DE2265235A1 - REMEDIES - Google Patents

Description

Remedies
(Removed from P 2 228 616)

Patent application 2 228 616 describes the production of two fractions which can be used as medicinal agents by the oxidation of O ^ - and ρ-pinene mixtures. The corresponding process for the preparation of oxidation products from d- and ß-pinene mixtures by oxidation with oxygen at temperatures of 50-90 ° C., subsequent alkali treatment and fractonated distillation under reduced pressure is characterized in that the oxidation is carried out in a manner known per se in the presence a free radical initiator for a period of 30-50 hours. The oxidation product is freed from the acidic constituents by chromatography or alkali washing, the fractional distillation is carried out at 14 Torr and the distillation is carried out at 40-60 ° C or 65-105 ° C wins over distilling fractions (1) or (2).

Fraction (2) has the following properties:

Crystal clear, straw-colored liquid with a camphor-like odor and bitter taste.

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Acidity: 3-4 η * ¹ - 1.47

Composition, based on the main ingredients:

a) Compounds with a carbonyl function (canpholene aldehyde with Traces of Myrtenal and Verbenon)

20-30 %

b) Compounds with an epoxy function * (c £.-pinene epoxide) 10 - 15 %

c) Compounds with an alcohol function (Verbenol, Myrtenol, Pinocarveol) 50 - 60 %

d) unrecognized connections: remainder up to 100 %.

This fraction is a valuable medicine; in particular it has an antibacterial effect; especially one on the airways.

It has now been found that drugs containing these fractions (2) together with an antibiotic have a synergistic effect Show effect.

For the preparation of the fractions (2) a mixture is preferred which has a dL-pinene content of 70-9096.

Compounds which have the ability to deliver free radicals in the solution can be used as initiators. In particular can acetylcyclohexanesulfonyl peroxide, 2,4 dichlorobenzene peroxide, Peroxit = tert-butyl pivalate, lauroyl peroxide, Dioctanoyl peroxide, diacetyl peroxide, A.cetylbenzene] jeroxide, p-chlorobenzene peroxide,

t-butyl peracetate, ^{2,2-azobis-C2,4-f} dimethyi.valeronitril), di-BenzQä-.peroxid, 2,2-bis (t-butylperoxy) butane, di-cumyl peroxide, di-t-butyl peroxide be used.

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Using such an initiator is quite a complete one Oxidation possible, so that the concentration of unreacted pinenes in the end products is negligible or negligible. is in any case far lower than with the previously known methods.

Compressed air is preferably used as the oxygen source, which is introduced directly into the reaction mixture, which is kept under vigorous stirring. After the oxidation, the reaction product freed from the acidic components. For this purpose, chromatography or an alkali wash is carried out.

According to the first embodiment, for example, chromatography is carried out in an Al ₂ O - * column (grade 3), a methanol-benzene mixture (with Λ% methanol) being used as the mobile phase. After evaporation of the solvent, an oily product is obtained, the degree of acidity of which is less than 1, while the degree of acidity of the oxidation products is generally around 21.

According to the second embodiment, the mixture obtained by the oxidation reaction is washed alkaline, preferably with a dilute, aqueous solution of sodium hydroxide. After vigorous shaking, two layers separate from which the aqueous phase is separated off, while the organic phase is trapped with ethyl ether in order to separate off any remaining aqueous phase. Then the organic phase is washed with water, _{dried over anhydrous Na 2} SO ^ and finally the ethyl ether is evaporated.

The purified product obtained in this way is then subjected to a fractional vacuum distillation, distilled at 40-6O ⁰ C or 65-103 ⁰ C at 14 Terr. The first fraction is separated and the second fraction is the fraction described above as used herein.

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It has now been verified through studies carried out in vivo and in vitro found that the above-mentioned fraction together with antibiotics, especially the tetracycline series, valuable, synergistic acting remedies supplies.

