Stable circulating histamine levels

Methods for obtaining beneficial stable levels of circulating histamine are disclosed for use in methods for enhancing natural killer cell cytotoxicity. In such methods, a beneficial level of circulating histamine is attained and an agent whose ability to enhance natural killer cell cytotoxicity is augmented by histamine is administered. Alternatively, stable beneficial levels of circulating histamine can be attained in subjects receiving chemotherapy or antiviral treatment. The invention may also be employed in treatments combining histamine, agents which enhance natural killer cell cytotoxicity, and chemotherapeutic agents.

BACKGROUND OF THE INVENTION 
The present invention relates to methods of treating cancer or infectious 
disease in which histamine is administered in conjunction with additional 
agents. The additional agent may be an agent which stimulates the 
cytotoxic activity of natural killer cells in a synergistic fashion with 
histamine. Alternatively, the additional agent may be a chemotherapeutic, 
antiviral, or antibiotic agent. Methods combining histamine, agents which 
act synergistically with histamine to increase NK cell cytotoxicity, and 
chemotherapeutic agents are also contemplated. 
The invention is based on the surprising observation that despite previous 
reports of histamine's short half life in the body, it is possible to 
attain stable beneficial levels of circulating blood histamine and to 
maintain these beneficial levels for hours or days after the last 
administration of histamine. This observation facilitates treatments in 
which histamine administration to obtain beneficial levels of circulating 
blood histamine is combined with treatment with other agents. A brief 
review of the observations leading to the present invention is provided 
below to place the present invention in context. 
A. Cell Types Involved in the Generation of an Immune Response 
Recent anticancer strategies have focussed on utilizing the host immune 
system as a means of cancer treatment or therapy. The immune system has 
evolved complex mechanisms for recognizing and destroying foreign cells or 
organisms present in the body of the host. Harnessing the body's immune 
mechanisms is an attractive approach to achieving effective treatment of 
malignancies. 
A wide array of effector cells, each having its own characteristics and 
role, implement the immune response. One type of effector cell, the B 
cell, generates antibodies targeted against foreign antigens encountered 
by the host. In combination with the complement system, antibodies direct 
the destruction of cells or organisms bearing the targeted antigen. 
Another type of effector cell, the T cell, is divided into subcategories 
which play different roles in the immune response. Helper T cells secrete 
cytokines which stimulate the proliferation of other cells necessary for 
mounting an effective immune response, while suppressor T cells down 
regulate the immune response. A third category of T cell, the cytotoxic T 
cell, is capable of directly lysing a targeted cell presenting a foreign 
antigen on its surface. 
An additional type of effector cell is the natural killer cell (NK cell), a 
type of lymphocyte having the capacity to spontaneously recognize and 
destroy a variety of malignant cell types. This characteristic of NK cells 
makes them an attractive candidate for exploitation in cancer treatments 
and therapies based on using the host's immune system as a weapon against 
malignant tumors. 
B. Cytokines Involved Mediating the Immune Response 
The interplay between the various effector cells listed above is influenced 
by the activities of a wide variety of chemical factors which serve to 
enhance or reduce the immune response as needed. Such chemical modulators 
may be produced by the effector cells themselves and may influence the 
activity of immune cells of the same or different type as the factor 
producing cell. 
One category of chemical mediators of the immune response is cytokines, 
molecules which stimulate a proliferative response in the cellular 
components of the immune system. 
Interleukin-2 (IL-2) is a cytokine synthesized by T cells which was first 
identified in conjunction with its role in the expansion of T cells in 
response to an antigen (Smith, K. A. Science 240:1169 (1988). Several 
studies have demonstrated that IL-2 has antitumor effects that make it an 
attractive agent for treating malignancies (see e.g. Lotze, M. T. et al, 
in "Interleukin 2", ed. K. A. Smith, Academic Press, Inc., San Diego, 
Calif., p237 (1988); Rosenberg, S., Ann. Surgery 208:121 (1988)). In fact, 
IL-2 has been utilized to treat subjects suffering from malignant 
melanoma, renal cell carcinoma, and acute myelogenous leukemia. 
(Rosenberg, S. A. et al., N. Eng. J. Med. 316:889-897 (1978); Bukowski, R. 
M. et al., J. Clin. Oncol 7:477-485 (1989); Foa, R. et al., Br. J. 
Haematol. 77:491-496 (1990)). 
It appears likely that NK cells are responsible for the anti-tumor effects 
of IL-2. For example, IL-2 rapidly and effectively augments the 
cytotoxicity of isolated human NK cells in vitro (Dempsey, R. A., et al., 
J. Immunol. 129:2504 (1982); Phillips, J. H., et al. J. Exp. Med. 170:291 
(1989)). Thus, the cytotoxic activity of NK cells treated with IL-2 is 
greater than the constitutive levels of cytotoxicity observed in untreated 
cells. Furthermore, depletion of NK-cells from animals eliminates IL-2's 
antitumor effects. (Mule, J. J. et al, J. Immunol. Invest. 139:285 (1987); 
Lotze, M. T. et al., supra). Additional evidence for the role of NK cells 
results from the observation that NK cells are the only resting human 
peripheral blood lymphocytes expressing the IL-2 receptor on their cell 
surface. (Caliguri, M. A. et al., J. Clin. Invst. 91:123-132 (1993). 
