CA2099427A1 - Indications for carnitine - Google Patents
Indications for carnitineInfo
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- CA2099427A1 CA2099427A1 CA 2099427 CA2099427A CA2099427A1 CA 2099427 A1 CA2099427 A1 CA 2099427A1 CA 2099427 CA2099427 CA 2099427 CA 2099427 A CA2099427 A CA 2099427A CA 2099427 A1 CA2099427 A1 CA 2099427A1
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- carnitine
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Abstract
Abstract of the Disclosure Novel therapeutic effects of carnitine are described:
1. An enhancing effect in relation to human phagocytes, more par-ticularly granulocytes and furthermore an opsonin-like effect, on the bacteria employed as target cells.
2. A strong and specific activation of stimulation, induced by Phythemagglutinin, of human T-lymphocytes, an effect which was observed less specifically in the case of human B-cells.
3. A strong membrane activating effect on human NK cells and the corresponding target cells.
1. An enhancing effect in relation to human phagocytes, more par-ticularly granulocytes and furthermore an opsonin-like effect, on the bacteria employed as target cells.
2. A strong and specific activation of stimulation, induced by Phythemagglutinin, of human T-lymphocytes, an effect which was observed less specifically in the case of human B-cells.
3. A strong membrane activating effect on human NK cells and the corresponding target cells.
Description
.
New Indications for Carnitine : :
The invention relates to novel applications for the inherently known pharmaceutical active substance carnitine.
~ ~ Carnitine is a normal and important constituent of the mammalian musculature, from which it was ~irst isolated and studi0d~from 1905 to 1907 by R. Krimberg and coworkers. It is a question of an amino acid deriva-tive,~a so-called betaine, ~-oxy-gamma-butyrobetaine, which also occurs in squids, in some shellfish and in the sardine. When dogs are fed with meat extract, L-carnitine appear~ in the urine. L-carnitine is commercially avai~lable,~ for instance in the form of the preparation Biocarn ~ of the Medice Company (Iserlohn).
L-carnitine is employed for the oxidative produciion of energy from :, fatty acids, which for their part are an important source of energy for athletes trained for endurance. This energy requirement, for example at the end of a~marathon run, may be met for up to 80 X by combustion of fats. i~
Such an~athlete~hence needs L-carnitine.~
A human being is a carnivore, that is to say a meat eater. In this manner he~may cover his need for L-carni~tine from normal nutrition. A
20 human is~howèver~also able to form L-carnit1né himself, a task~able to be ~;~
performed by different organs:~such L-carnitine may~be termed endogenous carn1tlne in~contradistinction to exogenous~L-carniti~ne supplied~in normal i~ nutrition.~Furthermore there~is L-carnitine supplied in the form of prepa-rations and not contained in~normal food.
~ The substitution of~minera1s~wi~11 serve~to make~clear a classical example for the replacement of lost e~lements during sporting endurance activities. Carbohydrates are listed after minerals in this connection.
As regards the necessity for a supply of protein or amino acids hardly anything was known until a few years ago when the s;ignificance of L-carnit-ine was dlscovered (26). (The numbers in brackets refer to the list of ., : .:. :. .:: . ,: ,, .,: . ." ",: , . . .
; '2 literature references herein).
L-carnitine is an essential co-factor in mitochondrial transport and in the oxidation oF long~chained fatty acids, more particularly in skeletal and cardiac muscle. The human organism synthesizes this amino acid deriva-tive itself or assimilates it from meat in food by an active and a passivetransport mechanism: endogenous and exogenous carnitine. Both serve to meet the needs of the organism, the latter more particularly in the case physical endurance exercise. In our view it is possible to distinguish between different L-carnitine deficiency conditions, it being clear that the therapeutic administration of L-carnitine constitutes a true substitu-tion of an essential biocarrier which primes metabolism and which can pre-vent damage to health. These carnitine deficiency conditions are listed in scheme1. From this there may be seen to be various different therapeutic aspects.
15 scheme l-Different forms of L-carnitine deficiencY Phenomena~ which necessitate a theraoy with L-carnitine substitution.
;~ I. Primary L-carnitine deficiency:
Hereditary, primary carnitine deficiency syndrome.
20 II. Secondary L-carnitine deficiency:
1) Endurance and high performance sport (26) 2) Diabetes mellitus and dialysis patients 3) AIDS patients (11) 4) Deficient uptake of L-carnitine (high performance vege-tarian athletes) or inhibition of transport to organs.
Furthermore the therapeutic application of L-carnitine may be em-ployad for some further purposes as compiled in scheme 2.
Scheme 2 .
Additive-suPportive substitution with a theraceutic effect.
1) For cardiac insufficiency (15) 2) In connection with weight reduction (1) 3) In the treatment of arteriosclerosis (lowering cholesterol level, increasing HDL, reduction in hypertriglyçedridemia in the case of dyslipidemias) (4; 17) 4) In Alzheimer's disease (8) 5) For the improvement of performance in the case of disturbances in peripheral circulation (9)~
New Indications for Carnitine : :
The invention relates to novel applications for the inherently known pharmaceutical active substance carnitine.
~ ~ Carnitine is a normal and important constituent of the mammalian musculature, from which it was ~irst isolated and studi0d~from 1905 to 1907 by R. Krimberg and coworkers. It is a question of an amino acid deriva-tive,~a so-called betaine, ~-oxy-gamma-butyrobetaine, which also occurs in squids, in some shellfish and in the sardine. When dogs are fed with meat extract, L-carnitine appear~ in the urine. L-carnitine is commercially avai~lable,~ for instance in the form of the preparation Biocarn ~ of the Medice Company (Iserlohn).
L-carnitine is employed for the oxidative produciion of energy from :, fatty acids, which for their part are an important source of energy for athletes trained for endurance. This energy requirement, for example at the end of a~marathon run, may be met for up to 80 X by combustion of fats. i~
Such an~athlete~hence needs L-carnitine.~
A human being is a carnivore, that is to say a meat eater. In this manner he~may cover his need for L-carni~tine from normal nutrition. A
20 human is~howèver~also able to form L-carnit1né himself, a task~able to be ~;~
performed by different organs:~such L-carnitine may~be termed endogenous carn1tlne in~contradistinction to exogenous~L-carniti~ne supplied~in normal i~ nutrition.~Furthermore there~is L-carnitine supplied in the form of prepa-rations and not contained in~normal food.
~ The substitution of~minera1s~wi~11 serve~to make~clear a classical example for the replacement of lost e~lements during sporting endurance activities. Carbohydrates are listed after minerals in this connection.
As regards the necessity for a supply of protein or amino acids hardly anything was known until a few years ago when the s;ignificance of L-carnit-ine was dlscovered (26). (The numbers in brackets refer to the list of ., : .:. :. .:: . ,: ,, .,: . ." ",: , . . .
; '2 literature references herein).
