Method of treating diseases associated with elevated levels of interleukin 1

The present invention relates to a method of treating diseases associated with elevated levels of interleukin 1 (IL-1) including inflammatory diseases such as arthritis, skin hypersensitivity and endotoxemia. More specifically, the invention relates to a method for the treatment of such diseases in warm-blooded animals, such as humans, which comprises the administration of a therapeutically effective amount of an aromatic diamidine, sufficient to inhibit IL-1 release from IL-1 producing cells. The aromatic diamidine can also be used to block interleukin 6 (IL-6) and tumor necrosis factor from cells producing these cytokines.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The present invention relates to a method for inhibiting the release of 
interleukin 1 (IL-1) from IL-1 producing cells. More specifically, the 
invention relates to the treatment of diseases associated with elevated 
levels of IL-1. 
2. Background Information 
IL-1, a polypeptide cytokine with multiple biological properties, is a key 
mediator in immunological reactions as well as in the body's response to 
microbial invasion, inflammation and tissue injury. Since IL-1 is also 
highly inflammatory, down-regulation of its production has been an area of 
intense investigation. Specific pathological conditions where Il-1 
diminution is beneficial include inflammatory diseases such as arthritis 
(where IL-1 is found in high concentrations in synovial fluid), 
endotoxemia (where, in conjunction with tumor necrosis factor the high 
concentration of IL-1 contributes to fever, hypothermia and hemodynamic 
shock), granulomatous diseases, fibrosis and hypersensitivity diseases (C. 
D. Dinarello, Review of Infect. Dis. 6, 51 (1986)). 
At present, the most effective therapeutic agents to treat diseases 
associated with elevated IL-1 levels are corticosteroids. These act by 
inhibiting IL-1 transcription, although they may stimulate IL-1 
translation of preformed IL-1 messenger RNA (mRNA). (P. J. Knudsen et al, 
J. Immunol. 139, 4129 (1987)). More recently, IL-1 receptor antagonists 
have been described that block the activity of IL-1 by binding to IL-1 
receptors (C. H. Hannum et al., Nature 343, 336 (1990)). 
It has now been found that aromatic diamidines are effective in treating 
diseases associated with elevated IL-1 levels. For example, 
1,5-bis(4-amidinophenoxy)pentane (pentamidine), an aromatic diamidine 
known for its effectiveness against AIDS related Pneumocystis carinii 
pneumonia, is a specific and effective inhibitor of cellular IL-1 release 
from macrophages. This may be associated with its putative ability to 
inhibit protease activity, since release of IL-1 from the membrane bound 
form is dependent upon the action of proteases which hydrolyze the peptide 
bond between the membrane anchoring sequence and the secreted form (K. 
Matsushima et al., J. Immunol. 136, 2883 (1986)). 
The use of pentamidine as a blocker of IL-1 is an improvement over the use 
of corticosteroids. Pentamidine allows the protein to be translated, but 
blocks IL-1 at the level of release while corticosteroids block IL-1 at 
the level of mRNA. By contrast, corticosteroids block the message of IL-1 
from being formed. This mechanism of action by corticosteroids is 
nonspecific, in that this class of drugs blocks the transcription of many 
biologically important proteins, as well as blocking IL-1 formation. 
In addition, the mechanism by which pentamidine appears to act (the 
alteration of a postranslational protein modification event) allows it to 
be much more selective when compared to a representative corticosteroid, 
for example, dexamethasone. Such selectivity by pentamidine serves to 
spare other components of the immune system and circumvent the overt broad 
immunotoxicity that often results in decreased host resistance in patients 
undergoing corticosteroid therapy. This mechanism is a vast improvement 
over the immunodepressive activity of the corticosteroids. 
1,5-di(4-imidazolinophenoxy)pentane, an aromatic substituted diamidine 
known for its effectiveness against Trypanosoma rhodesiense and Plasmodium 
berghei (E. Steck et al., Exp. Parasitol. 42, 404 (1981)), has also been 
found to be a specific and effective inhibitor of cellular IL-1 release 
from macrophages. This imidazoline is an analog to pentamidine and has a 
similar structure, with the exception that the terminal amidino groups are 
substituted with imidazoline moieties. Thus, this compound is believed to 
exert its effects in a way that is mechanistically similar to pentamidine. 
SUMMARY OF THE INVENTION 
The present invention relates to a method of treating diseases which are 
wholly or partly mediated by excess production of IL-1. More specifically, 
the present invention relates to a method of treating diseases associated 
with elevated levels of IL-1, comprising administering to warm blooded 
animals, including humans, in need of such treatment, a therapeutically 
effective amount of an aromatic diamidine. 
