HK1036409A - Use of calcium complex of 4-(4-ethoxybenzyl)-3,6,9-tris(carboxylatome acid - Google Patents

Description

Use of 4- (4-ethoxybenzyl) -3, 6, 9-tri (carboxylatomethyl) -3, 6, 9-triazaundecanedioic acid calcium complex

The invention relates to the use of calcium complexes of 4- (4-ethoxybenzyl) -3, 6, 9-tris (carboxylated methyl) -3, 6, 9-triazaundecanedioic acid and salts thereof for producing medicaments for reducing the effects caused by heavy metals.

In medicine, various coordination compounds are used, in particular, for the treatment of heavy metal poisoning, iron deficiency diseases and for the production of medicaments for diagnostic imaging.

In EP71564, the use of meglumine (meglumine) salts of Diethylene Triamine Pentaacetic Acid (DTPA) gadolinium (iii) complexes as contrast agents for NMR tomography is described. The trade name is Magnevist^(R)The formulations containing the complexes of (a) have gained global acceptance as first NMR contrast agents. This contrast agent disperses extracellularly after intravenous injection and is excreted by the kidney via glomerular secretion. Virtually no passage through an intact cell membrane was observed. Magnevist^_Is most suitable for imaging pathological parts (such as inflammation and tumor).

Compounds containing DTPA or Ca-DTPA are also used clinically for metal poisoning. Iron complexes are commonly used for iron deficiency anemia, while radioisotope complexes (e.g. iron-deficiency anemia)^99mTc-DT-PA) was used for the scintillation method.

When used as NMR and X-ray diagnostic contrast agents, EP405704 suggests that these compounds are excreted extra-renally and are thus suitable for liver, gall bladder and gastrointestinal imaging.

These known complexes and their salts can cause problems with compatibility and/or stability of the complexes in vivo in their clinical use. The heavy metal elements themselves have not hitherto been used advantageously as constituents of X-ray contrast agents, since the compatibility of these compounds is unsatisfactory. For the paramagnetic substances proposed so far and used in nuclear spin tomography, the difference between the effective and toxic dose in animal experiments is small.

Therefore, there is a need for drugs that can reduce the effects caused by heavy metals.

The object of the present invention is to efficiently prepare such compounds and drugs, and to provide a process for the preparation thereof. This object is achieved by the subject matter characterized in the claims.

It has been found that the calcium complex of 4- (4-ethoxybenzyl) -3, 6, 9-tris (carboxylated methyl) -3, 6, 9-triazaundecanedioic acid (Ca-EOB-DTPA) and its salts are particularly suitable for the preparation of medicaments which reduce the effects caused by heavy metals. In particular, Ca-EOB-DTPA is suitable for removing heavy metals deposited in the liver.

The term EOB-DTPA may use the following synonyms, i.e. the formation of the various enantiomers (4S or 4R) or any mixture thereof.

Ca-EOB-DTPA is surprisingly able to eliminate metal deposits, for example from the body, in particular from the liver, after heavy metal poisoning. As described in the examples, the amount of zinc in the liver and kidney can be reduced by administering Ca-EOB-DTPA.

It was also found that the addition of Ca-EOB-DTPA to Gd-EOB-DTPA for NMR diagnosis reduces the gadolinium content in liver and bone (see examples). The pharmacokinetic examination of animal models showed that Ca-EOB-DTPA … is similar to Gd-EOB-DTPA-surprisingly eliminated mainly by the liver (see examples). This is particularly surprising since the same 3-fold amount of Ca-DTPA added passively does not enter the liver. The addition of Ca-EOB-DTPA therefore also quantitatively eliminated non-coordinately bound gadolinium from the liver.

The present invention therefore relates to calcium complexes of 4- (4-ethoxybenzyl) -3, 6, 9-tris (carboxylated methyl) -3, 6, 9-triazaundecanedioic acid and to the salts of these complexes with physiologically compatible inorganic and/or organic cations, such as sodium, potassium, meglumine, ethanolamine, diethanolamine, morpholine, glucosamine, dimethylglucamine, lysine, arginine and/or ornithine.

