Process and substances for the release of a growth-regulating factor from endothelial cells

Abstract

A method of stimulating endothelial cells cultivated in a nutrient medium to release a growth-factor into the nutrient medium by addition of at least one pentacyclic oxindole alkaloid into the nutrient medium.

Description

CROSS-REFERENCE TO RELATED APPLICATION

This is a divisional application of application Ser. No. 09/788,888, filed Feb. 20, 2001, which was a continuation-in-part of application Ser. No. 09/341,607, which was a continuing application, under 35 U.S.C. § 120, of International application PCT/AT98/00008, filed Jan. 20, 1998; the application also claims the priority, under 35 U.S.C. § 119, of Austrian patent application No. A 73/97, filed Jan. 20, 1997; the prior applications are herewith incorporated by reference in their entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

It is known that pentacyclic oxindole alkaloids exert pharmacological effects on the immune system. Increased phagocytosis of granulocytes [H. Wagner, Kreutzkamp B., Jurcic K., (1985) Planta Med. 51, 419-423] and moderate inhibition of proliferation of leukemic cells [Stuppner H., Sturm S., Geisen G., Zillian U., Konwalinka G. (1993) Planta Med. 59, Suppl. A 583] have been demonstrated. A slight but significant lymphocytosis was observed in probands who had taken orally an alkaloid-containing extract of the root of Uncaria tomentosa (Willd.) DC. [Keplinger U. (1995) in Krallendorn: Extract from Radix Uncariae tomentosae (Willd.) DC., Information for physicians and pharmacists; Immodal Pharmaka GmbH, 3rd edn.]. From these findings it was deduced that pentacyclic oxindole alkaloids have immunostimulating or immunomodulating properties. Patents concerning this were granted [U.S. Pat. No. 5,302,611, WO 86/00524].

It is known that tetracyclic oxindole alkaloids act on the central nervous system, produce negatively chronotropic and negatively inotropic effects [Kanatani H., Kohda H., Yamasaki K., Hotta I., Nakata Y., Segawa T., Yamanaka E., Aimi N., Sakai S. I. (1984) J. Pharm. Pharmacol. 37, 401-404; Zhang W., Liu G. X. (1986) Act. Pharmacol. Sinica 7 (5), 426-428; Zhu Y., Guoxiong H. X. (1993) Chin. J. Pharmacol. Toxicol. 7 (2), 117-121], block Ca2+ transport [Sun A., Liu G., Wang X., Zhang W., Huang X. (1988) Chin. J. Pharmacol. Toxicol. 2 (2), 93-97; Zhang W., Liu G., Huang X. (1987) Act. Pharmacol. Sinica 8, 425-429], and inhibit the aggregation of blood platelets [Jin R. M., Chen C. X., Li Y. K., Xu P. K. (1991) Act. Pharmaceut. Sinica 26 (4), 246-249; Chen C. X., Jin R. M., Li Y. K., Zhong J., Yue L., Chen S. C., Zhou J. Y. (1992) Act. Pharmacol. Sinica 13 (2), 126-130].

It is also known that oxindole alkaloids undergo isomerization in solution. Only recently an analysis of the kinetics of the isomerization was reported [Laus G., Brössner D., Senn G., Wurst K. (1996) J.Chem.Soc., Perkin Trans. 2, 1931-1936]. The production of defined mixtures of isomers is known from U.S. Pat. No. 5,723,625. The alkaloids used in this work were isolated from the roots of Uncaria tomentosa. The alkaloid content of a number of these plants was investigated. It was found that two chemotypes of Uncaria tomentosa occur in nature. One chemotype of Uncaria tomentosa contains mainly the tetracyclic oxindole alkaloids rhynchophylline and isorhynchophylline, the other one contains the pentacyclic oxindole alkaloids pteropodine, isopteropodine, speciophylline, uncarine F, mitraphylline and isomitraphylline. Accordingly, they are designated as tetracyclic alkaloid-type or pentacyclic alkaloid-type [Laus G., Brössner D., Keplinger K. (1997) Phytochemistry 45, 855-860]. Transitional forms have also been found in some instances which contain both types of alkaloids in various ratios [Laus G., Keplinger D. (1994) J. Chromatogr. A 662, 243-249]. Therefore the tetracyclic as well as the pentacyclic alkaloids were used in the investigations which are described in the following.

General Structure of Pentacyclic Oxindole Alkaloids with Notation of Stereochemistry:

1	Pteropodine	3S, 7R, 15S, 19S, 20S
2	Isopteropodine	3S, 7S, 15S, 19S, 20S
3	Speciophylline	3R, 7S, 15S, 19S, 20S
4	Uncarine F	3R, 7R, 15S, 19S, 20S
5	Mitraphylline	3S, 7R, 15S, 19S, 20R
6	Isomitraphylline	3S, 7S, 15S, 19S, 20R

General Structure of Tetracyclic Oxindole Alkaloids with Notation of Stereochemistry:

7	Rhynchophylline	3S, 7R, 15S, 20R
8	Isorhynchophylline	3S, 7S, 15S, 20R

Experiments in Cell Cultures

The effect of the alkaloids was studied in endothelial cells because they are known for interactions with immunologic reactions [Kirchner H., Kruse A., Neustock P., Rink L. (1993) Cytokine und Interferone: Botenstoffe des Immunsystems, 61]. It was recognized that the pentacyclic alkaloids (c=1 μM) induced transformed EA.hy926 endothelial cells [Edgell C. -J. S., McDonald C. C., Graham J. B. (1983) Proc. Natl. Acad. Sci. USA 80, 3734-3737] as well as normal human umbilical vein endothelial cells (HUVEC, ATCC CRL-1730) to release a factor into the culture medium which significantly affects the proliferation of lymphocytes. In general, RPMI-1640 was used as the culture medium for EA.hy926 endothelial cells and lymphocytes, completed with 10% fetal calf serum, 2 mM glutamin, 50 units/ml penicillin G and 50 μg/ml streptomycin. For human umbilical vein endothelial cells HAM F12 (Sigma-Aldrich Company, St. Louis, USA) was used as the culture medium, completed with 10% fetal calf serum, 60 μg/ml Endothelial Cell Growth Supplement and 100 μg/ml heparin.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING

FIG. 1: Qualitative differentiation of the two chemotypes of Uncaria tomentosa by thin-layer-chromatography. Columns 1 and 2 are results from acid and alkaline solutions of Uncaria tomentosa containing pentacyclic oxindole alkaloids (IMM-2414), columns 3 and 4 are results from acid and alkaline solutions of Uncaria tomentosa containing tetracyclic oxindole alkaloids (IMM-2418), and colums 6 and 7 show results from acid and alkaline mixtures of both. Column 5 is a reference solution.

FIG. 2: Separation of pentacyclic and tetracyclic oxindole alkaloids by HPLC.

FIG. 3: Equilibria of isomerization Kaq, Korg and partition Korg/aq of two isomers (1) and (2) in a two-phase-system,
where $K_{\mathrm{org}}\left(1\to 2\right)=\frac{K_{\mathrm{org}/\mathrm{aq}}\left(2\right)}{K_{\mathrm{org}/\mathrm{aq}}\left(1\right)}{K}_{\mathrm{aq}}\left(1\to 2\right).$

FIG. 4: pH-dependence of equilibrium concentrations (in mol-%) in an octanol-water system containing mitraphylline and isomitraphylline as an example.