Another cytokine with promise as an anti-cancer agent is 
interferon-.alpha.. Interferon .alpha. (IFN-.alpha.) has been employed to 
treat leukemia, myeloma, and renal cell carcinomas. Isolated NK cells 
exhibit enhanced cytotoxicity in the presence of IFN-.alpha.. Thus, like 
IL-2, IFN-.alpha. also acts to augment NK cell mediated cytotoxicity. 
(Trinchieri, G. Adv. Immunol. 47:187-376 (1989)). 
C. In Vivo Results of Histamine and Histamine Agonist Treatments 
Histamine is a biogenic amine, i.e. an amino acid that possesses biological 
activity mediated by pharmacological receptors after decarboxylation. The 
role of histamine in immediate type hypersensitivity is well established. 
(Plaut, M. and Lichtenstein, L. M. 1982 Histamine and immune responses. In 
Pharmacology of Histamine Receptors., Ganellin, C. R. and M. E. Parsons 
eds. John Wright & Sons, Bristol pp. 392-435.) 
Examinations of whether histamine or histamine antagonists can be applied 
to the treatment of cancer have yielded contradictory results. Some 
reports suggest that administration of histamine alone suppressed tumor 
growth in hosts having a malignancy. (Burtin, Cancer Lett 12:195 (1981)). 
On the other hand, histamine has been reported to accelerate tumor growth 
in rodents (Nordlund, J. J. et al., J. Invest. Dermatol 81:28 (1983)). 
Similarly, contradictory results were obtained when the effects of 
histamine receptor antagonists were evaluated. Some studies report that 
histamine receptor antagonists suppress tumor development in rodents and 
humans (Osband, M. E. et al., Lancet 1 (8221) :636 (1981). Other studies 
report that such treatment enhances tumor growth and may even induce 
tumors (Barna, B. P. et al., Oncology 40:43 (1983)). 
D. Synergistic Effects of Histamine and IL-2 
Despite the conflicting results when histamine is administered alone, 
recent reports clearly reveal that histamine acts synergistically with 
cytokines to augment the cytotoxicity of NK cells. Thus, therapies 
employing the combination of histamine and cytokines represent an 
attractive approach to anti-cancer strategies based on using the host 
immune system to attack the malignancy. Similarly, antiviral treatments 
using any of the well known antiviral agents is also contemplated. 
Studies using histamine analogues suggest that histamine's synergistic 
effects are exerted through the H.sub.2 -receptors expressed on the cell 
surface of monocytes. For example, the H.sub.2 -receptor agonist dimaprit 
was capable of augmenting NK cell mediated cytotoxicity, while close 
structural analogues lacking biological activity failed to produce an 
effect. Additionally, H.sub.2 -receptor antagonists blocked the effects of 
histamine and dimaprit, implicating the H.sub.2 -receptor in the 
transduction of the histamine response. (Hellstrand, K. et al., J. 
Immunol. 137:656 (1986)). 
Histamine's synergistic effect when combined with cytokines is not the 
result of a direct positive effect of histamine on NK cell cytotoxicity. 
Rather, the synergistic effects result from the suppression of a down 
regulation of NK cell cytotoxicity mediated by other cell types present 
along with the NK cells. The discussion below provides some of the 
evidence suggesting that histamine's synergistic effects result from the 
suppression of negative signals exerted by other cell types. 
U.S. Pat. No. 5,348,739, which is incorporated herein by reference, 
discloses the synergistic effects of histamine and interleukin-2. As 
discussed above, IL-2 normally induces a cytotoxic response in NK cells. 
In vitro studies with NK cells alone confirm that cytotoxicity is 
stimulated when IL-2 is administered. However, in the presence of 
monocytes, the IL-2 induced enhancement of cytotoxicity of NK cells is 
suppressed. 
In the absence of monocytes, histamine had no effect or weakly suppressed 
NK mediated cytotoxicity. (U.S. Pat. No. 5,348,739; Hellstrand, K. et al., 
J. Immunol. 137:656 (1986); Hellstrand, K. and Hermodsson, S., Int. Arch. 
Allergy Appl. Immunol. 92:379-389 (1990)). However, NK cells exposed to 
histamine and IL-2 in the presence of monocytes exhibit elevated levels of 
cytotoxicity relative to that obtained when NK cells are exposed only to 
IL-2 in the presence of monocytes. Id. Thus, the synergistic enhancement 
of NK cell cytotoxicity by combined histamine and interleukin-2 treatment 
results not from the direct action of histamine on NK cells but rather 
from suppression of an inhibitory signal generated by monocytes. 
Without being limited to a particular mechanism, it is believed that the 
inhibitory effects of monocytes on NK mediated cytotoxicity result from 
the generation of H.sub.2 O.sub.2 by monocytes. It has been reported that 
the production of H.sub.2 O.sub.2 by monocytes suppresses NK cell 
cytotoxicity. (Van Kessel, K. P. M. et al., Immunology, 58:291-296 (1986); 
El-Hag, A. and Clark, R. A. J. Immuol. 133:3291-3297 (1984); Seaman, W. E. 
et al., J. Clin. Invest. 69:876-888 (1982)). Further evidence of the role 
of H.sub.2 O.sub.2 in suppressing NK cell cytotoxicity comes from in vitro 
studies showing that the addition of catalase, an enzyme which acts to 
remove H.sub.2 O.sub.2, to preparations of monocytes and NK cells exposed 
to IL-2 removes the inhibitory effects of the monocytes. (Seaman, supra.) 
Thus, histamine may exert its synergistic effects by reducing the level of 
H.sub.2 O.sub.2 produced by monocytes. Hellstrand, K., Asea, A., 
Hermodsson, S. Histaminergic regulation of antibody-dependent cellular 
cytotoxicity of granulocytes, monocytes and natural killer cells, J. 