The invention also relates to the use of Ca-EOB-DTPA and its salts for the preparation of pharmaceutical preparations, in particular antidotes against heavy metal poisoning. Preparation of the Compounds of the invention

The compounds of the invention are prepared starting from 4- (4-ethoxybenzyl) -3, 6, 9-tris (carboxylated methyl) -3, 6, 9-triazaundecanedioic acid-pentatert-butyl ester as described in EP405704A2 (example 8).

Firstly, the pentaester reacts with trifluoroacetic acid to prepare a free coordination agent. The complexes according to the invention are prepared from the complexing agents according to the methods disclosed in the patent specifications EP71564, EP 130934 and DE-OS 3401052.

This complexing agent is converted to a calcium complex (Ca-EOB-DTPA) by reaction with calcium hydroxide solution. The hydrogen atoms on the acid groups present are then replaced, if desired, by inorganic and/or organic bases or amino acids.

In this case, neutralization is carried out with inorganic bases (for example hydroxides, carbonates or hydrogen carbonates) such as sodium, potassium or lithium salts and/or organic bases such as primary, secondary and tertiary amines (for example ethanolamine, glucosamine, N-methyl-and N, N-dimethylglucamines) and basic amino acids such as lysine, arginine and ornithine.

Since the complex contains 3 free acid groups, it can form a neutral mixed salt containing both an inorganic cation and an organic cation as a counterion.

This can be achieved, for example, by reacting the complexing agent with calcium oxide or a calcium salt in aqueous suspension or solution to give the oxide or salt of the desired element, neutralizing with 1/3 or 2/3 amounts of the desired organic base, isolating the complex salt formed, optionally purifying, and then mixing with the desired amount of inorganic base to complete the neutralization. The order of addition of the bases may also be reversed.

Mixtures of Ca-EOB-DTPA and Gd-EOB-DTPA may be prepared by reacting Gd-EOB-DTPA with a calcium complex of DTPA at elevated temperature. By re-complexing, a mixture of the desired Ca-EOB-DTPA complex and Gd-EOB-DTPA can be obtained. The above neutralization may also be carried out in this process.

The medicaments according to the invention can be prepared by processes known in the art, i.e. by suspending or dissolving the complex compounds according to the invention, optionally together with customary additives for galenic preparations, in an aqueous medium and optionally sterilizing this suspension or solution. Suitable additives are, for example, physiologically harmless buffers (e.g. trimethylamine), electrolytes (e.g. sodium chloride) and antioxidants (e.g. ascorbic acid).

If the suspension or solution of the medicament of the invention is to be an aqueous or physiological salt solution for enteral administration or for other purposes, it is mixed with one or more adjuvants conventionally used in galenic formulations, such as methylcellulose, lactose or mannitol, and/or surfactants (e.g. lecithin, tween)^(R)Or Myri^(R)) And/or taste modifying substances (e.g., essential oils).

In principle, the medicaments according to the invention can even be prepared without isolation of the complex salts. However, specific protection must be taken to ensure chelation so that no toxic uncomplexed metal ions are actually present in the salts and salt solutions of the invention.

The medicament of the invention contains 1 mu mol/1-1 mol/1 coordination salt, preferably 0.5 mmol/l-250 mmol/l, and the general dosage is 0.005-2 mmol/kg body weight, preferably 50 mu mol/kg-500 mu mol/kg. The medicament is administered enterally and parenterally.

The invention therefore also relates to the above-mentioned processes for the preparation of these compounds and medicaments.

The following examples serve to explain the objects of the invention in more detail.

EXAMPLE preparation of calcium 4- (4-ethoxybenzyl) -3, 6, 9-tris (carboxylated methyl) -3, 6, 9-triazaundecanedioate Complex trisodium salt method 1a)4- (4-ethoxybenzyl) -3, 6, 9-tris (carboxylated methyl) -3, 6, 9-triazaundecanedioate

3.64 g of pentatert-butyl 4- (4-ethoxybenzyl) -3, 6, 9-tris (carboxylated-methyl) -3, 6, 9-triazaundecanedioate (for its preparation see EP0405704A2, Schering AG, Berlin, example 8a) are dissolved in 25ml of trifluoroacetic acid. This was stirred at room temperature for 1 hour, mixed with 150ml of diethyl ether and the precipitate filtered off with suction. The precipitate is dried under vacuum at 40 ℃ and then dissolved in 200ml of water and stirred with 2g of the anion exchanger Amberlite IRA 67 for 15 minutes. It was filtered and the solution was stirred with 0.5 g of activated carbon for 30 minutes. The charcoal was filtered off and lyophilized 3 times to remove residual trifluoroacetic acid to give 1.2 g of the desired compound as a white powder.

