Stable salts of S-adenosylmethionine and process for the preparation thereof

S-adenosyknethioniαe (SAMe) is physiological donor of methyl groups present in every living organism and employed in enzymatic transmethylation reactions.

Such substance therefore has a role of considerable biological importance, and is essentially clinically used as an anti-depressive.

By "SAMe", it is intended to indicate both die racemic mixture and die single diastereoisomers (RS)-(+)-S-adenosyl-L-methionine [(RS)-(+)-SAMe)] and (SS)-(+)-

S-adenosyl-L-methionine [(SS)-(+)-SAMe)], also in mixtures different from the racemic mixture.

The difficulty of using S-adenosylmethionine as drug and/or dietetic product ^" is however known, since it is extremely unstable at temperatures above 0°C or in the presence of moisture, both as degradation of the active ingredient intended as the sum of the two diastereoisomers and as transformation of active (SS)-(+)-S-adenosyl-

L-methionine into inactivate (RS)-(+)-S-adenosyl-L-mediionine (racemisation of die substance).

SAMe corresponds to the following formula:

SAMe participates in a great number of metabolic processes of fundamental importance for the human body, and its deficiency therefore underlies many organic malfunctions.

Even if the biological importance of this product has been known for a long time, the possibility to examine and use it as a drug and/or dietetic product has only developed recently, above all due to its extreme instability at temperatures above 0

⁰ C.

Only in 1975 was a sufficiently stable SAMe salt successively prepared at 25 ⁰ C.

(US3893999), followed afterward by other salts with good stability also at 45°C.

(US3954726 and US4057686).

More specifically, US3893999 describes tri-p-toluensulphonate of SAMe, US3954726 describes disulphate di-p-toluensulphonate of SAMe, US4057686 describes a group of SAMe salts which can be indicated overall as SAMe . 4RSO ₃ H or SAMe 3RSO ₃ H in which the RSO ₃ H indicates a disulphonic acid equivalent which can partly substitute the equivalents of sulphuric acid.

The US patent application No. 20020010147 describes a process for preparing salts

of (SS ₅ RS)-SAM in which the salified diastereoisomer RS(+) SAMe is present in a much lower amount than, the salified diastereoisomer SS(+) SAMe. It has now been surprisingly found that salts of SAMe having an improved stability over time are obtained by salifying the SAMe with 0.5 — 2.0 moles/mole of a strong inorganic acid with an acid dissociation constant (pKa) of less than 2.5 added with 0.5 — 1.0 moles/mole of an oxide and/ or salt. Said oxide and/ or salt is preferably selected from among calcium oxide, magnesium oxide, calcium chloride, magnesium chloride, calcium sulphate, magnesium sulphate and/ or a mixture thereof. Said salts of SAMe according to the present invention preferably contain a high percentage of SAMe. More preferably, the percentage of SAMe in the aforesaid salts is at least 70% by weight, and still more preferably is in the range of 75 - 90%. Salts of SAMe that contain lesser quantities of acid, oxide and/or salt are unacceptable for therapeutic use, since they are subject to degradation phenomena. It has in fact been noted how the presence even of small percentages of degradation products is unacceptable, not only since it leads to the loss of activity, but also and above all since it causes the formation of metabolites which have resulted to be toxic. The object of the present invention are therefore SAMe salts having the following general formula (I).

(I) where

HX is a strong mineral acid having an acid dissociation constant (pKa) of less than 2.5; n and m are independendy in the range of 0.5 - 2.0;

Y is a calcium oxide, magnesium oxide, calcium chloride, magnesium chloride, calcium iulphate, magnesium sulphate and/or a mixture thereof; Preferably, HX is an acid selected from among hydrochloric acid, sulphuric acid, phosphoric acid, phosphorous _cid, disulphoruc acid and/or 1,4 butanedisulphonic acid.

Examples of SλMe s.ilrs .lccording to the present invention preferably correspond to the following general formulas (II) and (HII)-

where

HX is a strong mineral acid having acid dissociation constant (pKa) of less than 2.5; Y is calcium oxide, magnesium oxide, calcium chloride, magnesium chloride, calcium sulphate, magnesium sulphate and/ or a mixture thereof.

Preferably, HX is an acid selected from among hydrochloric acid, sulphuric acid, phosphoric acid, phosphorous acid, disulphonic acid and/or 1,4 butanedisulphonic

acid.