FIG. 5: Increase of proliferation of normal human B lymphocytes, stimulated by supernatants of EA.hy926 cells which were grown in the presence of 1 μM IMM-2414, IMM-2417, IMM-2418.

FIG. 6: Inhibition of the proliferation of Jurkat cells (ATCC E6.1), treated with supernatants of EA.hy926 cells which were grown in the presence of 1 μM IMM-2414, IMM-2417, IMM-2418.

FIG. 7: Inhibition of the proliferation of highly activated human B and T lymphocytes (lymphoblasts) treated with supernatants of EA.hy926 cells (SN) which were grown in the presence of 1 μM IMM-2414.

FIG. 8: Inhibition of the proliferation of highly activated human T lymphocytes (lymphoblasts) treated with the alkaloids IMM-2414, IMM-2417, IMM-2435 or with supernatants of EA.hy926 cells (SN) which were grown in the presence of 1 μM IMM-2414, IMM-2417, IMM-2435.

FIG. 9: Proliferation of normal human T lymphocytes, treated with IMM-2414 and/or IMM-2418, or with supernatants of EA.hy926 cells (SN) which were grown in the presence of 1 μM IMM-2414 and/or IMM-2418.

FIG. 10: Antagonistic effect of tetracyclic oxindole alkaloids (TOA) on the biological activity on Raji and Jurkat cells caused by pentacyclic oxindole alkaloids (POA).

DETAILED DESCRIPTION OF THE INVENTION

Thin-layer Chromatographic Identification Test:

Thin layer chromatography provides an excellent method for the test of identity of the drug, especially when the characteristic pH-dependent isomerization behavior of the oxindole alkaloids is taken as an additional criterion. Ajmalicine is proposed as a reference substance because of its similar structure and commercial availability. In order to compensate for variations in chromatographic conditions the Rf values are referred to ajmalicine (hRajmalicine values, Table 1).

TABLE 1
hRajmalicine values of the alkaloids
Alkaloid           hRajmalicine
Speciophylline     12
Mitraphylline      26
Uncarine F         56
Isomitraphylline   75
Pteropodine        83
Isopteropodine     95
Rhynchophylline    22
Isorhynchophylline 91
Ajmalicine         100

Test solutions: 1 g of the drug is heated with 50 ml distilled water for 45 minutes at 85° C. The extract is decanted and the drug is washed with 20 ml water. The combined extracts are divided into two portions. One portion is acidified by the addition of 1 drop of hydrochloric acid 7% (approx. pH 4, solutions 1, 3 and 6) and refluxed for 24 h, the second portion is made alkaline with 1 drop of sodium hydroxide solution 8.5% (approx. pH 8, solutions 2, 4 and 7) and maintained at 50° C. for 24 h. Afterwards 2 drops of sodium hydroxide solution 8.5% are added to the acidic solution. All solutions are extracted with 3×5 ml chloroform, collecting at least 4 ml of the organic layer in each extraction step. The extracts are dried by the addition of anhydrous sodium sulfate and the solvent is evaporated. The residues are dissolved in 0.5 ml chloroform and the resulting solutions are used for thin layer chromatography. 1 mg ajmalicine (Fluka, Switzerland) is dissolved in 1 ml chloroform to give the reference solution (solution 5). Spots of 10 μl are applied to TLC-plastic sheets of silica 60 F254 (20×20 cm, 0.2 mm thickness of layer; Merck No. 5735). A mixture of ethyl acetate/n-hexane (95:5) is used to develop the chromatogramme, and fluorescence quenching at 254 nm is used for detection.

The possible cases are depicted in FIG. 1. In case of the pentacyclic alkaloid-type U. tomentosa spots of six alkaloids are observed. Speciophylline, mitraphylline and pteropodine dominate in solution 1 which was isomerized in acid (column 1), while isopteropodine and isomitraphylline prevail in solution 2 which was isomerized in alkali (column 2). The undesired alkaloids rhynchophylline and isorhynchophylline from tetracyclic alkaloid-type U. tomentosa can be seen clearly and exhibit similar dependance on the pH of the isomerized solutions 3 and 4 as mitraphylline and isomitraphylline. Column 5 is the reference compound ajmalicine. Columns 6 and 7 indicate typical drug mixtures. An HPLC chromatogramme which allows the analysis of pentacyclic and tetracyclic oxindole alkaloids is shown in FIG. 2. Method: LiChroCART 125 mm×4 mm (I.D.) columns packed with LiChrospher 100 RP-18 (5 μm) (Merck), thermostatted at 52° C., acetonitrile-aqueous phosphate buffer pH7 (40:60) with a flow of 1.3 ml/min. Detection at 247 nm.

Composition of Alkaloid Mixtures Used

As oxindole alkaloids undergo isomerization in aqueous solution, no single isomers but groups of isomers were employed. First, a mixture of pentacyclic alkaloids (IMM-2414) was used, then the isomer groups of mitraphylline (IMM-2417), rhynchophylline (IMM-2418) and pteropodine (IMM-2435) were used. The composition of the mixtures is given in Table 2. The composition of the alkaloid mixtures was determined by HPLC analyses.

TABLE 2
Composition of alkaloid mixtures used
	Code			IMM-
Alkaloid	IMM-2414	IMM-2417	IMM-2418	2435
Speciophylline	4%	—	—	4%
Uncarine F	6%	—	—	6%
Pteropodine	28%	—	—	30%
Isopteropodine	57%	—	—	60%
Mitraphylline	2%	33%	—	—
Isomitraphylline	3%	67%	—	—
Rhynchophylline	—	—	40%	—
Isorhynchophylline	—	—	60%	—
Comments:
The percent quotations are percents by weight.
The composition of the alkaloid mixtures was determined by HPLC analysis.

Simple derivatives were also used: the alkaloid carboxylic acids (IMM-2413) prepared by alkaline hydrolysis of the alkaloid mixture (IMM-2414), and the alkaloid N-oxides (IMM-2433) prepared by oxidation of the mixture (IMM-2414) using hydrogen peroxide.

Distribution of the Alkaloids in Biological Systems

Cells in a culture medium can be viewed as a two-phase system consisting of water and lipids. The distribution of the alkaloids in cell cultures and in mixtures of octanol and water was studied. It was found that the various isomers behave differently (Table 3). The alkaloids were partitioned between equal volumes of octanol and aqueous phosphate buffer pH 7 (0.01 M) at 20° C. The concentrations c of the alkaloids were determined by HPLC analysis and the coefficients of distribution $KO/W=\frac{c_{\left(\mathrm{in}\mathrm{control}\right)}}{c_{\left(\mathrm{in}\mathrm{water}\right)}}$

were calculated.