In one embodiment, the invention relates to a method of treating diseases 
associated with elevated levels of IL-1, comprising administering to 
warm-blooded animals, including humans, in need of such treatment, a 
therapeutically effective amount of 1,5-bis(4-amidinophenoxy)pentane 
(pentamidine), which can be in the form of pentamidine isethionate. 
In another embodiment, the invention relates to a method of treating 
diseases associated with elevated levels of IL-1 , comprising 
administering to warm-blooded animals, including humans, in need of such 
treatment, a therapeutically effective amount of an imidazoline, which can 
be in the form of 1,5-di(4-imidazolinophenoxy)pentane. 
Aromatic diamidines can also be used to inhibit the release of interleukin 
6 (IL-6) and tumor necrosis factor from cells producing these cytokines. 
It is a general object of the present invention to provide a method of 
treating a subject suffering from a disease associated with elevated 
levels of IL-1 , while avoiding the adverse side effects and lack of 
specificity associated with art-recognized corticosteroid therapy. 
Further objects and advantages of the invention will be clear to one 
skilled in the art from a reading of the description that follows.

DETAILED DESCRIPTION OF THE INVENTION 
The present invention is directed to a method of treating diseases 
associated with elevated levels of IL-1, comprising administering to 
warm-blooded animals, including humans, in need of such treatment, a 
therapeutically effective amount of an aromatic diamidine. 
In one embodiment, the present invention is directed to a method of 
treating diseases associated with elevated levels of IL-1 comprising 
administering to warm-blooded animals, including humans, in need of such 
treatment, a therapeutically effective amount of 
1,5-bis(4-amidinophenoxy)pentane (pentamidine). The pentamidine is 
advantageously in the form of pentamidine isethionate. 
In another embodiment, the present invention is directed to a method of 
treating diseases associated with elevated levels of IL-1, comprising 
administering to warm-blooded animals, including humans, in need of such 
treatment, a therapeutically effective amount of an imidazoline derivative 
of pentamidine, specifically, 1,5-di(4-imidazolinophenoxy) pentane. 
The diseases in which a diminution of IL-1 is beneficial include, but are 
not limited to, inflammatory diseases, such as arthritis, skin 
hypersensitivity, glomerulonephritis, septicemia including endotoxemia, 
pulmonary granulomas, pulmonary fibrosis, cirrhosis, sarcoidosis, 
tuberculosis, chronic granulomatous disease, dysfunctional clot formation, 
transplant rejection, and all diseases and conditions, including fever, 
associated with the release of acute phase reactants by hepatocytes. 
The pharmaceutical formulations of the present invention comprise, as an 
active ingredient, an aromatic diamidine, for example, pentamidine or its 
imidazoline substituted derivative, together with a pharmaceutically 
acceptable carrier. The active ingredient is present in the composition in 
an amount sufficient to wholly or partially block IL-1 release from IL-1 
producing cells. One skilled in the art would be able to determine when it 
would be desirable to wholly block IL-1 release and when it would be 
desirable to partially block IL-1 release. The composition of the 
invention can be formulated so as to be suitable, for example, for oral, 
nasal, parenteral, topical, transdermal or rectal administration. 
Administration of the pharmaceutical formulation may, for example, be in 
the form of aerosols, ointments, creams, gels, tablets, capsules, pills, 
coated tablets, suppositories, powder, dusting powder or in liquid form. 
The individual dosages are, for example: 
(a) up to 650 mg per dose, and advantageously about 300 mg per dose, in the 
case of medicinal forms for inhalation (aerosols or solutions), 
(b) 15 mg active ingredient/kg animal in the case of parenteral medicinal 
forms (for example, intravenous or intramuscular), and 
(c) up to 1% active ingredient in solution in the case of medicinal forms 
for dermal application. Appropriate individual dosage sizes can be readily 
determined by one skilled in the art. 
The aromatic diamidines can also be used to inhibit the release of IL-6 and 
tumor necrosis factor from cells producing these cytokines. 
The frequency of administration and the amount of active ingredient to be 
administered to effect treatment of a particular disease associated with 
elevated levels of IL-1 can readily be determined by one skilled in the 
art. 