Leukoc. Biol. 55:392-397 (1994). 
Monocytes are not the only cell type which negatively regulates NK cell 
cytotoxicity. Experiments have demonstrated that granulocytes suppress 
both the constitutive and IL-2 induced cytotoxic activity of NK cells in 
vitro. Like the monocyte mediated suppression discussed above, granulocyte 
mediated suppression is synergistically overcome by treatment with IL-2 
and histamine. (U.S. Pat. No. 5,348,739; Hellstrand, K., Asea, A., 
Hermodsson, S. Histaminergic regulation of antibody-dependent cellular 
cytotoxicity of granulocytes, monocytes and natural killer cells, J. 
Leukoc. Biol. 55:392-397 (1994). 
It appears that the H.sub.2 -receptor is involved in transducing 
histamine's synergistic effects on overcoming granulocyte mediated 
suppression. For example, the effect of histamine on granulocyte mediated 
suppression of antibody dependent cytotoxicity of NK cells was blocked by 
the H.sub.2 -receptor antagonist ranitidine and mimicked by the H.sub.2 
receptor agonist dimaprit. In contrast to the complete or nearly complete 
abrogation of monocyte mediated NK cell suppression by histamine and IL-2, 
such treatment only partially removed granulocyte mediated NK cell 
suppression. (U.S. Pat. No. 5,348,739; Hellstrand, K. et al., 
Histaminergic regulation of antibody dependent cellular cytotoxicity of 
granulocytes, monocytes and natural killer cells., J. Leukoc. Biol 
55:392-397 (1994). 
As suggested by the experiments above, therapies employing histamine and 
cytokines are effective anti-cancer strategies. U.S. Pat. No. 5,348,739 
discloses that mice given histamine and IL-2 prior to inoculation with 
melanoma cell lines were protected against the development of lung 
metastatic foci. This effect was a consequence of synergistic interaction 
between histamine and IL-2, as demonstrated by the significant reduction 
in metastatic foci observed in mice given histamine and IL-2 as compared 
to mice given histamine or IL-2 alone. 
In addition to the synergistic effects observed in the assay of lung 
metastatic foci, synergistic effects of histamine plus IL-2 treatment were 
also observed in assays in which NK cell cytotoxicity was measured by 
determining the ability of mice to kill malignant cell lines derived from 
both humans and mice which were injected into them. Id. 
The above results demonstrate that strategies employing a combination of 
histamine and IL-2 are an effective means of treating malignancies. 
E. Synergistic Effects of Histamine and Interferon-.alpha. 
Histamine also acts synergistically with interferon-.alpha. to overcome the 
suppression of NK cell cytotoxicity by monocytes (Hellstrand et al., 
Regulation of the NK cell response to interferon-alpha by biogenic amines, 
J. Interferon Res. 12:199-206 (1992)). Like IL-2, interferon-.alpha. 
augments NK cell constitutive NK cell cytotoxicity. Id. Monocytes suppress 
the interferon-.alpha. mediated enhancement of NK cell killing of 
malignant target cells in vitro. Monocyte mediated suppression of NK cell 
cytotoxicity was overcome by treatment with histamine and 
interferon-.alpha.. The effects of histamine were blocked by H.sub.2 
-receptor antagonists and mimicked by H.sub.2 -receptor agonists. 
Compounds bearing structural similarity to the H.sub.2 -receptor agonist 
dimaprit but lacking the activity of the agonist were unable to act 
synergistically with interferon-.alpha.. (Hellstrand et al. J., Interferon 
Res. 12:199-206 (1992)). 
F. Human Treatments Combining Histamine, Interleukin-2 and 
Interferon-.alpha. 
The in vitro and animal results discussed above suggested that 
histamine+IL-2+interferon-.alpha. was a promising method for treating 
human malignancies. In fact, combined histamine, IL-2, and 
interferon-.alpha. treatments have proven effective in the treatment of a 
variety of human malignancies, providing a 75% response rate significantly 
greater than that observed with IL-2 alone. (Hellstrand et al., Histamine 
in Immunotherapy of Advanced Melanoma: A Pilot Study, Cancer Immunology 
and Immunotherapy 39: 416-419 (1994). In the above study, subjects 
received a constant infusion of IL-2 (Proleukin.RTM., Eurocetus), 
18.times.10.sup.6 U/m.sup.2 on days 1-5 and 8-12, repeated every 4-6 
weeks, as well as interferon-.alpha. (6.times.10.sup.6 U daily, s.c.). In 
addition to the IL-2, eight of the subjects received histamine 
dihydrochloride (1 mg s.c.) twice daily. Id. 
Only one subject in the group receiving IL-2 and interferon-.alpha. 
exhibited a partial or mixed response, a response rate of 14% (1 of 7). In 
contrast, the group receiving both IL-2, interferon-.alpha. and histamine 
showed an corresponding response rate of 75% (6 of 8). Only two of these 
subjects failed to respond. Id. 
Thus, histamine+IL-2+interferon-.alpha. is an effective anti-cancer 
therapy. 
G. Human Treatments with Histamine+IL-2+Chemotherapeutic Agents 
Recently, the efficacy of treatments employing histamine, IL-2, and 
chemotherapeutic agents was examined in humans suffering from acute 
myelogenous leukemia (AML) (Brune and Hellstrand, Remission Maintenance 
Therapy with Histamine and Interleukin-2 in Acute Myelogenous Leukemia, 
Br. J. Haematology, March 1996). 