According to the present invention, the pKa of the aforesaid acids correspond to the following values:

HCl pKa < 0.5; H ₂ SO ₄ pKa _t < 0.5, pKa ₂ =1.92 (2° ionisation or dissociation constant); H ₃ PO ₄ PKa ₁ < 0.5, pKa ₂ =2.12 (2° ionisation or dissociation constant), pKa ₃ = 2.3 (3° ionisation or dissociation constant).

The improved stability of the salts of SAMe of the present invention is also directly correlated with the size and shape of the product itself in drying phase. This because the shape and size of the final powder influence the lrygroscopicity of the product, which determines the stability of the same to the extent that the closer the hygroscopicity value approaches zero, the greater the stability of the salt of SAMe.

In particular, the particle sizes of the salt according to the present invention are preferably in the range of 20 - 500 μm, more preferably in the range of 50 - 300 μm, and the particles are preferably in oval or spherical form, more preferably spherical.

The drying phase of the product according to the present invention occurs through a lyophilisation passage, preceded by a freezing passage by ultrasonic spray cooling.

Said freezing is preferably carried out according to the method described in

US707636.

The salts of SAMe according to the present invention moreover contain a high percentage of the active diastereoisomer, (SS)-(+)-S-adenosyl-L-methioniαe, of the

SAMe.

Said percentage of (SS)-(+)-S-adenosyl-L-methionine is preferably at least 80% by weight, more preferably in the range of 85 - 95% calculated with respect to the sum of the two diastereoisomers.

A further object of the invention is the use of at least of the salts of formula (I)

HX is a strong mineral acid having an acid dissociation constant (pKa) of less than 2 5, n and m are independently in the range of 0 5 - 20;

Y is a calcium oxide, magnesium oxide, calcium chloπde, magnesium chloride, calcium sulphate, magnesium sulphate and/or a mixture thereof for the preparation of a medicament for the treatment of depressive states.

Preferably, HX is an acid selected from among hydrochloric acid, sulphunc acid, phosphoric acid, phosphorous acid, djsulphoruc acid and/or 1,4 butanedisulphoruc acid

The following examples are present in order to better understand the invention, without in any manner limiting it EXAMPLES Example 1

In two 100 mL iliquots of distilled water, K) grams of SλMe were dissolved containing respectively 0 5 or 2 0 moles of sulphuric acid

Hie obtained solutions were filtered on 0 20 urn hirers up to complete tnnϊ>pirency To rhe iqueous solutions thub prepared, 0 5 or 1 0 moles of magnesium oxide were respectively idded iπd once ig.un filtered on i 0 20 μm tilter

Hie rwo salts thus ptepircd were subjected to quick freezing with the ^pray cooling method ind btibsequently subjected to lyophiliiition

In such a manner, two products were obtained with the following compositions:

SAMe . 0.5 H ₂ SO ₄ . 0.5Mg ₂ SO ₄ . 0.4 H ₂ O and SAMe . 2.0 H ₂ SO ₄ . 1.0Mg ₂ SO ₄ . 0.4

H ₂ O.

The salts have a white crystalline aspect with granulometry in the range of 50 - 300 μm and perfectly spherical form. They are extremely soluble in water up to about 60 mg/mL.

The high-resolution, thin-layer chromatography shows that the product is free of any impurity.

Table 1 reports the analytic data of the aforesaid two salts.

Example 2

Two aqueous solutions containing SAMe and sulphuric acid were prepared according to the method described in Example 1.

To the aqueous solutions thus prepared, 0.5 or 1.0 moles of magnesium chloride were respectively added and once again filtered on a 0.20 μm filter.

The two salts thus prepared were subjected to quick freezing with the spray cooling method and subsequent lyophilisation as in example 1.

In such a manner, six products were obtained with the following compositions:

SAMe . 0.5 H ₂ SO ₄ . 0.5 MgCl ₂ . 0.4 H ₂ O and SAMe . 2.0 H ₂ SO ₄ . 1.0 MgCl ₂ . 0.4

H ₂ O.

The salts have a white crystalline aspect with granulometry in the range of 50 - 300 μm and perfectly spherical form. They are extremely soluble in water up to about 60 mg/mL.

The high-resolution, thin-layer chromatography shows that the product is free of any impurity.

Table 1 reports the analytic data of the aforesaid two salts.