TABLE 3
Common logarithm of the partition coefficients K O/W at pH 7
Alkaloid           log K O/W
Pteropodine        2.9
Isopteropodine     3.2
Speciophylline     1.4
Uncarine F         3.1
Mitraphylline      2.5
Isomitraphylline   2.8
Rhynchophylline    2.7
Isorhynchophylline 3.1

As expected the equilibrium of isomers in a two-phase system (1aq1org; 2aq2org, FIG. 3) depends not only on the pH value of the aqueous phase but also on the amount and nature of the organic phase. For an example, the equilibria of mitraphylline and isomitraphylline in an octanol-water system (1:1) at pH values from 3 to 7 are shown (FIG. 4). It can be seen that in the pH range from 4 to 7 isomitraphylline is predominant in the octanol phase, with a maximum of 94 mol-% at approximately pH 5. However, below pH 3 mitraphylline begins to prevail in the aqueous phase. At no pH value is isomitraphylline produced in the aqueous phase to a reasonable extent. Therefore, the distribution of the isomers in a 2-phase system is clearly different compared to the situation in a purely aqueous solution.

In EA.hy926 endothelial cell cultures which were incubated with various alkaloid mixtures (c≈1 μM) a decline of the concentrations of isopteropodine or isomitraphylline, respectively, was observed after 7 days, whereas in contrast the concentration of isorhynchophylline remained nearly constant (Table 4). A RPMI-1640 culture medium (Sigma-Aldrich Company, St. Louis, USA) completed with 10% by volume fetal calf serum, 2 mM glutamin, 50 units/ml penicillin G, and 50 μg/ml streptomycin was used.

TABLE 4
Change of the alkaloid concentrations (mg/l) in the RPMI-1640
nutrient medium of EA.hy926 endothelial cell cultures after 7 days
Stimulant (concentration)	Alkaloid	Start	7 Days
IMM-2414 (1.0 μM)	Isopteropodine	0.22	0.12
	Pteropodine	0.10	0.16
	Total	0.32	0.28
IMM-2417 (1.0 μM)	Isomitraphylline	0.28	0.14
	Mitraphylline	0.10	0.11
	Total	0.38	0.25
IMM-2418 (1.1 μM)	Isorhynchophylline	0.27	0.27
	Rhynchophylline	0.15	0.13
	Total	0.42	0.40
IMM-2435 (1.4 μM)	Isopteropodine	0.35	0.18
	Pteropodine	0.11	0.20
	Total	0.46	0.38
Comments:
IMM-2414 = solution of standard mixture of pentacyclic alkaloids in medium
IMM-2417 = solution of mixture of isomers of mitraphylline in medium
IMM-2418 = solution of mixture of isomers of rhynchophylline in medium
IMM-2435 = solution of mixture of isomers of pteropodine in medium

The solubility of the alkaloids in cell membranes does not offer an explanation for the different changes of concentration. Rather, the decline in concentration is a consequence of physiological processes in the cytosol. Compared with pure medium, the isomerization takes a different course in the presence of endothelial cells. Within the 7 days of an experiment (EA.hy926 endothelial cells in RPMI-1640: pH 7.5 at the start, pH 8.1 after 7 days) an untypical mixture of isomers is formed which contains pteropodine and isopteropodine or mitraphylline and isomitraphylline, respectively, in a ratio of approx. 1:1, whereas in contrast rhynchophylline and isorhynchophylline isomerize to give a typical equilibrium mixture in a ratio of 1:2. Of course, activity of single isomers cannot be evaluated in this test model because of the isomerization. But it can be established that a turnover takes place in the case of the pentacyclic but not tetracyclic alkaloids. The effects of this factor on lymphocytes were investigated in detail. It was found that immortalized cells, e.g. the Epstein-Barr virus-transformed lymphoblastoid cell line Raji or the leukaemic cell line Jurkat, and normal human B and T lymphocytes (isolated from whole blood of normal donors) are affected by the factor in different ways:

1. Supernatants (SN) of endothelial cell cultures stimulated with IMM-2414 for 7 days were added to normal human non-activated or weakly activated B and T lymphocyte cultures in several concentrations. An increased proliferation of the lymphocytes was measured by [3H]thymidine uptake after 5 days (Table 5). Thus, the lymphocytes were treated with 1 μCi [3H]thymidine for 18 hours, harvested on nitrocellulose, and radioactivity was measured in a scintillation counter (cpm=counts per minute). Every assay was performed in triplicate.

TABLE 5
Proliferation (cpm after [3H]thymidine uptake) of normal human non-
activated or weakly activated B and T lymphocytes in med. RPMI-1640
Stimulant (dilution)	B lymphocytes	T lymphocytes
Medium	363 ± 213	349 ± 114
IMM-2414 (1 μM)	352 ± 323	332 ± 82
and stimulated with EA.hy926 endothelial cell culture supernatants
SN-Medium (1:4)	1015 ± 618	591 ± 252
SN-2414 (1:4)	1527 ± 540	1242 ± 752***
SN-Medium (1:8)	1381 ± 390	493 ± 278
SN-2414 (1:8)	2039 ± 530***	1084 ± 549****
SN-Medium (1:16)	1263 ± 299	371 ± 151
SN-2414 (1:16)	1795 ± 584*	549 ± 250**
Comments:
Medium = RPMI-1640 completed as specified above
IMM-2414 = solution of standard mixture of pentacyclic alkaloids
in medium
SN-Medium = supernatant of endothelial cell culture in medium,
diluted with medium in the ratio given
SN-2414 = supernatant of endothelial cell culture stimulated with
the standard mixture of pentacyclic alkaloids in medium for 7 days,
diluted with medium in the ratio given
Mean values ± standard deviation of at least 7 experiments are given.
Significance was evaluated with Student's t-test for paired samples:
*P < 0.05,
**P < 0.01,
***P < 0.005,
****P < 0.001.

It can be seen that the alkaloids alone do not have an effect compared to the blank medium. The supernatants of non-stimulated endothelial cell cultures (SN-Medium) increase the proliferation, and the supernatants of cells stimulated with IMM-2414 increase the proliferation even more. The maximum effect was obtained with T lymphocytes at a dilution of 1:8 and with B lymphocytes at 1:4 of the supernatant SN-2414.

2. Supernatants of endothelial cell cultures (SN-2414) stimulated with IMM-2414 for 7 days and non-stimulated endothelial cell cultures (SN-medium) were added to transformed cells (Raji ATCC CCL86 and Jurkat ATCC E6.1) in several concentrations. In contrast to the B and T lymphocytes, an inhibition of proliferation of the transformed cells was measured by [3H]thymidine uptake after 2 days (Table 6). Cultures of the myeloid cell line U937 (ATCC CRL1593.2) were also studied. The transformed cells were treated with 0.5 μCi [3H]thymidine for 5 hours, harvested on nitrocellulose, and radioactivity was measured in a scintillation counter (cpm=counts per minute).

