DE2228187A1 - drug - Google Patents

Description

PATENT LAWYERS IJiFl. O. DITTMANN K. L. SCHIFF DR. A. ν. FIVE DIPL. ING. P. STRBHL MUNICH ΘΟ MARIAHILFPLATZ 2 * 8

DA-4889

description
to the patent application

of the Lord

DARWIN JOHNSON PROCKOP
4628 Osage Avenue
Philadelphia, PA 19143
United States

concerning

drug

Priority: June 10, 1971 U.S.A. No. 151,986

The invention relates to pharmaceutical preparations containing certain analogs and homologues of proline or of lysine and contain a pharmaceutically acceptable carrier therefor and their use for regulating collagen synthesis and secretion Collagen across cells in animals, effective amounts of these compounds being administered.

The invention particularly relates to certain compounds which, when administered as medicaments, induce formation of collagen in the collagen-producing cells of animals and control the excretion of these collagens by these cells can, and therefore can be used to define certain Control effects that usually result from the synthesis and excretion of collagen.

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Collagen is a naturally occurring protein in all animals that is synthesized intracellularly from certain amino acids, including the amino acids proline and lysine. Collagen is a necessary naturally occurring substance in animals because it is the tough, fibrous "glue" that holds the tissues of the body together. The collagen-forming cells are mainly found in the skin, bones, lichen, tendons, muscles, cartilage and blood vessels of animals. In certain circumstances, excessive amounts of collagen are produced which can lead to significant undesirable results. So is. For example, excessive scarring in healing wounds caused by trauma or disease or surgery is the result of excessive collagen since the main constituent of scar tissue is collagen. ₍ The formation of extensive ugly masses of scar tissue can cause psychological difficulties in humans. The formation of scar tissue can also significantly disrupt physical functions in humans or animals Tendons of the wrist of the hand often significantly affect the functionality of the hand and can severely affect its use. Horses often experience a condition known as "wild flesh" as a result of a cut or other injury to the legs Flesh "is an excessive formation of scar tissue, and if it occurs near the ankle, it can use the legs for heavy work or running considerably. It is therefore desirable to reduce the amount of collagen produced by the collagen-forming Cells in specific areas of the body is formed and released to be able to control and limit during limited periods of time. Also because of the obvious association with certain organic blood vessel conditions, such as atherosclerosis, which is believed to result from excessive collagen production in the vessel walls, it; t; et; It is desirable to have a way of regulating and limiting the general formation and release of collagen that takes place in the organism by the collagen-forming cells

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can. In regulating the formation and release of collagen it is, however, extremely important that not those-in-them Cell production of other proteins is disturbed, which are necessary for the proper function of the body.

The object of the invention is to find a drug which contains compounds as active ingredients, the analogues of proline or lysine dars te Ilen> and introduced into the collagen-forming cells of animals by the systemic route or locally will be able to find certain "quasi-collagen compounds" to "form. Because the ·" quasi-collagens "are not carried by the collagen forming "cells are excreted, they accumulate in "these cells aii and inhibit after a certain Time for further collagen synthesis by these cells.

The invention thus provides a medicament for controlling collagen synthesis in cells, which. ■. ;

a) as active ingredient L-azetidine-2-carboxylic acid, cis-4 ~ hydroxy-L-proline, -3,4-Dehydro-L-proline, 2,6-diamino-4-hexenoic acid, allo-5-FluoThydroxy-L-lysine, allo-5-hydroxyr-L-lysine or the laevo- or cis isomers of compounds of the general formula

in which R stands for "OH, Cl, E, NH ₂ , CH ,, .OC (O) CH, OC (O) CH ₂ CH, SH, SCH ₅ , OCH ,, ONO ₂ , OSO ₅ H, H ₂ PO ₄ or COOH and

b) contains a pharmaceutical carrier,

Pas means according to the invention, the special analogues of proline or containing lysine in combination with pharmaceutically acceptable carriers can be administered to animals systemically or locally to limit the synthesis and excretion of collagen ^ Zen.

the application of the agent according to the invention can be agreed conditions in the animal and human body that are caused by excessive synthesis and excretion of collagen.

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stalls from coming can be prevented by adding effective amounts of the agent be administered, which the mentioned Proline or Lysia · * --.- analogues contain.