In one set of experiments, killing of AML blasts by NK cells was examined 
in vitro. IL-2 induced NK mediated cytotoxicity, but this effect was 
suppressed by monocytes. Histamine did not affect the IL-2 induced 
cytotoxic response in the absence of monocytes but blocked the suppressive 
effects of the monocytes. However, in the presence of the H.sub.2 
-receptor antagonist ranitidine, histamine was ineffective in overcoming 
the monocyte suppression. Id. 
Additionally, AML patients in remission were treated with 
histamine+IL-2+the chemotherapeutic agents cytarabine and thioguanine and 
the duration of remission was measured and compared to the length of 
remissions prior to initiating the treatment. Five of the patients 
receiving histamine+IL-2+cytarabine+ thioguanine remained in complete 
remission ranging in duration from 9-27 months at the time of publication. 
Two patients relapsed after remissions lasting 8 and 33 months. In the 
five patients who had undergone a remission followed by a relapse prior to 
initiation of the histamine+IL-2+cytarabine+ thioguanine regimen, the 
duration of remissions following initiation of the regimen exceeded the 
duration of the prior remission. Id. Such "inversion of remission time" is 
rare in the natural course of AML and reportedly only occurs in a small 
fraction (&lt;10%) of AML patients treated with IL-2 as the single agent. 
Shepherd et al, Phase II Study of Subcutaneous rHU IL-2 in Patients with 
Acute Myelogenous Leukemia in Partial or Complete Second Remission and 
Partial Relapse, Br. J. Haematol. S87, 205 (1994). 
Thus, histamine+IL-2+chemotherapeutic agents is an effective anti-cancer 
therapy. 
SUMMARY OF THE INVENTION 
The present invention relates to treatments combining the administration of 
histamine to achieve a beneficial level of circulating blood histamine 
with treatment with a second agent which enhances natural killer cell 
cytotoxicity. Alternatively, the invention relates to treatments which 
combine the administration of histamine to achieve beneficial levels of 
circulating histamine with treatment with a chemotherapeutic agent. 
Combinations of histamine, agents which enhance NK cell cytotoxicity, and 
chemotherapeutic agents are also contemplated. It will be appreciated that 
the beneficial stable levels of circulating blood histamine may be 
attained by administering histamine before, during, after or throughout 
the course of the administration of the second agent. As used herein 
"stable" means a level that is maintained for hours or preferably, days. 
Thus the present invention includes a method of augmenting the activity of 
an agent which enhances natural killer cell cytotoxicity comprising: 
a) administering a pharmaceutically acceptable form of histamine such that 
a stable blood histamine concentration sufficient to augment the 
cytotoxicity enhancing effect of said agent is achieved; and 
b) administering a beneficial amount of said agent, wherein the 
cytotoxicity enhancing effects of said agent are augmented. 
This invention also relates to a method of treating a subject having a 
malignancy with histamine and a second beneficial agent wherein the 
activity of said second beneficial agent is augmented by histamine 
comprising: 
a) administering a pharmaceutically acceptable form of histamine such that 
a beneficial blood histamine level is achieved; and 
b) administering said second beneficial agent. 
The invention also comprises a method of enhancing the cytotoxic activity 
of natural killer cells comprising: 
a) measuring blood histamine levels in a subject to determine whether the 
cytotoxic activity of said subject's natural killer cells could be 
enhanced by increasing the levels of blood histamine in said subject; 
b) administering a pharmaceutically acceptable form of histamine to a 
subject for whom said measuring step indicated that the cytotoxic activity 
of said subject's natural killer cells could be enhanced by increasing 
said subject's blood histamine levels, such that beneficial levels of 
blood histamine are achieved; and 
c) administering a second agent to said subject having beneficial levels of 
blood histamine, such that the cytotoxic activity of said subject's 
natural killer cells is enhanced. 
Another aspect of the invention is method of treating a malignancy 
comprising treating a subject having a malignancy with a chemotherapeutic 
agent and maintaining a beneficial stable level of circulating blood 
histamine by administering a pharmaceutically acceptable form of histamine 
in a dose sufficient to attain said beneficial stable levels of 
circulating blood histamine. 
A more complete appreciation of the invention and many of the attendant 
advantages thereof will be readily perceived as the same becomes better 
understood by reference to the following detailed description when 
considered in connection with the accompanying figures. 
Other objects, advantages and features of the present invention will become 
apparent to those skilled in the art from the following discussion.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
Prior to the present invention, histamine was believed to have an extremely 
short half life, on the order of five minutes, in the blood. Beaven, M. 
A., Factors regulating availability of histamine at tissue receptors in 
Pharmacology of Histamine Receptors, C. R. Ganellin and M. E. Parsons 
eds., Wright PSG, Bristol, U.K. pp. 103-145 (1982). The present invention 
arose from the unexpected finding that, contrary to the prior reports, it 
is possible to achieve stable levels of circulating blood histamine 
lasting several hours or even days from the time of histamine 
administration. The present invention is the first report of stable 
circulating blood histamine. Using regimens involving the administration 
of both H.sub.2 -receptor antagonists and histamine, Burtin was able to 
obtain normal levels of circulating blood histamine in cancer subjects 
experiencing stabilization of the disease. (Burtin et al., Eur. J. Clin. 