Pentamidine, the best known of the aromatic diamidine compounds currently 
used as protazoacidal agents, is used in the treatment of AIDS related 
Pneumocystis carinii pneumonia. The therapeutic use of pentamidine in the 
treatment of AIDS has recently utilized the pulmonary route of exposure 
via aerosol administration. Because the alveolar macrophage is among the 
first of the immune cells to come into contact with pentamidine, the 
pentamidine induced modulation of the immune functions of this cell were 
investigated. 
As discussed in detail below, Northern blot analysis demonstrates 
pentamidine's lack of effect on mRNA expression compared to the 
corticosteroid, dexamethasone. Western blot analysis of intracellular IL-1 
demonstrates that pentamidine allows proper translation of the protein, 
while Western blot analysis of secreted IL-1 demonstrates the nearly 
complete block in the release of the intracellular IL-1. 
Dose response studies were performed to determine the molar concentration 
at which pentamidine reduced viability in normal rat macrophages. No 
significant loss in cell viability is seen at concentrations 
.ltoreq.10.sup.-5 M. In normal human macrophages, pentamidine blocks IL-1 
secretion at a concentration of 10.sup.-5 M (see Example 5). This blockage 
was shown using an enzyme linked immunosorbant assay with specific 
antibody to IL-1.beta.. 
It has also been observed that pentamidine (10.sup.-5 M) blocks the 
secretion of tumor necrosis factor in rat macrophages (42 units/ml - 
pentamidine vs. 82 units/ml - control), as well as tumor necrosis factor 
and IL-6 in human macrophages, where at a concentration of 10.sup.-5 M 
pentamidine, tumor necrosis factor and IL-6 secretion are suppressed &gt;50%. 
These additional cytokines are also very influential mediators in 
inflammatory diseases. These data are the only known studies showing 
pentamidine's blockage of IL-6 and tumor necrosis factor. 
An imidazoline has also been shown to have the ability to block IL-1 and 
tumor necrosis factor in rat macrophages, and is less toxic to the cell 
with an in vitro LD.sub.50 &gt;10.sup.-4. 
Interleukin 1 release from pentamidine exposed rat pulmonary alveolar 
macrophages decreased in a concentration dependant manner in supernatant 
fluid 24 hours post-exposure to the stimulus lipolysaccharide (FIG. 1a). 
This observation was confirmed by Western blot analysis of secreted 
protein (FIG. 1b) which demonstrated the lack of a lipopolysaccharide 
inducible IL-1 band reacting with antibody to murine IL-1.alpha.. Since 
the classical inhibitors of IL-1, corticosteroids, have been shown to act 
at the level of gene transcription (P. J. Knudsen et al, J. Immunol. 139, 
4129 (1987)), the present inventors sought to determine whether 
pentamidine induces its suppressive activity in a similar manner. Specific 
transcription of IL-1.alpha. mRNA synthesis was assessed by incubating 
cells with and without 10 .mu.M pentamidine or 1 .mu.M of the 
corticosteroid, dexamethasone in the presence or absence of 
lipopolysaccharide (LPS). At the end of a 3 hour incubation period, the 
cells were lysed and the total cellular RNA was collected, blotted onto a 
nitrocellulose filter and probed with .sup.32 P-labeled IL-1.alpha. cDNA. 
FIG. 2 shows that both untreated cells and pentamidine treated cells 
expressed similar amounts of IL-1.alpha. mRNA, indicating that blockage of 
cytokine secretion is not at the level of transcription. In contrast, mRNA 
for IL-1.alpha. was not seen in dexamethasone treated cells. 
To determine the effect of pentamidine on cellular stores of IL-1, 
intracellular levels of IL-1 were quantitated by incubating cells with and 
without 10 .mu.M pentamidine and LPS for 24 hours. At the end of this 
incubation period, the cells were washed, lysed and the total cellular 
protein was collected for Western blot analysis. FIG. 3 shows a 
representative Western blot of such studies. In both control and 
pentamidine treated cells, antiserum to murine IL-1.alpha. bound to a 
protein that migrated to approximately 31 kD in the presence of LPS. This 
is in agreement with the generally accepted existence of a precursor form 
of IL-1 with a molecular weight in the range of 31-33 kD (J. Giri et al., 
J. Immunol. 134, 343 (1985)). In contrast to pentamidine, cells treated 
with dexamethasone did not express the precursor from of IL-1.alpha. (FIG. 
3). 
Kurt-Jones et al. (Proc. Natl., Acad. Sci. USA 82, 1204 (1985)) first 
demonstrated IL-1 activity on the plasma membrane (mIL-1) of peritoneal 
macrophages, and subsequent studies have similarly identified a membrane 
bound protein of approximately 31 kD with the biological and chemical 
characteristics of IL-1 (D. Brody et al., J. Immunol. 143, 1183 (1989)). 