By using "labeled" antibiotics it can be detected It will be noted that the combined administration markedly promotes passage of the antibiotics across cell membranes.

The following experiments and examples illustrate the invention.

Attempt;

Production of fraction (2);

. a) In a 5 1 fassendem reaction vessel equipped with a mechanical stirrer, a blowing tube for compressed air, a cooling coil and a ^"1 control thermometer is provided, are 1500 g of a mixture of o (- and ß-pinene (8C # o ^ - Pinene) "ing" - and about 1 % di-benzul peroxide added. The suspension is stirred and heated to a temperature of 75 and 85 ° C while compressed air is introduced. 30-50 hours is continued for.

The oxidation product (around 1200 g) appears air. a thick, oily, orange-yellow liquid with the following characteristics:

£ ¹ »1.4928 d | J = 0.9917

Acidity = 21
Peroxide number = 10

Then 10 g of the oxidation product are subjected to column chromatography (column diameter = 6.4 cm, column height «= 14.2 cm), Al ₂ O being used as a solid adsorbent and mixed with a methanol-benzene mixture (1 % methanol) is eluted. 200 ml of eluent are allowed to flow through the column, which are then separated out, then 3000 ml are allowed to flow through, from which, after evaporation of the solvent, 8 g of yellow oil with an acidity of less than 1 are obtained.

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50 g of this oil are subjected to fractional distillation under a vacuum of 14 mm Hg, whereby the two above-mentioned fractions with the following amounts:

(Fraction 1: 5g) Fraction 2: 15 g.-.

b) Part (a) was repeated instead of cleaning on the An alkali wash was carried out on the chromatographic column:

200 g of the oxidation product are mixed with approx. 150 ml

treated with a 5 # NaOH solution in a separatory funnel.

After vigorous shaking, the two phases can be separated and the aqueous phase is separated off. The organic phase is also trapped in a separating funnel with approx. 300 ml of ethyl ether, any remainder of the aqueous phase is removed, the organic phase is washed with water and _{dried on anhydrous Na 2 SO.} Finally the ethyl ether is allowed to evaporate. The acidity, which was 21 before the treatment, drops to 1 after washing.

With the washing, there is also a reduction in weight of 5-7 % compared to the initial weight. A similar treatment with NaHCO ^ solution causes a proportional reduction in weight of 4-6 % compared to the initial weight.

Examples (test results): Pharmacological-toxicological tests

1. Period of death of mice infected with Diplococcus pneumoniae.

Groups of 10 infected mice were given tetracycline chlorohydrate or treated with a mixture of fraction (2) and ι tetracycline in a ratio of 1:10. The results are shown in Table 1 below.

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Table 1 treatment

dose

Tetracycline

Fraction (2) + 700 mg / kg tetracycline

treated animals that have died
te animals by days

2 3 4-5

control 600 mg / kg 10 10 9 1 Tetracycline
HC1 700 mg / kg 10 O 9 1 Tetracycline
HC1 600 mg / kg 10 O 3 k Fraction (2) + 10 O

# The dose refers to tetracycline

5 0 3

cn cn ro

2. Effectiveness of the new remedy according to the method of the smallest Inhibitory Concentration (MIC).

To illustrate the synergetic, antibacterial effectiveness, the effectiveness of the above-mentioned combined administration on four types of bacteria was investigated in vitro, namely two types of the gram-proof type and two of the gram-free type. For grievous Staphylococcus aureus and Bacillus subtilis, and for grief-free Escherichia coli and Klebsielle pneumoniae. the Effectiveness was assessed in liquid soils by the method of the smallest inhibitory concentration of the antibiotic. The results are shown in Table 2 below.

Table 2

MIC (mcg / ml, mean + experimental error) of the tetracycline alone or in combination with fraction (2) in a ratio of 1:10.