Oncol. 24: 161-167 (1988)). However, the normal levels reported by Burtin 
were most likely a consequence of the stabilization of the cancer rather 
than an observation of stable circulating levels of histamine for a 
significant period following administration. Evidence that the levels 
reported in Burtin were a consequence of physiological stabilization comes 
from the fact that the normal levels of histamine reported by Burtin when 
the subjects experienced a remission dropped to below normal levels before 
the subjects' deaths. Burtin did not specify the time after administration 
at which circulating histamine levels were measured, but it seems likely 
that the normal levels reported by Burtin were a consequence of 
endogenously produced histamine associated with stabilization of the 
cancer rather than an observation of persistence of extrinsically 
administered histamine. 
Subjects suffering from cancer often exhibit decreased levels of 
circulating blood histamine. (Burtin et al. Decreased blood histamine 
levels in subjects with solid malignant tumors, Br. J. Cancer 47: 367-372 
(1983). Thus, the observation of stable and beneficial blood histamine 
levels lasting hours or days after histamine administration finds ready 
application to cancer treatments based on synergistic effects between 
histamine and agents which enhance NK cell mediated cytotoxicity. In such 
protocols, the cytotoxic activity of NK cells is enhanced by combining the 
administration of histamine to attain a stable level of circulating 
histamine sufficient to augment the activity of an agent which acts in 
synergy with histamine to increase NK mediated cytotoxicity with the 
administration of the agent. 
Additionally, chemotherapeutic treatments aimed at destroying the 
malignancy may result in lowered blood histamine levels. Example 1, below 
describes the decrease in blood histamine levels following treatment with 
the chemotherapeutic/cytostatic agents cytarabine and thioguanine. 
EXAMPLE 1 
In five patients with AML in remission, histamine levels in whole blood 
specimens were analyzed 1-5 days before and 1-2 weeks after treatment with 
cytarabine (16 mg/m.sup.2 /day subcutaneously) and thioguanine (40 mg/day 
orally) for 21 days or until the platelet count was 
.ltoreq.50.times.10.sup.9 /l. Histamine was analyzed in heparinized venous 
blood using the radioimmunoassay available from Biomerica Inc., Newport 
Beach, Calif. 92663 (catalog no. 1051) according to the instructions which 
accompanied the assay kit. 
In all patients, histamine levels declined after the treatment with 
cytostatics. (See Table below). 
______________________________________ 
Blood Histamine Before 
Blood Histamine after 
Patient Treatment (.mu.moles/l) 
Treatment (.mu.moles/l) 
______________________________________ 
1. 0.22 not detectable 
2. 0.12 not detectable 
3. 0.18 not detectable 
4. 0.93 0.38 
5. 0.24 0.14 
______________________________________ 
The present invention may be used to restore or maintain beneficial stable 
blood histamine levels in subjects whose blood histamine levels have 
decreased as a consequence of chemotherapy. 
Beneficial levels of circulating histamine can be achieved by administering 
histamine before, during, or after treatment with agents which enhance 
natural killer cell cytotoxicity or chemotherapeutic agents. 
Thus, this invention relates to a method of augmenting the activity of an 
agent which enhances natural killer cell cytotoxicity comprising 
administering histamine such that a stable blood histamine concentration 
sufficient to augment the cytotoxicity enhancing effect of said agent is 
achieved, and administering a beneficial amount of said agent, wherein the 
cytotoxicity enhancing effects of said agent are augmented. 
In one aspect of the present invention, the histamine is administered prior 
to the agent which enhances natural killer cell cytotoxicity. In another 
embodiment, the histamine is administered following the administration of 
the agent which enhances natural killer cell cytotoxicity. In a further 
embodiment, the histamine is administered during the course of 
administration of the agent which enhances natural killer cell 
cytotoxicity. In another embodiment, the histamine is administered prior, 
during, and after the administration of the agent which enhances natural 
killer cell cytotoxicity. 
In one embodiment, the histamine is administered at least 1 day prior to 
the administration of the agent which enhances natural killer cell 
cytotoxicity. In a preferred embodiment beneficial stable levels of 
circulating blood histamine are obtained by administering histamine at a 
dosage of 0.4 to 10 mg/day. In a further preferred embodiment, the 
histamine is administered over a period of 1 to 4 weeks. In a highly 
preferred embodiment, the histamine is administered for a period of 1-2 
weeks. In one embodiment of the invention, the beneficial stable level of 
circulating blood histamine concentration is at least 0.2 .mu.mol/L. 
In one embodiment of the present invention the NK cell cytotoxicity 
enhancing agent is at least one cytokine. Preferentially, the cytokine is 
interleukin-2. In a preferred aspect of the invention, the interleukin-2 
is administered in an amount of 0.5-50 .mu.g/kg/day. In a further 
preferred embodiment, the interleukin-2 is administered for a period of 1 
day to 4 weeks. 
In another embodiment of the invention, the cytokine is interferon-.alpha.. 
Preferentially, the interferon-.alpha. is administered at a dosage of 
10,000-200,000 U/kg/day. For treatment of primary melanoma, the preferred 
dosage of interferon-.alpha. is 200,000 U/kg/day. For treatment of other 
cancers, the preferred dosage of interferon-.alpha. is 50,000 U/kg/day. In 
a further embodiment, the interferon-.alpha. is administered for a period 
of up to 18 months. Preferentially, the interferon-.alpha. is administered 
for a period of between 2-6 weeks. 
In yet another embodiment, the natural killer cell cytotoxicity enhancing 
agent comprises interferon-.alpha. and interleukin-2. 