It has been postulated that since the immune response requires 
cell-to-cell interaction, mIL-1 may be a relevant form in the activation 
of lymphocytes (M. Hurme et al., Scand. J. Immunol. 27 (1988)). The 
observation of substantial levels of cell associated IL-1 with both 
Western blot analysis (FIG. 3) and bioassay raised the question of whether 
this biologically active precursor IL-1 was localizing and perhaps 
accumulating on the membrane of pentamidine treated macrophages. As shown 
in FIG. 4, the amount of bioactive mIL-1 was increased in a dose related 
manner in pentamidine exposed cells, a finding that correlates inversely 
with the suppression of the secreted form. 
In contrast to pentamidine, the immunodepressrve activity of 
corticosteroids has been known for many years (T. R. Cupps et al., 
Immunological. Rev 65, 133 (1982); A. S. Fauci, J. Immunopharmacol. 1, 1 
(1979)). Unfortunately, the pleotropic nature of steroids, influencing all 
components of the immune system, have often rendered their use tenuous 
considering the lowered resistance of steroid treated hosts to a variety 
of infectious agents. To assess other immunomodulatory effects of 
pentamidine in comparison to dexamethasone, two other measures of 
macrophage function were examined; phagocytosis and Ia antigen expression. 
As seen in Example 5, in Table 1, pentamidine did not modulate the 
phagocytic capacity of alveolar macrophages nor did it influence the mean 
concentration of Ia molecules on Ia positive cells. In contrast, 
dexamethasone clearly demonstrated its broad immunosuppressive action on 
these two macrophage functions. 
These data taken together indicate that the mechanism of pentamidine 
induced inhibition of IL-1 occurs via alteration in the post-translational 
modification of the protein. Specifically, the cellular target plays a 
role in the intracellular and/or membrane cleavage of the 31 kDa pro-IL-1 
to a 17 kDa secreted form. As yet, the mechanisms underlying IL-1 release 
are only partially understood, although the precursor form appears to 
undergo enzymatic cleavage prior to release from the cell (C. Gunther et 
al., Immunobiol. 178, 436, (1989); Y. Kobayashi et al., J. Immunol. 140, 
2279 (1988)). The intracellular proteases suggested to be responsible for 
this processing include cathepsins (C. Gunther et al., Immunobiol. 178, 
436 (1989)), tissue plasminogen activator and plasmin (K. Matsushima et 
al., J. Immunol. 136, 2883 (1986)), and trypsin (K. Matsushima et al., J. 
Immunol. 136, 2883 (1986); 0. Bakocuhe et al., J. Immunol. 138, 4249 
(1987)). Pentamidine is a well known protease inhibitor (S. Vonderfecht et 
al., J. Clin. Invest. 82, 2011 (1988); Y. Klemes et al., Differentiation 
27, 141 (1984)) and it is possible that its action on IL-1 release is 
mediated by this anti-protease activity. Regardless, the diminished IL-1 
secretion induced by pentamidine offers a mechanistically unique and 
relatively specific inhibitor of IL-1. 
At least two members of IL-1 (.alpha. and .beta.) have been identified 
(reviewed in J. J. Oppenheim et al., Immunol. Today, 7, 45 (1986)), and 
are the translation products of two distinct genes, each gene coding for a 
precursor of approximately 31kDa to 33kDa (J. Giri et al., J. Immunol. 
134, 343 (1985)) which are subsequently processed to the secreted form 
(17kDa). In the murine system, IL-1.alpha. appears to be the biologically 
active membrane bound form as well as the predominant form of released 
IL-1, and for these reasons, this form of IL-1 was focused on in the 
studies that led to this invention. As such, it is not necessarily 
possible to extrapolate such data to IL-1.beta.. However, in humans, 
IL-1.beta. is the predominant form of the secreted cytokine, and along 
these lines, decreased IL-1.beta. release by human macrophages at similar 
concentrations as those reported herein have been observed (Example 5) 
indicating that the mechanism of action of pentamidine in IL-1 inhibition 
is not species specific and is not limited to the .alpha. form of the 
cytokine. 
