Compositions and methods for decreasing IGIF and IFN-.gamma. production by administering an ICE inhibitor

The present invention relates to methods and pharmaceutical compositions for decreasing the production of interferon-gamma inducing factor (IGIF). The invention also relates to methods and pharmaceutical compositions for decreasing the production of interferon-gamma (IFN-.gamma.). The compositions comprise a therapeutically effective amount of a compound which inhibits interleukin-1.beta. converting enzyme (ICE) and a pharmaceutically acceptable carrier. The methods comprise the step of administering the above compositions to a subject. The present invention also relates to methods for treating or reducing the advancement, severity or effects of an IGIF- or IFN-.gamma.-mediated inflammatory, infectious or autoimmune condition.

TECHNICAL FIELD OF THE INVENTION 
The present invention relates to methods and pharmaceutical compositions 
for decreasing the production of interferon-gamma inducing factor (IGIF). 
The invention also relates to methods and pharmaceutical compositions for 
decreasing the production of interferon-gamma (IFN-.gamma.). The 
compositions comprise a therapeutically effective amount of a compound 
which inhibits interleukin-1.beta. converting enzyme (ICE) and a 
pharmaceutically acceptable carrier. The methods comprise the step of 
administering the above compositions to a subject. The present invention 
also relates to methods for treating or reducing the advancement, severity 
or effects of an IGIF- or IFN-.gamma.-mediated inflammatory, infectious or 
autoimmune condition. 
BACKGROUND OF THE INVENTION 
Interferon-gamma inducing factor (IGIF) is an approximately 18-kDa 
polypeptide that stimulates T-cell production of interferon-gamma 
(IFN-.gamma.). IGIF is produced by activated Kupffer cells and macrophages 
in vivo and is exported out of such cells upon endotoxin stimulation. 
Thus, a compound that decreases IGIF production would be useful as an 
inhibitor of such T-cell stimulation which in turn would reduce the levels 
of IFN-.gamma. production by those cells. 
IFN-.gamma. is a cytokine with immunomodulatory effects on a variety of 
immune cells. In particular, IFN-.gamma. is involved in macrophage 
activation and Th1 cell selection (F. Belardelli, APMIS, 103, p. 161 
(1995)). IFN-.gamma. exerts its effects in part by modulating the 
expression of genes through the STAT and IRF pathways (C. Schindler and J. 
E. Darnell, Ann. Rev. Biochem., 64, p. 621 (1995); T. Taniguchi, J. Cancer 
Res. Clin. Oncol., 121, p. 516 (1995)). 
Mice lacking IFN-.gamma. or its receptor have multiple defects in immune 
cell function and are resistant to endotoxic shock (S. Huang et al., 
Science, 259, p. 1742 (1993); D. Dalton et al., Science, 259, p. 1739 
(1993); B. D. Car et al., J. Exp. Md., 179, p. 1437 (1994)). Along with 
IL-12, IGIF appears to be a potent inducer of IFN-.gamma. production by T 
cells (H. Okamura et al., Infection and Immunity, 63, p. 3966 (1995); H. 
Okamura et al., Nature, 378, p. 88 (1995); S. Ushio et al., J. Immunol., 
156, p. 4274 (1996)). 
IFN-.gamma. has been shown to contribute to the pathology associated with a 
variety of inflammatory, infectious and autoimmune disorders and diseases. 
Thus, compounds capable of decreasing IFN-.gamma. production would be 
useful to ameliorate the effects of IFN-.gamma. related pathologies. 
The biological regulation of IGIF and thus IFN-.gamma. has not been 
elucidated. It is known that IGIF is synthesized as a precursor protein, 
called "pro-IGIF". It has been unclear, however, how pro-IGIF is cleaved 
and whether its processing has biological importance. 
Accordingly, compositions and methods capable of regulating the conversion 
of pro-IGIF to IGIF would be useful for decreasing IGIF and IFN-.gamma. 
production in vivo, and thus for ameliorating the detrimental effects of 
these proteins which contribute to human disorders and diseases. 
Another cytokine, IL-1.beta., is produced as an inactive precursor 
(pre-IL-1.beta.) which is proteolytically cleaved into an active, mature 
form (IL-1.beta.) by a cysteine protease called interleukin-1.beta. 
converting enzyme (ICE). ICE is a member of a larger family of cysteine 
proteases, called the ICE/CED-3 family, which share common structural and 
functional features. See, e.g., P. A. Henkarp, Immunity, 4, p. 195 (1996); 
D. W. Nicholson, Nature Biotechnology, 14, p. 297 (1996). 
However, ICE and other members of the ICE/CED-3 family have not previously 
been linked to the conversion of pro-IGIF to IGIF or to IFN-.gamma. 
production in vivo. 
SUMMARY OF THE INVENTION 
The present invention solves the problems above by providing methods and 
pharmaceutical compositions for decreasing the production of 
interferon-gamma inducing factor (IGIF) in vivo. The invention also 
provides methods and pharmaceutical compositions for decreasing the 
production of interferon-gamma (IFN-.gamma.) in vivo. The compositions of 
this invention comprise a therapeutically effective amount of a compound 
which inhibits interleukin-1.beta. converting enzyme (ICE) and a 
pharmaceutically acceptable carrier. The methods of this invention 
comprise the step of administering one or more of the above compositions 
to a subject. The present invention also provides methods for treating or 
reducing the advancement, severity or effects of an IGIF- or 
IFN-.gamma.-mediated inflammatory, infectious or autoimmune condition.

DETAILED DESCRIPTION OF THE INVENTION 
In order that the invention described herein may be more fully understood, 
the following detailed description is set forth. 
The following abbreviations and definitions are used throughout the 
application. 
The term "interferon gamma inducing factor" or "IGIF" refers to a factor 
which is capable of stimulating the endogenous production of IFN-.gamma.. 
The term "ICE inhibitor" refers to a compound which is capable of 
inhibiting the ICE enzyme. ICE inhibition may be determined using the 
methods described and incorporated by reference herein. The skilled 
practitioner realizes that an in vivo ICE inhibitor is not necessarily an 
in vitro ICE inhibitor. For example, a prodrug form of a compound 
typically demonstrates little or no activity in in vitro assays. Such 
prodrug forms may be altered by metabolic or other biochemical processes 
in the patient to provide an in vivo ICE inhibitor. 
The term "cytokine" refers to a molecule which mediates interactions 
between cells. 
The term "condition" refers to any disease, disorder or effect that 
produces deleterious biological consequences in a subject. 
The term "subject" refers to an animal, or to one or more cells derived 
from an animal. Preferably, the animal is a mammal, most preferably a 
human. Cells may be in any form, including but not limited to cells 
retained in tissue, cell clusters, immortalized cells, transfected or 
transformed cells, and cells derived from an animal that have been 
physically or phenotypically altered. 
The term "patient" refers to a subject which is a human. 
Other definitions are set forth in the specification where necessary. 
ICE and TX Cleave and Thereby Activate Pro-IGIF 
The ICE protease was identified previously by virtue of its ability to 
process inactive pro-IL-1.beta. to mature active IL-1.beta., a 
pro-inflammatory molecule, in vitro and in vivo. Here we show that ICE and 
its close homologue TX (Caspase-4, C. Faucheu et al., EMBO, 14, p. 1914 
(1995)) can proteolytically cleave inactive pro-IGIF. This processing step 
is required to convert pro-IGIF to its active mature form, IGIF. Cleavage 
of pro-IGIF by ICE, and presumably by TX, also facilitates the export of 
IGIF out of cells. 
We first used transient co-expression of plasmids transfected into Cos 
cells to determine whether any known members of the ICE/CED-3 protease 
family can process pro-IGIF to IGIF in cultured cells (Example 1) (FIG. 
1A). 
FIG. 1A demonstrates that ICE cleaves pro-IGIF in Cos cells co-transfected 
with plasmids that express pro-IGIF in the presence of active ICE. Cos 
cells were transfected with an expression plasmid for pro-IGIF alone (lane 
2) or in combination with the indicated expression plasmids encoding wild 
type or inactive mutants of ICE/CED-3 family of proteases (lanes 3-12). 
Cell lysates were prepared and analyzed for the presence of IGIF protein 
by immunoblotting with an anti-IGIF antiserum. Lane 1 contained lysates 
from mock transfected cells. 