Example 3

Two aqueous solutions containing SAMe and sulphuric acid are prepared according

to the method described in Example 1.

To the aqueous solutions thus prepared, 0.5 or 1.0 moles of CaC12 were respectively added and once again filtered on a 0.20 μm filter.

The solution was then frozen and lyophilised by spray cooling and subsequently subjected to lyophilisation.

In such a manner, six products were obtained with the following compositions:

SAMe . 0.5 H2 SO ₄ . 0.5 CaCl ₂ . 0.4 H ₂ O and : SAMe . 2.0 H ₂ SO ₄ . 1.0 CaCl ₂ . 0.4

H ₂ O.

The salts have a white crystalline aspect with granulometry in the range of 50 - 300 μm and perfectly spherical form. They are extremely soluble in water up to about 60 mg/mL.

The high-resolution, thin-layer chromatography shows that the product is free of any impurity.

Table 1 reports the analytic data of the aforesaid two salts.

Example 4

In two 100 roL aliquots of distilled water, 40 grams of SAMe were dissolved containing respectively 0.5 or 2.0 moles of hydrochloric acid.

The obtained solutions were filtered on 0.20 μtn filters up to complete transparency.

To the aqueous solutions thus prepared, 0.5 or 1.0 moles of magnesium sulphate were respectively added and once again filtered on a 0.20 μm filter.

The two salts thus prepared were subjected to quick freezing by spray cooling and subsequent lyophilisation.

In such a manner, two products were obtained with the following compositions:

SAMe . 0.5 HCl . 0.5Mg ₂ SO ₄ . 0.4 H ₂ O and SAMe . 2.0 HCl . 1.0 Mg ₂ SO ₄ . 0.4 H ₂

O.

The salts have a white crystalline aspect with granulometry in the range of 50 - 300 μm and perfectly spherical form. They are extremely soluble in water up to about 60

mg/mL.

The high-resolution, thin-layer chromatography shows that the product is free of any impurity.

Table 1 reports the analytic data of the aforesaid two salts.

Example 5

Two aqueous solutions containing SAMe and sulphuric acid are prepared according to the method described in Example 4.

To the aqueous solutions thus prepared, 0.5 or 1.0 moles of magnesium chloride were added and once again filtered on a 0.20 μm filter.

The solution was then frozen and lyophilised by spray cooling and subsequently lyophilised.

In such a manner, six products were obtained with the following compositions:

SAMe . 0.5 HCl . 0.5 MgCl ₂ . 0.4 H ₂ O and SAMe . 2.0 HCl . 1.0 MgCl ₂ . 0.4 H ₂ O.

The salts have a white crystalline aspect with granulometry in the range of 50 - 300 μm and perfectly spherical form. They are extremely soluble in water up to about 60 mg/mL.

The high-resolution, thin-layer chromatography shows that the product is free of any impurity.

Table 1 reports the analytic data of the aforesaid two salts.

Example 6

Two aqueous solutions containing SAMe and sulphuric acid are prepared according to the method described in Example 4.

To the aqueous solutions thus prepared, 0.5 or 1.0 moles of calcium chloride are added and once again filtered on a 0.20 μm filter.

The solution was frozen and lyophilised by spray cooling and subsequently lyophilised.

In such a manner, six products were obtained with the following compositions:

SAMe . 0.5 HCl . 0.5 CaCl ₂ . 0.4 H ₂ O and SAMe . 2.0 HCl . 1.0 CaCl ₂ . 0.4 H ₂ O.

The salts have a white crystalline aspect with granulometry in the range of 50 - 300 μm and perfectly spherical form. They are extremely soluble in water up to about 60 mg/mL.

The high-resolution, thin-layer chromatography shows that the product is free of any impurity.

Table 1 reports the analytic data of the aforesaid two salts.

Table 1

In the following Tables 2-13, the percentage values are reported which were obtained in the stability tests carried out on the products of examples 1-6. The stability tests were carried out in thermostated heaters at 40 ⁰ C and 75% R.H (tables 2-7) as well as

25°C and 60% R.H. (tables 8-13), preserving the samples in heat-sealed aluminium/ aluminium bags according to the following criteria: the active ingredient % was determined for every sampling point taken under consideration.

Table 2

Example 1

SAMe . 0.5 H ₂ SO ₄ . 0.5Mg ₂ SO ₄ . 0.4 H ₂ O and SAMe . 2.0 H ₂ SO ₄ . 1.0Mg ₂ SO ₄ . 0.4

H, O.