TABLE 6
Proliferation (cpm after [3H]thymidine uptake) of various cell lines in
medium RPMI-1640
Stimulant
(dilution)	Raji CCL86 a	Jurkat E6.1 a	U937 CRL1593.2 b
Medium	21621 ± 5755	35085 ± 13876	121349 ± 18653
IMM-	21698 ± 6299	35688 ± 14020	123346 ± 26412
2414
(1 μM)
and under the influence of EA.hy926 endothelial cell
culture supernatants
SN-	32967 ± 9652	26544 ± 17492	86115 ± 30792
Medium
(1:2)
SN-2414	4801 ± 3766****	4282 ± 3186**	84736 ± 33654
(1:2)
SN-	30919 ± 11134	34716 ± 17394	101093 ± 24813
Medium
(1:4)
SN-2414	9178 ± 7671****	7163 ± 6268***	94257 ± 30331
(1:4)
SN-	21976 ± 7443	41428 ± 16648	118634 ± 19980
Medium
(1:8)
SN-2414	11357 ± 5308*	8044 ± 3921***	107771 ± 30975
(1:8)
Comments:
Medium = RPMI-1640 completed as specified above
IMM-2414 = solution of standard mixture of pentacyclic alkaloids in medium
SN-Medium = supernatant of endothelial cell culture in medium, diluted with medium in the ratio given
SN-2414 = supernatant of endothelial cell culture stimulated with the standard mixture of pentacyclic alkaloids in medium for 7 days, diluted with medium in the ratio given
Mean values ± standard deviation of at least a 7 experiments, b 3 experiments are given.
Significance was evaluated with Student's t-test for paired samples:
*P < 0.05,
**P < 0.01,
***P < 0.005,
****P < 0.001.

It can be seen that the alkaloids alone do not have an effect compared to the blank medium. The supernatants of the IMM-2414-stimulated endothelial cell cultures inhibit the profileration of Raji and Jurkat cells dose-dependently, whereas the myeloid cell line U937 is not affected.

3. The anti-proliferative effect on Raji and Jurkat cells is not due to cytotoxicity, as shown by unchanged viability of the cells (Table 7).

TABLE 7
Viability (in %) of Raji and Jurkat cells after stimulation with IMM-
2414 or supernatants of EA.hy926 endothelial cell cultures which
were cultivated with IMM-2414
	Raji CCL86		Jurkat E6.1
Stimulant (dilution)	1st day	2nd day	1st day	2nd day
Medium	93.5	95.1	95.8	93.7
IMM-2414 (1 μM)	96.8	90.1	95.2	95.8
and under the influence of EA.hy926 endothelial cell culture supernatants
SN-Medium (1:2)	95.4	95.3	96.4	92.0
SN-2414 (1:2)	98.4	92.2	94.4	92.2
Comments:
Medium = RPMI-1640 completed as specified above
IMM-2414 = solution of standard mixture of pentacyclic alkaloids
in medium
SN-Medium = supernatant of endothelial cell culture in medium, diluted
with medium in the ratio given
SN-2414 = supernatant of endothelial cell culture stimulated with the
standard mixture of pentacyclic alkaloids in medium for 7 days, diluted
with medium in the ratio given

The viability of the cells was determined by trypan blue exclusion after 1 and 2 days of stimulation with IMM-2414. In all cases the viability was higher than 90%.

4. Supernatants of endothelial cell cultures stimulated with IMM-2414, IMM-2417 or IMM-2435 for 7 days (SN-2414, SN-2417, SN-2435) and non-stimulated (SN-medium) were added to cultures of human highly activated T lymphocytes (lymphoblasts) in several concentrations (Table 8).

TABLE 8
Proliferation (cpm after [3H]thymidine uptake) of human highly
activated T lymphocytes (lymphoblasts) in medium RPMI-1640
Stimulant (dilution) T lymphoblasts
Medium               4065
IMM-2414             4557
IMM-2417             3929
IMM-2435             4124
and under the influence of EA.hy926 endothelial cell supernatants
SN-medium (1:4)      3113
SN-2414 (1:4)        652
SN-2417 (1:4)        2285
SN-2435 (1:4)        1887
SN-medium (1:8)      3139
SN-2414 (1:8)        2628
SN-2417 (1:8)        1595
SN-2435 (1:8)        2380
SN-medium (1:16)     2320
SN-2414 (1:16)       1852
SN-2417 (1:16)       1913
SN-2435 (1:16)       1821
Comments:
Medium = RPMI-1640 completed as specified above
IMM-2414 = solution of standard mixture of pentacyclic alkaloids
in medium
IMM-2417 = solution of mixture of isomers of mitraphylline in medium
IMM-2435 = solution of mixture of isomers of pteropodine in medium
SN-medium = supernatant of endothelial cell culture in medium, diluted
with medium in the ratio given
SN-2414 = supernatant of endothelial cell culture stimulated with the
standard mixture of pentacyclic alkaloids in medium for 7 days, diluted
with medium in the ratio given
SN-2417 = supernatant of endothelial cell culture stimulated with the
mixture of isomers of mitraphylline in medium for 7 days, diluted with
medium in the ratio given
SN-2435 = supernatant of endothelial cell culture stimulated with the
mixture of isomers of pteropodine in medium for 7 days, diluted with
medium in the ratio given.
All values result from single experiments.

It can be seen that the alkaloids alone do not have an effect compared to the blank medium. The supernatants of the non-stimulated endothelial cell cultures (SN-medium) already inhibit the proliferation, the supernatants (SN-2414, SN-2417, SN-2435) of endothelial cell cultures stimulated with IMM-2414, IMM-2417, IMM-2435 further enhance this effect (FIG. 8). The dose dependance of the effect is clearly seen.

5. Supernatants of endothelial cell cultures (SN-2412) stimulated with IMM-2414 for 7 days and non-stimulated (SN-medium) were added to highly activated B or T lymphocyte cultures (lymphoblasts from peripheral blood or tonsils) in several concentrations. Table 9 shows an inhibition of proliferation of the lymphocytes, measured by [3H]thymidine uptake (cpm=counts per minute).

TABLE 9
Inhibition of the proliferation (cpm after [3H]thymidine uptake)
of highly activated human B and T lymphocytes (lymphoblasts)
in medium RPMI-1640
	Stimulant (dilution)	B lymphoblasts	T lymphoblasts
	Medium	17840	2186
	IMM-2414 (1 μM)	16610	2097
and under the influence of EA.hy926 endothelial cell culture supernatants
	SN-Medium (1:8)	9594	1254
	SN-2414 (1:8)	1527	884
	SN-Medium (1:16)	13865	1554
	SN-2414 (1:16)	1699	728
	SN-Medium (1:32)	13903	2049
	SN-2414 (1:32)	2534	720
Comments:
Medium = RPMI-1640 completed as specified above
IMM-2414 = solution of standard mixture of pentacyclic alkaloids
in medium
SN-Medium = supernatant of endothelial cell culture in medium,
diluted with medium in the ratio given
SN-2414 = supernatant of endothelial cell culture stimulated with
the standard mixture of pentacyclic alkaloids in medium for 7 days,
diluted with medium in the ratio given
All values result from single experiments.

It can be seen that the alkaloids alone do not have an effect compared to the blank medium. The supernatants of the non-stimulated endothelial cell cultures (SN-Medium) already inhibit the proliferation, the supernatants of the IMM-2414-stimulated endothelial cell cultures (SN-2414) further enhance this effect (FIG. 7). The dose dependance of the effect is clearly seen.

In another experiment, supernatants of HUVEC endothelial cell cultures stimulated with IMM-2414 for 7 days (SN-2414) and non-stimulated (SN-medium) were added to T lymphocyte cultures in several concentrations. The influence on proliferation of the lymphocytes was measured by [3H]thymidine uptake (cpm=counts per minute). The results are shown in Table 10.