By using the agent according to the invention, the formation of scar tissue on wounds is regulated and limited, which are caused by trauma, including burns, after illnesses, after surgery or by similar other circumstances excessive collagen accumulation, such as atherosclerosis, liver cirrhosis, rheumatoid arthritis or osteoarthritis and scleriasis (scleroderma).

Treatment of these diseases can be local or systemic Introduction of effective amounts of the agents according to the invention take place, the certain proline analogs or certain lysine analogs in combination with a pharmaceutically suitable, chemically inert carriers included. "■ ■ - - -

Suitable proline analogs for the purposes of the invention are below listed connections. Among such proline analogs suitable according to the invention, only the cis isomers are intended (if the compound has cis and trans isomers) but not the trans isomers. For both proline and lysine analogs, the invention applies only to the laevo isomers and not on the 'dextro isomers or not optically active Isomer is directed, with the exception of the case that the DL form of the compound is to be used because the L form cannot be economically isolated. The invention does not include the excluded isomers of those indicated Proline and lysone analogs, insofar as these excluded isomers are not used for the synthesis of "quasi-collagen" molecules by the Collagen-producing cells lead. Proline analogs suitable for the purposes of the invention include the cis and laevo isomers of compounds of the general formula

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in which R is OH, Cl, S ¹ , CH ₅ , NH ₂ , OC (O) CH ₅ , OC (OH) CH ₂ CH ₅ , SH, SCH ₃ , OCH ₃ , ONO ₂ , OSO H, H ₂ PO ₄ or COOH. Other proline analogs encompassed by the invention are L-pipecolic acid _f 1,2,3, 6-tetrahydro-L-picolinic acid e, 1,2,3,4 - ^ - tetrahydro-L-picolinic acid, 1,4 , 5-6-tetrahydro-L-picolinic acid, 1,2,5,6-tetrahydro-L-picolinic acid and 1,2-dihydro-L-.

"Picolinic acid. Further proline analogs suitable according to the invention are the laevo isomers of compounds, the general formula

.meln

Ϊ00Η
I.
Ji

wherein X is N, S or 0. The proline analogs also present in the agents according to the invention are L-azetidine-2-carboxylic acid, 3, ^ - dehydro-L-proline and 4,5-dehydro-L-proline.

4-

Suitable lysine analogs for the purposes of the invention are laevo isoins of compounds of the general structural formula

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E _o -C ° -SH,

H - C ⁵ - H

H - C ^ - NH ₀
C ¹ OOH

in which R ^, Ep, R _5> R /, »Rc and Rg any of the following radicals

mean: P ₁ Cl, OH, CH, NH ₃ , ONO ₂ , OSO-.H, OC (O) CH ₅ , SH, SCH ₇ ,

OCH ₃ , COOH, H ₂ PO ₄ or OC (O) CH ₂ CH ₅ . Other lysine analogs according to the invention are 2,6-diamino-5-hexenoic acid, 2,6-diamino-4-hexenoic acid ₍ and 2,6-diamino-3-hexenoic acid.

The lysine analogs preferred as a constituent of the agents according to the invention are 2,6 ~ diamino-4 ~ hexenoic acid, allo-5-fluorohydroxy-L ~ lysine and allo-5-hydroxy-L-lysine.

The expression "quasi-collagen" used in this description means a collagen-like protein molecule that is present in one or more of the places normally found in collagen Proline or hydroxyproline would be present, or on which lysine or hydroxylysine would be, on a case-by-case basis Contains a proline analog or a lysine analog. *

For the purposes of this description, what has been said in general for "animals" should also apply to humans.

The proline analogs preferred in the agents according to the invention are L-azetidine-2-urboxylic acid, cis-4-hydroxy-L-proline, 3,4-dehydro-L-proline, cis-4 ~ chlorine - L-proline and cis-4 ~ fluoro-L-proline. Those for use in the agents according to the invention most preferred compounds are cis - ^ - hydroxy-L-proline, L-azetidine-2-carboxylic acids and ^

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It should be mentioned - that in some cases it may be necessary can use the DL forms of the analogs when the pure L-form is not accessible by itself, even if the L-isomers of the proline analogs and lysine analogs listed are the only ones "are optical isomers suitable for the purposes of the invention.

-AIs components of the agents according to the invention are particularly valuable Lysine analogs are 2,6-diamino-4-hexenoic acid, - allO-5- * fluorohydroxy-L-lysine and allo-5-hydroxy-L-lysine.