IL-1 proteins are involved in a wide range of immunologic and inflammatory 
responses and also have endocrine function, which are attributed to their 
ability to modulate proliferation, maturation and functional activity of a 
broad spectrum of cell types (C. D. Dinarello, Review of Infect. Dis. 6, 
51 (1986)). Pharmacologic inhibition of cytokines, particularly IL-1, are 
expected to have a wide variety of therapeutic applications in 
inflammatory diseases including arthritis, granulomas of various organs, 
and fibrosis. Current therapeutics used for this purpose are the 
corticosteroids which, while effective inhibitors of IL-1, also affect 
macrophage functions as well as other immunological and nonimmunological 
responses. This lack of specificity often limit their therapeutic value. 
In contrast to the relative lack of immunologic specificity of 
corticosteroids, the immunologic specificity of pentamidine in inhibiting 
the secretion of IL-1 is demonstrated herein. 
The present invention will be illustrated in detail in the following 
examples. These examples are included for illustrative purposes and should 
not be considered to limit the present invention. 
EXAMPLE 1 
IL-1 Release From Pentamidine Exposed Rat Pulmonary Alveolar Macrophages 
Pulmonary alveolar macrophages were collected by lavaging the lungs as 
previously described (D. B. Warheit et al., Am. Rev. Resp. Dis. 134, 128 
(1986)). Once washed and resuspended, the macrophages were allowed to 
adhere to plastic plates in serum free RPMI 1640 containing 25 mM Hepes, 2 
mM L-glutamine, 50 .mu.g/ml gentamicin and streptomycin (media). Following 
adherence for 1 hour at 37.degree. C. in 5% CO.sub.2, the plates were 
washed once with warm media to remove nonadherent cells, cells were 
resuspended in media with 10% FCS and treated with or without pentamidine 
at the indicated concentration and LPS (5 ng/ml) for 24 hours). IL-1 
activity was assayed using a thymocyte co-stimulation assay (S. B. Mizel 
in Methods in Macrophage Biology, Editors, Herscowitz and Holden (1981)). 
Single cell thymocyte suspension from B6C3F1 mice (female, 5-9 weeks old) 
at a concentration of 2.0.times.10.sup.7 /ml in media containing 10% FCS, 
and 2.5.times.10.sup.-5 M 2-.beta. mercaptoethanol were added in 50 .mu.l 
aliquots to each well of flat bottom microtiter plates along with 50 .mu.l 
of PHA at 2.5 .mu.g/ml. Serial dilution of supernatants or cells were made 
in media with 10% FCS and added in 100 .mu.l aliquots. IL-1 activity was 
measured by quantifying the uptake of 3Hthymidine during the last 6 hours 
of a 72 hour culture at 37.degree. C. and 5% CO.sub.2. Cells were 
harvested onto glass fiber filters and filter associated radioactivity 
counted in a scintillation counter. 
The results are shown in FIG. 1a where it may be seen that IL-1 release 
from pentamidine exposed rat pulmonary alveolar macrophages decreased in a 
concentration dependent manner. 
EXAMPLE 2 
Northern Blot Analysis of IL-1.alpha. RNA Expression 
For determination of IL-1 mRNA levels, cells (4.times.10.sup.6) were 
cultured with or without pentamidine (10 .mu.M) or dexamethasone (1 .mu.M) 
in media with 10% FCS in the presence or absence of LPS (1 .mu.g/ml) for 3 
hours. Total cellular RNA was isolated by quanidinium-thiocyanate 
phenolchloroform extraction (P. Chomczynski et al., Anal. Bioch. 162, 156 
(1987)). RNA (10 .mu.g) was electrophoresed in 1.2% agarose gels as 
previously described (H. Leharch et al., Biochemistry 4743 (1977)). After 
electrophoresis, the gels were equilibrated in 10X SSC (1.5 M sodium 
chloride, 0.15M sodium citrate, pH 7.0) and transferred to Gene Screen 
Plus membrane (Du Pont) for capillary blot (E. M. Southern, J. Mol. Biol. 
98, 503 (1975)). The RNA was fixed by UV-crosslinking for 5 minutes at 300 
nm. The blot was hybridized with a 32P radiolabeled mouse IL-1.alpha. 
oligonucleotide probe by an oligonucleotide 3' end labeling system (Du 
Pont). Hybridization was carried out at 37.degree. C. in 50% formamide, 1M 
sodium chloride, 10% dextran sulfate and 1% SDS, heated denatured salmon 
sperm DNA (250 .mu.g/ml). The filter was washed 2.times. at room 
temperature in 1.0X SSC and 0.1% SDS and 2.times. in 0.1 XSSC and 1% SDS. 