TABLE 10
Proliferation (cpm after [3H]thymidine uptake) of human normal T
lymphocytes in medium HAM F12
Stimulant (dilution) T lymphocytes
Medium               884
IMM-2414 (2 μM)      885
and under the influence of supernatants from HUVEC endothelial cell
cultures
SN-Medium (1:4)      1339
SN-2414 (1:4)        1887
SN-Medium (1:8)      1106
SN-2414 (1:8)        1509
SN-Medium (1:16)     913
SN-2414 (1:16)       1279
Comments:
Medium = HAM F12 completed as specified above
IMM-2414 = solution of standard mixture of pentacyclic alkaloids
in medium
SN-Medium = supernatant of endothelial cell culture in medium,
diluted with medium in the ratio given
SN-2414 = supernatant of endothelial cell culture stimulated with
the standard mixture of pentacyclic alkaloids in medium for 7 days,
diluted with medium in the ratio given
All values result from single experiments.

It can be seen that the alkaloids alone do not have an effect compared to the blank medium. The supernatants of the non-stimulated endothelial cell cultures (SN-medium) already increase the proliferation, the supernatants of the IMM-2414-stimulated endothelial cell cultures (SN-2414) further enhance this effect. The dose dependance of the effect is clearly seen. Thus, activities produced by HUVEC culture supernatants are somewhat weaker but significant, too.

7. The release of the growth-factor was effected by the groups of isomers of the pentacyclic alkaloids pteropodine or mitraphylline (IMM-2414, IMM-2417 or IMM-2435), but not by the group of isomers of the tetracyclic alkaloid rhynchophylline (IMM-2418), as can be seen in Table 11. Supernatants of endothelial cell cultures stimulated with IMM-2414, IMM-2417 or IMM-2418 for 7 days (SN-2414, SN-2417, SN-2418) and non-stimulated (SN-medium) were added to cell cultures in several concentrations. The influence on proliferation of the cells was measured by [3H]thymidine uptake (cpm=counts per minute).

TABLE 11
Proliferation (cpm after [3H]thymidine uptake) of Jurkat cells (ATCC
E6.1) and normal human B lymphocytes in medium RPMI-1640
			B
Stimulant (dilution)	Jurkat E6.1 a	Stimulant (dilution)	lymphocytes b
Medium	32737	Medium	632
IMM-2414 (1 μM)	35688	IMM-2414 (1 μM)	560
IMM-2417 (1 μM)	33700	IMM-2417 (1 μM)	606
IMM-2418 (1 μM)	31440	IMM-2418 (1 μM)	501
and stimulated with supernatants of EA.hy926 cells
SN-Medium (1:2)	21673	SN-Medium (1:8)	1639
SN-2414 (1:2)	4282	SN-2414 (1:8)	2082
SN-2417 (1:2)	3953	SN-2417 (1:8)	2183
SN-2418 (1:2)	15724	SN-2418 (1:8)	1908
SN-Medium (1:4)	25288	SN-Medium (1:16)	1306
SN-2414 (1:4)	7163	SN-2414 (1:16)	1617
SN-2417 (1:4)	6068	SN-2417 (1:16)	2289
SN-2418 (1:4)	26132	SN-2418 (1:16)	1474
SN-Medium (1:8)	28124	SN-Medium (1:32)	1231
SN-2414 (1:8)	8044	SN-2414 (1:32)	1505
SN-2417 (1:8)	11783	SN-2417 (1:32)	2258
SN-2418 (1:8)	30190	SN-2418 (1:32)	1437
Comments:
Medium = RPMI-1640 completed as specified above
IMM-2414 = solution of standard mixture of pentacyclic alkaloids
in medium
IMM-2417 = solution of mixture of isomers of mitraphylline in medium
IMM-2418 = solution of mixture of isomers of rhynchophylline in medium
SN-medium = supernatant of endothelial cell culture in medium,
diluted with medium in the ratio given
SN-2414 = supernatant of endothelial cell culture stimulated with the
standard mixture of pentacyclic alkaloids in medium for 7 days, diluted
with medium in the ratio given
SN-2417 = supernatant of endothelial cell culture stimulated with the
mixture of isomers of mitraphylline in medium for 7 days, diluted with
medium in the ratio given
SN-2418 = supernatant of endothelial cell culture stimulated with the
mixture of isomers of rhynchophylline in medium for 7 days, diluted with
medium in the ratio given
a Mean values of at least 3 parallel experiments.
b Results of single experiments.

The alkaloids alone do not have an effect compared to the blank medium. The supernatants of the non-stimulated endothelial cell cultures (SN-medium) already inhibit the proliferation of the Jurkat cells (FIG. 6) and increase the proliferation of the B lymphocytes (FIG. 5). The supernatants of the IMM-2414 and IMM-2417-stimulated endothelial cell cultures (SN-2414, SN-2417) further enhance this effect. The dose dependance of these effects are clearly seen. It is to notice that supernatants of endothelial cell cultures stimulated with IMM-2417 increase the proliferation of B lymphocytes even when diluted in the ratio 1:16 or 1:32, whereas the activity of endothelial cell cultures stimulated with IMM-2414 already decreases (SN-2414). The supernatants of the IMM-2418-stimulated endothelial cell cultures (SN-2418) produce no effect compared to supernatants of non-stimulated endothelial cell cultures (SN-medium).

8. Rather it was shown that the tetracyclic alkaloids act antagonistically on the production and/or release of the growth-factor. This could be connected with their known capability of blocking Ca2+ transport. Furthermore, they reduce the influence of the factor on the proliferation of T-lymphocytes in a dose-dependent manner (addition of 1 pM IMM-2418 to an active supernatant reduces the activity by 10%, 10 μM by 20%). Results are shown in Table 12. Supernatants of endothelial cell cultures stimulated with IMM-2414 and/or IMM-2418 for 7 days (SN-2414, SN-2418, SN-2414/2418 and SN-2414/10×2418) and non-stimulated (SN-medium) were added to T lymphocyte cultures in several concentrations. The influence on proliferation of the lymphocytes was measured by [3H]thymidine uptake (cpm=counts per minute).

TABLE 12
Proliferation (cpm after [3H]thymidine uptake) of human normal T
lymphocytes in medium RPMI-1640
Stimulant (dilution)         T lymphocytes
Medium                       598 ± 429
IMM-2414 (1 μM)              590 ± 420
IMM-2418 (1 μM)              564 ± 409
IMM-2414 (1 μM)/2418 (1 μM)  536 ± 416
IMM-2414 (1 μM)/2418 (10 μM) 602 ± 504
and under the influence of EA.hy926 endothelial cell supernatants
SN-Medium (1:4)              1900 ± 1603
SN-2414 (1:4)                2694 ± 1662
SN-2418 (1:4)                1956 ± 1618
SN-2414/2418 (1:4)           1890 ± 1712
SN-2414/10 × 2418 (1:4)      1479 ± 1191
SN-Medium (1:8)              1581 ± 1448
SN-2414 (1:8)                2144 ± 1402
SN-2418 (1:8)                1588 ± 1393
SN-2414/2418 (1:8)           1698 ± 1644
SN-2414/10 × 2418 (1:8)      1692 ± 1422
Comments:
Medium = RPMI-1640 completed as specified above
IMM-2414 = solution of standard mixture of pentacyclic alkaloids in
medium
IMM-2418 = solution of mixture of isomers of rhynchophylline in medium
SN-Medium = supernatant of endothelial cell culture in medium, diluted
with medium in the ratio given
SN-2414 = supernatant of endothelial cell culture stimulated with the
standard mixture of pentacyclic alkaloids in medium for 7 days, diluted
with medium in the ratio given
SN-2418 = supernatant of endothelial cell culture stimulated with the
mixture of isomers of rhynchophylline in medium for 7 days, diluted with
medium in the ratio given
SN-2414/2418 = supernatant of endothelial cell culture stimulated with
the standard mixture of pentacyclic alkaloids and the mixture of isomers
of rhynchophilline in equal parts in medium for 7 days, diluted with
medium in the ratio given
SN-2414/10 × 2418 = supernatant of endothelial cell culture stimulated
with the standard mixture of pentacyclic alkaloids and the mixture of
isomers of rhynchophilline (1:10) in medium for 7 days, diluted with
medium in the ratio given.
The values represent mean values + standard deviation of 7 experiments.