Co-expression of pro-IGIF with ICE or TX resulted in the cleavage of 
pro-IGIF into a polypeptide similar in size to the naturally-occurring 
18-kDa mature IGIF. This processing event is blocked by single point 
mutations that alter the catalytic cysteine residues and thus inactivate 
ICE and TX (Y. Gu et al., EMBO, 14, p. 1923 (1995)). 
Co-expression with CPP32 (Caspase-3), a protease involved in programmed 
cell death (T. Fernandes-Alnemri et al., J. Biol. Chem., 269, p. 30761 
(1994); D. W. Nicholson et al., Nature, 376 p. 37 (1995)), resulted in the 
cleavage of pro-IGIF into a smaller polypeptide, while co-expression with 
CMH-1 (Caspase-7), a close homolog of CPP32 (J. A. Lippke et al., J. Biol. 
Chem., 271, p. 1825 (1996)), failed to cleave pro-IGIF to any significant 
extent. Thus, ICE and TX appear to be capable of cleaving pro-IGIF into a 
polypeptide similar in size to the naturally-occurring 18 kDa IGIF. 
We next examined the ability of these cysteine proteases to cleave pro-IGIF 
in vitro using a purified, recombinant (His).sub.6 -tagged pro-IGIF as a 
substrate (Example 1). 
FIG. 1B demonstrates that pro-IGIF is cleaved in vitro by ICE. Purified 
recombinant (His).sub.6 -tagged pro-IGIF (2 .mu.g) was incubated with the 
indicated cysteine protease in the presence or absence of ICE or CPP32 
inhibitors as described in Example 1. The cleavage products were analyzed 
by SDS-PAGE and Coomassie Blue staining. The proteases and inhibitors used 
were: lane 1, buffer control; lane 2, 0.1 nM ICE; lane 3, 1 nM ICE; lanes 
4 and 5, 1 nM ICE with 10 nM 
Cbz-Val-Ala-Asp-[(2,6dichlorobenzoyl)oxy]methyl ketone and 100 nM 
Ac-Tyr-Val-Ala-Asp-aldehyde, respectively; lanes 6 and 7, 15 nM CPP32 with 
and without 400 nM Ac-Asp-Glu-Val-Asp-aldehyde (SEQ ID No. 2) (D. W. 
Nicholson et al., Nature, 376, p. 37 (1995)), respectively; lane 8, 100 nM 
CMH-1; lane 9, 10 units/ml granzyme B; and M, molecular weight markers in 
kDa. 
ICE cleaved the 24 kDa pro-IGIF into two polypeptides of approximately 
18-kDa and 6-KDa. N-terminal amino acid sequencing of the ICE cleavage 
products indicated that the 18-kDa polypeptide contains the same 
N-terminal amino acid residues (Asn-Phe-Gly-Arg-Leu) (SEQ ID No. 3) as the 
naturally occurring IGIF. This shows that ICE cleaves pro-IGIF at the 
authentic processing site (Asp35-Asn36) (H. Okamura et al., Infection and 
Immunity, 63, p. 3966 (1995); H. Okamura et al., Nature, 378, p. 88 
(1995)). N-terminal amino acid sequencing of the CPP32 cleavage products 
indicated that CPP32 cleaved pro-IGIF at Asp69-Ile7O. 
The cleavage by ICE of pro-IGIF is highly specific with a catalytic 
efficiency (k.sub.cat /K.sub.M) of 1.4.times.10.sup.7 M.sup.-1 s.sup.-1 
(K.sub.M =0.6.+-.0.1 .mu.M; k.sub.cat =8.6.+-.0.3 s.sup.-1) and is 
inhibited by specific ICE inhibitors (Ac-Tyr-Val-Ala-Asp-aldehyde, and 
Cbz-Val-Ala-Asp-[(2,6-(dichlorobenzoyl)oxy]methylketone, (N. A. Thornberry 
et al., Nature, 356, p. 768 (1992); R. E. Dolle et al., J. Med. Chem., 37, 
p. 563 (1994)). 
FIG. 1C demonstrates that ICE cleavage in vitro activates pro-IGIF. 
Uncleaved pro-IGIF, ICE- or CPP32-cleaved products of pro-IGIF, or 
recombinant mature IGIF (rIGIF) were each added to A.E7 cell cultures to a 
final concentration of 12 ng/ml (open bar) or 120 ng/ml (hatched bar) 
(Example 1). Eighteen hours later, IFN-.gamma. in the cultural medium was 
quantified by ELISA. While the uncleaved pro-IGIF had no detectable 
IFN-.gamma. inducing activity, ICE-cleaved pro-IGIF was active in inducing 
IFN-.gamma. production in Th1 cells. 
Like ICE, the ICE homolog TX also cleaved pro-IGIF into similarly sized 
polypeptides. However, its catalytic efficiency was about two orders of 
magnitude lower than that shown for ICE. 
Consistent with the observations from the Cos cell experiments above, CPP32 
cleaved pro-IGIF at a different site (Asp69-Ile7O) and the resulting 
polypeptides had little IFN-.gamma. inducing activity (FIG. 1C). CMH-1 and 
granzyme B each failed to cleave pro-IGIF to any significant extent. 
Together, these results demonstrate that, both in Cos cells and in vitro, 
ICE and TX are capable of processing the inactive pro-IGIF precursor at 
the authentic maturation site to generate a biologically active IGIF 
molecule. 
Processing of Pro-IGIF by ICE Facilitates Its Export 
IGIF is produced by activated Kupffer cells and macrophages in vivo and is 
exported out of the cells upon stimulation by endotoxin (H. Okamura et 
al., Infection and Immunity, 63, p. 3966 (1995); H. Okamura et al., 
Nature, 378, p. 88 (1995). We used the Cos cell co-expression system 
(Example 1) to examine whether the intracellular cleavage of pro-IGIF by 
ICE would facilitate the export of mature IGIF from the cell. Such is the 
case for pro-IL-1.beta. when it is cleaved by ICE into active IL-1.beta. 
(N. A. Thornberry et al., Nature, 356, p. 768 (1992)). 
In FIG. 2A, Cos cells transfected with an expression plasmid for pro-IGIF 
alone (lanes 2 and 6) or in combination with an expression plasmid 
encoding wild type (lanes 3 and 7) or inactive mutant ICE (lanes 4 and 8) 
were metabolically labeled with .sup.35 S-methionine (Example 2). Cell 
lysates (left) and conditioned medium (right) were immunoprecipitated with 
an anti-IGIF antiserum. The immunoprecipitated proteins were analyzed by 
SDS-PAGE and fluorography (FIG. 2A). 
An 18 kDa polypeptide corresponding in size to mature IGIF was detected in 
the conditioned medium of Cos cells co-expressing pro-IGIF and ICE, while 
Cos cells co-expressing pro-IGIF and an inactive ICE mutant (ICE-C285S), 
or pro-IGIF alone (-) exported only very low levels of pro-IGIF and no 
detectable mature IGIF. We estimate that about 10% of the mature IGIF was 
exported from co-transfected cells, while greater than 99% of pro-IGIF was 
retained within the cells. 
We also measured the presence of IFN-.gamma. inducing activity in cell 
lysates and in the conditioned medium of the above transfected cells 
(Example 2). IFN-.gamma. inducing activity was detected in both cell 
lysates and the conditioned medium of Cos cells co-expressing pro-IGIF and 
ICE, but not in cells expressing either pro-IGIF or ICE alone (FIG. 2B). 
These results indicate that ICE cleavage of pro-IGIF facilitates the export 
of mature, active IGIF from cells. 
Pro-IGIF is a Physioloaical Substrate of ICE In Vivo 
To study the role of ICE in the proteolytic activation and export of IGIF 
under physiological conditions, we examined the processing of pro-IGIF and 
export of mature IGIF from lipopolysaccharide (LPS)-activated Kupffer 
cells harvested from Propiobacterium acnes-elicited wild type and ICE 
deficient (ICE-/-) mice (Example 3). 
As shown in FIG. 3A, Kupffer cells from ICE-/- mice are defective in the 
export of IGIF. Kupffer cell lysates of wild type and ICE-/- mice 
contained similar amounts of IGIF as determined by ELISA. IGIF, however, 
could be detected only in the conditioned medium of wild type but not of 
the ICE-/- cells. Thus, ICE-deficient (ICE-/-) mice synthesize pro-IGIF, 
but fail to export it as extracellular pro- or mature IGIF. 
