GB2037161A - Preparations containing amino acids - Google Patents

Abstract

Preparations usable to treat hepatic encephalopathy, comprise an aqueous solution of branched chain essential amino acids, e.g. leucine, isoleucine and valine. The preparations are administered parenterally.

Description

SPECIFICATION Injectable pharmaceutical preparation containing amino acids The present invention relates to an injectable pharmaceutical preparation containing amino acids which is particularly useful in curing the hepatic encephalopathy.

During severe hepatic insufficencies involving risk for the noblest nervous functions, namely in case of encephalopathies of hepatic origin, it is customary to drastically reduce or stop the alimentary intake of proteins, yet maintaining the necessary caloric intake merely by means of carbohydrates.

This unbalanced alimentary intake, although it allows the symptoms of encephalic pain to be reduced to some extent, has nevertheless the obvious drawback of progressive lowering of the proteic patrimony of the organism.

On the other hand it was recently found that during certain hepatopathies, particularly in the cirrhosis, changes of the aminoacidemic configuration in man and animals take place, which are represented by a progressive increasing of aromatic amino acids such as tryptophan, phenylalanine, tyrosine and methionine, as well as by a lessening of branched chain amino acids such as leucine, isoleucine, valine.

Such changes are found also in cephalorachidian fluid and accompanied by the appearance or increase in concentration of substances qualified as "false neurotransmitters", i.e. octopamine and phenylethanolamine.

These alterations reach highly significant values at the appearance of those neurologic symptoms which are known undertheterm of"hepatic encephalopathy".

Similar hematic, humoral and clinical patterns take place in animals, particularly in dogs, which are subjected to portacaval shunt operations. As a logic consequence of the initial remarks the administration (venoclysis) of standard solutions, that is of balanced solution with regard to the proteic intake, of amino acids both to man and animals under the above-stated conditions makes worse the neurological and biochemical pattern of the syndrome.

On the basis of above remarks concerning the aminoacidemicconfiguration of ailing subjects, it has been conceived to use solutions of amino acids in which the amount of branched-chain amino acids (leucine, isoleucine, valine) is higher than the amount of other amino acids. The administration of these selective solutions, such as those described in US-PS 3.950.529, which are characterized by a reduced concentration of methionine and aromatic amino acids and by an increased content of branched-chain amino acids, causes a remarkable clinical and bio-humoral improvement as well as a favourable change of the electroencephalogram.

Taking advantage of the acquired knowledge and with regard to observations suggesting that the getting through the blood-brain barrier is controlled by the ratio between branched-chain and aromatic amino acids, pharmacological and clinical tests have been carried out by applicants using solutions in which said ratio was stressed in favour of branched-chain amino acids.

As a result of these tests the applicants have provided a solution to be used parenterally in which the aminoacidic portion consists of leucine, isoleucine and valine only.

The pharmaceutical preparation according to this invention substantially consists of a sterile acqueous solution wherein leucine, isoleucine and valine are dissolved in suitable amount (solution A).

In order to couple the advantages issuing from the administration of carbohydrates, to this solution of said three essential amino acids a sterile aqueous solution of glucose (solution B) is mixed at the time of use.

The maintenance of the amino acid solution and glucose solution in two separate bottles up to the moment of use aims to avoid the Maillard's reactions, occurring on steam sterilization and storage, which would lead to the formation of brown reaction products adversely affecting the stability of the compound solution.

By carrying out the preparation of the solutions and the filling of bottles under nitrogen and suitably adjusting the pH value of the content in at least one of the two bottles, there are obtained solutions which remain practically colourless during steam sterilization and storage, have a very good and lasting stability and are suitable to be injected.

The amino acid solution A according to the present invention contains 1.30 to 1.80% (w/v) leucine, 1.00 to 1.50% isoleucine and 0.90 to 1.30% valine, more preferably about 1.57% leucine, 1.28% isoleucine and 1.15%valine (all percentages being expressed as weightlvolume ratio). At the given concentrations quite clear solutions are obtained which remain unchanged even on possible cooling down toOC.

The present invention is hereinafter described by way of some embodying examples given only for the sake of illustration and not limitation.

Example 1 (preparation of amino acid solution) In 600 ml water for injectable preparations which was saturated with nitrogen and heated to 80"C, 11 g leucine, 9 g isoleucine and 8 g valine were dissolved upon stirring.

The pH of the solution was adjusted to 7.5 by means of NaOH, then the volume was increased up to 700 ml by further addition of sterile water. The solution thus obtained was first filtered through asbest plates, then through a cellulose membrane having pores of 0.45 Cl and the filtrate was collected in a 11 bottle.

The solution contained in the bottle was again saturated with suitably filtered nitrogen gas and the vessel was sealed and sterilized at 121"C for 40 minutes.

Example2 (preparation of glucose solution) In 150 ml water for injectable preparations which was saturated with nitrogen gas and heated to 800C, 200 g glucose monohydrate were dissolved under stirring, the volume was then increased up to 300 ml by a further addition of water for injectable preparations.