The bWörzugte compound as part of the erfindungsgemäs-: sen agent i t-Gis - ^ - hydroxy-L-prolin.- This compound is - _. Commercially available in limited quantities, occurs in dereliction and is - probably from the leaves and the pericarp

, a species of the sandwood tree, Santalum album, native to India common, extractable. The connection can be found in these leaves in their natural form and can be extract according to procedures outlined in Biochemical Journal, -80,

"(1961), and Biochemical Journal 58, 57- (195 ^ Oi are described" C-is-4-fluoro-L-proline can be synthesized by the methods which are described in Biochemistry, 4, 25o7 (1965), L-azetidine-2-carboxylic acid can from the sheets of vojn. Lilies of the valley are extracted according to procedures outlined in Biochemical Journal, · ■ 64, 323 (1956). 3 »4- ~ dehydro-L-proline can according to the in-J, AcC. S., 84, 1697 (1962) .synthesized. The synthesis of 2,6-diamino-4-hexcaic acid can be done according to the procedures -the -in Chemical Reports 93, 2282 (1960).

It is believed that the mode of action of the invention Can be described by means as follows. When introducing an agent according to the invention, which is a suitable • Proline analog or lysine analog contains, in an animal, the takes place either locally or systemically, the administered analog is inter alia in the collagen-producing cells of the Animal either locally or systemically. During the collagen synthesis in the cells concerned, depending on the case, the proline analog or lysine analog administered

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incorporated into some of the collagen molecules that are synthesized in the cell, forming "quasi-fo>llage" molecules. Of the. one thousand amino acid molecules into a single normal collagen molecule be incorporated are occupied about 23o of the amino acid sites of proline or hydroxyproline, and about to Jo ^ o Ask siiid by lysine or hydroxylysine occupied.

When using the agent according to the invention, if a proline analog is administered, "quasi-collagen" - -Molecules. synthesized at certain * points where proline or hydroxyproline would be present in a normal collagen molecule containing the analog. When in use of the agent according to the invention administered a lysine analog so will be in the collagen-producing cells that produce the lysine analog contain "quasi-collagen" molecules from the cells synthesized · made in certain places where in a normal Collagen molecule would be lysine or hydroxylysine contain the analogue. The number of sites at which the proline or lysine analog is bound in this way, varies from a few places in each molecule to more than half of the available positions for proline or hydroxyproline or lysine or hydroxylysine, as the case may be. These "quasi-collagen" molecules inhibit that Formation and excretion of plollagen in the collagen-producing cells, starting about four or more hours after the Agents containing the proline analog or lysine analog were administered. The duration of this inhibition of collagen formation and excretion depends on factors such as the particular analog administered, the amount administered, whether the administration is local or systemic, and the frequency with which the dosing is repeated. '

The inhibition of the formation and excretion due to the synthesis of the "quasi-collagens" probably arises from the following reasons given. The transport system for excreting collagen from the collagen-producing cells is apparent

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.Particularly trained, and some of the proline residues must be in . flydroxyproline, some of the lysine residues need to be in hydroxylysine . must be transferred, and some of the hydroxylysine residues must

be substituted with a sugar before excretion every collagen molecule from the collagen-producing cell can take place ~. The "quasi-collagen" molecules are different sufficient of normal collagen molecules so that they are not excreted from the collagen-producing cells. These "Quasi-collagen" molecules therefore accumulate in the cells to the extent that they are formed. After the "quasi-collagen" molecules during e-twa. two to four hours at- * have enriched, the concentration of these molecules within the cells has apparently reached a critical value, so that a noticeable diminution - the rate of another synthesis ■ is caused by ~ collagen. Because the "quasi-collagen" molecules _ accumulate only in cells that are highly specialized in To form collagens, the agents according to the invention cannot substantially synthesize proteins other than collagen by not interfering with collagen-producing cells, though · those here listed analogues may to some extent not Collagen-producing cells are introduced.

It is quite important to establish that it is a temporal one Delay of about two hours to about four hours between the time at which the agents according to the invention administered, and the time occurs at which a substantial decrease in the deposition of collagen by the affected cells occurs. The exact value of this time delay will vary depending on factors such as the particular proline or lysine analog administered, the amount, the analog administered and whether, for example the administration is local or systemic.