Analysis (versus anhydrous material):

calculated values: c52.36% H6.31% N7.97%

Measured value: c52.21% H6.39% N7.84% b) calcium 4- (4-ethoxybenzyl) -3, 6, 9-tris (carboxylated methyl) -3, 6, 9-triazaundecanedioate complex trisodium salt

4.22 g of 4- (4-ethoxybenzyl) -3, 6, 9-tris (carboxylated-methyl) -3, 6, 9-triazaundecanedioic acid (8mmol) are mixed in 100ml of water with 0.59 g (8mmol) of calcium hydroxide. This was stirred for 15 minutes while mixing with 24ml of 1N sodium hydroxide solution. The resulting solution was filtered through a membrane filter to remove a small amount of suspended materials, and then lyophilized. 4.9 g of the desired compound are obtained in the form of a white powder with a water content of 9.4%.

Analysis (versus anhydrous material): calculated values: c43.74% H4.47% Ca 6.35% N6.65% Na 10.32% found: c43.91% H4.60% Ca 6.27% N6.61% Na 10.62%

Melting point: the complex did not melt until 300 ℃.

Optical rotation: [ alpha ] to]_DCalcium 4- (4-ethoxybenzyl) -3, 6, 9-tris (carboxylated methyl) -3, 6, 9-triazaundecanedioate complex meglumine salt (= + 7.2 ° (c = 1.0 in methanol) = c)

If 24ml of 1N methylglucamine solution is used instead of the sodium hydroxide solution in example b, the corresponding trimigramine salt is obtained. Method 2

Another method starts from 4- (4-ethoxybenzyl) -3, 6, 9-tris (carboxylated methyl) -3, 6, 9-triazaundecanedioic acid-pentatert-butyl ester, in which the ligand 4- (4-ethoxybenzyl) -3, 6, 9-tris (carboxylated methyl) -3, 6, 9-triazaundecanedioic acid is not isolated but is likewise converted in solution further into 4- (4-ethoxybenzyl) -3, 6, 9-tris (carboxylated methyl) -3, 6, 9-triazaundecanedioic acid calcium complex trisodium salt:

a solution of 21.4 g (26.5 mmol) of 4- (4-ethoxybenzyl) -3, 6, 9-tris (carboxylated-methyl) -3, 6, 9-triazaundecanedioic acid-pentatert-butyl ester in 150ml of methanol is refluxed for 5 hours together with 16ml of 15N sodium hydroxide solution.

Methanol was removed in vacuo on a rotary evaporator, mixed with 190ml of water and a sufficient amount of the cation exchanger IR-120 was added in portions with stirring until a pH of 3.0 was reached. The ion exchanger is filtered off, the residue is washed with water and the filtrate is purified.

The 4- (4-ethoxybenzyl) -3, 6, 9-tris (carboxylated methyl) -3, 6, 9-triazaundecanedioic acid-sodium salt solution thus obtained was mixed with 1.96 g of calcium hydroxide and the pH was adjusted to 10.2 by addition of 40% sodium hydroxide solution. The solution was stirred with activated carbon at room temperature for 1 hour and then filtered through a nitrocellulose filter (0.2 μm).

After lyophilization, 17.84 g of the trisodium salt of the calcium 4- (4-ethoxybenzyl) -3, 6, 9-tris (carboxylated-methyl) -3, 6, 9-triazaundecanedioate complex are obtained as a white powder with a water content of 7.5%.

The other analysis data are consistent with the values of the first method.