Hybridizing species were detected by autoradiography at -70.degree. C., 
using Kodak XAR film with an intensifying screen. The total RNA levels per 
lane on the gel was assessed by monitoring 28S and 18S, and expression of 
.alpha.-tubulin mRNA. Accumulation of .alpha.-tubulin mRNA was determined 
by probing the same blot with .sup.32 P labeled 30 mer oligonucleotide (Du 
Pont). 
The results set forth in FIG. 2 show that both untreated cells (lane A) and 
pentamidine treated cells (lane B) expressed equal amounts of L-1.alpha. 
mRNA, indicating that blockage of cytokine secretion by pentamidine is not 
at the level of transcription. 
EXAMPLE 3 
Western Blot Analysis of Cell Associated IL-1.alpha. 
Cells (4.times.10.sup.6 cells) were oultured with pentamidine (10.mu.M) or 
dexamethasone (1.mu.M) in media with 10% fetal calf serum (FCS) in the 
presence or absence of LPS (1 .mu.g/ml) for 24 hours. The cells were 
washed twice in HBSS and then scraped in homogenizing buffer (HBSS 
containing 0.1 mM EGTA, 1 mM pmsf, 10 .mu.g/ml leupeptin, 1 KIU/ml 
aprotinin, pH 7.4). The cell suspension was left on ice for 10 minutes at 
which time the swollen cells were disrupted by sonication. The nuclei and 
undisrupted cells were pelleted by centrifugation at 800 rpm for 10 
minutes and were discarded. The supernatant was centrifugated at 39,000 
rpm for 1 hour at 4.degree. C. After centrifugation, the supernatants 
containing cytosolic components of the cell was carefully removed and was 
used as the cytosol fraction. The pellet was resuspended in homogenizing 
buffer and used as the particulate fraction. The sample were boiled in 
Laemmli sample buffer for 5 minutes and then electrophoresed (20 .mu.g of 
protein) into a 10-20% SDS polyacrylamide gel with a 4% stacking gel. (U. 
K. Laemmli, Nature 277, 680 (1970)). After electrophoresis, the gel was 
equilibrated for 30 minutes in 20 mM tris, 100 mM glycine, 20% methanol, 
pH 8.8 (tris buffer) and was transferred to a nitrocellulose membrane 
overnight at 100 mA using tris buffer. After transferring proteins to 
membrane, IL-1.alpha. was visualized by using rabbit antimurine 
IL-1.alpha. as the primary antibody (1:100 diluted) and goat anti-rabbit 
immunoglobulin conjugated with alkaline phosphatase as the secondary 
antibody (Immuno-blot assay kit, Bio-Rad). The primary antibody was tested 
previously for neutralization of rat IL-1.alpha. and found to inhibit in a 
dose related manner bioactive IL-1.alpha. as assayed by thymocyte 
co-stimulation assay. Molecular weight references were determined by 
running one lane with pre-stained molecular weight standards. 
The results shown in the Western blot analysis of FIG. 3 demonstrate that 
pentamidine allows proper translation of the protein, while dexamethasone 
does not. 
EXAMPLE 4 
Effect of Pentamidine on Membrane IL-1 
Membrane IL-1 was assayed as previously described (E. A. Kurt-Jones et al, 
Proc. Natl. Acad. Sci. USA, 82, 1204 (1985)). 10.sup.5 cells were cultured 
in media and 10% FCS with or without pentamidine at varying concentrations 
and LPS at 5 ng/ml for 24 hours and then fixed to the bottom of the 96 
well plates with paraformaldehyde. The IL-1 bioassay was then performed as 
described in Example 1. 
The results are shown in FIG. 4, where it may be seen that the amount of 
bioactive mIL-1 was increased in a dose related manner in pentamidine 
exposed cells. 
EXAMPLE 5 
Human Alveolar Macrophage Studies 
Four normal healthy human volunteers were lavaged and their pulmonary 
macrophages cultured in vitro in the presence of 10.sup.-5 M pentamidine 
and the IL-1 inducer, lipopolysaccharide. Following 24 hours of 
incubation, the supernatant fluid was collected and stored at -20.degree. 
C. for IL-1 determination using an enzyme linked immunosorbant assay with 
specific antibody to human IL-1. The results are shown in Table 1 below. 