Another aspect of the invention is a method of treating a subject having a 
malignancy with histamine and a second beneficial agent wherein the 
activity of said second beneficial agent is augmented by histamine 
comprising administering a pharmaceutically acceptable form of histamine 
such that a beneficial blood histamine level is achieved and administering 
said second beneficial agent. 
In one embodiment of this aspect, the histamine is administered prior to 
the second beneficial agent. In a preferred embodiment of this aspect, the 
histamine is administered at least 1 day prior to the administration of 
the second beneficial agent. 
In another embodiment of this invention, the histamine is administered 
after the second beneficial agent. In yet another embodiment, the 
histamine is administered during the course of administration of said 
second beneficial agent. In another aspect of the invention, the histamine 
is administered prior, during, and after the second beneficial agent. 
Preferentially, the second beneficial agent acts to stimulate the cytotoxic 
activity of natural killer cells. 
A third aspect of the present invention is a method of enhancing the 
cytotoxic activity of natural killer cells comprising 
a) measuring blood histamine levels in a subject to determine whether the 
cytotoxic activity of said subject's natural killer cells could be 
enhanced by increasing the levels of blood histamine in said subject. 
b) administering a pharmaceutically acceptable form of histamine to a 
subject for whom said measuring step indicated that the cytotoxic activity 
of said subject's natural killer cells could be enhanced by increasing 
said subject's blood histamine levels, such that beneficial levels of 
blood histamine are achieved. and 
c) administering a second agent to said subject having beneficial levels of 
blood histamine, such that the cytotoxic activity of said subject's 
natural killer cells is enhanced. 
In one aspect of the invention, the histamine is administered prior to the 
second agent. In a preferred version of this aspect, the histamine is 
administered at least 1 day prior to the administration of the second 
agent. 
In another aspect of the invention, histamine is administered after the 
second agent. In another embodiment, histamine is administered during the 
administration of the second agent. In another embodiment, the histamine 
is administered prior, during, and after the administration of the second 
agent. In each instance, one embodiment of the invention includes an 
antiviral or antimicrobial agent as the second agent. 
The present invention also includes a method of treating a malignancy 
comprising treating a subject having a malignancy with a chemotherapeutic 
agent and achieving and maintaining a beneficial stable level of 
circulating blood histamine by administering histamine in a dose 
sufficient to attain said beneficial stable levels of circulating blood 
histamine. 
In one embodiment, the histamine is administered before the 
chemotherapeutic agent. In another embodiment, the histamine is 
administered after the chemotherapeutic agent. In another aspect of the 
invention, the histamine is administered during treatment with the 
chemotherapeutic agent. In yet another embodiment, the histamine is 
administered prior, during, and after treatment with the chemotherapeutic 
agent. 
It will be appreciated that the subject's circulating blood histamine 
levels may be monitored during the course of treatment and boosted to 
beneficial levels whenever levels drop below beneficial levels or approach 
the lower limits of beneficial levels. For example, in this embodiment, 
histamine may be administered whenever the subject's histamine levels drop 
below 0.2 .mu.mole/L. 
Alternatively, it will be appreciated that histamine may be administered at 
periodic intervals at dosages sufficient to establish and maintain 
beneficial levels. 
Routes and carrier compositions for administering histamine and cytokines 
have been disclosed in U.S. Pat. No. 5,348,739, which is incorporated 
herein by reference. Additionally, methods for administering 
chemotherapeutic agents are well established. 
For the purposes of the above treatments, beneficial levels of blood 
histamine are at least 0.2 .mu.mole/L. 
Stable Circulating Blood Histamine Levels 
As described above, the present invention is based on the observation that 
it is possible to obtain beneficial and stable levels of circulating blood 
histamine. Example 2 depicts the stable beneficial levels of circulating 
blood histamine obtained following histamine administration. Surprisingly, 
beneficial levels of circulating blood histamine persisted for 
considerable periods after histamine administration ceased. 
EXAMPLE 2 
Five patients with AML in remission received treatment with histamine 
dihydrochloride diluted in sterile sodium chloride purchased from 
Apoteksbolaget, Umea, Sweden and human recombinant interleukin-2 
(Proleukin.RTM.) obtained from the commercially available vial (Eurocetus, 
the Netherlands) Histamine and IL-2 were administered morning and night at 
separate subcutaneous injection sites over a period of 21 consecutive 
days. The histamine was given as subcutaneous injections using 1 ml 
syringes containing 0.1 mg of histamine/ml. The histamine treatment was 
given twice daily (morning and night) at a dosage of 0.4-0.7 mg histamine 
per injection (i.e. a daily total dose of histamine of 0.8-1.4 mg/day). 
The IL-2 was given as subcutaneous injections using separate 1-ml syringes. 
The IL-2 syringes contained 50 .mu.g of IL-2/ml. The IL-2 treatment was 
given twice daily (morning and night), and the dose was 35-50 .mu.g of 
IL-2 per injection, i.e. a daily total dose of 70-100 .mu.g/day. 
Peripheral blood venous samples were drawn in 10 ml heparinized test tubes 
before the onset of treatment and weekly thereafter. The samples were 
drawn at least 8 hours after the last injections of histamine and IL-2. 
The concentration of histamine in the whole blood samples was analyzed by 
use of a double antibody radioimmunoassay kit obtained from Biomerica, 
Inc., Newport Beach, Calif. 92663 (catalog no. 1051). The manufacturer's 
instructions provided with the kit, dated June, 1989, were followed. Blood 
histamine levels were measured at the indicated times. 
FIG. 1 shows the results of the experiments described above. Data are given 
as the concentration of histamine in micromoles/1 (mean.+-.standard error 
of the mean). 