It is therefore believed that the mechanism of action of the invention Means is as follows. By administering an agent containing any of the aforementioned Proliri Containing analogs or lysine analogs, the analog becomes intracellular locally or systemically introduced into the collagen-forming zones and replaced in the

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some of the proline or hydroxyproline (in the case of proline -: Analogs) or some of the lysine or hydroxy lysine units (in the case of lysine analogs) that would otherwise turn into a normal collagen molecule were installed. This replacement occurs with some, however not necessarily on all collagen molecules that are "synthesized by the cell. As well as these" quasi-collagen "molecules accumulates within a cell, this disrupts the excretion of collagen, and the gene molecules accumulate within the cell, which causes possibly the further collagen synthesis and excretion by the cell, depending on the "dose used," the method of administration and the like, inhibited "or pre- ' is terminated temporarily. In this way, the means according to the invention can control production and excretion of collagen caused by collagen-producing cells in animals.

The mechanism of the formation of "quasi-collagen" when in use the means according to the invention takes place, "serves" in addition, during a certain period of time the synthesis and excretion of collagen by individual collagen-forming agents Substantially degrading cells, however, does not affect the production of other proteins by the same cells or by non-collagen producing cells. This is very important for the effective function of the agent according to the invention to limit or control collagen formation because in the usual situation means that the synthesis of protein by interfering with one type of animal cell, affecting the synthesis of all proteins in the animal and in this way produce a fourth range of systemic effects, many of which can be harmful.

As will be explained in more detail below, those of the present invention have Agent Efficacy to inhibit collage formation in laboratory animals and are therefore considered useful considered to regulate the formation of scar tissue in wounds, those caused by trauma or illness or by surgical interventions! in animals, while the growth of Epilhel-

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tissue is made possible. "It is noteworthy that in the normal Healing of wounds or cuts once a thin layer of new epithelial tissue is exposed over the exposed surface. laid tissue is grown i-st, the subsequent formation "of Scar tissue from the mesenchyme cells is extremely little, if it takes place at all. When using the Agents according to the invention for preventing the formation of scar tissue can contain a limited amount of the agent according to the invention By means of being applied locally for a limited period of time, until a noticeable formation of epithelial tissue has occurred, after which no further application of the inventive

is required. There. the formation of scar tissue during Wounds caused by trauma, illness or surgery "should not be borrowed before the end of a v / es Period after the occurrence of the wound or

When the incision begins (about five days in humans), the administration of the agents according to the invention must only begin at the stage of the healing process in which the formation of scar tissue is just occurring. It is also important to note that the agent of the present invention, when used to prevent scarring, does not necessarily stop the excretion of collagen by the collagen-producing cells to which the agents of the present invention are administered. Rather, by precise control the dose amount and the. . Dosisbäufigkeit be ensured that these cells continue to synthesize and excretion of a limited amount of collagen, which is important -for the normal Punktion- the weave, ^x exerted by the collagen. This possibility of using the agent according to the invention for regulating scarring is of particular importance in certain human races whose members show a genetic tendency towards keloid formation on wounds or incisions as a result of excessive collagen accumulation.

The means according to the invention are also used for other purposes in addition to regulating the formation of larval tissue following injuries or surgical interventions. A particularly important application of the

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inventive means is the prevention of excessive collagen deposits in pathological conditions ^ —in — not in them Collagen-containing tissues can be replaced by collagen. So For example, in cirrhosis of the liver, hardening of the liver occurs as a result of the .- ^ B

through collagen. The triggering damage to the liver can be caused by certain viruses, excessive alcohol consumption or certain Toxine. If only moderate liver damage -nol-e — liver cells replace the damaged tissue. WenET-JSttoclr a swine * © * - damage _

; .- has occurred, the damaged tissue is not caused by

in this PcdriHpleibt in which -Patients functional collagen replaced before DLE normal liver cells can be regenerated, a ^ nftii-rftir.h · pr> f nnkt; i on wiriifv Tf, fjjp

.-often leads to death. These states caused by excessive Collagen deposits can be caused by systemic or local introduction of sufficient quantities of the Prottn ^ nalOga or containing lysine analogs Agents are treated, whereby the amount of collagen that is supplied to the diseased organs is significantly reduced will. Also, in some cases, acute liver damage, the by Toocine, like CCl ^, caused v / ird, kasi ^^ i ^ e-V-e3? abr & i— - ■ - ■ the preparation of the means according to the invention for a limited period of time

Suppress excessive collagen formation in the liver

possible that the damaged liver cells are replaced by regenerating healthy liver cells.