TABLE 1 
______________________________________ 
Control* 
Pentamidine* 
______________________________________ 
Individual 1 7.7 1.3 
Individual 2 4.6 1.0 
Individual 3 6.2 4.3 
Individual 4 4.6 2.2 
______________________________________ 
*values represent mg/ml IL1 per 10.sup.6 human cells 
IL-1 production by pentamidine treated cells from these four human subjects 
cumulatively demonstrated a 62.1% suppression in the amount of IL-1 
secreted (control values derive from macrophages of the same individual 
that were not exposed to drug). These data clearly demonstrate that the 
inhibitory effects of pentamidine on IL-1 are not confined to rodents and 
cross species barriers effectively. 
EXAMPLE 6 
Immunomodulation by Pentamidine and Dexamethasone 
TABLE 2 
______________________________________ 
Is Antigen.sup.a 
Phagocytic 
Expression 
Index 
(MFI) (%) 
______________________________________ 
Control 459 32 
Pentamidine (10.sup.-5 M) 
464 32 
Dexamethasone (10.sup.-6 M) 
253* 25* 
______________________________________ 
*denotes statistically different from control values (p value &lt; 0.025) as 
determined by Students T test. 
.sup.a Ia antigen expression - Alveolar macrophages were diluted to 
1.times.10.sup.6 cells/ml in RPMI (supplemented as above). Three mls. 
(3.times.10.sup.6 cells) were added to sterile teflon vials and treated 
with pentamidine or dexamethasone for 4 hours at 37.degree. C., 5% 
CO.sub.2, followed by the addition of rat .sub..gamma. IFN at 100 U/ml. 
Cells were incubated for 18 hours, pelleted, counted and adjusted to 
1.times.10.sup.7 cells/ml in ice cold standard buffer (HBSS, 0.1% Na 
azide, 1% BSA). 
Aliquots of cells (50 .mu.l in quadruplicate) from the various treatment 
groups were plated to a 96 well round bottom plate (Linbro) and incubated 
for 45 minutes on ice with 50 .mu.l aliquots of one of the following: 
monoclonal anti-rat OXS (Sera-lab), monoclonal anti-mouse OX6 (Sera-lab), 
both at 0.5 .mu.g/5.times.10.sup.5 cells, or HBSS (control). Cells were 
washed 3.times. with standard buffer and incubated for 45 minutes on ice, 
in the dark, with 50 .mu.l of phycoerythrin-labeled goat anti-mouse IGGI 
(Fisher Biotech) at 1 .mu.g/5.times.10.sup.5 cells. Cells were washed 
3.times. with standard buffer, resuspended in 0.4 ml of standard buffer 
and analyzed via flow cytometry. 
.sup.b Phagocytosis - Pulmonary alveolar macrophages were adjusted to 
1.times.10.sup.6 cells/ml in RPMI 1640 containing 10% FCS (supplemented as 
above). One ml aliquots were added to sterile teflon vials (Nalgene) along 
with pentamidine or dexamethasone (Sigma) as indicated, and incubated for 
4 hours at 37.degree. C., 5% CO.sub.2. Fluoresbrite carboxylate beads, 
1.73 .mu.m (Polysciences) were used to achieve a 100:1 bead to cell ratio 
and the suspension was incubated for 45 minutes at 37.degree. C., 5% 
CO.sub.2 with gentle agitation. Cell suspensions were then layered over 3 
mls. of RPMI/FCS+0.3% BSA, and cells pelleted at 150 xg for 10 minutes. 
Supernates were discarded and cells resuspended in 1 ml of RPMI/FCS for 
analysis via flow cytometry. The data presented above is the mean of the 
percentage of cells taking up one or more beads of triplicate cultures. 
EXAMPLE 7 
Endotoxemia Following Pentamidine Administration 
Hypothermia and Survival 
Female B6C3F1 mice weighing approximately 24 g were dosed with pentamidine 
(15 mg/kg.; i.v.) at both 24 hours and 1 hour prior to LPS administration 
(50 mg/kg; i.p.). Temperature response indices (TRI) were determined for 
the period between time zero and +12 hours in groups of 5 mice. Table 3 
below also shows the mean rectal temperatures at time zero (Tr.sub.0) and 
12 hours (Tr.sub.12). Animals used for body temperature measurements were 
treated identically and on the same day as those animals for which 
survival was measured, although the survival of these animals is not 
included in the Table below. Survival data shown in Table 3 is following 
24 hours of observation. 