To determine whether ICE-deficient (ICE-/-) mice process intracellular 
pro-IGIF but fail to export IGIF, Kupffer cells from wild type and ICE-/- 
mice were metabolically labeled with .sup.35 S-methionine and IGIF 
immunoprecipitation experiments were performed on cell lysates and 
conditioned media as described in Example 3. These experiments 
demonstrated that unprocessed pro-IGIF was present in both wild type and 
ICE-/- Kupffer cells. However, the 18 kDa mature IGIF was present only in 
the conditioned medium of wild type and not ICE-/- Kupffer cells (FIG. 
3B). This shows that active ICE is required in cells for the export of 
processed IGIF out of the cell. 
In addition, conditioned medium from wild type but not from ICE-/- Kupffer 
cells contained IFN-.gamma. inducing activity that was not attributed to 
the action of IL-12 because it was insensitive to a neutralizing 
anti-IL-12 antibody. The absence of IGIF in the conditioned medium of 
ICE-/- Kupffer cells is consistent with the finding in Cos cells that the 
processing of pro-IGIF by ICE is required for the export of active IGIF. 
FIGS. 3C and 3D show that, in vivo, ICE-/- mice have reduced serum levels 
of IGIF and IFN-.gamma., respectively. Wild type (ICE+/+) and ICE-/- mice 
(n=3) primed with heat-inactivated P. acnes were challenged with LPS 
(Example 4), and the levels of IGIF (FIG. 3C) and IFN-.gamma. (FIG. 3D) in 
the sera of challenged mice were measured by ELISA three hours after LPS 
challenge (Example 3). 
The sera of ICE-/- mice stimulated by P. acnes and LPS contained reduced 
levels of IGIF (FIG. 3C) and no detectable IFN-.gamma. inducing activity 
in the presence of an anti-IL-12 antibody. The reduced serum levels of 
IGIF likely accounts for the significantly lower levels of IFN-.gamma. in 
the hera of ICE-/- mice (FIG. 3D), because we have observed no significant 
difference in the production of IL-12 in ICE-/- mice under these 
conditions. Consistent with this interpretation is the finding that 
non-adherent splenocytes from wild type and ICE-/- mice produced similar 
amounts of IFN-.gamma. when stimulated with recombinant active IGIF in 
vitro. Thus the impaired production of IFN-.gamma. is not due to any 
apparent defect in the T cells of the ICE-/- mice. 
Taken together, these results establish a critical role for ICE in 
processing the IGIF precursor and in the export of active IGIF both in 
vitro and in vivo. 
To examine in more detail the relationship between serum levels of 
IFN-.gamma. and ICE activity in vivo, a time course after challenge of 
wild type and ICE-deficient mice with LPS was performed (Example 4) (FIG. 
4). 
FIG. 4 shows a time course increase of serum IFN-.gamma. in wild type mice, 
with sustained levels of .gtoreq.17 ng/ml occurring from 9-18 hrs after 
LPS challenge. As predicted by the experiments discussed above, serum 
IFN-.gamma. levels were significantly lower in ICE-/- mice, with a maximum 
of 2 ng/ml achieved over the same time period, which is approximately 15% 
of the level observed in wild type mice (FIG. 4). 
Animals were also observed for clinical signs of sepsis and body 
temperature was measured at 4-hour intervals in wild type and ICE-/- mice 
challenged with 30 mg/kg or 100 mg/kg LPS (ICE-/- only). Results in FIG. 4 
show that wild type mice experienced a significant decrease in body 
temperature (from 36.degree. C. to 26.degree. C.) within 12 hours of LPS 
challenge. Signs of clinical sepsis were evident and all animals expired 
within 24-28 hours. 
In contrast, ICE-/- mice challenged with 30 mg/kg LPS experienced only a 
3.degree.-4.degree. C. decrease in body temperature with minimal signs of 
distress and with no observed lethality. ICE-/- mice challenged with 100 
mg/kg LPS experienced clinical symptoms, a decrease in body temperature, 
and mortality similar to wild type mice at the 30 mg/kg LPS dose. 
The ICE Inhibitor Ac-YVAD-CHO (SEQ ID No. 1) is an Equipotent Inhibitor of 
IL-1.beta. and IFN-.gamma. Production 
Since the processing and secretion of biologically active IGIF is mediated 
by ICE, we compared the activity of a reversible ICE inhibitor 
(Ac-YVAD-CHO (SEQ ID NO: 1)) on IL-1.beta. and IFN-.gamma. production in a 
peripheral blood mononuclear cell (PBMC) assay (Examples 5 and 6). 
Results in FIG. 5 show a similar potency for the ability of the Ac-YVAD-CHO 
(SEQ ID No. 1) ICE inhibitor to decrease IL-1.beta. and IFN-.gamma. 
production in human PBMCs, with an IC.sub.50 of 2.5 .mu.M for each. 
Similar results were obtained in studies with wild type mouse splenocytes. 
These findings provide additional evidence that pro-IGIF is a physiological 
substrate for ICE and suggest that ICE inhibitors will be useful tools for 
controlling physiological levels of IGIF and IFN-.gamma.. 
In summary, we have found that ICE controls IGIF and IFN-.gamma. levels in 
vivo and in vitro and that ICE inhibitors can decrease levels of IGIF and 
IFN-.gamma. in human cells. 
Compositions and Methods for Controlling IGIF and IFN-.gamma. 
The pharmaceutical compositions and methods of this invention will be 
useful for controlling IGIF and IFN-.gamma. levels in vivo. The methods 
and compositions of this invention will thus be useful for treating or 
reducing the advancement, severity of effects of IGIF- and 
IFN-.gamma.-mediated conditions. 
Accordingly, one embodiment of this invention provides a method for 
decreasing IGIF production in a subject comprising the step of 
administering to the subject a pharmaceutical composition comprising a 
therapeutically effective amount of an ICE inhibitor and a 
pharmaceutically acceptable carrier. 
Another embodiment of this invention provides a method for decreasing 
IFN-.gamma. production in a subject comprising the step of administering 
to the subject a pharmaceutical composition comprising a therapeutically 
effective amount of an ICE inhibitor and a pharmaceutically acceptable 
carrier. 
In another embodiment, the methods of this invention comprise the step of 
administering to a subject a pharmaceutical composition comprising an 
inhibitor of an ICE-related protease that is capable of cleaving pro-IGIF 
to active IGIF, and a pharmaceutically acceptable carrier. One such 
ICE-related protease is TX, as described above. This invention thus 
provides methods and pharmaceutical compositions for controlling IGIF and 
IFN-.gamma., levels by administering a TX inhibitor. 
Other ICE-related proteases capable of processing pro-IGIF into an active 
IGIF form may also be found. Thus it is envisioned that inhibitors of 
those enzymes may be identified by those of skill in the art and will also 
fall within the scope of this invention. 
Pharmaceutical compositions of this invention comprise an ICE inhibitor or 
a pharmaceutically acceptable salt thereof and a pharmaceutically 
acceptable carrier, adjuvant or vehicle. Such compositions may optionally 
comprise an additional therapeutic agent. Such agents include, but are not 
limited to, an anti-inflammatory agent, a matrix metalloprotease 
inhibitor, a lipoxygenase inhibitor, a cytokine antagonist, an 
immunosuppressant, an anti-cancer agent, an anti-viral agent, a cytokine, 
a growth factor, an immunomodulator, a prostaglandin or an anti-vascular 
hyperproliferation compound. 
If the pharmaceutical composition comprises only the ICE inhibitor as the 
active component, such methods may additionally comprise the step of 
administering to the subject an additional agent. Such agents include, but 
are not limited to, an anti-inflammatory agent, a matrix metalloprotease 
inhibitor, a lipoxygenase inhibitor, a cytokine antagonist, an 
immunosuppressant, an anti-cancer agent, an anti-viral agent, a cytokine, 
a growth factor, an immunomodulator, a prostaglandin or an anti-vascular 
hyperproliferation compound. 
The term "pharmaceutically acceptable carrier or adjuvant" refers to a 
carrier or adjuvant that may be administered to a subject, together with a 
compound of this invention, and which does not destroy the pharmacological 
activity thereof and is nontoxic when administered in doses sufficient to 
deliver a therapeutic amount of the compound. 
