U.S. patent application number 09/974408 was filed with the patent office on 2002-03-28 for method for treatment of cancer and infectious disease.
Invention is credited to Hellstrand, Kristoffer, Hermodsson, Svante.
Application Number | 20020035998 09/974408 |
Document ID | / |
Family ID | 26709293 |
Filed Date | 2002-03-28 |
United States Patent
Application |
20020035998 |
Kind Code |
A1 |
Hellstrand, Kristoffer ; et
al. |
March 28, 2002 |
Method for treatment of cancer and infectious disease
Abstract
In a method of treating a viral infection involving activation
of NK cells, the improvement comprising administering to a patient
receiving such treatment an effective NK-cell-activity facilitating
amount of a histamine H.sub.2 or 5-HT.sub.1A receptor agonist.
Inventors: |
Hellstrand, Kristoffer;
(Goteborg, SE) ; Hermodsson, Svante; (Molndal,
SE) |
Correspondence
Address: |
KNOBBE MARTENS OLSON & BEAR LLP
620 NEWPORT CENTER DRIVE
SIXTEENTH FLOOR
NEWPORT BEACH
CA
92660
US
|
Family ID: |
26709293 |
Appl. No.: |
09/974408 |
Filed: |
October 9, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09974408 |
Oct 9, 2001 |
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09574874 |
May 19, 2000 |
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6305380 |
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09574874 |
May 19, 2000 |
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09465432 |
Dec 21, 1999 |
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6155266 |
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09465432 |
Dec 21, 1999 |
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09033110 |
Mar 2, 1998 |
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6003516 |
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09033110 |
Mar 2, 1998 |
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08374787 |
May 8, 1995 |
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5728378 |
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Current U.S.
Class: |
128/898 |
Current CPC
Class: |
A61K 38/212 20130101;
A61K 38/212 20130101; A61K 2300/00 20130101; A61K 45/06 20130101;
A61P 31/12 20180101 |
Class at
Publication: |
128/898 |
International
Class: |
A61B 019/00 |
Claims
What is claimed is:
1. A method for the treatment of cancer in a patient through
activation of NK cells, the improvement comprising administering to
the patient an effective NK-cell-activity facilitating amount of a
histamine H.sub.2 or 5-HT.sub.1A receptor agonist.
2. The method of claim 1, wherein the receptor agonist is
histamine.
3. The method of claim 1, wherein the receptor agonist is a
histamine congener.
4. The method of claim 1, wherein the receptor agonist is
serotonin.
5. The method of claim 1, wherein the receptor agonist is selected
from the group consisting of dimaprit, clonidine, tolazoline,
impromadine, 4-methylhistamine, betazole, 8-OH-DPAT
(8-hydroxy-2-(di-n-propylamino)tet- ralin), ALK-3
(cis-8-hydroxy-1-methyl-2-(di-n-propylamino)tetralin), BMY 7378
(8[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4,5]decane-
-7,9-dione), NAN 190
(1-(2-methoxyphenyl-4-[4-(2-phthalimmido)butyl]pieraz- ine HBr),
lisuride, d-LSD, flesoxinan, DHE (dihydroergotamine), MDL 72832
(8-[4-91,4-benzodioxan-2-ylmethyl-amino)butyl]-8-azaspiro[4,5]
decane-7,9-dione), 5-CT (5-carboxamidotryptamine), DP-5-CT
(N,N-dipropyl-5-carboxamidotryptamine), ipsapirone, WB 4101
(2-[[[2-(2,6-dimethoxyphenoxy)ethyl]amino]methyl]-1,4-benzodioxane),
ergotamine, buspirone, metergoline, spiroxatrine, PAPP
(1-[2-(4-aminophenyl)ethyl]-4-(3-trifluoromethylphenyl)
piperazine), SDZ (-) 21009
((4(3-terbutylamino-2-hydroxypropoxy)indol-2-carbonic-acidisopr-
opylester), and butotenine.
6. The method of claim 1, wherein said receptor agonist is
administered in a daily dose of between about 0.1 and 10 mg.