L-carnitine is an essential co-factor in mitochondrial transport and in the oxidation oF long~chained fatty acids, more particularly in skeletal and cardiac muscle. The human organism synthesizes this amino acid deriva-tive itself or assimilates it from meat in food by an active and a passivetransport mechanism: endogenous and exogenous carnitine. Both serve to meet the needs of the organism, the latter more particularly in the case physical endurance exercise. In our view it is possible to distinguish between different L-carnitine deficiency conditions, it being clear that the therapeutic administration of L-carnitine constitutes a true substitu-tion of an essential biocarrier which primes metabolism and which can pre-vent damage to health. These carnitine deficiency conditions are listed in scheme1. From this there may be seen to be various different therapeutic aspects.
15 scheme l-Different forms of L-carnitine deficiencY Phenomena~ which necessitate a theraoy with L-carnitine substitution.
;~ I. Primary L-carnitine deficiency:
Hereditary, primary carnitine deficiency syndrome.
20 II. Secondary L-carnitine deficiency:
1) Endurance and high performance sport (26) 2) Diabetes mellitus and dialysis patients 3) AIDS patients (11) 4) Deficient uptake of L-carnitine (high performance vege-tarian athletes) or inhibition of transport to organs.
Furthermore the therapeutic application of L-carnitine may be em-ployad for some further purposes as compiled in scheme 2.
Scheme 2 .
Additive-suPportive substitution with a theraceutic effect.
1) For cardiac insufficiency (15) 2) In connection with weight reduction (1) 3) In the treatment of arteriosclerosis (lowering cholesterol level, increasing HDL, reduction in hypertriglyçedridemia in the case of dyslipidemias) (4; 17) 4) In Alzheimer's disease (8) 5) For the improvement of performance in the case of disturbances in peripheral circulation (9)~
6) For the stimulation of regeneration of the liver following partial hepatectomy in experiments on animals (7) 7) In order to protect cardiac mitochondria against the cytotoxic e~fect , : , .
2 ~
of adriamycine (2).
Some brief observations will now be made on these therapeutic ef-fects. In the treatment of cardiac and circulatory diseases there is not only the benefit of the energetic effect for the heart muscle but further-more there is a general effect on the body fats which may be termed anti-arteriosclerotic, which however furthermore emphasizes an important preven-tive aspect, the improvement in the degradation of fat during therapeutic fasting also having a favorable effect (table 2).
Th0 neurological mechanisms are of interest, which can be affected by L-carnitine and acetyl-L-carnitine (14). On the basis of the stereospecif-ic similarity with acetylcholine workers have not only suspected a cholino-mimetic activity but furthermore actually detected it (19). The influence of L-carnitine on cholinergic pathophysiology also plays a part in Alzheim-er's disease, as has already been alluded to by Janri et al. (14) and has even given rise to therapeutic concepts (8).
One object of the present invention is to indicate novel therapeutic effects of carnitine, in addition to these inherently known effects there-of, which will provide novel applications therefor.
This object is to be attained by the applications as defined in more detail in the claims.
In accordance with the invention the phagocytosis function of mono-cytes and granulocytes and furthermore the stimulation of T- and B-lympho-cytes was investigated. These effects may be attributed to a membrane effect of L-carnitine.
Materials and Methods L-carnitine: Biocarn ~ of the Medice Company (Iserlohn).
T- and B-cell stimulation tests: in accordance with Schumacher and coworkers (20) using Phythemagglutinin (PHA) (Welcome Diagnostics) and pokeweed mitogen (Sigma, L-9397).
Macrophage function tests: as described by Van Mil and Uhlenbruck (25) and Uhlenbruck et al. (23).
Granulocyte function tests: as described by Oben and Foreman (18), the evaluation being performed microscopically.
Measurement of the phagocytosis with the aid of chemiluminescence: in this case the method described by Beuth et al. (5) was employed.
Chromium release test with natural killer (NK) cells: This test procedure was performed as described by Cosentino and Catheart (10).
Free-flow cell electrophoresis: this was performed as described by Hannig (12), that is to say once for isolation of human B-lymphocytes (3) and once to be able to prove a membrane effect of L-carnitine. For this .. . .
æ~
purpose human erythrocytes and blood cells as employed in the preceding tests were incubated ~or 30 minutes with a 1 % solution of L-carnitine at 37 C, then centrifuged and washed with the corresponding electrophoresis buffer (12).
Results:
The ~ranulocvte function and L-carnitine:
Firstly the granulocytes of 10 healthy donors were tested as regards their ability to phagocytize bacteria of the type Micrococcus Lysodeikticus (ML), it turning out that the values varied considerably from individual to individual, for the result was able to be reproduced by double assays with an offset in time. 2 x 200 cells were evaluated with the aid of micro-scopic counting. The pre-incubation of the granulocytes (human granulocy-tes = MG) with L-carnitine was performed For one hour at 37' C in a shaken water bath.
For this on the one hand the MG were pretreated with 0.1 % L-carnit-ine and on the other hand the MG were pretreated in the same manner (second and third items in table 1, wherein these results are show as a whole).
The first column o-F table 1 contains the normal values as percentage of the phagocytizing cells. Pre-incubation of the MG with L-carnitine increases the phagocytosis rate substantially, but howeYer it is increased even fur-ther if it is not the MG but the ML to be phagocytized which are pre-incu-bated with 0.1 ~ L-carnitine.
The whole is represented once again graphically in figure 1 which also demonstrates that L-carnitine has a direct effect 1ncreasing phagocy-tosis both on the MG and also on the ML.
Table 1. Increase of bacterio-phagocytosis by granulocytes with the aid of L-carnitine in pre-incubation tests.
Donor MG + ML MG/L-carnitine MG/ML/L-carnitine 0.15 ~ ML 0.1 %
1 36 % 58 % 71 %
2 38 % 54 % 70 %
3 39 % 47 % 63 %
4 54 % 75 ~ 88 %
35 5 56 % 78 % 89 %
6 63 % 87 % 92 %
7 57 % 80 % 79 %
2 ~
of adriamycine (2).
Some brief observations will now be made on these therapeutic ef-fects. In the treatment of cardiac and circulatory diseases there is not only the benefit of the energetic effect for the heart muscle but further-more there is a general effect on the body fats which may be termed anti-arteriosclerotic, which however furthermore emphasizes an important preven-tive aspect, the improvement in the degradation of fat during therapeutic fasting also having a favorable effect (table 2).
Th0 neurological mechanisms are of interest, which can be affected by L-carnitine and acetyl-L-carnitine (14). On the basis of the stereospecif-ic similarity with acetylcholine workers have not only suspected a cholino-mimetic activity but furthermore actually detected it (19). The influence of L-carnitine on cholinergic pathophysiology also plays a part in Alzheim-er's disease, as has already been alluded to by Janri et al. (14) and has even given rise to therapeutic concepts (8).
One object of the present invention is to indicate novel therapeutic effects of carnitine, in addition to these inherently known effects there-of, which will provide novel applications therefor.
This object is to be attained by the applications as defined in more detail in the claims.
In accordance with the invention the phagocytosis function of mono-cytes and granulocytes and furthermore the stimulation of T- and B-lympho-cytes was investigated. These effects may be attributed to a membrane effect of L-carnitine.
