DD222782A1 - METHOD FOR PRODUCING STABLE SOLUTIONS OF UNDECAPEPTIDES AND ITS PARTIAL SEQUENCES - Google Patents

Abstract

The invention relates to a process for the preparation of stable solutions of undecapeptides, especially those of the substance P series and the substance P partial sequences. The peptides are dissolved in acids and neutralized with bases, whereby acids are used, which guarantee the p H-Konstanz by buffer effect after addition of the bases. The solutions are suitable for nasal application. Field of application is the pharmaceutical industry.

Description

With solutions of substance P, their partial sequences and analogues prepared according to the invention, it was possible to

a) achieve a plasma level of the peptide which is sufficient for pharmacological efficacy and

b) to detect the already known from other types of application for substance P anti-stress effects for the first time after nasal application.

The invention will be explained in more detail with reference to exemplary embodiments.

Exemplary embodiments; _v '.

Example 1 _:; .. ·.

1g substance. P is stirred with TOmI 0.02 M phosphoric acid and completely dissolved with further addition of phosphoric acid (90 ml, 0.02 M). It is then adjusted to pH 7.0 with 0.01 M sodium hydroxide solution and dissolved in peptide solution 100 mg of benzalkonium chloride. '. '

Example 2:.

, 1 g of substance P is stirred with 10 ml of 0.02 M phosphoric acid and the peptide is dissolved with continued stirring with further addition of phosphoric acid (90 ml, 0.02 M) completely. This peptide solution is adjusted to pH 7.0 with 0.01 M sodium hydroxide solution and preserved with 100 mg of benzalkonium chloride. This solution is filled up with a clarified 3 percent solution of Meth'ylcellulose to 1000ml. \,,

Step Example! 3:. · ₁ ~~

Analogously to Example 2, 0.8 g of N-terminal tetrapeptide from substance P (1-4) or other N- or C-terminal partial sequences or analogs are dissolved in 100 ml of 0.02M hydrochloric acid and made up to 0.02 M with trishydr'oxymethylaminomethane solution pH 7.0. Make up to 1000 ml with a 2.5% hydroxyethylcellulose / dextran solution containing 2 mg of methionine, 100 mg of benzalkonium chloride and sodium ethylenediaminetetraacetate. '

Example 4: - '. '

With solution prepared according to application example 1 or 2 of SP-undecapeptide (SP _1-11 ) rats in a volume of 40μ.Ι at a total dose of 125 / u, g / kg substance P applied. For plasma level determination, a specific radioimmunoassay is used. The substance P content in the plasma is determined before and at defined times after peptide delivery. Between the 1st and 3rd minute after the application, an increase in the plasma level compared to the content before the Peptidgabe is observed at 8 to 10 times. At the same time, in comparison with the behavior after intravenous administration, a prolongation of the plasma half-life occurs. The plasma level of substance P thus achieved is sufficient for the achievement of pharmacological effects of the peptide.

Example 5: ·

Preparations of SP _1-11,. SP ₁ ^ bZw. In other effective subsequences or analogs according to Examples 1-3, rats with stress-related sleep disorders are administered substance P peptides once a day for at least four days. The success of the administration of paptides is assessed by normalizing the previously disturbed sleep profile, ie the proportion of the individual sleep stages in the total sleep time and the latencies for the occurrence of the individual sleep phases. ·

Tab. T proportion of sleep stages (in%) in total sleep in rats after chronic stress without and with simultaneous nasal SP administration,

Sleep stages.

I 'll Ill IV V

normal animals 15.8 10.4 23.5 42.0. 8.3

stressed, _v , · 22,6. 23.4 29.7. 21.2 3,1 '

Rats with ', -'

Sleep disorders ' . '

stressed / "'10.0 14.0 26.0 40.9' 9.1

Rats. ".

Nasal SP application. ... '.

nasal) -. , ..-. _(|

Tab.2 Latency periods of the sleep phases (min) before and after 125 / g.g / kg Substance P intranasal in sleep-disordered rats. 'Sleep phases

Il 5.4 II! IV   ν- 12.5 13.1 27.8 55.5 32.0 44.3 ^r 84.7

normal animals, stressed

Rats with -. .

Sleep disorders . ,

stressed rats 4.8 18.5 25.0 54.2; ^:

with additional,. , '' '

nasal SP application (4X125Mg / kg · ..., nasal)

In particular, the values given for stages IV and V can be significantly adjusted to those of experimental animals without insomnia after intranasal administration of substance P-Psptiden. Thus, SP normalizes in nasal application occurring in rats after chronic stress sleep disorders such that a largely normal sleep profile is restored. The same efficacy as with the preparation of the undecapeptide SP _1-11 (Example 1 and 2) is with Beispie! 3 prepared solution of the N-terminal tetrapeptide substance P ₁ ^ ₁ achieved. '-.