7. The method of claim 1, wherein said cancer is a myeloma.
8. The method of claim 1, wherein said cancer is a renal
cancer.
9. The method of claim 1, wherein said cancer is a leukemia.
10. The method of claim 1, wherein said cancer is a melanoma.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 09/574,874, filed on May 19, 2000, now U.S.
Pat. No. ______, which is a continuation of U.S. patent application
Ser. No. 09/465,432, filed on Dec. 21, 1999, now U.S. Pat. No.
6,155,266, which is a continuation of U.S. patent application Ser.
No. 09/033,110, filed Mar. 2, 1998, now U.S. Pat. No. 6,000,516,
which is a continuation of U.S. patent application Ser. No.
08/374,787, filed May 8, 1995, now U.S. Pat. No. 5,728,378.
FIELD OF THE INVENTION
[0002] The present invention concerns a pharmaceutical preparation
or system for activation of natural killer cells (NK-cells), in
order, for example, to treat tumors or virus infections.
BACKGROUND OF THE INVENTION
[0003] Natural killer cells (NK-cells) are a group of spontaneously
cytotoxic lymphocytes that destroy tumor cells by lysis with no
antigen specificity or restriction by histocompatibility molecules.
Monocytes are involved in the regulation of the NK-cell's function,
both through mechanisms involving cell contact and through
providing soluble NK cell-regulating mediators. Recently, a cell
contact-mediated mechanism has been described whereby monocytes
regulate NK-cells. This type of monocyte-mediated regulation is
exerted by monocytes that are obtained directly from peripheral
blood through counterflow centrifugal elutriation (CCE) and is
regulated by the biogenic amines histamine and serotonin
(Hellstrand and Hermodsson, 1986, J. Immunol. 137, 656-660;
Hellstrand and Hermodsson, 1987, J. Immunol. 139, 869-875;
Hellstrand and Hermodsson, 1990, Scand. J. Immunol. 31, 631-645;
Hellstrand and Hermodsson, 1990, Cell. Immunol. 127, 199-214;
Hellstrand, Kjellson and Herrnodsson, 1991, Cell. Immunol., 138,
44-54). These NK-cell regulating mechanisms caused by biogenic
amines should be of importance to the NK-cell-mediated defense
against metastatic tumors in vivo (Hellstrand, Asea and Hermodsson
(1990), J. Immunology 145, 4365-4370).
[0004] Interferon-.alpha. (IFN-.alpha.) is an important regulating
factor for NK cells. It effectively enhances the NK cell's
cytotoxicity (NKCC) both in vivo and in vitro (Trinchieri, 1989,
Adv. Immunol. 47,187-376; Einhorn, Blomgren and Strander, 1978,
Int. J. Cancer 22, 405-412; Friedman and Vogel, 1984, Adv.
Immunol., 34, 97-140).
[0005] Owing to the high rate of cancer and the only partially
successful treatment methods available today, there is a constant
demand for other improved methods of treatment of tumors. There is
also a great demand for improved treatment methods for virus
infections.
SUMMARY OF THE INVENTION
[0006] The goal of the invention is to create a pharmaceutical
preparation or system that effectively stimulates NK cells, e.g.,
in order to treat tumors, primarily myelomas, renal cancer,
leukemias and melanoma, or to treat virus infections, primarily
chronic hepatitis B and hepatitis C. The preparation or system
according to the invention involves a first composition, containing
interferon-.alpha. or analogues thereof, and a second composition
containing at least one substance with histamine H.sub.2, or
serotonin 5-HT.sub.1A receptor agonist activity, whereby said first
and second compositions are either mixed in a preparation or
supplied in separate doses in an amount sufficient for the intended
treatment. The invention also comprises a method for treatment of
viral or neoplastic disease comprising the step of coadministering
interferon-.alpha. and an effective amount of a histamine H.sub.2
receptor agonist or a serotonin 5-HT.sub.1A receptor agonist.