The subjects exhibited blood histamine levels of less than 0.2 .mu.mole/L 
at the start of the experiment. Following histamine administration, 
circulating blood histamine levels rose to beneficial levels. 
Surprisingly, the circulating blood histamine levels remained elevated for 
sustained periods of time, even after histamine administration was 
discontinued. 
Treatments Employing a Combination of Histamine and Interleukin-2 
The stable blood histamine levels discussed above find application in 
treatments in which natural killer cell cytotoxicity is augmented through 
the synergistic effects of histamine and agents which enhance NK cell 
cytotoxicity. As discussed above, one such enhancer of NK cytotoxicity is 
interleukin-2. Example 3 describes methods of treatment in which a stable 
and beneficial level of histamine is achieved which augments the activity 
of IL-2. 
EXAMPLE 3 
0.5 mg/day of histamine in a pharmaceutically acceptable form is injected 
subcutaneously in a sterile carrier solution into subjects having a 
malignancy. One week later, after circulating blood histamine levels have 
increased to at least 0.2 .mu.mole/L, IL-2 administration is begun. Human 
recombinant IL-2 (Proleukin.RTM., Eurocetus) is administered by continuous 
infusion of 27 .mu.g/kg/day on days 1-5 and 8-12. 
The above procedure is repeated every 4-6 weeks until an objective 
regression of tumor disease is observed. The therapy may be continued even 
after a partial or complete response has been observed. In patients with 
complete responses, the therapy may be given with longer intervals between 
cycles. 
The treatment can additionally include monitoring circulating levels of 
blood histamine periodically and, when circulating histamine levels drop 
below 0.2 .mu.mole/L, injecting 0.5 mg/day of a pharmaceutically 
acceptable form of histamine to restore circulating blood histamine levels 
above 0.2 .mu.mole/L . 
The treatment can also include periodically boosting circulating blood 
histamine levels by administering 0.5 mg/day histamine over a period of 
one to two weeks at regular intervals, such as bi-weekly, in order to 
maintain circulating blood histamine levels above 0.2 .mu.mole/L. 
Combination of Histamine and Interferon-.alpha. 
Another enhancer of NK cell cytotoxicity is interferon-.alpha.. Example 4 
describes methods of treatment in which a stable and beneficial level of 
histamine is achieved which augments the activity of interferon-.alpha.. 
EXAMPLE 4 
0.5 mg/day of histamine in a pharmaceutically acceptable form is injected 
subcutaneously in a sterile carrier solution into subjects having a 
malignancy or who have had a primary tumor surgically removed. One week 
later, after circulating blood histamine levels have increased to at least 
0.2 .mu.mole/L, interferon-.alpha. administration is begun. Interferon is 
administered in doses of 50,000 U/kg/day in a suitable carrier solution 
for 2-6 weeks. 
The above procedure is repeated at intervals of sometimes for 18 months or 
3 times a week for 18 months until an objective regression of the tumor is 
observed. The therapy with histamine, IL-2 and interferon may be continued 
even after a partial or complete response has been observed. In patients 
with complete responses, the therapy may be given with longer intervals 
between cycles. 
The treatment can additionally include monitoring circulating levels of 
blood histamine periodically and, when circulating histamine levels drop 
below 0.2 .mu.mole/L, injecting 0.5 mg/day of a pharmaceutically 
acceptable form of histamine to restore circulating blood histamine levels 
above 0.2 .mu.mole/L. 
The treatment can also include periodically boosting circulating blood 
histamine levels by administering 0.5 mg/day histamine over a period of 
one to two weeks at regular intervals, such as bi-weekly, in order to 
maintain circulating blood histamine levels above 0.2 .mu.mole/L. 
Additionally, the frequency of interferon-.alpha. administration may be 
varied depending on the patient's tolerance of the treatment and the 
success of the treatment. For example, interferon may be administered 
three times per week, or even daily, for a period of up to 18 months. 
Those skilled in the art are familiar varying interferon treatments to 
achieve both beneficial results and patient comfort. 
Combination of Histamine, IL-2 and Interferon-.alpha. 
Beneficial stable levels of circulating blood histamine can also be 
employed in conjunction with treatments involving several enhancers of NK 
cell cytotoxicity. Example 5 describes how to administer such treatments. 
EXAMPLE 5 
0.5 mg/day of histamine in a pharmaceutically acceptable form is injected 
subcutaneously into subjects having a malignancy in a sterile carrier 
solution. One week later, after circulating blood histamine levels have 
increased to at least 0.2 .mu.mole/L, human recombinant IL-2 
(Proleukin.RTM., Eurocetus) is administered by continuous infusion of 27 
.mu.g/kg/day on days 1-5 and 8-12. Additionally, subjects also receive a 
daily dose of 6.times.10.sup.6 U interferon-.alpha. administered by 
subcutaneous injection. 
The above procedure is repeated every 4-6 weeks until an objective 
regression of the tumor is observed. The therapy may be continued even 
after a partial or complete response has been observed. In patients with 
complete responses, the therapy may be given with longer intervals between 
cycles. 
The treatment can additionally include monitoring circulating levels of 
blood histamine periodically and, when circulating histamine levels drop 
below 0.2 .mu.mole/L, injecting 0.5 mg/day of a pharmaceutically 
acceptable form of histamine for a period of one to two weeks to restore 
circulating blood histamine levels above 0.2 .mu.mole/L . 