Materials and Methods L-carnitine: Biocarn ~ of the Medice Company (Iserlohn).
T- and B-cell stimulation tests: in accordance with Schumacher and coworkers (20) using Phythemagglutinin (PHA) (Welcome Diagnostics) and pokeweed mitogen (Sigma, L-9397).
Macrophage function tests: as described by Van Mil and Uhlenbruck (25) and Uhlenbruck et al. (23).
Granulocyte function tests: as described by Oben and Foreman (18), the evaluation being performed microscopically.
Measurement of the phagocytosis with the aid of chemiluminescence: in this case the method described by Beuth et al. (5) was employed.
Chromium release test with natural killer (NK) cells: This test procedure was performed as described by Cosentino and Catheart (10).
Free-flow cell electrophoresis: this was performed as described by Hannig (12), that is to say once for isolation of human B-lymphocytes (3) and once to be able to prove a membrane effect of L-carnitine. For this .. . .
æ~
purpose human erythrocytes and blood cells as employed in the preceding tests were incubated ~or 30 minutes with a 1 % solution of L-carnitine at 37 C, then centrifuged and washed with the corresponding electrophoresis buffer (12).
Results:
The ~ranulocvte function and L-carnitine:
Firstly the granulocytes of 10 healthy donors were tested as regards their ability to phagocytize bacteria of the type Micrococcus Lysodeikticus (ML), it turning out that the values varied considerably from individual to individual, for the result was able to be reproduced by double assays with an offset in time. 2 x 200 cells were evaluated with the aid of micro-scopic counting. The pre-incubation of the granulocytes (human granulocy-tes = MG) with L-carnitine was performed For one hour at 37' C in a shaken water bath.
For this on the one hand the MG were pretreated with 0.1 % L-carnit-ine and on the other hand the MG were pretreated in the same manner (second and third items in table 1, wherein these results are show as a whole).
The first column o-F table 1 contains the normal values as percentage of the phagocytizing cells. Pre-incubation of the MG with L-carnitine increases the phagocytosis rate substantially, but howeYer it is increased even fur-ther if it is not the MG but the ML to be phagocytized which are pre-incu-bated with 0.1 ~ L-carnitine.
The whole is represented once again graphically in figure 1 which also demonstrates that L-carnitine has a direct effect 1ncreasing phagocy-tosis both on the MG and also on the ML.
Table 1. Increase of bacterio-phagocytosis by granulocytes with the aid of L-carnitine in pre-incubation tests.
Donor MG + ML MG/L-carnitine MG/ML/L-carnitine 0.15 ~ ML 0.1 %
1 36 % 58 % 71 %
2 38 % 54 % 70 %
3 39 % 47 % 63 %
4 54 % 75 ~ 88 %
35 5 56 % 78 % 89 %
6 63 % 87 % 92 %
7 57 % 80 % 79 %
8 52 % 75 % 90 %
9 46 ~ 78 % 85 %
40 10 45 % 87 % 91 %
:.. ;.
" ~
~ 2 7 , MG - human granulocytes ML = Micrococcus lysodeictus It will be seen that pre incubation for one hour of the MG with 0.1 of L-carnitin~ increases the phagocytosic capacity of the MG by 20 to 9~ %
(MW - 40 %). This is dependent on the lower value: the lower the base value, the higher the increase being, as will be seen from the following tests.
It is of interest that the pre-incubation of the FITC-marked ML bac-teria leads to a substantially increased rate of the phagocyti7ing MG: 50-100 % (MW - 70 %).
It was now to be examined whether this enhancing effect of L-carnit-ine is specific with respect to the bacterio-phagocytosis by MG or whether the acetylcholine employed is able to produce a similar effect. For this purpose four more granulocyte samples from the donors were examined. The result is to be seen in table 2. It will be clearly seen that acetylcho-line has no phagocytosis enhancing e-~fect. In this respect it is again made clear that the increase in rate due to L-carnitine is dependent on the initial value: the lower the latter, the more efficient the increase.
Table 2: direct increase in granulocyte bacterio-phagocytosis by L-carnitine, but without using acetylcholine pre-incubation.
Donor MG + ML MG/L-carnitine MG~acetylcholine 0.1 % + ML 0.1 % + ML
1 32 % 60 % 30 %
2 27 % 52 % 25 æ
3 56 % 66 % 51 %
4 61 % 7~ % 62 %
Chemiluminescence tests for the Phagocvtosis ca~acitY of ~ranulocvtes and monocvtes.
The increase found in the preceding tests in bacterio-phagocytosis by L-carnitine was examined again with the aid of the chemilumines-cence method.
In this respect it was to be seen from preliminary tests that human monocytes are not directly stimulated in their function by incubation with L-carnitine. As an example human monocytes are used, whose phagocytosis function was not capable of being substantially modified with respect to EAC (charged with erythrocyte/antibody/complement), with respect to E-anti-D (human erythrocytes sensitized with incomplete anti-D) and with respect to ERDE (ovine erythrocytes treated with neuraminidase) (25) either by pre-incubation with L-carnitine or by acetylcholine (see figure 3). Here as S
.
,, . , " .
, ' : ' well the percentages of phagocytizing cells are given.
The individual valu0s for figure 3 may be seen from table 3, the monocytes having been pre-incubated -for cne hour at 37 C in an incubator (flushed with C02) with L-carnitine and, respectively, acetylcholine (1 %).
The phagocytosis index is derived from the number of phagocytized erythro-cytes in relation to the phagocytosis activated monocytes.
Table 3: Phagocytosis function test3with human monocytes and pre-incubation with L-carnitine and acetylcholine.
Medium L-carnitine 0.~ % acetylcholine 0.1 %
æ pos. No. Index % pos. No. Index X pos. No. Index monoc. ery. monoc. ery. monoc. ery.
EAC 85 236 2.8 90 244 2.7 80 161 2.0 EAnti D 75 122 1.6 73 115 1.6 77 133 1.7 15 ERDE 82 i93 2.4 88 238 2.7 83 200 2.4 After these preliminary investigations a chemiluminescence test was employed to determine whether monocytes are not directly activatable by pre-incubation with 0.1 ~ L-carnitine or, respectively, acetylcholine with-out opsonization with serum. The result is shown in figure 4. It will be gathered that monocytes, which undergo pre-incubation with 0.1 % L-carnit-ine, achieve 73 X of the zymosan stimulation, whereas monocytes, which underwent pre-incubation with 0.1 % acetylcholine only achieved 24 % of the zymosan stimulation.
In the case of the MG there was no such direct activation by L-carni-tine or acetylcholine, whereas however the functional capacity after pre-incubation both of the MG and also of the ML was significant-ly stimulated by L-carnitine.
In the case of the human monocytes just the reverse is the case.
They are directly activated by L-carnitine, whereas their function could ; 30 not be substantially improved by the methodic test conditions employed by us .