Another example of the excessive collagen deposition in certain pathological conditions against which the inventive Means applicable is pulmonary fibrosis, which is caused by inhalation of foreign materials, such as silica or - asbestos, or which occurs in the elderly for unknown reasons. Under the circumstances, it could an incipient pulmonary fibrosis by administration of the e.r invention, Containing proline analogs or lysine analogs Means are treated to prevent further port stepping.

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The medicaments according to the invention are also used for Treatment of specific blood vessel cranes

the

rose of unknown etiology or / specificη type-of-atherosclerosis (Atherosclerosis) that occurs in the small blood vessels of diabetics occurs, whereby the walls of the bloom g ^ fä "ßl" thicken. It is believed that the thickening of the blood vessel walls is caused by excessive collagen build-up, and "this condition could also be achieved by administration" of the invention Means to be affected. .. ·. ....

Examples are given below that illustrate the application, the Means according to the invention for spreading the Sy ^ tKes-e-round separation of collagen in animals, as well as making a number more representative of the agent according to the invention

Example 1 ■ - "-

Chicken embryos grow rapidly between the ages of 8 days and 12 days, and during this period they tend to have a pronounced increase in the collagen content. The effects _r of the proline analog L-azetidine-2-carboxylic acid test for the collagen ■ synthesis, 8-day-old chickens were embryo's day with the analogue treated ,, and their Gewebjr ^ would by:.: JU_ & dey 5 days examined. .- - - ■■ "'-" -

L-Azetidiii-2-carboxylic acid was dissolved in distilled water, and o, 2 ml of an aqueous solution, either 375 or 5oo Micrograms contained * on the air sacs of the chicken embryos injected daily from the eighth day to the twelfth day. Control embryos received injections in the same manner with 0.2 ml of distilled water daily. About 90% of those with 500 micrograms. treated per day for five days Embryos survived. About a fifth of the surviving embryos showed small bone deformities or small ones Petechiae, but the remaining embryos treated with this dose appeared normal with the exception of that

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- 14 - 2228Ί87

their tissues showed lower strength. Embryos treated at 375 micrograms per day for five days showed none noticeable mortality and indistinguishable from control embryos except that their tissues were a showed somewhat lower strength. ·

Numerous tissues from the embryos were examined by optical microscopy examined. For embryos with 500 micrograms per Day after treatment, there was no evidence of non-specific toxicity in liver tissue, kidney tissue or others Tissues found when sections of these tissues were stained with hematoxylin and eosin. When dyeing jaiiL _Gomori 's Beticulum dye, however, became a marked reduction indicated by reticulum in the liver, and staining by the Gomori one-step trichrome method showed pronounced Reduction of collagen fibers in periosteum, perichondrium -. and most other connective tissues, including the examination from. JBaSt and tendons by 'electron microscopy after staining this tissue with phosphotungstic uranyl acetate indicated that in embryos treated with 500 micrograms per day essentially no striated KoI - .. lagenfibrilLen ^ Xorlagen, although in control embryos these tissues were filled with striated collagen fibrils.

Treatment with the analog, especially at a dose of 500 micrograms, had a marked effect on total wet weight and dry weight of the embryos:

Table 1

Effect of L-azetidine-2-carboxylic acid on the growth of

Age of dose of analog wet weight, ^{a /} dry weight, ^{a /}

Embryos, micrograms / day grams milligrams

Days _-

8 o, 95 + o, o5 66 + 7

1o ',' 2, o3 + o, 14 -

1o 375 1.60 + o, 29

Ίο 5oo - 1.43 + o, 26

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Age of Dose of the Analogous, Wet Weight, Dry Weight ^

Embryos, micrograms / day. Gram .milligram

Days.

12 _Ί 3.88 + ο, 21 3oo + 62

12 375 3, o1 + o, 21 2oo + 23

12th 5oo '2.75 ± o, 2o 181 +

a) The values give the mean and standard error of the mean for five embryos. The wet weights were measured after the embryos were removed from the eggs and briefly dabbed with tissue paper. the Dry weights were measured after placing the embryos in a desiceator under vacuum with Drierite for one week been held waz'en.