Pharmaceutically acceptable carriers, adjuvants and vehicles that may be 
used in the pharmaceutical compositions of this invention include, but are 
not limited to, ion exchangers, alumina, aluminum stearate, lecithin, 
self-emulsifying drug delivery systems (SEDDS) such as d.alpha.-tocopherol 
polyethyleneglycol 1000 succinate, or other similar polymeric delivery 
matrices, serum proteins, such as human serum albumin, buffer substances 
such as phosphates, glycine, sorbic acid, potassium sorbate, partial 
glyceride mixtures of saturated vegetable fatty acids, water, salts or 
electrolytes, such as protamine sulfate, disodium hydrogen phosphate, 
potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal 
silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based 
substances, polyethylene glycol, sodium carboxymethylcellulose, 
polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, 
polyethylene glycol and wool fat. Cyclodextrins such as .alpha.-, .beta.-, 
and .gamma.-cyclodextrin, or chemically modified derivatives such as 
hydroxyalkylcyclodextrins, including 2- and 
3-hydroxypropyl-.beta.-cyclodextrins, or other solubilized derivatives may 
also be advantageously used to enhance delivery of compounds of formulae 
I-V. 
The pharmaceutical compositions of this invention may be administered 
orally, parenterally, by inhalation spray, topically, rectally, nasally, 
buccally, vaginally or via an implanted reservoir. We prefer oral 
administration or administration by injection. The pharmaceutical 
compositions of this invention may contain any conventional non-toxic 
pharmaceutically-acceptable carriers, adjuvants or vehicles. In some 
cases, the pH of the formulation may be adjusted with pharmaceutically 
acceptable acids, bases or buffers to enhance the stability of the 
formulated compound or its delivery form. The term parenteral as used 
herein includes subcutaneous, intracutaneous, intravenous, intramuscular, 
intra-articular, intrasynovial, intrasternal, intrathecal, intralesional 
and intracranial injection or infusion techniques. 
The pharmaceutical compositions may be in the form of a sterile injectable 
preparation, for example, as a sterile injectable aqueous or oleaginous 
suspension. This suspension may be formulated according to techniques 
known in the art using suitable dispersing or wetting agents (such as, for 
example, Tween 80) and suspending agents. The sterile injectable 
preparation may also be a sterile injectable solution or suspension in a 
non-toxic parenterally-acceptable diluent or solvent, for example, as a 
solution in 1,3-butanediol. Among the acceptable vehicles and solvents 
that may be employed are mannitol, water, Ringer's solution and isotonic 
sodium chloride solution. In addition, sterile, fixed oils are 
conventionally employed as a solvent or suspending medium. For this 
purpose, any bland fixed oil may be employed including synthetic mono- or 
diglycerides. Fatty acids, such as oleic acid and its glyceride 
derivatives are useful in the preparation of injectables, as are natural 
pharmaceutically-acceptable oils, such as olive oil or castor oil, 
especially in their polyoxyethylated versions. These oil solutions or 
suspensions may also contain a long-chain alcohol diluent or dispersant 
such as those described in Ph. Helv. (Pharmacopeia Helvetica) or a similar 
alcohol. 
The pharmaceutical compositions of this invention may be orally 
administered in any orally acceptable dosage form including, but not 
limited to, capsules, tablets, and aqueous suspensions and solutions. In 
the case of tablets for oral use, carriers which are commonly used include 
lactose and corn starch. Lubricating agents, such as magnesium stearate, 
are also typically added. For oral administration in a capsule form, 
useful diluents include lactose and dried corn starch. When aqueous 
suspensions are administered orally, the active ingredient is combined 
with emulsifying and suspending agents. If desired, certain sweetening 
and/or flavoring and/or coloring agents may be added. 
The pharmaceutical compositions of this invention may also be administered 
in the form of suppositories for rectal administration. These compositions 
can be prepared by mixing a compound of this invention with a suitable 
non-irritating excipient which is solid at room temperature but liquid at 
the rectal temperature and therefore will melt in the rectum to release 
the active components. Such materials include, but are not limited to, 
cocoa butter, beeswax and polyethylene glycols. 
Topical administration of the pharmaceutical compositions of this invention 
is especially useful when the desired treatment involves areas or organs 
readily accessible by topical application. For application topically to 
the skin, the pharmaceutical composition should be formulated with a 
suitable ointment containing the active components suspended or dissolved 
in a carrier. Carriers for topical administration of the compounds of this 
invention include, but are not limited to, mineral oil, liquid petroleum, 
white petroleum, propylene glycol, polyoxyethylene polyoxypropylene 
compound, emulsifying wax and water. Alternatively, the pharmaceutical 
composition can be formulated with a suitable lotion or cream containing 
the active compound suspended or dissolved in a carrier. Suitable carriers 
include, but are not limited to, mineral oil, sorbitan monostearate, 
polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, 
benzyl alcohol and water. The pharmaceutical compositions of this 
invention may also be topically applied to the lower intestinal tract by 
rectal suppository formulation or in a suitable enema formulation. 
Topically-transdermal patches are also included in this invention. 
The pharmaceutical compositions of this invention may be administered by 
nasal aerosol or inhalation. Such compositions are prepared according to 
techniques well-known in the art of pharmaceutical formulation and may be 
prepared as solutions in saline, employing benzyl alcohol or other 
suitable preservatives, absorption promoters to enhance bioavailability, 
fluorocarbons, and/or other solubilizing or dispersing agents known in the 
art. 
Dosage levels of between about 0.01 and about 100 mg/kg body weight per 
day, preferably between about 0.5 and about 75 mg/kg body weight per day 
of the ICE inhibitor compounds described herein are useful in a 
monotherapy for the prevention and treatment of IGIF or IFN-.gamma. 
mediated conditions, diseases, or effects. Typically, the pharmaceutical 
compositions of this invention will be administered from about 1 to about 
5 times per day or alternatively, as a continuous infusion. Such 
administration can be used as a chronic or acute therapy. The amount of 
active ingredient that may be combined with the carrier materials to 
produce a single dosage form will vary depending upon the host treated and 
the particular mode of administration. A typical preparation will contain 
from about 5% to about 95% active compound (w/w). Preferably, such 
preparations contain from about 20% to about 80% active compound. 
When the compositions of this invention comprise a combination of an ICE 
inhibitor and one or more additional therapeutic or prophylactic agents, 
both the ICE inhibitor and the additional agent should be present at 
dosage levels of between about 10% to 100%, and more preferably between 
about 10% to 80% of the dosage normally administered in a monotherapy 
regimen. 
When the subject is a patient, upon improvement of the patient's condition, 
a maintenance dose of a compound, composition or combination of this 
invention may be administered, if necessary. Subsequently, the dosage or 
frequency of administration, or both, may be reduced, as a function of the 
symptoms, to a level at which the improved condition is retained when the 
symptoms have been alleviated to the desired level, treatment should 
cease. Patients may, however, require intermittent treatment on a 
long-term basis upon any recurrence of symptoms. 
As the skilled artisan will appreciate, lower or higher doses than those 
recited above may be required. When the subject is a patient, specific 
dosage and treatment regimens for any particular patient will depend upon 
a variety of factors, including the activity of the specific compound 
employed, the age, body weight, general health status, sex, diet, time of 
administration, rate of excretion, drug combination, the severity and 
course of the infection, the patient's disposition to the infection and 
the judgment of the treating physician. 
The methods of this invention may be used for treating, or reducing the 
advancement, severity or effects of an IGIF- or IFN-.gamma.-mediated 
inflammatory, autoimmune, infectious, proliferative, destructive bone, 
necrotic, and degenerative conditions, including diseases, disorders or 
effects, wherein the conditions are characterized by increased levels of 
IGIF or IFN-.gamma. production. 
Inflammatory conditions which may be treated or prevented include, but are 
not limited to osteoarthritis, acute pancreatitis, chronic pancreatitis, 
asthma, rheumatoid arthritis, inflammatory bowel disease, Crohn's disease, 
ulcerative collitis, cerebral ischemia, myocardial iscemia and adult 
respiratory distress syndrome. 
Preferably, the inflammatory condition is rheumatoid arthritis, ulcerative 
collitis, Crohn's disease, hepatitis and adult respiratory distress 
syndrome. 
Infectious conditions which may be treated or prevented include, but are 
not limited to, infectious hepatitis, sepsis, septic shock and 
Shigellosis. 