The chemiluminescence test in the case of the MG on the other hand show ~ an increase of bacterio-cytosis by 34 % with the use of ML, some-thing that is graphically illustrated in figure 5. Acetylcholine does not exhibit this effect. Accordingly the initially indicated results are con-firmed.
T- and B-lymphocvte stimulation test.
Firstly T-lymphocyte stimulation tests were performed in the conven-tional manner with the aid of PHA, whereas stimulation tests were performed in parallel with the same cells, after the lymphocytes had been incubated . . ~ .
,.: ,,: - :
.
~ ~ ~3 ~
with different concentrations of L-carnitine. As a control pre-incubation with acetylcholine was again employed. The pre-incubation steps lasted one hour and took place at 37 C in a C02 incubator. The result of these tests is indicated in one example in table 4.
Table 4: Stimulation of T-lymphocytes by PHA and the influence there-on by L-carnitine and acetylcholine.
Concentration L-carnitine Acetylcholine PHA
40 10 45 % 87 % 91 %
:.. ;.
" ~
~ 2 7 , MG - human granulocytes ML = Micrococcus lysodeictus It will be seen that pre incubation for one hour of the MG with 0.1 of L-carnitin~ increases the phagocytosic capacity of the MG by 20 to 9~ %
(MW - 40 %). This is dependent on the lower value: the lower the base value, the higher the increase being, as will be seen from the following tests.
It is of interest that the pre-incubation of the FITC-marked ML bac-teria leads to a substantially increased rate of the phagocyti7ing MG: 50-100 % (MW - 70 %).
It was now to be examined whether this enhancing effect of L-carnit-ine is specific with respect to the bacterio-phagocytosis by MG or whether the acetylcholine employed is able to produce a similar effect. For this purpose four more granulocyte samples from the donors were examined. The result is to be seen in table 2. It will be clearly seen that acetylcho-line has no phagocytosis enhancing e-~fect. In this respect it is again made clear that the increase in rate due to L-carnitine is dependent on the initial value: the lower the latter, the more efficient the increase.
Table 2: direct increase in granulocyte bacterio-phagocytosis by L-carnitine, but without using acetylcholine pre-incubation.
Donor MG + ML MG/L-carnitine MG~acetylcholine 0.1 % + ML 0.1 % + ML
1 32 % 60 % 30 %
2 27 % 52 % 25 æ
3 56 % 66 % 51 %
4 61 % 7~ % 62 %
Chemiluminescence tests for the Phagocvtosis ca~acitY of ~ranulocvtes and monocvtes.
The increase found in the preceding tests in bacterio-phagocytosis by L-carnitine was examined again with the aid of the chemilumines-cence method.
In this respect it was to be seen from preliminary tests that human monocytes are not directly stimulated in their function by incubation with L-carnitine. As an example human monocytes are used, whose phagocytosis function was not capable of being substantially modified with respect to EAC (charged with erythrocyte/antibody/complement), with respect to E-anti-D (human erythrocytes sensitized with incomplete anti-D) and with respect to ERDE (ovine erythrocytes treated with neuraminidase) (25) either by pre-incubation with L-carnitine or by acetylcholine (see figure 3). Here as S
.
,, . , " .
, ' : ' well the percentages of phagocytizing cells are given.
The individual valu0s for figure 3 may be seen from table 3, the monocytes having been pre-incubated -for cne hour at 37 C in an incubator (flushed with C02) with L-carnitine and, respectively, acetylcholine (1 %).
The phagocytosis index is derived from the number of phagocytized erythro-cytes in relation to the phagocytosis activated monocytes.
Table 3: Phagocytosis function test3with human monocytes and pre-incubation with L-carnitine and acetylcholine.
Medium L-carnitine 0.~ % acetylcholine 0.1 %
æ pos. No. Index % pos. No. Index X pos. No. Index monoc. ery. monoc. ery. monoc. ery.
EAC 85 236 2.8 90 244 2.7 80 161 2.0 EAnti D 75 122 1.6 73 115 1.6 77 133 1.7 15 ERDE 82 i93 2.4 88 238 2.7 83 200 2.4 After these preliminary investigations a chemiluminescence test was employed to determine whether monocytes are not directly activatable by pre-incubation with 0.1 ~ L-carnitine or, respectively, acetylcholine with-out opsonization with serum. The result is shown in figure 4. It will be gathered that monocytes, which undergo pre-incubation with 0.1 % L-carnit-ine, achieve 73 X of the zymosan stimulation, whereas monocytes, which underwent pre-incubation with 0.1 % acetylcholine only achieved 24 % of the zymosan stimulation.
In the case of the MG there was no such direct activation by L-carni-tine or acetylcholine, whereas however the functional capacity after pre-incubation both of the MG and also of the ML was significant-ly stimulated by L-carnitine.
In the case of the human monocytes just the reverse is the case.
They are directly activated by L-carnitine, whereas their function could ; 30 not be substantially improved by the methodic test conditions employed by us .
The chemiluminescence test in the case of the MG on the other hand show ~ an increase of bacterio-cytosis by 34 % with the use of ML, some-thing that is graphically illustrated in figure 5. Acetylcholine does not exhibit this effect. Accordingly the initially indicated results are con-firmed.
T- and B-lymphocvte stimulation test.
Firstly T-lymphocyte stimulation tests were performed in the conven-tional manner with the aid of PHA, whereas stimulation tests were performed in parallel with the same cells, after the lymphocytes had been incubated . . ~ .
,.: ,,: - :
.
~ ~ ~3 ~
with different concentrations of L-carnitine. As a control pre-incubation with acetylcholine was again employed. The pre-incubation steps lasted one hour and took place at 37 C in a C02 incubator. The result of these tests is indicated in one example in table 4.
Table 4: Stimulation of T-lymphocytes by PHA and the influence there-on by L-carnitine and acetylcholine.
Concentration L-carnitine Acetylcholine PHA
10 ~gPHA/culture 1 % 0.1 æ 0.01 % 0.1 %
_ Stimulation index 0.01 7 3 9 10 6 15 0.1 24 11 26 31 21 0.2 36 14 67 96 32 0.5 19 10 34 56 20 1.0 13 8 27 38 14 2.0 12 6 24 31 1 20 5.0 12 6 21 27 9 10.0 9 5 16 19 7 20.0 8 3 6 5 6 Medium These findings are presented once again in figure 6 graphically.
It will be clearly seen that acetylcholine has practically the same curve with PHA, whereas a concentration (1 %t clearly suppresses normal PHA
~; stimulation. Therefore the result with concentrations of L-carnitine ofthe order of 0.1 % and 0.01 % was all the more unexpected. These values are made apparent in the two upper curves, a surprising increase of lympho-cyte stimulation by PHA having to be noted. It seems that there is a con-centration-dependent process at the T-lymphocyte membrane, which inhibits PHA stimulation at high dosages, whereas on the other hand at low ones it is responsible for a significant increase in PHA stimulation.