The obtained solution was then filtered and handled according to Example 1 by finally collecting the glucose solution in a 500 ml bottle.

The glucose solution is poured in the 1 I bottle containing the amino acid solution just prior to use.

Before the experimental use of the thus prepared amino acid solution was made, an evaluation of the toxicity was carried out. One single intravenous administration of massive doses did neither cause in rats and mice any toxic symptoms nor any immediate or belated mortality.

The administration of the solution containing the three branched-chain amino acids was effected in dog during a period of four weeks at a dose of 20 mllkgldaythrough intravenous route at a rate of five running days each week. In the course of the tests no death was observed nor any particular symptom was pointed out which could be ascribed to the treatment.

Also the various biochemical constants as measured by means of hematochemical and hematological analyses were kept within standard limits, except a slight increase of glycemia and an equally slight decrease of cholesterolemia; nevertheless it should be noted that such changes were noticed to the same extent also in control group.

Dogs of both sexes averaging 15 kg were subjected to termino-lateral portacaval shunt with a view to cause hepatic encephalopathy. After about three weeks the peculiar neurologic symptoms and biochemical changes of hepatic encephalopathy appeared, in particular an increase in plasma or aromatic amino acids tryptophan, tyrosine and phenylalanine as well as octopamine and phenylethanolamine, and of ammoniemia.

The animals thus treated were divided in two groups, one of which was subjected to a treatment with a solution according to this invention and the other to a treatment with a standard aminoacidic solution as commercially available.

A solution of this kind is for instance as follows: Synthetic L-amino acids: Essential Not essential L-isoleucine 3.80 g/l L-alanine 10.00 g/l L-leucine 5.80 g/l L-glutamic acid 2.00 gll L-lysine (base) 5.44 girl Glycine 4.40 g/l L-methionine 4.80 g/l L-proline 12.00 g/l L-phenylalanine 6.88 gll L-ornithine L-aspartate 2.00 gll L-threonine 3.20 9/l L-serine 2.40 g/l L-tryptophan 1.40 g/l Total N 12.40 gll L-valine 4.48 9/l L-malic acid 4.6 g/l Semi-essential Electrolytes:: L-arginine 9.20 9/l Na+ 35 mEqll L-histidine 2.20 g/l .K+ 30 mEq/l Mg++ 5 mEqll Cl- 65 mEq/l Acetate- 5 mEqll The therapeutic treatment was started three weeks afterthe shunt operation. The amino acid solution was administered intravenously in the amount of 1 I a day through 5 to 6 running days each week.

The biochemical observations in plasma were carried out prior to portacaval shunt operation, three weeks after it and at the 3rd, 6th and 21st day of the care period with amino acids. In parallel with the biochemical tests, blood gas analysis, hemoglobin and hematocrit were also evaluated; the weight of the animals was checked twice a week.

The group which was treated with standard amino acids showed an increasing deterioration of encephalopathy together with high percentage of deaths during the three weeks treatment; to these clinical symptoms made a pendant a progressive aggravation of biochemical values also.

In the group which was treated with solutions according to the present invention no deaths were observed, while the biochemical balance and the disappearance of neurogical symptoms were reached in a very short time.

For sake of comparison, tests were carried out also with other kinds of solutions always containing three amino acids but partially or totally different from the preceding ones (for instance 69 leucine, 10 9 alanine, 9 g arginine in 1000 ml). The effectiveness of such alternative solutions turned out to be poor or of no value, whereas an aggravation of the syndrome was on the contrary noticed.

Solutions according to the present invention were subsequently employed in the symptomatic care of grave conditions of hepatic encephalopathy in humans suffering from advanced cirrhosis. Six patients which were subjected to observation and treatment were classified as pertaining to the 3rd and 4th level of hepatic encephalopathy on the ground of their clinical pattern, bio-humoral checkings and electroencephalographic control.

To these six patients the solutions were intraveneously administered at a rate of one phleboclysis each day in the course of 14-16 hours and repeated for 3-4 days. The used solutions according to this invention allowed to achieve in all of cared subjects a substantial clinical improvement which was characterized by the revival from coma and important bio-humoral and electroencephalographic changes. The favourable changes happened in some cases in a dramatically quick way even 5-6 hours afterthe beginning of phleboclysis only. The tolerance was good in each case.

Claims (5)

1. A sterile aqueous solution to be administered by intravenous route, characterized in that it is substantially an aqueous solution of essential branched-chain amino acids.

2. The solution of claim 1, characterized in that said essential branched-chain amino acids are leucine, isoleucine and valine.

3. The solution of claim 2, characterized in that the weightlvolume percentages of leucine, isoleucine and valine are in the range 1.30 to 1.80%, 1.00 to 1.50% and 0.90 to 1.30 respectively.

4. A solution according to the preceding claims, characterized in that it contains 11 g leucine, 9 g isoleucine and 8 g valine in 1000 ml.

5. The solution of claim 4, further characterized in that it is prepared by mixing just before the therapeutic use a solution of leucine, isoleucine and valine with an injectable solution of glucose.