Microorganisms MIC MIC

Tetracycline fraction (2) +

Tetracycline

Staphylococcus aureus N 5 0.22+ 0.04 0.10 + 0.03 *

Bacillus subtilis ATCC 9466 2.05 + 0.26 1.13 + 0.21 *

Escherichia coli 266 0.98 + 0.11 0.67 + 0.09 *

Klebsieila pneumoniae 132 1.60 + 0.06 1.37 + 0.07 *

* The dose refers to tetracycline.

3. Acute toxicity

The DLcQ of fraction (2) for the mouse with ip injection is 443 mcg / kg. When administered in combination with tetracacline, the DLc _{0 of} the tetracycline administered in connection with fraction (2) was found to be smaller than that of the same antibiotic used alone. On the one hand, this result cannot raise any concerns, because the dose intended and tested for human therapy

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is 32 to 48 times smaller than the DLc ₀ of oral administration in the rat; on the other hand, when administered orally, it confirms a greater uptake localized to the intestinal tract and liver. However, if the administration takes place intraperineally, no difference in the toxicity of the tetracycline alone and the tetracycline in combination with fraction (2) can be determined.

4. Chronic toxicity

In the case of chronic treatment carried out on the rat for 180 days, oral doses higher than those recommended for human therapy are not objective in animals Changes have been observed; the weight gain in the treated animals compared to the control animals not changed. In addition, during the autopsy examination of the most important trunk and abdominal organs, neither macroscopic nor microscopic ones could be found Detect changes and the hystological examination showed no pathological disorders; it is therefore it is clear that the increased intake of the tetracycline caused by the new remedy does not cause any harm, too if the dose is more than twice the usual clinical dose.

None of the test animals is involved in the pharmacodynamic tests for acute tolerability - even after intravenous ones Administration of higher doses (up to 300 mg / kg tetracycline in combination with 30 mg / kg fraction (2), which is 30 times the highly recommended for clinical use when administered orally Dose corresponds) - died within 4 hours after administration. Also no changes in arterial pressure as well as the cardiac and respiratory activity could be determined compared to the control test.

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5. Pharmacokinetics

In this study - using labeled tetracycline - the fixation effect of the tetracycline on the Bacterial bodies in combination with the prescribed terpene fraction (2) and their ability to penetrate when attracted of a culture of Bacillus subtilis. In addition, the absorption through the intestines and the distribution examined on the tissues. The results of these tests are summarized in Tables 3, 4 and 5 below.

Table 3

Fixation of the tritiated tetracycline to isolated microorganisms from a bacterial culture of 18 Hours that were subsequently added for 2 hours with tetracycline (as a control) or with fraction (2) + tetracycline (mean + Experimental error) was incubated.

• number of
Determine
gene Tetracycline H ^
ipm / mg of
bacteria
proteins Treatments
Controls Controls
Treatments 6th
6th 2750 + 254
5036 + 359 Table 4

Effect of fraction (2) on the uptake of tetracycline by the intestine (expressed values: for the liver as i.p.m. Per g of the organ, for the intestinal contents as total i.p.m.) (Mean + experimental error).

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number of - 10 - 2265235 ■ Determine
gene Tetracycline H ³ 6th
6th liver total
intestinal contents Controls
Treatments 7083 ±
12433 + 1152 238000 + 9335
1146 120166 + 12270 Table 5

Effect of the terpene fraction (2) on tissue distribution.

The values are expressed as i.p.m./g of the organ (mean + experimental error)

organs number of Controls 16 Treatments 19th Provisions 3177 2046 blood 6th 656 + 1337 733 + 1843 lung 6th 40600 + 1231 54600 + 5761 liver 6th 31900 + 29016 + kidney 6th 236500 + 245650 +

From the above results one can conclude that a decisive Increase in the fixation values of the antibiotic on the bacillus subtilis is present.

It can also be seen that fraction (2) increased The uptake of the tetracycline in the intestinal tract after oral administration causes the antibiotic to be fixed at the same time Am. Lung tissue promotes if the administration is endovinous.