This result was produced again and again with other T-lymphocyte samples: inhibition by 1 % L-carnitine pre-incubation and an increase of PHA stimulation by lower concentrations of L-carnitine (from 0.5 % upwards) was always to be observed. It is interesting that the increase is dosage-dependent (dilution series 0.01 % - 20 ~m/culture) and is specific, since acetylcholine does not exhibit this effect.
In comparison with the T-lJmphocytes human B-lymphocytes were also .. . . ,, . .. ., ~ . , ~9~27 stimulated with pokeweed mitogen (PWM) and tested to see if this stimula-tion was to be influenced positively in the same way by L-carnitine. The result is shown in figure 7 and in table 5.
Table 5: B-lymphocyte stimulation test with PWM and pre-incubation of L-carnitine or, respectively, acetylcholine. The pre-incubation was per-formed in the same manner as with the T-lymphocytes.
Concentration L-carnitine Acetylcholine PWM
~gPHA/culture 0.01 ~ 0.1 æ o. 1 Stimulation index (cpm sample: cpm med.) .
0.1 8 20 10 16 Medium Figure 7 also indicates that there is a substantial increase in lym-phocyte stimulation by L-carnitine at a concentration of 0.01 %. However the control with acetylcholine also increases the PWM stimulation of B-lymphocytes, whereas at a concentration of 0.1 % L-carnitine practically coincides with the normal stimulation curve, that is to say it fails to have any effect at this concentration. It is to be expected that 1 % L-carnitine would inhibit stimulation again. The B-lymphocyte population was produced with the aid of cell electrophoresis pr~paratively.
Since these tests provide a clear indication that L-carnitine is ~ responsible for an increase in the biological function by a membrane ef-fect, which frequently may e~en be non-specific, as is shown by the posi-tive control tests with acetylcholine, human erythrocytes, T- and B-lympho-cytes, as well as various different bacteria were then investigated to see whether the electrophoretic migration velocity, that is to say the zeta potential, was changed afterincubation with L-carnitine and, respectively, acetylcholine.
The tests so far performed have, in the case of human blood cells after pretreatment with these substances not shown that there are any modi-fications in the electrophoretic migration velocity. Furthermore different bacteria employed for phagocytosis did not, after pretreatment with L-carn-itine, show any basic modification in the zeta potential, although in some ~ . , , , , . , :
~ ' ' .
.
cases small departures (a decrease of up to 5 % in mobility) were observed.NK cell tests and the effect thereon of L-carnitine and acetYlcho-line~
In order to make a still ~urther investigation of the suspected cell membrane effect of L-carnitine or, respectively, acetylcholine, with the aid of chromium release tests human killer cells (NK cells) were investi-gated as regards their effect on target cells (T). The result is indicated in figure 8. It will be seen from the first column (I) that the normal target cell Iysis by NK cells amounts to 38 % (see furthermore table 6).
10 The pretreatment of the NK cells with L-carnitine and acetylcholine (0.1 ~) leads to a significant increase in NK cell lysis. If on the other hand the target cells are treated with chromium5~ and if then L-carnitine or, respectively, acetylcholine are added, values will be obtained which are distinctly lower (column III). It is therefore to be seen that one hour's pre-incubation of the NK cells significantly steps up lysis, whereas pre-incubation with chromium5~ and L-carnitine or, respectively, ace~ylcho-line is less effective. It will hence be seen that these two substances attack both the membrane of the NK cells as well as the membrane of the target cells, the chromium loss from the target cel-ls owing to the pre-incubation being taken into account as well. The notion that both sub-stances, L-carnitine and furthermore acetylcholine, are responsible for a membrane effect in the case of these cells, is confirmed by the two columns IV in figure 8. Here it was apparent that a pre-incubation of the target cells with 0.1 % L-carnitine followed by the addition of chromium51 led to a 129 % increase in lysis, whereas in the case of 0.1 % acetylcholine pre-incubation also led to an increase in lysis by 100 ~. The corresponding figures are presented in table 6.
Table 6: chromium release tests after pre-incubation with L-carnitine and acetylcholine.
Form of test (NK:T - 25:1) ~ % lysis % increase I NK : T 38 II NK + L-carnitine 0.1 %:T + Cr5~ 54 42 NK + acetylcholine 0.1 % + Cr51 53 39 III NK : T + Cr51 + L-carnitine 0.1 % 45 18 NK : T + CrS1 + acetylcholine 0.1 % 43 13 IV NK : T + L-carnitine 0.1 % + Cr51 87 129 NK : T + acetylcholine 0.1 % + CrS1 80 110 40 It would appear that pre-incubation in IV does not cause an enhanced . : -. .
uptake of chromium5l (there being the same values after SDS lysis), and it is rather a ques~ion of the NK cells lysin~ more target cells owing to a membrane effect. qhis being an effect which may even be employed for im-proving the chromium release test and for economizing in the use of radio-S active chromium, while the pre-incubation in III, as is shown by measure-ments in the washing solution, also based on a membrane effect, in the case of which however radioactive chromium is lost.
20 years ago it was pointed out for the first time (22~ that the significant consequences of regular endurance training included "non-spe-cific stimulation of the immune-competent body defense mechanism', in con-nection with any protection caused which is effective against infections and cancer. In the meantime we have now learned that moderate endurance training may reduce the risk of infections and also the risk of cancer ~16), whereas excessive high performance stress may have the opposite re-sult.
Therefore there has been no lack of concepts and systems for counter-acting debility of the immune system by performance orientated popular sport (24), be it in the form of immune stimulation or substitution (admin-istration of immunoglobulins etc.) or by mental training. Therefore it always appeared important for the inventors for all substances not affected by the doping controversy, which are offered to high performance sportsman and -women for substitution, stabilization of performance and for regenera-tion, to be investigated as regards their effect on the immune system. In this respect it was possible to show that aspirin as sometimes employed on the triathlon scene does not have any effect on the immune system.
In the investigations reported on here with L-carnitine i~ was possi-ble to show in accordance with the invention that this amino-acid deriva-tive does in fact have an influence on the most different immunocompetent cells. On the basis of the results produced here it is possible to note three main effects, of which there has so far been no knowledge:
1. A phagocytosis increasing effect after action on human granulo-cytes, and furthermore on the bacteria employed as target cells: a sort of pseudo-opsonin effect, which must be specifi-cally attributed to L-carnitine.
35 2. An effect activating stimulation of T-lymphocytes which is specific too and which is also dosage rate-dependent and is specific for L-carnitine, whereas a directly activating effect was to be seen in the case of human monocytes. Less specific was the action in human B-lymphocyte stimulation tests, as also acetylcholine led to a clearer but however weaker effect.
2 ~ 2 ~
3. An interesting membrane effect was shown by L-carnitine in the case of human killer cells (NK) or, respectively, the corre-sponding target cells. Since acetylcholine exhibited the same effect, these results must be interpreted as being non-speci~-ic. A similar membrane permeability effect has furthermore been assumed by other authors in the case of the mitochondrial membrane (2~.