It can be seen that the alkaloids alone do not have an effect compared to the blank medium. The supernatants of the non-stimulated endothelial cell cultures (SN-medium) already increase the proliferation, the supernatants of the IMM-2414-stimulated endothelial cell cultures (SN-2414) further enhance this effect. The supernatants of the IMM-2418 stimulated endothelial cell cultures (SN-2418) do not have an effect compared to the supernatants of non-stimulated endothelial cell cultures (SN-medium). The supernatants of the IMM-2414 and IMM-2418-stimulated endothelial cell cultures (SN-2414/2418) do not have an effect, either, compared to the supernatants of non-stimulated endothelial cell cultures (SN-medium). IMM-2418 therefore cancels the effect of IMM-2414 (FIG. 9). Highly concentrated (diluted 1:4) supernatants of endothelial cell cultures stimulated with tenfold concentrated IMM-2418 (SN-2414/10×2418) produce of slight inhibition of proliferation compared to supernatants of non-stimulated cultures (SN-medium).

9. Admixture of 0.01, 0.1 and 1 μM tetracyclic oxindole alkaloids to 1 μM pentacyclic oxindole alkaloids (pteropodine isomers as well as mitraphylline isomers) as stimulant reduced the effect of the supernatants on Raji and Jurkat cells in a dose-dependent manner. Supernatants (SN) of endothelial cell cultures stimulated with IMM-2417, IMM-2435 and/or IMM-2418 for 7 days and non-stimulated (SN-medium=control) were added to transformed lymphoblastoid Raji and Jurkat cell cultures. The inhibition of proliferation of the cells was measured by [3H]thymidine uptake (cpm=counts per minute). The tetracyclic oxindole alkaloids act antagonistically on the pentacyclic oxindole alkaloids in a dose-dependent manner (FIG. 10). This could be connected with their known capability of blocking Ca2+ transport. Furthermore, they reduce the influence of the factor on the proliferation of T-lymphocytes in a dose-dependent manner (addition of 1 μM IMM-2418 to an active supernatant reduced the activity by 10%, 10 μM by 20%). In Table 13, the values are given in % of the control (SN-Medium) of the proliferation as effected by the mix-supernatants in 3 dilutions (1:4, 1:8, and 1:16).

TABLE 13
Proliferation of lymphoblastoid cell lines Raji CCL86 and Jurkat
E6.1 after treatment with EA.hy936 endothelial cell culture
supernatants (% of control ± s.d.)
	Jurkat E6.1	Raji CCL86
SN-2417 (1 μM)	52 ± 2**	36 ± 6***
SN-[2417 (1 μM) + 2418 (0.01 μM)]	74 ± 4**	57 ± 1***
SN-[2417 (1 μM) + 2418 (0.1 μM)]	88 ± 3*	73 ± 3**
SN-[2417 (1 μM) + 2418 (1 μM)]	89 ± 19	85 ± 16
SN-2418 (1 μM)	104 ± 1	99 ± 5
SN-2435 (1 μM)	50 ± 2*	32 ± 3***
SN-[2435 (1 μM) + 2418 (0.01 μM)]	78 ± 24	53 ± 4***
SN-[2435 (1 μM) + 2418 (0.1 μM)]	83 ± 16	67 ± 6**
SN-[2435 (1 μM) + 2418 (1 μM)]	87 ± 23	82 ± 16
SN-2418 (1 μM)	113 ± 11	100 ± 5
SN-Medium - control	100	100
Comments:
SN-Medium = supernatant of endothelial cell culture in medium, diluted with medium in the ratio given
SN-2417 = supernatant of endothelial cell culture stimulated with the mixture of isomers of mitraphylline in medium for 7 days in the concentration given
SN-2418 = supernatant of endothelial cell culture stimulated with the mixture of isomers of rhynchophylline in medium for 7 days in the concentration given
SN-2435 = supernatant of endothelial cell culture stimulated with the mixture of isomers of pteropodine in medium for 7 days, diluted with medium in the ratio given
Significantly different from control (Student's t-test):
*p < 0.01,
*p < 0.005,
***p < 0.001;
n = 6.

• 10. Alkaloids alone were added to lymphocytes to exclude the possibility of a direct effect. Endothelial cells were grown without alkaloids and the alkaloids were added to the supernatant in order to prove that stimulation of the cells by the alkaloids is necessary for the production and/or release of the factor. Both experiments showed that neither the alkaloids alone nor in combination with a supernatant of untreated endothelial cells exert an effect on the proliferation of lymphocytes. Thus it was shown that the pentacyclic isomers do not affect directly the proliferation but rather induce endothelial cells to produce and/or release a factor which influences the proliferation of lymphocytes. It is assumed that endothelial cells produce a similar factor even without stimulation because the supernatants of unstimulated cultures do influence the proliferation, although to a minor degree. A lower dosage of pentacyclic oxindole alkaloids (0.1 μM) did not induce the production and/or release of the factor anymore (Table 14).

Supernatants of endothelial cell cultures stimulated with IMM-2414 (c=0.1 μM) for 7 days (SN-2414) and non-stimulated (SN-medium) were added to lymphoblastoid cell cultures in several concentrations. The proliferation of the transformed cells was measured by [3H]thymidine uptake (cpm=counts per minute) after 2 days.

TABLE 14
Proliferation (cpm after [3H]thymidine uptake) of lymphoblastoid
cell lines in medium RPMI-1640
	Stimulant (dilution)	Raji CCL86	Jurkat E6.1
	Medium	27617	42536
	IMM-2414 (0.1 μM)	28708	42779
and under the influence of EA.hy926 endothelial cell culture supernatants
	SN-Medium (1:2)	38556	35917
	SN-2414 (1:2)	36397	35653
	SN-Medium (1:4)	36227	38982
	SN-2414 (1:4)	28558	34048
	SN-Medium (1:8)	30785	40389
	SN-2414 (1:8)	22661	39902
Comments:
Medium = RPMI-1640 completed as specified above
IMM-2414 = solution of standard mixture of pentacyclic alkaloids in
medium
SN-Medium = supernatant of endothelial cell culture in medium, diluted
with medium in the ratio given
SN-2414 = supernatant of endothelial cell culture stimulated with the
standard mixture of pentacyclic alkaloids in medium for 7 days, diluted
with medium in the ratio given

It can be seen that the alkaloids alone do not have an effect compared to the blank medium. The supernatants of low concentration-stimulated endothelial cell cultures (SN-2414) exhibit only a weak influence on the proliferation of the Raji and Jurkat cells. The proliferation-regulating factor binds to interferon-□-antiserum (from sheep) and can be isolated on sepharose.