The wet weights of the treated embryos were 21 to 29% less than that of the comparison embryos, and there was an even greater effect on the dry weights of the Embryos. However, both the treated and control embryos showed a marked increase in wet weight than also of the dry weight during the five-day period.

To determine whether the analog had a more pronounced effect on collagen than on other proteins, the collagen content and the content of the "ff-collagen" proteins of the embryos were examined. (The experimental method is given in Table 2.) The results indicated that after three days of treatment (day 10) the non-collagen protein content of the embryos was only 3 to 1p% less than that of the control embryos, and that after After five days of treatment (day 12) the non-collagen protein content was 23 to 28% less than that of the control embryos. The treatment therefore had a far greater effect on the collagen content than on the non-collagen protein content. On the day 1o of collagen content was 39-5> 8 ° / ° less than the control values, and on day 12, he was 61 to 72% less · than the control values -

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The effect was evident even when it was pure fortification of non-collagen proteins and collagen was measured.

Table 2
Effect of L-Ac ^ tidine-2-carboxylic acid on pure enrichment

Dose of Reine Anrei- Reine Anrei- Reine An-L-.A zetidin-2- ■ assurance of enrichment enrichment carboxylic acid day non-collagen collagen -'of not- of colla-micrograms / gene-protein ' Micromoles of collagen gen Tag _ micromoles of amino acid protein % of

Amino acid% of the control

trolls

without 1o 375 1o 5oo ^; 1o without 12th 375 12th 5oo 12th

143

133 111

662 472 426

16.4

7.14 2.56

88.7 3o _L 6 2o, 1

93
78

64

44 16

35 23

a) The values were calculated by subtracting the initial content on day 8 from the level on day 10 or day 12. The embryos were homogenized in distilled water, and the homogenates were dialyzed to remove free amino acids and peptides. Then they were in 6 N HCL at 116 G hydrolyzed for 24 hours. The hydrolysates were evaporated and with the help of a ninhydrin method for their total amino acid content and checked for hydroxyproline using a specific colorimetric method. The collagen content was calculated based on the fact that Kolla-. gen "contains 93 residues of hydroxyproline per 1,000 amino acid residues. The content of non-collagen proteins was calculated as the total content of non-dialysable amino acids minus the content of non-dialysable amino acids that were taken into account for collagen. The values give the mean

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value of two collective samples, each containing 6 embryos. -.

The results indicate that the proline analog specifically inhibits the synthesis and accumulation of collagen in the growing chick embryos. The specificity of the effect was most pronounced in the embryos treated with 375 micrograms per day for 3 days (day 10) * These embryos (see Table 2) enriched 133 micromoles of non-collagen protein (93 ° / ° of the comparison values); However, they had synthesized less than half the amount of egg lagen that had been synthesized in the control embryos (.44% of the comparison values). Treatment with larger doses for a longer duration caused some inhibition of the. Synthesis of non-collagen proteins; the effect on the collagen synthesis, however, was always at least twice as great as the effect on other proteins.

The effect of the proline analogue ci-s-4-hydroxy-L-proline in Rats were examined by the growth of collagen granulomas in rats "with carrageenan, an extract of seaweed, - ■ was induced.

Male Sprague-Dawley rats were placed on a diet containing all of the nutrients necessary for normal growth but which was free of proline. After the rats were kept on this diet for three days, they became 5 nil of 4% carrageenan in sterile physiological saline solution injected subcutaneously (test day 1). Received from day 5 to day 13 the treated rats daily 2 subcutaneous injections of cis-4-hydroxy-L-proline in 1.0 ml of 0.05m Tris-HCl buffer, pH 7 »4, and control rats received 1 ml of the same buffer, which contained 0.1m sodium chloride. On day 14- the granulomas were removed, weighed and checked for collagen content

analyzed. A series of experiments was carried out on young adults

Rats carried out at the beginning of the test each about 15o g weighed, and a similar set of experiments was carried out on adults

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Rats, each weighing about 300 g at the beginning of the test.

The collagen content was determined by hydrolyzing the tissue in 6 η HCl for 24 hours at 116 ⁰ C and then. after the hydroxyproline content was analyzed by a chemical method specific for trans-hydroxyproline. The collagen content of the tumors has been determined due to the fact

/ Von - calculates that normal collagen is about 93 residues / trans-hydroxyproline contains per 1,000 residues.

