50

The data show a counteractive effect of IP against this type of HIV-induced infection.

Example 4

Viral isolates from HIV-infected patients were used in order to induce an infection by adding peripheral blood mononuclear cells (PBMC) to a medium containing different concentrations of the sodium salt of D-myo-inositol-1,2,6-trisphosphate (IP ) 25 CCID (50 % cell culture infections dose) of PBMCs from HIV-infected patients were incubated with three different concentrations of IP₃; 0.125 mg/ml, 0.250 mg/ml and 0.5 mg/ml. A fourth preparation, without any IP served as a control. The growth medium consisted of 10 % fetal calf serum, 2 μM glutamine, 100 IU/ml penicillin, 100 IU/ml streptomycin and 20 μg/ml gentamicin. The concentration of

5 cells were 2 x 10 per ml. All preparations were incubated for 1 hour at 37°C.

PBMCs from healthy donors were then added to the prepara¬ tion. Before addition, these cells were stimulated for three days with phytohemagglutinin (PHA) . 0.5 x 10 PHA-stimulated PBMCs were added to each preparation, followed by incubation for 3 hours at 37°C. After extensive washing the cells were resuspended in growth medium supplemented with 10 IU/ml of interleukin-2 and seeded in quadruplicates of 100.000 cells in a 96-well microtite plate before further cultivation for 7 days. The HIV-antigen production was assayed at the seventh day using an ELISA-technique. The obtained values were norma¬ lized and are summarized in the following table:

Concentration of IP HIV-infection (%) mg/ml

0 100

0.125 89

0.250 16

0.500 0

In this example, where the HIV-infection is induced by clinical isolates from HIV-infected patients, IP shows a strong effect to counteract the infection. At the concentration of 0.5 mg/ml of IP the infection is totally abolished.

Claims

1. The use of at least one isomer of inositoltrisphosphate ( (IIPP )) ffoorr tthhee pprreeppaaring of a medicament effective against retroviral diseases

2. The use according to claim 1 wherein the disease is acquired immuno deficiency syndrome (AIDS) or an AIDS- related disease.

3. The use according to anyone of claims 1-2 wherein said isomer of inositoltrisphosphate is in salt form.

4. The use according to claim 3 wherein said inositoltris¬ phosphate is a salt of sodium, potassium, calcium or zinc.

5. The use according to anyone of claims 1-4 wherein the medicament is in unit dosage forms comprising tablets, granules, capsules, solutions or suspensions.

6. The use according to anyone of claims 1-5 wherein said inositoltrisphosphate is D-myo-inositol-1,2,6-trisphos- phate.