Furthermore, the invention includes a method for the treatment of
viral infection comprising administering a histamine H.sub.2
receptor agonist or a 5-HT.sub.1A receptor agonist.
[0007] The invention shall be described in greater detail below,
making reference to reported in vitro experiments.
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] FIG. 1 shows in graph form the synergistic NK cell
activation against cultured target cells produced by IFN-.alpha.
and histamine or serotonin for various concentrations of
IFN-.alpha. (0- 100 U/ml).
[0009] FIG. 2 shows in graph form the synergistic NK cell
activation produced against freshly recovered human leukemic cells
by IFN-.alpha. and histamine for various concentrations of
IFN-.alpha. (0-100 U/ml).
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0010] The invention is based on the unexpected discovery that
IFN-.alpha. and the biogenic amines histamine and/or serotonin
produce a synergistic activation of NK cells.
[0011] The experiments reported hereafter show that eluted
monocytes effectively suppress the activation of NK cells induced
by IFN-.alpha.. Furthermore, it is shown that histamine or
serotonin, which act through defined bioaminergic receptors, remove
the monocyte induced suppression and thereby restore the ability of
the NK cells to respond to IFN-.alpha..
[0012] Analogues of histamine with H2-receptor agonist activity or
other compounds with H2-receptor agonist activity and analogues of
serotonin with 5-HT.sub.1A-receptor agonist activity or other
compounds with 5-HT.sub.1A-receptor agonist activity that are
suitable for use in the present invention are known within the art
and shall not be described more closely here. For example, these
analogues can have a chemical structure resembling that of
histamine or serotonin, but modified by addition of groups that do
not negatively affect the H.sub.2 or 5-HT.sub.1A receptor
activities. Known H.sub.2-receptor agonists include histamine,
dimaprit, clonidine, tolazoline, impromadine, 4-methylhistamine,
betazole and histamine congener derivatives such as: 1
[0013] described as compounds 1, 6, and 9 in Khan et al., J.
Immunol., Vol. 137 pp. 308-315. Known serotonin 5-HT.sub.1A
receptor agonists include 8-OH-DPAT
(8-hydroxy-2-(di-n-propylamino)tetralin), ALK-3
(cis-8-hydroxy-1-methyl-2-(di-n-propylamino)tetralin), BMY 7378
(8[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4,5]decane-7,9--
dione), NAN 190
(1-(2-methoxyphenyl-4-[4-(2-phthalimmido)butyl]pierazine HBr),
lisuride, d-LSD, flesoxinan, DHE (dihydroergotamine), MDL 72832
(8-[4-91,4-benzodioxan-2-ylmethyl-amino)butyl]-8-azaspiro[4,5]
decane-7,9-dione), 5-CT (5-carboxamidotryptamine), DP-5-CT
(N,N-dipropyl-5-carboxamidotryptamine), ipsapirone, WB 4101
(2-[[[2-(2,6-dimethoxyphenoxy)ethyl]amino]methyl]-1,4-benzodioxane),
ergotamine, buspirone, metergoline, spiroxatrine, PAPP
(1-[2-(4-aminophenyl)ethyl]-4-(3-trifluoromethylphenyl)
piperazine), SDZ (-) 21009
((4(3-terbutylamino-2-hydroxypropoxy)indol-2-carbonic-acidisopr-
opylester), and butotenine.
[0014] IFN-.alpha. and histamine/serotonin can be administered
separately or in the same preparation. The method of administration
can be either local or systemic injection or infusion. Other
methods of administration can also be suitable.
[0015] The compounds can even be administered intraperitoneally or
in another parenteral method. Solutions of the active compounds in
the form of free acids or pharmaceutically acceptable salts can be
administered in water with or without a tenside such as
hydroxypropylcellulose. Dispersions making use of glycerol, liquid
polyethyleneglycols, or mixtures thereof with oils can be used.
Antimicrobial compounds can also be added to the preparation.