Supplementing endogenous and exogenous L-carnitine by an additional supply of L-carnitine for high performance sportsmen and sportswomen is consequently not only free of objections from the immunological point of view but furthermore appears to have a reinforcing and activating effect, in a manner which has so far not been elucidated, on immunological defense mechanisms both specifically and non-specifically, by producing an effect at the membrane of phagocytes, NK cells and monocytes in concentrations, 1~ which may become established in the case of supply by way of substitution in blood as well, albeit temporarily (0.01 ~. Therefore it is an advan-tage that acetylcholine, which is sometimes effective in the same manner (NK cells) does not come into question for "therapeutic" purposes. The fact that L-carnitine is effective at relatively low dosage rates, at the 2~ same time gives the lie to arguments which seek to attribute this effect to various different preserving agents present in commercial preparations.
By way of conclusion it may be said that the two fields of applica-tion so far ~or L-carnitine are to be supplemented by a third one (scheme 3).
Scheme 3. The positive effects of an exogenous L-carnitine supply. 5 1. Cardiac, circulation and metabolic effects (cardiac insuffi-ciency, diabetes, arteriosclerosis, diet, high per~ormance sport), 2. Neurological ef~ect (AIDS, Alzheimer's disease, similarity to acetylcholine), and 0 3. ~Membrane effects in the case of phagocytes, immunocytes and bacteria (effect enhancing the immune reaction).
In this connection it must be of interest that in high performance spurt it is possible in this manner to not only stimulate metabolism (6) but furthermore to activate those cells in the immune system, which partic-ularly become involved in connection with inflammatory and acute phaseevents due to stress in sport: monocytes, granulocytes and NK cells. This might provide a third argument in support o~ "theoretical therapy" (1; 13) which is not accepted in all quarters.
Reference (21) relates to the action of L-carnitine as an aggregation 4û inhibitor in the case of myxomycetes and cells of the organism and erythro-.:
. .
,~ ,. .
' ',' , .
cytes. In accordance with the invention it was possible to show that onthe administration of L-carnitine macrophages are no longer able to adhere and can be dètached from tissue culture dishes with the aid of L-carnitine.
The inhibition tests described in the invention using L~carnitine, even with different sets of c011s, would appear to be based on a direct membrane effect. The fat metabolism aspect appears to play no part at all and rather seems to be an electrostatic effect, which however would not occur on the outer cell surface, for otherwise it would have been noticed during cell electrophoresis during tests performed in acc~rdance with the invention.
The activation described in the above for the first time of immuno-competent cells constitutes a significant consequence of this membrane effect, which may also be employed therapeutically, something which can hardly fail to be of importance for sport medicine, since in this case experience with the administration of L-carnitine in high performance sport is available.
Thus it has been found in accordance with the invention that L-carni-tine influences biological cell membranes in such a way, that is to say by direct accumulation which leads to an activation of the membrane surface, this in turn meaning that aggregation phenomena are inhibited, agglutina-tion events are influenced, adhesion mechanisms are changed and cell mem-brane metabolism events are caused to take place differently. A further point to be considered is that owing to this membrane action the uptake of substances into the cell and the release of substances from the cell is improved or, respectively, changed, something which was proved by the above ; described chromium release tests. Moreover cells may be better stimulated~by pretreatment with L-carnitine, for instance by mitog0ns, lectines etc.
or they may be inhibited, for instance by cytostatica. From these results it is possible to show that there are the following possibilities of appli-cation:
1. L-carnitine as an a~regation inhibitor~ The aggregation o~ thrombo-cytes, erythrocytes and white blood corpuscles is inhibited. This is im-portant for the anti-arteriosclerotic effect of L-carnitine, far the pro-phylactic treatment of cardiac thrombosis and more particularly for inhibi-tion of metastatic spread of tumor cells by an influence of the membranestructure and also however fluidity, including the "docking" cells. Osmot-ic resistance is increased.
2. Activation of the cell membrane plays a decisive role in the uptake of medicaments. L-carnitine improves the uptake of different substances into the cell, for instance of radioactively marked chromium into natural , :
","
. . . " . .
: :. .. ~ . .; . ., ' -~9~2~
killer cells. In the case of the concentration of L-carnitine which may be produced by administration, namely 0.01 to 0.001 ~, this uptake enhancing effect would sésm to be a substantial advantage for the treatment of tumors with cytostatica; more particularly leukemia and Hodgkin cells come to mind in this connection. Carnitine leads to an opening of pores of cells and therefore facilitates the entry of medicaments. This applies not only for an influence on tumor cells, but furthermore in principle as an enhancing effect as regards the action of other medicaments in connection with other cells. We assume that this property plays a part in the case of the neuro-logical effects of carnitine (on Alzheimer's disease, AIDS etc). Use inthe case of hemolytic anemia is also recommended.
3. The indirect immuno-modulatorv effect of carnitine: here there is an activation of phagocytosis, such pseudo-opsonin effect being in relation to bacteria on the one hand, which are better phagocytized and on the other hand to the phagocytes themselves, which are also stimulated as regards their phagocytic activity. A further point to be considered is that there is an activation of T- and B-lymphocyte functions, of NK cells and of gran-ulocytes: their functions and activities are improved with therapeutic doses of carnitine, both quantitatively and qualitatively. It activates immunocytes so that they are better able to fulfill their defense tasks.
, - ..
. ' .:
,.
. . ~
.
_ Stimulation index 0.01 7 3 9 10 6 15 0.1 24 11 26 31 21 0.2 36 14 67 96 32 0.5 19 10 34 56 20 1.0 13 8 27 38 14 2.0 12 6 24 31 1 20 5.0 12 6 21 27 9 10.0 9 5 16 19 7 20.0 8 3 6 5 6 Medium These findings are presented once again in figure 6 graphically.
It will be clearly seen that acetylcholine has practically the same curve with PHA, whereas a concentration (1 %t clearly suppresses normal PHA
~; stimulation. Therefore the result with concentrations of L-carnitine ofthe order of 0.1 % and 0.01 % was all the more unexpected. These values are made apparent in the two upper curves, a surprising increase of lympho-cyte stimulation by PHA having to be noted. It seems that there is a con-centration-dependent process at the T-lymphocyte membrane, which inhibits PHA stimulation at high dosages, whereas on the other hand at low ones it is responsible for a significant increase in PHA stimulation.
This result was produced again and again with other T-lymphocyte samples: inhibition by 1 % L-carnitine pre-incubation and an increase of PHA stimulation by lower concentrations of L-carnitine (from 0.5 % upwards) was always to be observed. It is interesting that the increase is dosage-dependent (dilution series 0.01 % - 20 ~m/culture) and is specific, since acetylcholine does not exhibit this effect.
In comparison with the T-lJmphocytes human B-lymphocytes were also .. . . ,, . .. ., ~ . , ~9~27 stimulated with pokeweed mitogen (PWM) and tested to see if this stimula-tion was to be influenced positively in the same way by L-carnitine. The result is shown in figure 7 and in table 5.
Table 5: B-lymphocyte stimulation test with PWM and pre-incubation of L-carnitine or, respectively, acetylcholine. The pre-incubation was per-formed in the same manner as with the T-lymphocytes.