Autoimmune conditions which may be treated or prevented include, but are 
not limited to, glomerulonephritis, systemic lupus erythematosus, 
scleroderma, chronic thyroiditis, Graves'disease, autoimmune gastritis, 
insulin-dependent diabetes mellitus (Type I), juvenile diabetes, 
autoimmune hemolytic anemia, autoimmune neutropenia, thrombocytopenia, 
myasthenia gravis, multiple sclerosis, psoriasis, lichenplanus, graft vs. 
host disease, acute dermatomyositis, eczema, primary cirrhosis, hepatitis, 
uveitis, Behcet's disease, acute dermatomyositis, atopic skin disease, 
pure red cell aplasia, aplastic anemia, amyotrophic lateral sclerosis and 
nephrotic syndrome. 
Preferably the autoimmune condition is glomerulonephritis, 
insulin-dependent diabetes mellitus (Type I), juvenile diabetes, 
psoriasis, graft vs. host disease, including transplant rejection, and 
hepatitis. 
Destructive bone disorders which may be treated or prevented include, but 
are not limited to, osteoporosis and multiple myeloma-related bone 
disorder. 
Proliferative conditions which may be treated or prevented include, but are 
not limited to, acute myelogenous leukemia, chronic myelogenous leukemia, 
metastatic melanoma, Kaposi's sarcoma, and multiple myeloma. 
The neurodegenerative conditions which may be treated or prevented by the 
compounds of this invention include, but are not limited to, Alzheimer's 
disease, Parkinson's disease and Huntington's disease. 
ICE Inhibitors 
ICE inhibitors which may be used according to the embodiments of this 
invention include those described in published PCT Application WO 
95/35308, in particular, see pages 77-89, 92-93, 96-97, 102-103, 107-108, 
and 125-127; and co-pending U.S. applications Ser. Nos. 08/575,641, filed 
Dec. 20, 1995, in particular, see pages 50-53, 55-63, and 76-126; Ser. No. 
08/575,648, filed Dec. 20, 1995, in particular, see pages 20-24 and 25-26; 
and Ser. No. 08/598,332, filed Feb. 2, 1996, in particular, see pages 
50-53, 56-63, 77-126, 166-167, 172-173 and 206. 
Further examples of ICE inhibitors which may be used according to the 
embodiments of this invention are those found in U.S. Pat. Nos. 5,008,245; 
5,411,985; 5,416,013; 5,430,128; 5,434,248; 5,462,939; 5,486,623; 
5,498,616 and 5,498,695; PCT published applications WO 91/15577; WO 
93/05071; WO 93/09135; WO 93/14777; WO 93/16710; WO 94/03480; WO 95/05192; 
WO 95/26958; WO 95/29672; WO 95/33751 and WO 96/03982; Foreign patent 
documents EP 519,748; EP 528,487; EP 529,713; EP 533,226; EP 547,699; EP 
618,223; EP 623,592; EP 623,606; EP 628,550; EP 644,197; EP 644,198; AU 
64514/94; DE 195 34 164; and GB 2,292,149; and other documents, such as M. 
Ator, "Peptide and Non-peptide Inhibitors of Interleukin-1.beta. 
Converting Enzyme", Cambridae Healthtech Institute (Inflammatory Cytokine 
Antaaonists Targets, Strategies, and Indication), (1994), see pyridazines, 
pages 2-4; peptides, pages 5-13; M. Ator and R. Dolle, 
"Interleukin-1.beta. Converting Enzyme: Biology and the Chemistry of 
Inhibitors", Curr. Pharm. Design, I, pp. 191-210 (1995); K. Chapman, 
"Synthesis of a Potent, Reversible Inhibitor of Interleukin-1.beta. 
Converting Enzyme", Bioorg. Med. Chem. Lett., 2, pp. 613-618 (1992); R. 
Dolle et al., "Aspartyl 
.alpha.-((1-Phenyl-3-(trifluoromethyl)-pyrazol-5-yl)oxy)methyl Ketones as 
Interleukin-1.beta. Converting Enzyme Inhibitors. Significance of the 
P.sub.1 and P.sub.3 Amido Nitrogers for Enzyme-Peptide Inhibitor Binding", 
J. Med. Chem., 37, pp. 3863-3865 (1994), see page 364; R. Dolle et al., 
"Aspartyl .alpha.-((Diphenylphosphinyl)oxy)methyl Ketones as Novel 
Inhibitors of Interleukin-1.beta. Converting Enzyme. Utility of the 
Diphenylphosphinic Acid Leaving Group for the Inhibition of Cysteine 
Proteases", J. Med. Chem., 38, pp. 220-222 (1995), see page 221; R. Dolle 
et al., "P.sub.1 Aspartate-Based Peptide 
.alpha.-((2,6-Dichlorobenzoyl)oxy)methyl Ketones as Potent Time-Dependent 
Inhibitors of Interleukin-1.beta.-Converting Enzyme", J. Med. Chem., 37, 
pp. 563-564 (1994), see page 563; R. Dolle et al., "First Examples of 
Peptidomimetic Inhibitors of Interleukin-1.beta. Converting Enzyme", J. 
Med. Chem., 39, pp. 2438-2440 (1996); P. Elford et al., "Reduction of 
Inflammation and Pyrexia in the Rate by Oral Administration of SDZ 
224-015, an Inhibitor of the Interleukin-1.beta. Converting Enzyme", Brit. 
J. Pharm., 115, pp. 601-606 (1995); I. Fauszt et al., "Inhibition of 
Interleukin-1.beta. Converting Enzyme by Peptide Derivatives", Proc. of 
the 13th Am. Peptide Symp., June 20-25, 1993, Hodges, R. S. and Smith, J. 
A., Eds., Peptides, pp. 589-591 (1994); T. Graybill et al., "Preparation 
and evaluation of peptidic aspartyl hemiacetals as reversible inhibitors 
of interleukin-1.beta. converting enzyme (ICE)", Int. J. Peptide Protein 
Res., 44, pp. 173-182 (1994); T. Graybill et al., "Synthesis and 
Evaluation of Diacylhydrazines as Inhibitors of the Interleukin-1.beta. 
Converting Enzyme (ICE)", Bioorg. Med. Chem, Lett., 5, pp. 1197-1202 
(1995); B. Miller et al., "Inhibition of Mature IL-1.beta. Production in 
Murine Macrophages and a Murine Model of Inflammation by WIN 67694, an 
Inhibitor of IL-1.beta. Converting Enzyme", J. Immunol., 154, pp. 
1331-1338 (1995), see page 1332; A. Mjalli et al., "Phenylalkyl Ketones as 
Potent Reversible Inhibitors of Interleukin-1.beta. Converting Enzyme", 
Bioorg, Med. Chem. Lett., 3, pp. 2689-2692 (1993); A. Mjalli et al., 
"Synthesis of a Peptidyl 2,2-Difluoro-4-Phenylbutyl Ketone and its 
Evaluation as an Inhibitor of Interleukin-1.beta. Converting Enzyme", 
Bioorg. Med. Chem. Lett., 3, pp. 2693-2698 (1993); A. Mjalli et al., 
"Activated Ketones as Potent Reversible Inhibitors of Interleukin-1.beta. 
Converting Enzyme", Bioorg Med. Chem. Lett., 4, pp. 1965-1968 (1994), see 
page 1967; M. Mullican et al., "The Synthesis and Evaluation of Peptidyl 
Aspartyl Aldehydes as Inhibitors of ICE", Bioorg. Med. Chem. Lett., 4, pp. 
2359-2364 (1994), see page 2362; C. Ray et al., "Viral Inhibition of 
Inflammation: Cowpox Virus Encodes an Inhibitor of the Interleukin-1.beta. 
Converting Enzyme", Cell, 69, pp. 597-604 (1992); R. Robinson and K. 
Donahue, "Synthesis of a Peptidyl Difluoro Ketone Bearing the Aspartic 
Acid Side Chain: An Inhibitor of Interleukin-1.beta. Converting Enzyme", 
J. Org. Chem., 57, pp. 7309-7314 (1992), see page 7309; M. Salvatore et 
al., "L-741,494, A Fungal Metabolite that is an Inhibitor of 
Interleulin-1.beta. Converting Enzyme", J. Nat. Prods., 57, pp. 755-760 
(1994); S. Schmidt et al., "Synthesis and Evaluation of aspartyl 
.alpha.-Chloro-, .alpha.-Aryloxy-, and .alpha.-Arylacyloxymethyl Ketones 
as Inhibitors of Interleukin-1.beta. Converting Enzyme", Am. Chem. Soc. 