[0016] Injectable preparations may include sterile water-based
solutions or dispersions and powders that can be dissolved or
suspended in a sterile medium prior to use. Carriers such as
solvents or dispersants containing, e.g., water, ethanolpolyols,
vegetable oils and the like can also be added. Coatings such as
lecithin and tensides can be used to maintain suitable fluidity of
the preparation. Isotonic substances such as sugar or sodium
chloride can also be added, as well as products intended to retard
absorption of the active ingredients, such as aluminum monostearate
and gelatin. Sterile injectable solutions are prepared in the
familiar way and filtered before storage and/or administration.
Sterile powders can be vacuum-dried or freeze-dried from a solution
or suspension.
[0017] All substances added to the preparation must be
pharmaceutically acceptable and essentially nontoxic in the
quantities used. The preparation and formulations that produce a
delayed release are also part of the invention.
[0018] The preparation is supplied in dosage units for a uniform
dosage and to facilitate administration. Each dosage unit contains
a predetermined quantity of active components to produce the
desired therapeutic effect, along with the requisite quantity of
pharmaceutical carriers.
[0019] IFN-.alpha. can be administered in a quantity of around 1000
to 300,000 U/kg/day, preferably around 3000 to 100,000 U/kg/day and
more preferably, around 10,000 to 50,000 U/kg/day.
[0020] The compounds with H.sub.2, and 5-HT.sub.1A receptor agonist
activity can be administered in a of quantity of around 0.1 to 10
mg/day, preferably around 0.5 to 8 mg/day and more preferably,
around 1 to 5 mg/day. However other quantities can be administered
with IFN-.alpha., as decided by the treating physician. For
substances other than biogenic amines with corresponding receptor
activity, doses producing an equivalent pharmacological effect
shall be used.
[0021] Although it is stated in the examples that the
administration was given in a single dose, it is obvious that the
compounds can be distributed over longer periods of time for
treatment of virus infections or tumors.
[0022] The daily dose can be administered as a single dose or it
can be divided into several doses, should negative effects
occur.
EXAMPLES
In Vitro Studies of IFN-.alpha. and Histamine/Serotonin
[0023] This example illustrates the effect of human recombinant
IFN-.alpha. and histamine/serotonin, separately and in combination,
on the NK cell cytotoxicity (NKCC) for human mononuclear cells
(MNC).
[0024] MNC were obtained from peripheral venous blood from healthy
human blood donors by Ficoll-Hypaque centrifuging, followed by
Percoll density-gradient fractionation (Timonen and Saksela, 1980,
J. Immunol. Methods 36, 285-291; Hellstrand and Hermodsson, 1990,
Scand. J. Immunol. 31, 631-645).
[0025] In the respective Percoll fractions, the high-density MNC
(Percoll fractions 1-4) were small lymphocytes with low baseline
cytotoxicity against K562 target cells. After removal of the
monocytes, the low-density fractions 6-10 displayed high NKCC,
consistent with earlier studies. (Timonen and Saksela, 1980, J.
Immunol. Methods 36, 285-291).
[0026] The target cells used in these experiments were K562, an
NK-cell sensitive erythroleukemic cell line, or Daudi, a relatively
NK-insensitive EBV-transformed B-cell lymphoblastoid cell line.
[0027] The NKCC was determined six times as the specific
.sup.51Cr-release for a NMNC: target-cell ratio of between 30:1 and
3.8:1 in two-fold dilution gradients. The suspensions of MNC/target
cells were incubated in microplates at 37.degree. C. for 6 hours
(Daudi) or 16 hours (K562). The supernatant solution was then
collected and examined for radioactivity in a gamma counter. The
maximum .sup.51Cr-release was measured in target cell cultures
treated with Triton X-100. The NKCC was calculated as the cell
lysis % by the formula 100.times.(experimental release-spontaneous
release/maximum release-spontaneous release)=cell lysis %.
[0028] A low-density Percoll fraction was separated by counterflow
centrifuge elusion (CCE) in a monocyte and in a lymphocyte
fraction. The monocyte fraction was concentrated to >90% purity
whereupon the contaminating cells consisted of large lymphocytes.