Concentration L-carnitine Acetylcholine PWM
~gPHA/culture 0.01 ~ 0.1 æ o. 1 Stimulation index (cpm sample: cpm med.) .
0.1 8 20 10 16 Medium Figure 7 also indicates that there is a substantial increase in lym-phocyte stimulation by L-carnitine at a concentration of 0.01 %. However the control with acetylcholine also increases the PWM stimulation of B-lymphocytes, whereas at a concentration of 0.1 % L-carnitine practically coincides with the normal stimulation curve, that is to say it fails to have any effect at this concentration. It is to be expected that 1 % L-carnitine would inhibit stimulation again. The B-lymphocyte population was produced with the aid of cell electrophoresis pr~paratively.
Since these tests provide a clear indication that L-carnitine is ~ responsible for an increase in the biological function by a membrane ef-fect, which frequently may e~en be non-specific, as is shown by the posi-tive control tests with acetylcholine, human erythrocytes, T- and B-lympho-cytes, as well as various different bacteria were then investigated to see whether the electrophoretic migration velocity, that is to say the zeta potential, was changed afterincubation with L-carnitine and, respectively, acetylcholine.
The tests so far performed have, in the case of human blood cells after pretreatment with these substances not shown that there are any modi-fications in the electrophoretic migration velocity. Furthermore different bacteria employed for phagocytosis did not, after pretreatment with L-carn-itine, show any basic modification in the zeta potential, although in some ~ . , , , , . , :
~ ' ' .
.
cases small departures (a decrease of up to 5 % in mobility) were observed.NK cell tests and the effect thereon of L-carnitine and acetYlcho-line~
In order to make a still ~urther investigation of the suspected cell membrane effect of L-carnitine or, respectively, acetylcholine, with the aid of chromium release tests human killer cells (NK cells) were investi-gated as regards their effect on target cells (T). The result is indicated in figure 8. It will be seen from the first column (I) that the normal target cell Iysis by NK cells amounts to 38 % (see furthermore table 6).
10 The pretreatment of the NK cells with L-carnitine and acetylcholine (0.1 ~) leads to a significant increase in NK cell lysis. If on the other hand the target cells are treated with chromium5~ and if then L-carnitine or, respectively, acetylcholine are added, values will be obtained which are distinctly lower (column III). It is therefore to be seen that one hour's pre-incubation of the NK cells significantly steps up lysis, whereas pre-incubation with chromium5~ and L-carnitine or, respectively, ace~ylcho-line is less effective. It will hence be seen that these two substances attack both the membrane of the NK cells as well as the membrane of the target cells, the chromium loss from the target cel-ls owing to the pre-incubation being taken into account as well. The notion that both sub-stances, L-carnitine and furthermore acetylcholine, are responsible for a membrane effect in the case of these cells, is confirmed by the two columns IV in figure 8. Here it was apparent that a pre-incubation of the target cells with 0.1 % L-carnitine followed by the addition of chromium51 led to a 129 % increase in lysis, whereas in the case of 0.1 % acetylcholine pre-incubation also led to an increase in lysis by 100 ~. The corresponding figures are presented in table 6.
Table 6: chromium release tests after pre-incubation with L-carnitine and acetylcholine.
Form of test (NK:T - 25:1) ~ % lysis % increase I NK : T 38 II NK + L-carnitine 0.1 %:T + Cr5~ 54 42 NK + acetylcholine 0.1 % + Cr51 53 39 III NK : T + Cr51 + L-carnitine 0.1 % 45 18 NK : T + CrS1 + acetylcholine 0.1 % 43 13 IV NK : T + L-carnitine 0.1 % + Cr51 87 129 NK : T + acetylcholine 0.1 % + CrS1 80 110 40 It would appear that pre-incubation in IV does not cause an enhanced . : -. .
uptake of chromium5l (there being the same values after SDS lysis), and it is rather a ques~ion of the NK cells lysin~ more target cells owing to a membrane effect. qhis being an effect which may even be employed for im-proving the chromium release test and for economizing in the use of radio-S active chromium, while the pre-incubation in III, as is shown by measure-ments in the washing solution, also based on a membrane effect, in the case of which however radioactive chromium is lost.
20 years ago it was pointed out for the first time (22~ that the significant consequences of regular endurance training included "non-spe-cific stimulation of the immune-competent body defense mechanism', in con-nection with any protection caused which is effective against infections and cancer. In the meantime we have now learned that moderate endurance training may reduce the risk of infections and also the risk of cancer ~16), whereas excessive high performance stress may have the opposite re-sult.
Therefore there has been no lack of concepts and systems for counter-acting debility of the immune system by performance orientated popular sport (24), be it in the form of immune stimulation or substitution (admin-istration of immunoglobulins etc.) or by mental training. Therefore it always appeared important for the inventors for all substances not affected by the doping controversy, which are offered to high performance sportsman and -women for substitution, stabilization of performance and for regenera-tion, to be investigated as regards their effect on the immune system. In this respect it was possible to show that aspirin as sometimes employed on the triathlon scene does not have any effect on the immune system.
In the investigations reported on here with L-carnitine i~ was possi-ble to show in accordance with the invention that this amino-acid deriva-tive does in fact have an influence on the most different immunocompetent cells. On the basis of the results produced here it is possible to note three main effects, of which there has so far been no knowledge:
1. A phagocytosis increasing effect after action on human granulo-cytes, and furthermore on the bacteria employed as target cells: a sort of pseudo-opsonin effect, which must be specifi-cally attributed to L-carnitine.
35 2. An effect activating stimulation of T-lymphocytes which is specific too and which is also dosage rate-dependent and is specific for L-carnitine, whereas a directly activating effect was to be seen in the case of human monocytes. Less specific was the action in human B-lymphocyte stimulation tests, as also acetylcholine led to a clearer but however weaker effect.
2 ~ 2 ~
3. An interesting membrane effect was shown by L-carnitine in the case of human killer cells (NK) or, respectively, the corre-sponding target cells. Since acetylcholine exhibited the same effect, these results must be interpreted as being non-speci~-ic. A similar membrane permeability effect has furthermore been assumed by other authors in the case of the mitochondrial membrane (2~.
Supplementing endogenous and exogenous L-carnitine by an additional supply of L-carnitine for high performance sportsmen and sportswomen is consequently not only free of objections from the immunological point of view but furthermore appears to have a reinforcing and activating effect, in a manner which has so far not been elucidated, on immunological defense mechanisms both specifically and non-specifically, by producing an effect at the membrane of phagocytes, NK cells and monocytes in concentrations, 1~ which may become established in the case of supply by way of substitution in blood as well, albeit temporarily (0.01 ~. Therefore it is an advan-tage that acetylcholine, which is sometimes effective in the same manner (NK cells) does not come into question for "therapeutic" purposes. The fact that L-carnitine is effective at relatively low dosage rates, at the 2~ same time gives the lie to arguments which seek to attribute this effect to various different preserving agents present in commercial preparations.