(208th Natl. Mtg.), MEDI 4, Aug. 21-25 (1994); N. Thornberry et al., 
"Inactivation of Interleukin-1.beta. Converting Enzyme by Peptide 
(Acyloxy)methyl Ketones", Biochemistry, 33, pp. 3934-3940 (1994), see 
pages 3937-3938; E. Tsukuda et al., "EI-1507 and -2, Novel 
Interleukin-1.beta. Converting Enzyme Inhibitors Produced by Streptomyces 
sp. E-1507", J. Antibiotics, 49, pp. 333-339 (1996). 
All of the cited documents are incorporated by reference herein. The ICE 
inhibitors cited in these documents may be used alone or in combination, 
in one or more embodiments of this invention. 
Selected examples of such ICE inhibitors include but are not limited to the 
following compounds: A1, A4, A5, A6, A7, A8, A9, A1, A11, A12, A13, A14, 
A15, A16, A17, A18, A19, A20, A22, A23, A24, A25, A27, A28, 213e, 214c, 
214e, 214e, 217c, 217d, 217e, 220b, 223b, 223e, 226e, 227e, 246, 257, 265, 
280, 281, 282, 283, 284, 285, 286, 287, 302, 304a, 308a, 308b, 404, 405, 
406, 407, 408, 409, 410, 411, 412, 413, 415, 416, 417, 418, 419, 420, 421, 
422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 
437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 
452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 
467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 
481s, 482, 482s, 483, 484, 485, 486, 487, 488, 489, 490, 491, 493, 494, 
495, 496, 497, 498, 499, 605a, 605b, 605c, 605d, 605e, 605f, 605g, 605h, 
605i, 605j, 605m, 605n, 605o, 605p, 605q, 605s, 605t, 605v, 609a, 609b, 
619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 
634, 635, 813e, 814c, 817c, 817d, 817e, 820b, 823b, 823e, 826e, 827e, 880, 
881, 882, 883, 884, 885, 886, 887, 902, 904a, 907a, 907b, 1004, 1005, 
1006, 1007, 1008, 1009, 1010, 1011, 1012, 1013, 1015, 1016, 1017, 1018, 
1019, 1020, 1021, 1022, 1023, 1024, 1025, 1026, 1027, 1028, 1029, 1030, 
1031, 1032, 1033, 1034, 1035, 1036, 1037, 1038, 1039, 1040, 1041, 1042, 
1043, 1044, 1045, 1046, 1047, 1048, 1049, 1050, 1051, 1052, 1053, 1054, 
1055, 1056, 1057, 1058, 1059, 11060, 1061, 1062, 1063, 1064, 1065, 1066, 
1067, 1068, 1069, 1070, 1071, 1073, 1074, 1075, 1076, 1077, 1078, 1079, 
1080, 1081, 1081s, 1082, 1082s, 1083, 1084, 1085, 1086, 1087, 1088, 1089, 
1090, 1091, 1093, 1094, 1095, 1096, 1097, 1098, 1099, 2001, 2002, 2100a, 
2100b, 2100c, 2100d, 2100e and 2201. 
At this point in time, a more preferred pharmaceutical composition 
comprises compound 412. 
The structures of the above selected compounds may be found in FIG. 6. 
These compounds may be prepared by standard methodologies. Further details 
about the preparation of such compounds may be found in the documents 
incorporated herein (in particular, see the Examples in published PCT 
Applications WO 95/35308 and WO 95/33751 and copending U.S. applications 
Ser. Nos. 08/575,641, filed Dec. 20, 1995, Ser. No. 08/575,648, filed Dec. 
20, 1995 and Ser. No. 08/598,332, filed Feb. 2, 1996. 
Preferred ICE inhibitors which may be used according to the embodiments of 
this invention are those of formula (I): 
##STR1## 
wherein: R.sub.1 is selected from the group consisting of the following 
formulae: 
##STR2## 
ring C is chosen from the group consisting of benzo, pyrido, thieno, 
pyrrolo, furano, thiazolo, isothiazolo, oxazolo, isoxazolo, pyrimido, 
imidazolo, cyclopentyl, and cyclohexyl; 
R.sub.2 is: 
##STR3## 
m is 1 or 2; each R.sub.5 is independently selected from the group 
consisting of: 
--C(O)--R.sub.10, 
--C(O)O--R.sub.9, 
--C(O)--N(R.sub.10)(R.sub.10) 
--S(O).sub.2 --R.sub.9, 
--S(O).sub.2 --NH--R.sub.10, 
--C(O)--CH.sub.2 --O--R.sub.9, 
--C(O)C(O)--R.sub.10, 
--R.sub.9, 
--H, 
--C(O)C(O)--OR.sub.10, and 
--C(O)C(O)--N(R.sub.9)(R.sub.10); 
X.sub.5 is 
##STR4## 
Y.sub.2 is H.sub.2 or O; R.sub.6 is selected from the group consisting of 
--H and --CH.sub.3 ; 
R.sub.8 is selected from the group consisting of: 
--C(O)--R.sub.10, 
--C(O)O--R.sub.9, 
--C(O)--N(H)--R.sub.10, 
--S(O).sub.2 --R.sub.9, 
--S(O).sub.2 --NH--R.sub.10, 
--C(O)--CH.sub.2 --OR.sub.10, 
--C(O)C(O)--R.sub.10 ; 
--C(O)--CH.sub.2 N(R.sub.10)(R.sub.10), 
--C(O)--CH.sub.2 C(O)--O--R.sub.9, 
--C(O)--CH.sub.2 C(O)--R.sub.9, 
--H, and 
--C(O)--C(O)--OR.sub.10 ; 
each R.sub.9 is independently selected from the group consisting of 
--Ar.sub.3 and a --C.sub.1-6 straight or branched alkyl group that is 
optionally substituted with Ar.sub.3, wherein the --C.sub.1-6 alkyl group 
is optionally unsaturated; 
each R.sub.10 is independently selected from the group consisting of --H, 
--Ar.sub.3, a --C.sub.3-6 cycloalkyl groap, and a --C.sub.1-6 straight or 
branched alkyl group optionally substituted with Ar.sub.3, wherein the 
--C.sub.1-6 alkyl group is optionally unsaturated; 
R.sub.13 is selected from the group consisting of H, Ar.sub.3, and a 
C.sub.1-6 straight or branched alkyl group optionally substituted with 
Ar.sub.3, --CONH.sub.2, --OR.sub.5, --OH, --OR.sub.9, or --CO.sub.2 H; 
each R.sub.51 is independently selected from the group consisting of 
R.sub.9, --C(O)--R.sub.9, --C(O)--N(H)--R.sub.9, or each R.sub.51 taken 
together forms a saturated 4-8 member carbocyclic ring or heterocyclic 
ring containing --O--, --S--, or --NH--; 
each R.sub.21 is independently selected from the group consisting of --H or 
a --C.sub.1-6 straight or branched alkyl group; 
each Ar.sub.3 is a cyclic group independently selected from the set 
consisting of an aryl group which contains 6, 10, 12, or 14 carbon atoms 
and between 1 and 3 rings; and an aromatic heterocycle group containing 
between 5 and 15 ring atoms and between 1 and 3 rings, said heterocyclic 
group containing at least one heteroatom group selected from --O--, --S--, 
--SO--, SO.sub.2, .dbd.N--, and --NH--, said heterocycle group optionally 
containing one or more double bonds, said heterocycle cgroup optionally 
comprising one or more aromatic rings, and said cyclic group optionally 
being singly or multiply substituted by --Q.sub.1 ; 
each Q.sub.1 is independently selected from the group consisting of 
--NH.sub.2, --CO.sub.2 H, --Cl, --F, --Br, --I, --NO.sub.2, --CN, .dbd.O, 
--OH, --perfluoro C.sub.1-3 alkyl, R.sub.5, --OR.sub.5, --NHR.sub.5, 
OR.sub.9, --N(Rg)(R.sub.10), R.sub.9, --C(O)--R.sub.10, and 
##STR5## 
provided that when --Ar.sub.3 is substituted with a --Q.sub.1 group which 
comprises one or more additiona --Ar.sub.3 groups, said additional 
--Ar.sub.3 groups are not substituted with another --Ar.sub.3. 