The lymphocyte fractions obtained by CCE contained <3%
monocytes, determined by morphology and Leu-M3 (CD14) antigen
expression. The lymphocytes were CD3.sup.-/16.sup.+/56.sup.+ T
cells (45-50%), CD3.sup.-/16.sup.-/56.sup.-- NK cells (35-40%) ,
CD3.sup.+/16.sup.-/56.sup.- T cells (45-50%),
CD3.sup.+/16.sup.+/56.sup.+ cells (1-5%), determined by flow
cytometry.
[0029] J The eluted monocytes and/or the NK cell-concentrated
low-density lymphocytes were treated with IFN-.alpha. and
histamine/serotonin. The compounds were added, separately or in
combination, to mixtures of MNC and K562 target cells at the start
of a 16-hour .sup.51Cr-release assay. The cytotoxicity against K562
in the NK cell-concentrated lymphocyte fraction was increased by
IFN-.alpha. and unaffected by histamine or serotonin. The eluted
monocyte fraction exhibited a low baseline cytotoxicity and was
slightly induced by histamine/ IFN-.alpha. or serotonin/
IFN-.alpha.; this cytotoxicity resulted from the low fraction of
contaminating lymphocytes (data not given). The addition of eluted
monocytes to the NK cell concentrated lymphocytes suppressed the
baseline cytotoxicity to K562. Furthermore, the eluted monocytes
almost totally inhibited the activation of the cytotoxicity by
means of IFN-.alpha. (Table 1) .
[0030] Histamine and serotonin restored the basal cytotoxicity of
lymphocytes in mixtures of monocytes and lymphocytes. Furthermore,
both histamine and serotonin eliminated the monocyte induced
inhibition of the NK cell response to IFN-.alpha.. Hence,
IFN-.alpha. plus histamine or serotonin synergistically enhance the
cytotoxicity in mixtures of monocytes and NK cell-enriched
lymphocytes (Table 1).
[0031] In the experiments reported in Table 1, eluted lymphocytes
were mixed with monocytes as shown in the table, in a total volume
of 150 .mu.l. The data are NKCC (mean .+-.SEM) of six
determinations. Serotonin 10.sup.-4 M and/or IFN-.alpha. (25 U/ml)
was added at the start of a 16-hour microcytotoxicity test against
10.sup.4 K562 target cells.
1TABLE 1 Suppression of NK Cell Cytotoxicity by Monocytes and
Elimination of This Effect with Serotonin NK CELL CYTOTOXICITY
AFTER TREATMENT WITH (.times.10.sup.-4) Lymphocytes
(.times.10.sup.-4) Control Serotonin IFN Serotonin + IFN 0 12 34
.+-. 1 34 .+-. 3 58 .+-. 3 60 .+-. 2 6 12 10 .+-. 2 31 .+-. 2 17
.+-. 1 52 .+-. 2 12 12 9 .+-. 1 31 .+-. 2 10 .+-. 1 52 .+-. 2
[0032] Table 2 shows the synergistic activation of NK cells by
combined treatment with IFN-.alpha. and histamine. Monocytes were
recovered along with NK cells in low-density Percoll fractions. In
the experiment shown in Table 2, IFN-.alpha. and/or histamine was
added to MNC obtained from these monocyte-containing Percoll
fractions. As was the case with mixtures of eluted monocytes and
low-density lymphocytes, IFN-.alpha. was relatively ineffective in
these cell fractions, while histamine increased the cytotoxicity.
Treatment of monocyte-containing cells with histamine (10.sup.-4 -
10.sup.-6 M) and IFN-.alpha. (25 U/ml) produced a synergistic
NK-boosting response against K562 and against Daudi target cells. A
similar result was obtained when histamine was replaced by
serotonin.
[0033] In the results shown in Table 2, MNC from five different
donors were used. All compounds were added to mixtures of MNC and
target cells at the start of a 6 h (Daudi) or 16 h (K562) effecter
and target cell incubation. The effecter cells were obtained from
Percoll fractions 7-8, containing 33-55% monocytes.