The treatment can also include periodically boosting circulating blood 
histamine levels by administering histamine at regular intervals, such 
bi-weekly. 
Additionally, the frequency of interferon-.alpha. administration may be 
varied depending on the patient's tolerance of the treatment and the 
success of the treatment. For example, interferon may be administered 
three times per week, or even daily, for a period of up to 18 months. 
Those skilled in the art are familiar varying interferon treatments to 
achieve both beneficial results and patient comfort. 
Combination of Histamine and Chemotherapeutic Agents 
Histamine may also be used in conjunction with chemotherapeutic agents. 
Typically, levels of circulating histamine decline during chemotherapy. 
Low levels of circulating histamine may result in the suppression of NK 
cell cytotoxicity by monocytes. This monocyte mediated suppression may be 
eliminated by administration of histamine prior, during, following or 
throughout chemotherapy in order to return circulating histamine levels to 
normal. 
Accordingly, the present invention contemplates the restoration of 
circulating blood histamine levels to normal levels in conjunction with 
chemotherapeutic agents. Additionally, the treatment may also include 
administration of IL-2 and/ or interferon-.alpha.. 
Representative compounds used in cancer therapy include cyclophosphamide, 
choroambucil, melphalan, estramustine, iphosphamide, prednimustin, 
busulphan, tiottepa, carmustin, lomustine, methotrexate, azathioprine, 
mercaptopurine, thioguanine, cytarabine, fluorouracil, vinblastine, 
vincristine, vindesine, etoposide, teniposide, dactinomucin, doxorubin, 
dunorubicine, epirubicine, bleomycin, nitomycin, cisplatin, carboplatin, 
procarbazine, amsacrine, mitoxantron, tamoxifen, nilutamid, and 
aminoglutemide. Procedures for employing these compounds against 
malignancies are well established. In addition, other cancer therapy 
compounds may also be utilized in the present invention. 
Malignancies against which the treatment may be directed include, but are 
not limited to, primary and metastatic malignant tumor disease, 
hematological malignancies such as acute and chronic myelogenous leukemia, 
acute and chronic lymphatic leukemia, multiple myeloma, Waldenstroms 
Macroglobulinemia, hairy cell leukemia, myelodysplastic syndrome, 
polycytaemia vera, and essential thrombocytosis. 
As described above, histamine+IL-2 has proven an effective combination with 
traditional chemotherapeutic methods in treating acute myelogenous 
leukemia. (Brune and Hellstrand, Br. J. Haematology, March 1996). 
Procedures for using the present invention in combination with 
chemotherapeutic agents and IL-2 are presented in Example 6. It will be 
appreciated that beneficial stable levels of circulating histamine may 
also be employed in treatments using only chemotherapeutic agents. 
EXAMPLE 6 
Subjects with AML in first, second, subsequent or complete remission are 
treated in 21-day courses with IL-2 35-50 .mu.g (equivalent to 
6.3-9.times.10.sup.5 IU) s.c. twice daily!, repeated with six-week 
intermissions and continued until relapse. In cycle #1, patients receive 
three weeks of low dose chemotherapy consisting of 16 mg/m2/day 
cytarabine, and 40 mg/day thioguanine. Thereafter, patients are injected 
subcutaneously with 0.5 mg/day of a pharmaceutically acceptable form of 
histamine for a period of 1 week to boost circulating histamine to a 
stable beneficial level above 0.2 .mu.mole/L. Thereafter, patients receive 
100 .mu.g of interleukin-2/day for three weeks. Circulating histamine 
levels are boosted to beneficial levels by administering 0.5 mg/day of a 
pharmaceutically acceptable form of histamine during the second week of 
this period. Thereafter, the subjects are allowed to rest for one week. 
After the rest period at the end of cycle 1, cycle #2 is initiated. 0.5 
mg/day of a pharmaceutically acceptable form of histamine in a sterile 
carrier solution is injected subcutaneously until circulating blood 
histamine levels of at least 0.2 .mu.mole/L are achieved. Cytarabine (16 
mg/m.sup.2 /day s.c.) and thioguanine (40 mg/day orally) are given for 21 
days (or until the platelet count is .ltoreq.50.times.10.sup.9 /1). In the 
middle week, patients receive 0.5 mg/day of a pharmaceutically acceptable 
form of histamine to boost circulating histamine to beneficial levels. At 
the end of the three week chemotherapy treatment, patients receive 0.5 
mg/day of a pharmaceutically acceptable form of histamine for a week. 
Thereafter, patients receive 100 .mu.g/day of interleukin-2 for three 
weeks. Circulating histamine levels are boosted in the middle week of the 
three week IL-2 treatment as described above. Patients are permitted to 
rest for two weeks. 
Thereafter, cycle #3 is initiated. Cycle #3 is identical to cycle #2. 
Alternatively, circulating histamine levels may be periodically monitored 
throughout the above procedure and histamine may be administered whenever 
circulating levels drop below a desirable level in order to maintain a 
beneficial level of blood histamine above 0.2 .mu.mole/L. Additionally, 
histamine may be administered at regular intervals during the treatment to 
maintain beneficial circulating levels. Another alternative is to provide 
histamine in a depot or controlled release form. Numerous controlled 
release vehicles are known, including biodegradable or bioerodable 
polymers such as polylactic acid, polyglycolic acid, and regenerated 
collagen. Implantble or injectalbe polymer matrices from which active 
ingredients are slowly released are also well known. Finally, transdermal 
patches or formulations, with or without, use of a penetration enhancer 
such as DMSO or Azone can be used in the present invention.