TABLE 3 
__________________________________________________________________________ 
Body Temp .degree.C. (.degree.C. .+-. SE) 
TRI Tr.sub.0 
Tr.sub.12hr 
(.DELTA..degree.C. .+-. SE) 
(.degree.C. .+-. SE) 
(.degree.C. .+-. SE) 
Survival 
__________________________________________________________________________ 
VEHICLE -1.3 38.2 .+-. 0.1 
36.9 .+-. 1.4 
10/10 
PENTAMIDINE -2.4 37.4 .+-. 0.6 
35.0 .+-. 2.9 
9/9 
LPS -13.3 38.6 .+-. 0.4 
25.3 .+-. 0.5 
0/10 
PENTAMIDINE + LPS 
-8.3* 37.3 .+-. 0.4 
29.0 .+-. 2.2* 
10/10* 
__________________________________________________________________________ 
Denotes statistically different from LPS group alone (P &lt; 0.01). 
EXAMPLE 8 
Effect of Pentamidine Isethionate on the Ear Swelling Response to Oxazolone 
All groups were sensitized with 25 .mu.l of 2.0% oxazolone on day 0 on the 
shaved dorsal surface of the back. On day 5, each group was treated with 
10 .mu.l of pentamidine on each side of the right ear immediately prior to 
challenge with 10 .mu.l of 0.5% oxazolone on each side of the right ear. 
The left ear was treated at same time with the oxazolone vehicle (4:1 
acetone:olive oil) and in the same manner as the challenged ear. Ear 
thickness was determined before, 24 and 48 hours after challenge. The 
concentration of pentamidine for each group was as follows: 
Group 1--80 .mu.g/ear pentamidine 
Group 2--40 .mu.g/ear pentamidine 
Group 3--20 .mu.g/ear pentamidine 
Group 4--10 .mu.g/ear pentamidine 
Group 5--5 .mu.g/ear pentamidine 
Group 6--no treatment (oxazolone positive control) 
The results shown in FIG. 5 indicate that treating mice with pentamidine 
eipcutaneously at the time of challenge resulted in a statistically 
significant (p&lt;0.001) reduction in the hypersensitivity response as 
measured by ear swelling. It is concluded that dermal application of 
pentamidine at the time of challenge with oxazolone is sufficient to 
produce a statistically significant reduction in ear swelling as a measure 
of delayed-type hypersensitivity. 
EXAMPLE 9 
Alveolar Macrophage Viability Following Exposure to Pentamidine 
Alveolar macrophage viability was assessed by incubating the cells in RPMI 
1640 media with 10% fetal calf serum in the presence of pentamidine at the 
indicated concentrations. Twenty-four hours later the cells were analyzed 
for viability using ethidium bromide staining. A minimum of 10,000 cells 
were analyzed by flow cytometry on a FACSCAN analyzer. 
The results are shown in FIG. 6. These observations have been confirmed 
using microscopic analysis via trypan blue dye exclusion. 
FIG. 6(a) shows the pulmonary alveolar macrophage viability following 24 
hour exposure to pentamidine at a molar concentration in the range of 0 to 
10.sup.-4 M. 
FIG. 6(b) is an expanded view of the FIG. 6(a) graph showing the pulmonary 
alveolar macrophage viability following 24 hour exposure to pentamidine at 
a molar concentration in the range of 10.sup.-4 M to 10.sup.-5 M. 
EXAMPLE 10 
Effect of an Imidazoline on Secretion of IL-1 and Tumor Necrosis Factor 
In vitro studies were performed by taking alveolar macrophages from normal 
healthy rat lungs and culturing them in the presence of an imidazoline 
with the inducer lipopolysaccharide for 24 hours. The standard bioassay 
for IL-1 was performed (see above). As shown in FIG. 7, the imidazoline 
blocked IL-1 in a dose related manner at 10.sup.-6 M, 10.sup.-5 M and 
10.sup.-4 M. No effect was observed at 10.sup.-8 M. Similar observations 
were observed with bioassays for tumor necrosis factor. 
TABLE 4 
______________________________________ 
Tumor Necrosis Factor (units/ml at 10.sup.-5 M Imidazoline) 
Control 
Imidazoline 
______________________________________ 
83 .+-. 11 
46 .+-. 19 
______________________________________ 
In addition, Western blot analysis of IL-1 on cellular protein derived from 
imidazoline treated cells was recently performed. This method definitively 
identifies the presence or absence of the protein using electrophoretic 
techniques to separate by molecular weight IL-1 from other proteins 
followed by staining with specific antibody fog IL-1. This very sensitive 
technique confirmed the bioassay determination that this compound blocks 
the secretion of IL-1 in a similar manner to pentamidine. 
The invention having been described, it will be appreciated by those 
skilled in the art, that various modifications in form and detail can be 
made without departing from the true scope of the invention.