[0034] FIG. 1 shows the synergistic NK cell activation by
IFN-.alpha. and histamine/serotonin for different concentrations of
IFN-.alpha. (0-100 U/ml). Cells from the monocyte-containing
Percoll fraction 8 were incubated with culture medium, histamine
(10.sup.-4 M) or serotonin (10.sup.-4 M) in the presence of
IFN-.alpha. (0-100 U/ml). The data shown are NKCC (cell lysis %;
mean.+-.SEM of six determinations). The compounds were added at the
start of a 16 h microcytotoxicity test against K 562 target
cells.
2TABLE 2 Synergistic Activation of NK Cells by Histamine and
IFN-.alpha. MNC/ target NKCC (cell lysis % .+-. SEM) Target cell
Histamine concentration Exp cell ratio Treatment 0 10.sup.-4 M
10.sup.-5 M 10.sup.-6 M 1 K562 15:1 Medium 33.1 .+-. 0.5 55.5 .+-.
1 54.7 .+-. 1 39.2 .+-. 1 IFN 25 U/ml 33.1 .+-. 1 76.4 .+-. 3 74.1
.+-. 1 66.0 .+-. 2 2 K562 15:1 Medium 20.7 .+-. 0.4 32.4 .+-. 1
27.4 .+-. 1 23.2 .+-. 2 IFN 25 U/ml 27.4 .+-. 1 67.9 .+-. 2 66.2
.+-. 1 55.4 .+-. 1 3 K562 15:1 Medium 31.4 .+-. 1 43.3 .+-. 1 38.6
.+-. 1 29.4 .+-. 1 IFN 25 U/ml 32.5 .+-. 1 71.9 .+-. 1 66.5 .+-. 2
56.3 .+-. 2 4 Daudi 30:1 Medium 1.0 .+-. 0.4 4.4 .+-. 1 3.5 .+-. 2
1.1 .+-. 0.3 IFN 25 U/ml 1.1 .+-. 0.5 31.7 .+-. 1 28.3 .+-. 1 14.1
.+-. 1 5 Daudi 30:1 Medium 2.2 .+-. 1 13.5 .+-. 1 9.7 .+-. 1 2.5
.+-. 1 IFN 25 U/ml 2.7 .+-. 1 61.3 .+-. 3 52.3 .+-. 2 31.7 .+-.
1
[0035] The effect of histamine on monocyte-induced suppression of
resting and IFN-.alpha.-activated NK cells was completely blocked
by simultaneous treatment with the specific H.sub.2R antagonist
ranitidine and imitated by the H.sub.2R agonist dimaprit, which is
shown in Table 3. This means that the effect of histamine on the NK
cell's response to IFN-.alpha. is H.sub.2R-specific.
3TABLE 3 Effects of Histamine and H.sub.2R Agonist Dimaprit and
H.sub.2-Antagonist Ranitidine on NK Cells NKCC (Cell Lysis %) .+-.
SEM AFTER TREATMENT WITH Treatment Control Ran IFN Ran + IFN
Control 0.1 .+-. 0.1 0.0 .+-. 0.1 0.1 .+-. 0.1 0.0 .+-. 0.1
Histamine 9.4 .+-. 0.3 1.5 .+-. 0.3 31.7 .+-. 0.3 1.6 .+-. 0.2
Dimaprit 6.4 .+-. 1 0.4 .+-. 0.4 32.6 .+-. 1 0.5 .+-. 0.5
[0036] In the experiment shown in Table 3, culture medium
(control), histamine (10.sup.-4 M), dimaprit (10.sup.-4 M),
ranitidine (ran) (10.sup.-4 M) and/or IFN-.alpha. (25 U/ml) were
added at the start of a 6-hour .sup.51Cr release assay using Daudi
target cells. The data are representative of three similar
experiments. NKCC is given as mean cell lysis %.+-.SEM of six
determinations. The effecter cells were recovered from a
low-density Percoll fraction 8, containing around 40%
monocytes.