Method for preparing Gd-EOB-DTPA and Ca-EOB-DTPA mixture

20ml of 250. mu. mol/ml (5000. mu. mol) Gd-EOB-DTPA in water and 20ml of 1. mu. mol/ml (20. mu. mol) CaNa₃-mixing with an aqueous DTPA solution. After heat treatment at 121 ℃ for 20 minutes, a quasi-complete reassortment in 20. mu. mol Ca-EOB-DTPA, 4980. mu. mol Gd-EOB-DTPA and 20. mu. mol Gd-DTPA was observed.

The following examples are specific examples of the mode of action of the novel compounds.

Pharmacokinetics: male rats were injected intravenously with a 25. mu. mol/kg dose¹⁴C-labelled Ca-EOB-DTPA/Na₃. The concentration in the blood of each organ was measured at different times 24 hours after injection. Urine and feces excretion were measured in another group of animals by peritoneal injection for up to 7 days.

The concentration in the blood dropped rapidly with a half-life of 8 minutes. A distribution volume of 0.45L/kg indicates accumulation in cells, possibly in hepatocytes.

Only 4.2% of the dose remained in vivo 24 hours after administration, with the highest values in kidney and liver.

Doses of 2/3 were excluded from the feces and 1/3 was collected in the urine.

The observed values are very similar or in practical agreement with Gd complexes (G.schuhmann-Giamperi, H.schmitt-Willich, W. -R.press, C.Negishi, H. -J.Qeinmann, and U.Speck, Radiolgy, 1992, 183, 59).

Reduction of metal concentration in organisms by intravenous injection of Ca-EOB-DTPA:

examples of Zinc

Male rats were injected intravenously with 200. mu. mol/kg of Ca-EOB-DT-PA/Na₃Aqueous solution, 2 days later, liver and kidney by ICP-AESThe concentration of zinc.

Organ	Concentration of	Zn[μmol/q]
			Liver disease	Before injection	5.3
After injection of Ca-EOB-DTPA	4.4
		Kidney (Kidney)		Before injection	0.9
After injection of Ca-EOB-DTPA	0.5

Ca-EOB-DTPA/Na injection₃The metal concentration of zinc in the liver and kidney is reduced.

In this regard, the known antidote Ca-DTPA/Na₃Is ineffective because the substance does not pass through the hepatocytes.

Reduction of the concentration of metals in organisms by addition of Ca-EOB-DTPA to NMR contrast agents: examples of Gd-EOB-DTPA

Male rats were injected intravenously with Gd-EOB-DT-PA/Na at a dose of 250. mu. mol/kg₂The Gd concentration in bone and liver was determined 7 days later by ICP-AES as an aqueous solution (solution A, B, C, D). Solution A did not contain any additives, while B, C, D had Ca-EOB added at different concentrations-DTPA／Na₃

Solution A: 0 mg/ml

Solution B: 0.1 mg/ml

Solution C: 0.5 mg/ml

Solution D: 1.0 mg/ml

Gd content [ nmol/g]	A	B	C	D
					Liver disease	1.12±0.014	1.14±0.028	0.90±0.13	0.79±0.15
Bone	1.51±0.08	1.39±0.13	1.04±0.12	0.67±0.09

By adding the Ca complex, the concentration of Gd in the liver and the bone is significantly reduced.

This finding is very advantageous because the relatively small amount of Gd already left in the organism also causes a contraindication reaction of the conjugation. Since the novel Ca complex also actually enters the liver cells unexpectedly at the same time, it is ensured that the influence of the possible release of Gd from the complex is minimized.

Ca-EOB-DTPA improves the elimination rate of Gd-EOB-DTPA and may reduce the concentration of toxic metals in the organism.

Claims (2)

1. Use of calcium 4- (4-ethoxybenzyl) -3, 6, 9-tris (carboxylated methyl) -3, 6, 9-triazaundecanedioate complexes and their salts with physiologically compatible cations for the preparation of a medicament for reducing the effects caused by heavy metals.
2. Use of calcium 4- (4-ethoxybenzyl) -3, 6, 9-tris (carboxylated methyl) -3, 6, 9-triazaundecanedioate complexes and their salts with physiologically compatible cations for the preparation of a medicament against heavy metal poisoning.