As the factor enhances the proliferation of normal B and T lymphocytes, it is assumed that it also increases the release of other factors which are normally produced by lymphocytes, e.g. interferon-□, various interleukins or a granulocyte-macrophage-stimulating-factor. Even if the activity of the factor is controlled by immunological regulatory circuits, it can be useful to limit this activity in a specific way. The tetracyclic alkaloids can be employed for this purpose because of their dose-dependent inhibition of the activity of the factor.

In addition, experiments were performed using simple derivatives of the pentacyclic alkaloids. From the mixture (IMM-2414) the corresponding carboxylic acids (IMM-2413) were prepared by alkaline hydrolysis, and alkaloid N-oxides (IMM-2433) by oxidation with hydrogen peroxide. Supernatants of endothelial cell cultures stimulated with IMM-2414, IMM-2413 or IMM-2433 for 7 days (SN-2414, SN-2413, SN-2433) and non-stimulated endothelial call cultures (SN-medium) were added to T lymphocyte cultures in several concentrations. It was demonstrated that the carboxylic acids had only weak activity and the N-oxides almost none (Table 15). Probably these derivatives, due to their higher polarity compared with the parent alkaloids, cannot enter the cells. It is also possible that the free amine and the methyl ester are essential pharmacophores.

TABLE 15
Proliferation (cpm after [3H]thymidine uptake) of human normal
T lymphocytes in medium RPMI-1640
Stimulant (dilution) T lymphocytes
Medium               1724
IMM-2414 (1 μM)      1309
IMM-2413 (1 μM)      876
IMM-2433 (1 μM)      1305
and under the influence of EA.hy926 endothelial cell culture supernatants
SN-Medium (1:4)      2366
SN-2414 (1:4)        4864
SN-2413 (1:4)        2717
SN-2433 (1:4)        2418
SN-Medium (1:8)      2079
SN-2414 (1:8)        3103
SN-2413 (1:8)        2850
SN-2433 (1:8)        2327
SN-Medium (1:16)     2062
SN-2414 (1:16)       2493
SN-2413 (1:16)       2049
SN-2433 (1:16)       1994
Comments:
Medium = RPMI-1640 completed as specified above
IMM-2414 = solution of standard mixture of pentacyclic alkaloids in
medium
IMM-2413 = solution of carboxylic acids prepared from the standard
mixture of pentacyclic alkaloids in medium
IMM-2433 = solution of N-oxides prepared from from the standard
mixture of pentacyclic alkaloids in medium
SN-Medium = supernatant of endothelial cell culture in medium, diluted
with medium in the ratio given
SN-2414 = supernatant of endothelial cell culture stimulated with the
standard mixture of pentacyclic alkaloids in medium for 7 days, diluted
with medium in the ratio given
SN-2413 = supernatant of endothelial cell culture stimulated with the
alkaloid carboxylic acids for 7 days, diluted with medium in the
ratio given
SN-2433 = supernatant of endothelial cell culture stimulated with the
alkaloid N-oxides for 7 days, diluted with medium in the ratio given.
All values result of single experiments.

It can be seen that the alkaloids alone do not have an effect compared to the blank medium. The supernatants of the non-stimulated endothelial cell cultures (SN-medium) already increase the proliferation, the supernatants of the IMM-2414-stimulated endothelial cell cultures (SN-2414) further enhance this effect. The supernatants of the IMM-2413-stimulated endothelial cell cultures (SN-2413) produce only weak effects, the supernatants of the IMM-2433-stimulated endothelial cell cultures (SN-2433) have no effect compared to supernatants of the non-stimulated endothelial cell cultures (SN-medium).

From these investigations and considerations it is seen that the composition of the mixture of isomers cannot be left to chance when a certain action on endothelial cells within a definite time is desired. Elimination from living organisms has to be considered. The different solubility of the isomers in water and lipids has also to be considered when a galenic form is developed. In order to obtain a specific induction of release of the factor the pentacyclic isomers have to be administered in proportions which are adjusted to the physiological equilibrium composition.

In general, RPMI-1640 was used as the culture medium for EA.hy926 endothelial cells and lymphocytes, completed with 10% fetal calf serum, 2 mM glutamin, 50 units/ml penicillin G and 50 μg/ml streptomycin. For HUVEC cultures HAM F12 was used, completed with 10% fetal calf serum, 60 μg/ml Endothelial Cell Growth Supplement and 100 μg/ml heparin. Supernatants of the endothelial cell cultures, stimulated with oxindole alkaloids for 7 days, were diluted with the medium and added to lymphocyte cultures in several concentrations. Proliferation of the lymphocytes was assayed by [3H]thymidine uptake. Thus, normal cells were treated with 1 μCi [3H]thymidine for 18 hours, and transformed cells were treated with 0.5 μCi [3H]thymidine for 5 hours. They were harvested on nitrocellulose, and radioactivity was measured in a scintillation counter. Every assay was performed in triplicate.

In vivo Experiments

An extract of Uncaria tomentosa root containing pentacyclic oxindole alkaloids was administered orally to rats and human volunteers, and the effect on the lymphocyte numbers was studied. The numbers of lymphocytes increased in patients with a suppressed immune system, whereas the lymphocyte count decreased in patients with a highly activated immune system.

EXAMPLES

Normal human umbilical vein endothelial cells (HUVEC, ATCC CRL-1730) are cultivated for 7 days at 37° C. in HAM F12 nutrient medium which was completed with 10% fetal calf serum, 60 μg/ml Endothelial Cell Growth Stimulant and 100 μg/ml heparin. Then the supernatant is taken, filtered sterile, diluted 1:4, and added to cultures of normal human T lymphocytes. The presence of the factor released from the endothelial cells leads within 5 days to an increase of the proliferation of the lymphocytes by 50%.

Transformed EA.hy926 endothelial cells are cultivated in RPMI-1640 nutrient medium which was completed with 10% fetal calf serum, 2 mM glutamine, 50 units/ml penicilline G, and 50 μg/ml streptomycin. Then the supernatant is taken, filtered sterile, diluted 1:4, and added to cultures of normal human B lymphocytes. The presence of the factor released from the endothelial cells leads within 5 days to an increase of the proliferation of the lymphocytes by 180%.

Normal human umbilical vein endothelial cells (HUVEC, ATCC CRL-1730) are cultivated for 7 days at 37° C. in HAM F12 nutrient medium which was completed with 10% fetal calf serum, 60 μg/ml Endothelial Cell Growth Stimulant, 100 μg/ml heparin, and which contains pentacyclic oxindole alkaloids (c=0.4 mg/l). Then the supernatant is taken, filtered sterile, diluted 1:4, and added to cultures of normal human T lymphocytes. The presence of the factor released from the endothelial cells leads within 5 days to an increase of the proliferation of the lymphocytes by 110%.