More preferably: 
m is 1; 
R.sub.13 is H or a --C.sub.1-4 straight or branched alkyl group optionally 
substituted with --Ar.sub.3, --OH, --OR.sub.9, or --CO.sub.2 H, wherein 
the R.sub.9 is a --C.sub.1-4 branched or straight alkyl group, wherein 
Ar.sub.3 is morpholinyl or phenyl, wherein the phenyl is optionally 
substituted with Q.sub.1 ; 
R.sub.21 is --H or --CH.sub.3 ; 
R.sub.51 is a C.sub.1-6 straight or branched alkyl group optionally 
substituted with Ar.sub.3, wherein Ar.sub.3 is phenyl, optionally 
substituted by --Q.sub.1 ; 
each Ar.sub.3 cyclic group is independently selected from the set 
consisting of phenyl, naphthyl, thienyl, quinolinyl, isoquinolinyl, 
pyrazolyl, thiazolyl, isoxazolyl, benzotriazolyl, benzimidazolyl, 
thienothienyl, imidazolyl, thiadiazolyl, benzo[b]thiophenyl, pyridyl, 
benzofuranyl, and indolyl, and said cyclic group optionally being singly 
or multiply substituted by --Q.sub.1 ; 
each Q.sub.1 is independently selected from the group consisting of 
--NH.sub.2, --Cl, --F, --Br, --OH, --R.sub.9, --NH--R.sub.5 wherein 
R.sub.5 is --C(O)--R.sub.10 or --S(O).sub.2 --R.sub.9, --OR.sub.5 wherein 
R.sub.5 is --C(O)--R.sub.10, --OR.sub.9, --N(R.sub.9)(R.sub.10), and 
##STR6## 
wherein each R.sub.9 and R.sub.10 are independently a --C.sub.1-6 
straight or branched alkyl group optionally substituted with Ar.sub.3 
wherein Ar.sub.3 is phenyl; 
provided that when --Ar.sub.3 is substituted with a --Q.sub.1 group which 
comprises one or more additional --Ar.sub.3 groups, said additional 
--Ar.sub.3 groups are not substituted with another --Ar.sub.3. 
Most preferably the ICE inhibitors of formula (I) are those wherein R.sub.1 
is (w2) and the other substituents are as described above. 
Other most preferred ICE inhibitors of formula (I) are those wherein 
R.sub.1 is (e10) and X.sub.5 is CH. 
Alternatively, in these most preferred ICE inhibitors, R.sub.1 is (e10) and 
X.sub.5 is N. 
Other preferred ICE inhibitors that may be used according to the 
embodiments of this invention are those of formula (II): 
##STR7## 
wherein: m is 1 or 2; 
R.sub.1 is selected from the group consisting of the following formulae: 
##STR8## 
ring C is chosen from the group consisting of benzo, pyrido, thieno, 
pyrrolo, furano, thiazolo, isothiazolo, oxazolo, isoxazolo, pyrimido, 
imidazolo, cyclopentyl, and cyclohexyl; 
R.sub.3 is selected from the group consisting of: 
--CN, 
--C(O)--H, 
--C(O)--CH.sub.2 --T.sub.1 --R.sub.11, 
--C(O)--CH.sub.2 --F, 
--C.dbd.N--O--R.sub.9, and 
--CO--Ar.sub.2 ; 
each R.sub.5 is independently selected from the group consisting of: 
--C(O)--R.sub.10, 
--C(O)O--R.sub.9, 
--C(O)--N(R.sub.10)(R.sub.10) 
--S(O).sub.2 --R.sub.9, 
--S(O).sub.2 --NH--R.sub.10, 
--C(O)--CH.sub.2 --O--R.sub.9, 
--C(O)C(O)--R.sub.10, 
--R.sub.9, 
--H, 
--C(O)C(O)--OR.sub.10, and 
--C(O)C(O)--N(R.sub.9)(R.sub.10); 
X.sub.5 is 
##STR9## 
Y.sub.2 is H.sub.2 or O; each T.sub.1 is independently selected from the 
group consisting of --O--, --S--, --S(O)--, and --S(O).sub.2 --; 
R.sub.6 is selected from the group consisting of --H and --CH.sub.3 ; 
R.sub.8 is selected from the group consisting of: 
--C(O)--R.sub.10, 
--C(O)O--R.sub.9, 
--C(O)--NH--R.sub.10, 
--S(O).sub.2 --R.sub.9, 
--S(O).sub.2 --NH--R.sub.10, 
--C(O)--CH.sub.2 --OR.sub.10, 
--C(O)C(O)--R.sub.10, 
--C(O)--CH.sub.2 --N(R.sub.10)(R.sub.10) 
--C(O)--CH.sub.2 C(O)--O--R.sub.9, 
--C(O)--CH.sub.2 C(O)--R.sub.9, 
--H, and 
--C(O)--C(O)--OR.sub.10 ; 
each R.sub.9 is independently selected from the group consisting of 
--Ar.sub.3 and a --C.sub.1-6 straight or branched alkyl group that is 
optionally substituted with Ar.sub.3, wherein the --C.sub.1-6 alkyl group 
is optionally unsaturated; 
each R.sub.10 is independently selected from the group consisting of --H, 
--Ar.sub.3, a C.sub.3-6 cycloalkyl group, and a --C.sub.1-6 straight or 
branched alkyl group optionally substituted with Ar.sub.3, wherein the 
--C.sub.1-6 alkyl group is optionally unsaturated; 
each R.sub.11 is independently selected from the group consisting of: 
--Ar.sub.4, 
--(CH.sub.2).sub.1-3 --Ar.sub.4, 
--H, and 
--C(O)--Ar.sub.4 ; 
R.sub.15 is selected from the group consisting of --OH, --OAr.sub.3, 
--N(H)--OH, and a --OC.sub.1-6 straight or branched alkyl group optionally 
substituted with --Ar.sub.3, --CONH.sub.2, --OR.sub.5, --OH, --OR.sub.9, 
or --CO.sub.2 H; 
each R.sub.21 is independently selected from the group consisting of --H or 
a --C.sub.1-6 straight or branched alkyl group; 
Ar.sub.2 is independently selected from the following group, in which any 
ring may optionally be singly or multiply substituted by --Q.sub.1 : 
##STR10## 
wherein each Y is independently selected from the group consisting of O 
and S; 
each Ar.sub.3 is a cyclic group independently selected from the set 
consisting of an aryl group which contains 6, 10, 12, or 14 carbon atoms 
and between 1 and 3 rings; and an aromatic heterocycle group containing 
between 5 and 15 ring atoms and between 1 and 3 rings, said heterocyclic 
group containing at least one heteroatom group selected from --O--, --S--, 
--SO--, SO.sub.2, .dbd.N--, and --NH--, --N(R.sub.5)--, and --N(R.sub.9)-- 
said heterocycle group optionally containing one or more double bonds, 
said heterocycle group optionally comprising one or more aromatic rings, 
and said cyclic group optionally being singly or multiply substituted by 
--Q.sub.1 ; 
each Ar.sub.4 is a cyclic group independently selected from the set 
consisting of an aryl group which contains 6, 10, 12, or 14 carbon atoms 
and between 1 and 3 rings, and a heterocycle group containing between 5 
and 15 ring atoms and between 1 and 3 rings, said heterocyclic group 
containing at least one heteroatom group selected from --O--, --S--, 
--SO--, SO.sub.2, .dbd.N--, --NH--, --N(R.sub.5)--, and --N(R.sub.9)-- 
said heterocycle group optionally containing one or more double bonds, 
said heterocycle group optionally comprising one or more aromatic rings, 
and said cyclic group optionally being singly or multiply substituted by 
--Q.sub.1 ; 
each Q.sub.1 is independently selected from the group consisting of 
--NH.sub.2, --CO.sub.2 H, --Cl, --F, --Br, --I, --NO.sub.2, --CN, .dbd.O, 
--OH, --perfluoro C.sub.1-3 alkyl, R.sub.5, --OR.sub.5, --NHR.sub.5, 
OR.sub.9, --N(R.sub.9)(R.sub.10), R.sub.9, --C(O)--R.sub.10, and 
##STR11## 
provided that when --Ar.sub.3 is substituted with a --Q.sub.1 group which 
comprises one or more additional --Ar.sub.3 groups, said additional 
--Ar.sub.3 groups are not substituted with another --Ar.sub.3. 