Claims (4)

-1- 259413 7 Invention claims: 1. A process for the preparation of stable solutions of undecapeptides and their partial sequences, characterized in that substance P-Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-MetN ^ - their analogs and their partial sequences in Acids dissolves and then neutralized with bases, for substance P and its analogues, an acid is used, which guarantees the pH-stability by buffering and adding adjuvants to the solution after addition of a base.man, the peptide solutions are largely outside the range colloid Solutions holds. 2. The method according to item 1, characterized in that is used as the acid phosphoric acid and thereby after neutralization with a base is a buffer system. 3. The method according to item 1, characterized in that one chooses the concentration of the excipients so that colloidal peptide solutions are largely avoided. 4. The method according to item 1-3, characterized in that preparations of the substance P-undecapeptide SP _1-11 in nasal administration sufficient for the pharmacological effect substance P plasma levels! to reach. Field of application of the invention The invention relates to a process for preparing stable solutions of substance P-series peptides for therapeutic applications. Substance P is an undecapeptide of the following structure: Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-MetNH. ₂ This compound, its analogs and their partial sequences possess a wide range of biological activities. Field of application of the invention is the pharmaceutical industry. Characteristic of the known technical solutions _ It is known that Substance P, its analogs and partial sequences are harnessed in therapy. So z. For example, the treatment of stress-related damage has been described (Oehme, P., K. Hecht, L. Piesche, M. Poppei in: CA Marsan and WC Traczyk (eds.), IBRO-Symp. Series, vol.7: Neuropeptides and Neural Transmission, Raven Press, New York, 1980, pp.73-89 . Hecht, K., P.Oehme, Jakolometseva, JPLjovschina, M.Poppei and MGAirapetjanz,: Acta biol. med. germ. 29 (1980), 141). Γ '''' * '' ..- -. '- "^;' - ^; 'V /' · '.' · ..; Peptides can be administered orally only in exceptional cases, as they are subject to rapid degradation in the gastrointestinal tract. In most cases, parenteral administration is necessary. A nasal administration of peptides is least burdensome for the patient. Thus, it is known that nasal oxytocin, vasopressin and their analogs are advantageously administered (AMA Drug Evaluations, 3rd Edition, Publishing Sciences Group, Inc. Littleton, Mass. USA, 619, 622). Also, the releasing hormone of the luteinizing hormone (LHRH) can be administered by this route (Fink, G. Gennser, P. Liedholm, J. Thorell and J. Mulder, J. Endocrinol 63, 351-360 (1974). .) Since only small volumes can be applied nasally, it is necessary to use relatively concentrated solutions of the peptides. This is to be done in the cases described above without difficulty, since it is easy to dissolve in water substances. In contrast, the undecapeptide substance P has a much lower solubility in water. Even in organic solvents, their solubility is low. Furthermore, in substance P-peptides, the phenomenon of formation of multimer solutions is observed. These are clear solutions in which by self-association of the peptide molecules there are particles with a broad size distribution up to a few hundred nanometers diameter. These initially clear solutions can form clouding and precipitation during prolonged storage by aging processes of the colloid particles, which can not be resolved by heating, again. Difficulties occasionally arise also in the dissolution of lyophilisates of the substance P-peptides, which were stored for a long time. In the attempt to dissolve in aqueous media remains a seemingly insoluble residue, but consists of chemically unaltered peptide. Evidently similar aging processes play a role here as with the multimer solutions. Solutions for nasal application have a number of requirements. So it is required that their pH should be kept neutral. This is possible by the use of suitable buffer solutions, whereby the solubility in the peptide solution is increased. However, high ionic strength promotes the formation of multimers and decreases the solubility for substance P peptides in this medium. The same effect is observed even with the addition of neutral water-soluble polymers, which are usually added to nasal administration forms in order to increase the viscosity of the solutions. It also comes with prolonged storage of the solutions to undesirable turbidity and precipitation. Object of the invention It is the object of the invention to make the nasal application of peptides of the substance P-series possible and to give the pharmaceutical industry a stable neutral solution of these peptides over the years. Explanation of the essence of the invention The invention has for its object to develop a method by means of which the Schwierigketten in the solution of peptides of the substance P series, especially the formation of multimer solutions, the incomplete dissolution of lyophilisates of these peptides and the tendency to Unwanted turbidity and precipitation with prolonged storage of the solution after the addition of polymers, bypass lasseh. The object has been achieved by dissolving substance P-series peptides (the undecapeptide substance P, its analogs and its partial sequences) in an acid and then neutralizing it with a base using an acid for substance P. and its analogues, which guarantees the .pH constancy by buffer effect after addition of a base. The peptide solutions are largely kept outside the range of colloidal solutions. - Preferably, phosphoric acid is used and then adjusted to pH 7 with sodium hydroxide solution. This achieves a good resolution while maintaining a minimum salt concentration. The concentration of peptide active substance, buffer substance and excipients is adjusted so that the area in which peptide multimers are present is largely avoided. With the help of known methods, such as filtration and sterilization, low-germ solutions of the peptides can be produced. The solutions prepared according to the invention are suitable for nasal administration. They remain clear and stable for years without showing signs of peptide precipitation.