Table 3

Example 2

SAMe . 0.5H ₂ SO ₄ . 0.5MgCl ₂ and SAMe . 2.0H ₂ SO ₄ . 1.0MgCl ₂

Table 4

Example 3

SAMe . 0.5H ₂ SO ₄ . 0.5CaCl ₂ and SAMe . 2.0H ₂ SO ₄ . 1.0CaCl ₂

Table 5

Example 4

SAMe . 0.5HC1 . 0.5MgSO ₄ and SAMe . 2.0HC1 . 1.0MgSO ₄

Table 6

Example 5

SAMe . 0.5HC1 . 0.5MgCl ₂ and SAMe . 2.0HC1 . 1.0MgCl ₂

Table 7

Example 6

SAMe . 0.5HC1 . 2.0CaCl ₂ and SAMe . 2.0HC1 . 1.0CaCl ₂

Table 8

Example 1

SAMe . 0.5 H ₂ SO ₄ . 0.5Mg ₂ SO ₄ . 0.4 H ₂ O and SAMe . 2.0 H ₂ SO ₄ . 1.0Mg ₂ SO ₄ . 0.4

H2 O.

Table 9

Example 2

SAMe . 0.5H ₂ SO ₄ . 0.5MgCl ₂ and SAMe . 2.0H ₂ SO ₄ . 1.0MgCl ₂

Table 10

Example 3

SAMe .0.5HSO .0.5CaCl and SAMe .2.0HSO ■ 1.0CaCl

Table 11

Example 4

SAMe .0.5HC1.0.5M SO and SAMe .2.0HC1.1.0M SO

Table 12

Example 5

SAMe .0.5HC1.0.5M Cl and SAMe .2.0HC1.1.0MgCl ₂

Table 13

Example 6

SAMe . 0.5HC1 . 2.QCaCl ₂ and SAMe . 2.0HC1 . 1.0CaCl ₂

In the following Tables 14-25, the percentage values are reported of the degradation of the active stereoisomer S,S calculated at 4O ⁰ C and 75% R.H. (Tables 14-19) and at

25°C and 65% R.H.

Table 14

Example 1

SAMe . 0.5 H ₂ SO ₄ . 0.5Mg ₂ SO ₄ . 0.4 H ₂ O and SAMe . 2.0 H ₂ SO ₄ . 1.0Mg ₂ SO ₄ . 0.4

H ₂ O.

Table 15

Example 2

SAM . 0.5H ₂ SO ₄ . 0.5MgCl ₂ and SAMe . 2.0H ₂ SO ₄ . 1.0MgCl ₂

Table 16

Example 3

SAMe . 0.5H SO . 0.5CaCl and SAMe . 2.0H SO . 1.0CaCl

Table 17

Example 4

SAMe . 0.5HC ₁ . 0.5MgSO ₄ and SAMe . 2.0HC1 . 1.0MgSO ₄

Table 18

Example 5

SAMe . 0.5HC1 . 0.5M Cl and SAMe . 2.0HC1 . 1.0M Cl

Table 19

Example 6

SAMe . 0.5HC1 . 2.0CaCl ₂ and SAMe . 2.0HC1 . 1.0CaCl ₂

Table 20

Example 1

SAMe . 0.5 H ₂ SO ₄ . 0.5Mg ₂ SO ₄ . 0,4 H ₂ O and SAMe . 2.0 H ₂ SO ₄ . 1.OMg ₂ SO ₄ . 0,4

Table 21

Example 2

SAMe . 0.5H ₂ SO ₄ . 0.5MgCl ₂ and SAMe . 2.0H ₂ SO ₄ . 1.0MgCl ₂

Table 22

Example 3

SAMe . 0.5H ₂ SO ₄ . 0.5CaCl ₂ and SAMe . 2.0H ₂ SO ₄ . 1.0CaCl ₂

Table 23

Example 4

SAMe . 0.5HC1 . 0.5MgSO ₄ and SAMe . 2.0HC1 . 1.0MgSO ₄

Table 24

Example 5

SAMe . 0.5HC1 . 0.5MgCl _? and SAMe . 2.0HC1 . 1.OMgCl.

Table 25

Example 6

SAMe . 0.5HC1 . 2.0CaCl ₂ and SAMe . 2.0HC1 . 1.0CaCl ₂