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Clinical trials

A first series of experiments were carried out on 40 people. Half of these were given tetracycline chlorohydrate (250 mg per dose) and the other half with the combination Tetracycline hydrochloride (same dose) and fraction (2) (25 mg) treated.

The following diseases were found to be effective: Chronic, asthmatic bronchitis, chronic, obstructive bronchopneumopathy, chronic bronchopneumopathy that has become acute again, chronic bronchitis which has become active again, acute bronchitis, pleuropneumonia, which has become acute again, asthmatic bronchitis, emphysema, chronic bronchitis.

The capsules with the combination of tetracycline + fraction (2) were well tolerated in almost all cases.

The patients treated with the above combination showed a noticeable decrease or absence of the Explanation regarding the patients treated with tetracycline only. The comparison was made on the basis of the recording of the vitality of the Teffenau index and the "mixing time". It was clearly shown that the presence of fraction (2), even in small doses, had very beneficial and significant effects would have.

A second group of 36 patients in two subgroups of 18 shared each and with the same diseases as that of the in the first group, both in the acute stage with fever and in the subacute and chronic stage with or without fever, was treated with tetracycline chlorohydrate and fraction (2) in the same dose. The following assessment parameters were created:

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- body temperature

- amount of excrement

- Cough

In this test, too, tolerability was excellent. In only two cases are dyspeptic disorders or other Signs of gastric intolerance occurred. It is worth mentioning that the fever is faster in those with the Combination of tetracycline and fraction (2) treated patients goes down. Also the daily amount of excrement experienced a greater reduction in the patients treated with the combination and, in parallel, there was an improvement found in the tests of the respiratory function. Eventually, with the improvement in respiratory function, there was a significantly greater decrease in coughing achieved.

Another experiment was carried out on a group of 31 children suffering from acute febrile forms of inflammation of the Respiratory system suffered. The drug used for this experiment in drop form was in the dose of 20-40 mg tetracycline Administered 3-4 times a day per kg body weight and 2-4 mg torpen fraction (2). The 31 test children were like divided as follows:

1st group: 11 infants aged 6-12 months who are at

suffered acute febrile inflammation in the larynotracheobronchial region;

2nd group: 14 toddlers aged 1-3 years with acute,

febrile forms of inflammation of the bronchial tree;

3rd group: 6 children with laryngo-tracheo-bronchitis.

The following results were achieved:

The tolerability of the drug was excellent in all treated patient groups, except for one case. There are

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no symptoms of gastric intolerance, kidney problems, or changes in blood composition occurred. All Patients healed in a very short time. In the tested patient groups, the drug had a beneficial effect on the Respiratory symptoms from: improvement of dyspnea, cough, bronchial discharge. The body temperature dropped to normal.

Similar experiments have been made in the field of diseases of the genital and urinary apparatus. The clinical trials show that the combination of fraction (2) with antibiotics, especially with tetracycline, thanks to the preferential uptake on the intestinal tract, primarily when administered orally, antibiotic therapy in cases of pelvic inflammation, Cystitis, cystopyelitis, ureteritis, through Hummingbird bacteria and proteins accompanying urethritis and pyonephritis are considerably supported.

Also in this case is the greater effectiveness of the antibiotic due to the vehicle function and cytopermeabilizing action of the terpene fraction, which also has its own Has a bacteriostatic effect.

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Claims (2)

Claims [iy- remedy containing an antibiotic and a distillation fraction obtained by treating a mixture of eC- and ß-pinene with oxygen at temperatures of
50 ~ 90 ° C. oxidized in a manner known per se in the presence of a free radical initiator for 30-50 hours, the oxidation product by chromatography or alkali washing
freed from the other constituents, then distilled and the distilled at 14 Torr at 65-105 ⁰ C. Group
wins . 2. Remedy according to claim 1, characterized in that
the antibiotic is a tetracycline. The patent attorney: 609851/1087