Transformed EA.hy926 endothelial cells are cultivated for 7 days in RPMI-1640 nutrient medium which was completed with 10% fetal calf serum, 2 mM glutamine, 50 units/ml penicilline G, 50 μg/ml streptomycin, and which contains pentacyclic oxindole alkaloids (c=0.4 mg/l). Then the supernatant is taken, filtered sterile, diluted 1:4, and added to cultures of normal human B lymphocytes. The presence of the factor released from the endothelial cells leads within 5 days to an increase of the proliferation of the lymphocytes by 330%.

Transformed EA.hy926 endothelial cells are cultivated for 7 days in RPMI-1640 nutrient medium which was completed with 10% fetal calf serum, 2 mM glutamine, 50 units/ml penicilline G, 50 μg/ml streptomycin, and which contains pteropodine and isopteropodine (c=0.4 mg/l). Then the supernatant is taken, filtered sterile, diluted 1:4, and added to cultures of leukemic Jurkat cells (ATCC E6.1). The presence of the factor released from the endothelial cells leads within 2 days to an inhibition of the proliferation of the lymphocytes by 80%.

Transformed EA.hy926 endothelial cells are cultivated for 7 days in a nutrient medium which contains mitraphylline and isomitraphylline (c=0.4 mg/l). Then the supernatant is taken, filtered sterile, diluted 1:4, and added to cultures of highly activated T lymphocytes (lymphoblasts). The presence of the factor released from the endothelial cells leads within 2 days to an inhibition of the proliferation of the lymphocytes by 40%.

A dose of 1 g/kg bodyweight of an extract from the root of Uncaria tomentosa mod. pent. which contains approximately 1% pentacyclic oxindole alkaloids is administered orally to healthy rats. Within 28 days a significant (relative and absolute) lymphocytosis develops (in 5 male rats from 86.8% to 90.4%, from 8.4 G/l to 8.5 G/l; in 5 female rats from 83.4% to 88.4%, from 4.9 G/l to 5.7 G/l).

A tumor patient whose lymphocyte count is diminished (relative and absolute lymphopenia, share of lymphocytes: 18% of leucocytes, 1.1 G/l) by chemotherapy (Taxol) is given an extract from the root of Uncaria tomentosa in a dose corresponding to 0.6 mg pentacyclic oxindole alkaloids daily. In spite of continued chemotherapy the lymphocyte counts rise significantly within 1 month (share of lymphocytes: 21% of leucocytes, 1.4 G/l).

A group of patients (n=30) with autoimmune disease received an extract from the root of Uncaria tomentosa corresponding to 0.6 mg pentacyclic oxindole alkaloids daily for one year. Initially, they had a mean total leucocyte count of 8.44 G/l (share of lymphocytes: 21.5%, 1.82 G/l). After half a year of treatment the leucocyte count was 8.66 G/l, the lymphocytes dropped to 18.2%, 1.57 G/l. After one year the leucocyte count was 8.50 G/l, and the lymphocytes remained stable at 18.5%, 1.57 G/l.

Transformed EA.hy926 endothelial cells are cultivated for 7 days at 37° C. in a nutrient medium which contains mitraphylline and isomitraphylline (c=0.4 mg/l) or pteropodine and isopteropodine (c=0.4 mg/l). Then the supernatant is taken, filtered sterile, diluted (1:8, 1:16, 1:32) and added to cultures of normal human B lymphocytes. The supernatant which has been obtained from the mitraphylline-stimulated cells increases the proliferation of the lymphocytes by 260% in all dilutions, whereas the supernatant which has been obtained from the pteropodine-stimulated cells shows a dose-dependent activity (increase of proliferation by 250%, 170%, and 150%, respectively).

Normal human umbilical vein endothelial cells (HUVEC, ATCC CRL-1730) are cultivated for 7 days at 37° C. in HAM F12 nutrient medium which was completed with 10% fetal calf serum, 60 μg/ml Endothelial Cell Growth Stimulant, 100 μg/ml heparin, and which contains pentacyclic oxindole alkaloids (c=0.4 mg/l). Then the supernatant is taken and filtered sterile. 50 μl interferon-□-antiserum (from sheep) in sterile water (c=19000 units/ml) is added per ml of supernatant. The mixture is shaken and incubated for 1 hour at 4° C. Then 25 μl of a 10% protein-A-sepharose suspension is added, incubated for 30 minutes at 4° C. and centrifuged at 2000 g.

From the sediment the factor-antiserum-complex is eluted with a 0.1 M solution of glycin hydrochloride at pH 2.6.

Extracts from the root of Uncaria tomentosa are tested for the absence of tetracyclic oxindole alkaloids by High Performance Liquid Chromatography (HPLC). A RP-18 (5 μm) column (125×4 mm) is used. A mixture of acetonitrile and 0.01 M phosphate buffer pH 7 (40:60) at a flow of 1.3 ml/min is used as the eluent at 52° C. A relable separation of tetracyclic and pentacyclic oxindole alkaloids is achieved. Detection is carried out at 247 nm. Only extracts which contain solely pentacyclic oxindole alkaloids are further processed.

Although endothelial cells are not part of the immune system, they possess the ability to release soluble factors into their environment which affect the behaviour of immune-related cells. It is the object of the present invention to effect the release of such a factor which increases the proliferation of resting or weakly activated lymphocytes and decreases the proliferation of highly activated lymphocytes and transformed lymphoblasts without reducing their viability. It is a further object of this invention to effect the release of this factor by stimulating endothelial cells with pentacyclic oxindole alkaloids. It is yet another object of this invention to limit the release of this factor by the simultaneous administration of tetracyclic oxindole alkaloids which act as antagonists. The production and use of this new proliferation-regulating factor are claimed.

Claims

1. A method of inducing endothelial cells to release a growth-factor that is able to regulate proliferation of lymphocytes, said method comprising stimulating said endothelial cells with at least one pentacyclic oxindole alkaloid.

2. The method according to claim 1, wherein said at least one pentacyclic oxindole alkaloid is selected from the group consisting of pteropodine, isopteropodine, speciophylline, uncarine F, mitraphylline and isomitraphylline.

3. The method according to claim 1, wherein said endothelial cells are in a living subject and said at least one pentacyclic oxindole alkaloid is administered to the living subject.

4. The method according to claim 1, wherein said endothelial cells are cultivated in a nutrient medium containing said at least one pentacyclic oxindole alkaloid.

5. A method of producing a preparation containing a growth-factor that is able to regulate proliferation of lymphocytes, said method comprising stimulating endothelial cells with at least one pentacyclic oxindole alkaloid, whereby said endothelial cells are stimulated to release said growth-factor, and removing the endothelial cells.

6. The method according to claim 5, wherein, when said endothelial cells are cultivated in a nutrient medium, said at least one pentacyclic oxindole alkaloid is added to said nutrient medium to induce a release of said growth-factor into said nutrient medium, and removing the endothelial cells from said nutrient medium.

7. The method according to claim 5, wherein said at least one pentacyclic oxindole alkaloid is selected from the group consisting of pteropodine, isopteropodine, speciophylline, uncarine F, mitraphylline and isomitraphylline.