T a b e 1 1 e 3

Effect of cis-hydroxyproline on the development of C ar rage e na n-

Dose of the wet weight ^ of the collagen content Analogs granuloma a) of granuloma a)

mg / kg / day grams

none 4.21 + o, 2o 64.5 + 1.91. I00 2.69 + 0.39 32.3 + 4.3o

2oo 1,8o + o, "26 26,2 + 3, o3

a) Mean and standard error for the mean of 5 or 6 granulomas. ■

Corresponding effects on the weight or the collagen content of the granulomas were observed in experiments performed with adults Rats under the same conditions were performed.

The final weight of the treated rats in all experiments was 10 to 15% less than that of the control animals; Control experiments in which food intake was limited, however, showed that limiting the growth of rats by 10 % had no noticeable effect on the total weight or the collagen content of the granulomas.

The results therefore showed that the synthesis of collagen in Carrageenan granulomas in rats can be specifically inhibited by administration of cis-4-hydroxy-L-proline.

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Example_3

The effect of various proline analogs and one lysine analog the collagen synthesis was investigated by adding mixtures, which the analogs together with embryonic cartilage oil -, - the Collagen is synthesized at a rapid rate, held up to four hours were incubated in vitro · As the inhibition of collagen synthesis by the analogs is delayed The effect is that cannot be precisely demonstrated within four hours, the cartilage system could not be examined the inhibition of synthesis can be used. However, it is a convenient system. To determine "whether amino acid analogues are incorporated into collagen and whether the incorporation of the analogue into the collagen is its subsequent — excretion from the cells into the extracellular matrix.

Cartilaginous tibiae from 10 to 11 day old chick embryos were placed in 2.5 ml Krebs II A medium containing inorganic salts, glucose and phosphate buffer. Samples were incubated in a water bath at 37 ° G for the specified duration.

Twenty tibiae were tested at 25 to 50 micrograms per ml (5 to

Ί U-

1o microcuria) C-labeled preparations of L-azetidine-2-carboxylic acid, 3,4-dehydro-L-proline or cis-4-hydroxy-L-proline incubated for two hours at 37 ° G. The tissues were homogenized and the homogenates became exhaustive dialyzed. The dialyzed homogenates were hydrolyzed at 116 G in 6 η HCl or in 2 M NaOH at 10 8 ° G for 18 hours, and the hydrolyzates were chromatographed on the long column of a Spinco Model 116 amino acid analyzer.

14th

Determinations of \ G in the eluates from the column indicated that the collagen and other proteins made by the tissues

14 synthesized in vitro, the corresponding C-labeled Proline analog included.

When the tibiae were incubated with the Lyson analogue, 4-, 5-dehydro-L-lysine (2,6-diamino- ^/ 4-hexenoic acid), quantitative amounts of the analogue were determined by amino acid analysis of the

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Preserve proteins in the tissues. With 2.5 millimolar dehydro-L-lysine, up to 80 % of the lysine residues in newly synthesized proteins were replaced by the analogue.

It has been shown that the collagen ^. that-any of the indicated four analogues contains, in terms of molecular weight '(determined by gel filtration), the tendency to the formation of insoluble aggregates and the ability to be broken down by bacterial collagenase - the same normal collagen that d-u-rch das — tissue synthesizes will. . - ~

In further experiments, the cartilage material was treated for two to four hours with 1o to 5o microcuria <H-3,4 ~ proline and with 10o "to 3 ° o micrograins per ml of one of the analogues of proline given in Example 3 or of 4.5- Dehydro-L-lysine was incubated. Autoradiographii were then made of the cartilage. In comparison samples it was found that "a broad fraction of the% was distributed in the matrix. Since <? O to 60 % of the radioactive proline incorporated into the tissue was ± in the collagen, the results indicated that the ^ H-3,4-proline was incorporated into the collagen and that the largest Part of the ^ H-collagen has been excreted into the extracellular matrix. In the samples that had been incubated with one of the analogs, most of the .H-profile was localized in the cells and only a small proportion of the radioactivity was distributed in the extracellular matrix. Comparative experiments indicated, when the synthesis of normal ^ H collagen was first allowed in the tissue, that the addition of the analogs did not interfere with normal ^ li collagen excretion. The same results were obtained with another proline analog, 'cis-4-fluoro-L-proline. When the cartilage material was incubated with 50 microcurie * Έ-3,4 - oil in and 2oo ivlicrograms per ml of ice-4-fluoro-L-proline, autoradiographs indicated that most of the% collagen was not excreted was. As not a radioactively labeled preparation