By way of conclusion it may be said that the two fields of applica-tion so far ~or L-carnitine are to be supplemented by a third one (scheme 3).
Scheme 3. The positive effects of an exogenous L-carnitine supply. 5 1. Cardiac, circulation and metabolic effects (cardiac insuffi-ciency, diabetes, arteriosclerosis, diet, high per~ormance sport), 2. Neurological ef~ect (AIDS, Alzheimer's disease, similarity to acetylcholine), and 0 3. ~Membrane effects in the case of phagocytes, immunocytes and bacteria (effect enhancing the immune reaction).
In this connection it must be of interest that in high performance spurt it is possible in this manner to not only stimulate metabolism (6) but furthermore to activate those cells in the immune system, which partic-ularly become involved in connection with inflammatory and acute phaseevents due to stress in sport: monocytes, granulocytes and NK cells. This might provide a third argument in support o~ "theoretical therapy" (1; 13) which is not accepted in all quarters.
Reference (21) relates to the action of L-carnitine as an aggregation 4û inhibitor in the case of myxomycetes and cells of the organism and erythro-.:
. .
,~ ,. .
' ',' , .
cytes. In accordance with the invention it was possible to show that onthe administration of L-carnitine macrophages are no longer able to adhere and can be dètached from tissue culture dishes with the aid of L-carnitine.
The inhibition tests described in the invention using L~carnitine, even with different sets of c011s, would appear to be based on a direct membrane effect. The fat metabolism aspect appears to play no part at all and rather seems to be an electrostatic effect, which however would not occur on the outer cell surface, for otherwise it would have been noticed during cell electrophoresis during tests performed in acc~rdance with the invention.
The activation described in the above for the first time of immuno-competent cells constitutes a significant consequence of this membrane effect, which may also be employed therapeutically, something which can hardly fail to be of importance for sport medicine, since in this case experience with the administration of L-carnitine in high performance sport is available.
Thus it has been found in accordance with the invention that L-carni-tine influences biological cell membranes in such a way, that is to say by direct accumulation which leads to an activation of the membrane surface, this in turn meaning that aggregation phenomena are inhibited, agglutina-tion events are influenced, adhesion mechanisms are changed and cell mem-brane metabolism events are caused to take place differently. A further point to be considered is that owing to this membrane action the uptake of substances into the cell and the release of substances from the cell is improved or, respectively, changed, something which was proved by the above ; described chromium release tests. Moreover cells may be better stimulated~by pretreatment with L-carnitine, for instance by mitog0ns, lectines etc.
or they may be inhibited, for instance by cytostatica. From these results it is possible to show that there are the following possibilities of appli-cation:
1. L-carnitine as an a~regation inhibitor~ The aggregation o~ thrombo-cytes, erythrocytes and white blood corpuscles is inhibited. This is im-portant for the anti-arteriosclerotic effect of L-carnitine, far the pro-phylactic treatment of cardiac thrombosis and more particularly for inhibi-tion of metastatic spread of tumor cells by an influence of the membranestructure and also however fluidity, including the "docking" cells. Osmot-ic resistance is increased.
2. Activation of the cell membrane plays a decisive role in the uptake of medicaments. L-carnitine improves the uptake of different substances into the cell, for instance of radioactively marked chromium into natural , :
","
. . . " . .
: :. .. ~ . .; . ., ' -~9~2~
killer cells. In the case of the concentration of L-carnitine which may be produced by administration, namely 0.01 to 0.001 ~, this uptake enhancing effect would sésm to be a substantial advantage for the treatment of tumors with cytostatica; more particularly leukemia and Hodgkin cells come to mind in this connection. Carnitine leads to an opening of pores of cells and therefore facilitates the entry of medicaments. This applies not only for an influence on tumor cells, but furthermore in principle as an enhancing effect as regards the action of other medicaments in connection with other cells. We assume that this property plays a part in the case of the neuro-logical effects of carnitine (on Alzheimer's disease, AIDS etc). Use inthe case of hemolytic anemia is also recommended.
3. The indirect immuno-modulatorv effect of carnitine: here there is an activation of phagocytosis, such pseudo-opsonin effect being in relation to bacteria on the one hand, which are better phagocytized and on the other hand to the phagocytes themselves, which are also stimulated as regards their phagocytic activity. A further point to be considered is that there is an activation of T- and B-lymphocyte functions, of NK cells and of gran-ulocytes: their functions and activities are improved with therapeutic doses of carnitine, both quantitatively and qualitatively. It activates immunocytes so that they are better able to fulfill their defense tasks.
, - ..
. ' .:
,.
. . ~
.
Claims (11)
1. The use of carnitine for the activation of cell membranes of human and animal origin.
2. The use of carnitine as claimed in claim 1 as a cell aggregation inhibitor with respect to human and animal cells.
3. The use of carnitine as claimed in claim 1 or in claim 2 for inhi-bition of the aggregation of thrombocytes, erythrocytes and leucocytes and more particularly of macrophages.
4. The use of carnitine as claimed in claim 1 for increasing and facilitating the uptake of chemical and more particularly pharmaceutically active substances, in cells of human or animal origin.
5. The use of carnitine as claimed in claim 1 or in claim 4, the cells being tumor cells and/or cells of the hemopoietic system or cells of the immune system.
6. The use of carnitine as claimed in any one or more of the claims and through 5 in the chromium release test.
7. The use of carnitine as claimed in claim 1 for the therapy and prophylactic treatment of immunodeficiency conditions.
8. The use of carnitine as claimed in claim 7 for the activation of immune cells of human or animal origin.
9. The use of carnitine as claimed in claim 7 for the activation of leucocytes in the T- and B-lymphocyte, granulocyte, monocyte and killer cell group.
10. The use of carnitine as claimed in any one of more of the preced-ing claims in the form of L-carnitine and/or D-carnitine.
11. The use of carnitine as claimed in any one of more of the preced-ing claims in an amount equal to 0.01 to 0.001 %.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE4232469 | 1992-09-28 | ||
| DEP4232469.6 | 1992-09-28 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2099427A1 true CA2099427A1 (en) | 1994-03-29 |
Family
ID=6469025
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2099427 Abandoned CA2099427A1 (en) | 1992-09-28 | 1993-06-30 | Indications for carnitine |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA2099427A1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6403124B1 (en) * | 1997-04-16 | 2002-06-11 | Sigma-Tau Industrie Farmaceutiche Riunite S.P.A. | Storage and maintenance of blood products including red blood cells and platelets |
-
1993
- 1993-06-30 CA CA 2099427 patent/CA2099427A1/en not_active Abandoned
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6403124B1 (en) * | 1997-04-16 | 2002-06-11 | Sigma-Tau Industrie Farmaceutiche Riunite S.P.A. | Storage and maintenance of blood products including red blood cells and platelets |
| AU774252B2 (en) * | 1999-06-08 | 2004-06-24 | Sigma-Tau Industrie Farmaceutiche Riunite S.P.A. | Improved storage and maintenance of blood products including red blood cells and platelets |
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