[0037] Serotonin acted synergistically with IFN-.alpha. and had an
effect corresponding to that of histamine. Ranitidine ( 10.sup.-4
M) did not alter the effect of serotonin. The specific synthetic
5-HT.sub.1A R-agonists 8-OH-DPAT and (+) - ALK-3, which lack
activity for 5-HT.sub.18R; 5-HT.sub.10R, 5-HT.sub.2R or
--HT.sub.3R, intensified the baseline NKCC and restored the NK
cell's response to IFN-.alpha. with a potency and effect comparable
to that of serotonin. This is shown in Table 4. Ketanserin and
ondansetron, which are antagonists of 5-HT.sub.2R and 5-HT.sub.3R,
respectively, did not influence the effect of serotonin in
equimolar concentrations.
4TABLE 4 The Effect of Serotonin and 5-HT.sub.1AR Agonists on NK
Cells NKCC AFTER TREATMENT WITH Treatment Medium IFN Medium 1.1
.+-. 1 0.5 .+-. 0.3 Serotonin 10.sup.-4 M 10.4 .+-. 1 44.3 .+-. 1
Serotonin 10.sup.-5 M 4.5 .+-. 0.3 33.2 .+-. 1 Serotonin 10.sup.-6
M 2.2 .+-. 0.4 12.3 .+-. 1 8-OH-DPAT 10.sup.-4 M 8.8 .+-. 1 43.3
.+-. 1 (+)-ALK-3 10.sup.-4 M 9.1 .+-. 1 40.4 .+-. 1
[0038] In the experiment shown in Table 4, culture medium
(control), serotonin, 8-OH-DPAT (+)-ALK and/or IFN-.alpha. (25
U/ml) were added at the start of a 6-hour .sup.51Cr-release assay
against Daudi target cells. The NKCC is given as cell lysis
%.+-.SEM of six determinations. The effector cells were recovered
from the low-density Percoll fraction 7, containing around 36%
monocytes.
[0039] Similar experiments were then performed using freshly
recovered human tumor cells as target cells, rather that the
cultured tumor cell lines used as target cells in the experiments
described above.
[0040] MNC were obtained from peripheral venous blood by
Ficoll-Hypaque centrifuging and the mononuclear cells were
separated into monocytes and NK-cell-enriched lymphocytes
(Hellstrand et al., J. Interferon Res., 12, 199-206 1992). Seventy
thousand NK-cell-enriched lymphocytes were mixed with 70,000
monocytes and 20,000 .sup.51Cr-labeled leukemic target cells (97%
pure acute myelogenous leukemic cells) in a total volume of 150
.mu.l. The cells were treated with culture medium (control) or
histamine dihydrochloride at a final concentration of 10.sup.-4 M,
during a 16 hour .sup.51Cr-release assay to determine killed target
cells.
[0041] The results are shown in FIG. 2. The data are the mean
percent cell lysis of six determinations .+-.SEM. The recorded
cytotoxicity was completely depleted after removal of NK-cells
using DYNABEADS coated with anti-CD56, but not by removal of
T-cells using beads coated with anti-CD3 (Hellstrand et al., Scand.
J. Immunol., 37:7-18 (1993). As seen in FIG. 2, treatment with
interferon alone does not induce killing of leukemic target cells
unless histamine is present. In addition, it has been shown that
the cytotoxic effects obtained with histamine and
interferon-.alpha. are seen not only in cultured tumor cells, but
in freshly recovered human leukemic cells as well.
[0042] Thus, in conclusion, it can be affumed that the
above-described in vitro experiments demonstrate that the biogenic
amines histamine, through H.sub.2 type receptors, and serotonin,
through 5-HT.sub.1A type receptors, abolish the monocyte-induced
suppression of resting and IFN-.alpha. activated NK cells.
Treatment with IFN-.alpha. and compounds with H.sub.2 or HT.sub.1A
receptor agonist activity thus produces a synergistic activation of
NK cells, which can be used in connection with tumor treatment or
treatment of virus infections.
* * * * *