* through the cells

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was accessible from cis-4-fluoro-L-proline, it was not possible to show directly that this proline analog is incorporated into collagen became. However, trans - ^ - fluoro-L-proline had no effect on the excretion of collagen, and it therefore seemed possible that cis- ^ h-fluoro-L-proline was the excretion inhibited by incorporating it into "the" quasi-collagen "molecule became. ·. .

Attempts in which the cartilage material with other radioactive Amino acids incubated as proline or lysine indicated that the analogs increased the rate of collagen synthesis within of the four-hour period required for this

- "Search could be used in vitro, not significantly

diminished. The observation that the collagen containing the analog was not excreted to any significant extent in four hours therefore indicates that there was a strong intracellular accumulation of the "Q, uasi ~ collagen" molecules.

The results of the preceding examples show that cells synthesizing collagen have certain proline and lysine analogs incorporate into collagen. The collagen containing any of the aforementioned analogs is that synthesized by the cells normal collagen same; however, it cannot be excreted from the cells at the normal rate and accumulates within the cells.

Suitable pharmaceutical preparations for controlling the synthesis and the release of collagen in animals become beneficial and convenient by combining the indicated pro-lin analogs or lysine analogs with pharmaceutically suitable, chemically inert fillers, carriers, extenders and Excipients, such as those commonly used in the manufacture of pharmaceuticals substances that are administered orally or parenterally, or that are to be injected locally, and that are in in this description and the claims are generally and collectively referred to as "pharmaceutical carriers", manufactured. The compounds according to the invention can be in the form

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of tablets, powders, capsules, suspensions, solutions, emulsions and similar dosage forms or compounded be formulated. The preparations can be mixed by ^^ h the specific proline or lysine analogs, preferably in water-soluble form, with these common diluents or tablet additives, such as cellulose powder, corn starch, Lactose, talc, stearic acid, magnesium stearate, gums or the like, can be made by conventional manufacturing methods known in the art. - -

If the product is to be administered parenterally — or locally injected — the agents according to the invention can preferably be combined in their non-toxic, water-soluble form with carriers, such as water, saline, glue solution or the like. . _v .. ~ "

Effective amounts of any of the compounds used in the present invention can be administered to the body of a living animal by any of several methods, for example orally, such as in capsules or tablets, parenterally in the form of sterile solutions or suspensions, intravenously in sterile solution, or locally in the form of sterile solutions or suspensions.

Suitable dosage units of the agent according to the invention for Parenteral or topical administration is in the range of about one-half milligrams to about 500 milligrams of the proline analog or lysine analog per dose unit per kilogram of body weight.

Zv / ar are small amounts of the active substances according to the invention effective when limited, local control of collagen formation and deposition is to be carried out, or if it tells patients with relatively low body weight> - "~ be enough; however, usually the unit dosages are 5 milligrams or more and preferably 100 to 500 Milligrams per kilogram or even more, which of course depends on the type of situation and the specific

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desired result depends. The exact individual doses as well as the daily doses in a special case become natural determined by the management of a doctor or veterinarian according to established medical principles.

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Claims (2)

Pat enfra η sayings Medicines that affect collagen synthesis by cells, marked by the fact that it a) L-azetidine-2-carboxylic acid, cis-4-hydroxy-L-proline, 3> 4 ~ Dehydro-L-proline, 2,6-diamino-4-hexenoic acid, allo-5-fluorohydroxy-L-lysine, allo-5-hydroxy-L-lysine and / or laevo- and / or cis-isoratures of compounds of the general Formula ^ • N ^ \ GOOH II
in der'R for OH, Cl, F, HH ₂ , CH ₃ , OG (O) CH ₇ , OC (O) C1I _? GH ₇₅ SH, SGHv, OGH ₇ , ONO ₂ , OSO ₇ H, H _? PO _{/ +} or GOOH and b) contains a pharmaceutical carrier. 2. Means according to claim 1, characterized in that that it contains cis-4-LIydj.'oxy-L-proline as active ingredient. 20985 1/1263