More preferred ICE inhibitors of formula (II) are those wherein R.sub.1 is 
(w2) and the other substituents are as described above. 
Most preferably in these more preferred ICE inhibitors: 
m is 1; 
ring C is benzo, pyrido, or thieno; 
R.sub.3 is selected from the group consisting of --C(O)--H, 
--C(O)--Ar.sub.2, and --C(O)CH.sub.2 --T.sub.1 --R.sub.11 ; 
R.sub.5 is selected from the group consisting of: 
--C(O)--R.sub.10, wherein R.sub.10 is --Ar.sub.3 ; 
--C(O)O--R.sub.9, wherein R.sub.9 is --CH.sub.2 --Ar.sub.3 ; 
--C(O)C(O)--R.sub.10, wherein R.sub.10 is --Ar.sub.3 ; 
--R.sub.9, wherein R.sub.9 is a C.sub.1-2 alkyl group substituted with 
--Ar.sub.3 ; and 
--C(O)C(O)--OR.sub.10, wherein R.sub.10 is --CH.sub.2 Ar.sub.3 ; 
T.sub.1 is O or S; 
R.sub.6 is H; 
R.sub.8 is selected from the group consisting --C(O)--R.sub.10, 
--C(O)--CH.sub.2 --OR.sub.10, and --C(O)CH.sub.2 --N(R.sub.10)(R.sub.10) 
wherein R.sub.10 is H, CH.sub.3, or --CH.sub.2 CH.sub.3 ; 
R.sub.11 is selected from the group consisting of --Ar.sub.4, 
--(CH.sub.2).sub.1-3 --Ar.sub.4, and --C(O)--Ar.sub.4 ; 
R.sub.15 is --OH or --OC.sub.1-4 straight or branched alkyl group 
optionally substituted with --Ar.sub.3, --OH, --OR.sub.9, or --CO.sub.2 H, 
wherein the R.sub.9 is a --C.sub.1-4 branched or straight alkyl group, 
wherein Ar.sub.3 is morpholinyl or phenyl, wherein the phenyl is 
optionally substituted with Q.sub.1 ; 
Ar.sub.2 is (hh); 
Y is O; 
each Ar.sub.3 cyclic group is independently selected from the set 
consisting of phenyl, naphthyl, thienyl, quinolinyl, isoquinolinyl, 
thiazolyl, benzimidazolyl, thienothienyl, thiadiazolyl, benzotriazolyl, 
benzo[b]thiophenyl, benzofuranyl, and indolyl, and said cyclic group 
optionally being singly or multiply substituted by --Q.sub.1 ; 
each Ar.sub.4 cyclic group is independently selected from the set 
consisting of phenyl, tetrazolyl, naphthyl, pyridinyl, oxazolyl, 
pyrimidinyl, or indolyl, said cyclic group optionally being singly or 
multiply substituted by --Q.sub.1 ; 
each Q.sub.1 is independently selected from the group consisting of 
--NH.sub.2, --Cl, --F, --Br, --OH, --R.sub.9, --NH--R.sub.5 wherein 
R.sub.5 is --C(O)--R.sub.10 or --S(O).sub.2 --R.sub.9, --OR.sub.5 wherein 
R.sub.5 is --C(O)--R.sub.10, --OR.sub.9, --N(R.sub.9)(R.sub.10), and 
##STR12## 
wherein each R.sub.9 and R.sub.10 are independently a --C.sub.1-6 
straight or branched alkyl group optionally substituted with Ar.sub.3 
wherein Ar.sub.3 is phenyl; 
provided that when --Ar.sub.3 is substituted with a --Q.sub.1 group which 
comprises one or more additional --Ar.sub.3 groups, said additional 
--Ar.sub.3 groups are not substituted with another --Ar.sub.3. 
Other more preferred ICE inhibitors are those wherein R.sub.1 is (e10), 
X.sub.5 is CH and the other substituents are as described above. 
More preferably, in these more preferred ICE inhibitors R.sub.3 is 
--CO--Ar.sub.2 and the other substituents are as described above. 
Alternatively, in these more preferred ICE inhibitors, wherein R.sub.3 is 
--C(O)--CH.sub.2 --T.sub.1 --R.sub.11 and R.sub.11 is --(CH.sub.2).sub.1-3 
--Ar.sub.4 and the other substituents are as described above. 
Alternatively, in these more preferred ICE inhibitors, R.sub.3 is 
--C(O)--CH.sub.2 --T.sub.1 --R.sub.11, T.sub.1 is O, and R.sub.11, is 
--C(O)--Ar.sub.4 and the other substituents are as described above. 
Alternatively, in these more preferred ICE inhibitors, R.sub.3 is --C(O)--H 
and the other substituents are as described above. 
Alternatively, in these more preferred ICE inhibitors, R.sub.3 is 
--CO--CH.sub.2 --T.sub.1 --R.sub.11 and R.sub.11 is --Ar.sub.4 and the 
other substituents are as described above. 
Most preferably, in these more preferred ICE inhibitors: 
m is 1; 
T.sub.1 is O or S; 
R.sub.13 is H or a --C.sub.1-4 straight or branched alkyl group optionally 
substituted with --Ar.sub.3, --OH, --OR.sub.9, or --CO.sub.2 H, wherein 
the R.sub.9 is a --C.sub.1-4 branched or straight alkyl group, wherein 
Ar.sub.3 is morpholinyl or phenyl, wherein the phenyl is optionally 
substituted with Q.sub.1 ; 
R.sub.21 is --H or --CH.sub.3 ; 
R.sub.51 is a C.sub.1-6 straight or branched alkyl group optionally 
substituted with Ar.sub.3, wherein Ar.sub.3 is phenyl, optionally 
substituted by --Q.sub.1 ; 
Ar.sub.2 is (hh); 
Y is O, and 
each Ar.sub.3 cyclic group is independently selected from the set 
consisting of phenyl, naphthyl, thienyl, quinolinyl, isoquinolinyl, 
pyrazolyl, thiazolyl, isoxazolyl, benzotriazolyl, benzimidazolyl, 
thienothienyl, imidazolyl, thiadiazolyl, benzo[b]thiophenyl, pyridyl, 
benzofuranyl, and indolyl, and said cyclic group optionally being singly 
or multiply substituted by --Q.sub.1 ; 
each Ar.sub.4 cyclic group is independently selected from the set 
consisting of phenyl, tetrazolyl, pyridinyl, oxazolyl, naphthyl, 
pyrimidinyl, or thienyl, said cyclic group being singly or multiply 
substituted by --Q.sub.1 ; 
each Q.sub.1 is independently selected from the group consisting of 
--NH.sub.2, --Cl, --F, --Br, --OH, --R.sub.9, --NH--R.sub.5 wherein 
R.sub.5 is --C(O)--R.sub.10 or --S(O).sub.2 --R.sub.9, --OR.sub.5 wherein 
R.sub.5 is --C(O)--R.sub.10, --OR.sub.9, --NHR.sub.9, and 
##STR13## 
wherein each R.sub.9 and R.sub.10 are independently a --C.sub.1-6 
straight or branched alkyl group optionally substituted with Ar.sub.3 
wherein Ar.sub.3 is phenyl; 
provided that when --Ar.sub.3 is substituted with a --Q.sub.1 group which 
comprises one or more additionaL --Ar.sub.3 groups, said additional 
--Ar.sub.3 groups are not substituted with another --Ar.sub.3. 
Other more preferred ICE inhibitors are those wherein R.sub.1 is (e10), 
X.sub.5 is N and the other substituents are as described above. 
More preferably, in these more preferred ICE inhibitors R.sub.3 is 
--CO--Ar.sub.2 and the other substituents are as described above. 
Alternatively, in these more preferred ICE inhibitors, R.sub.3 is 
--C(O)--CH.sub.2 --T.sub.1 --R.sub.11 and R.sub.11 is --(CH.sub.2).sub.1-3 
--Ar.sub.4 and the other substituents are as described above. 
Alternatively, in these more preferred ICE inhibitors, R.sub.3 is 
--C(O)--CH.sub.2 --T.sub.1 --R.sub.11, T.sub.1 is O, and R.sub.11 is 
--C(O)--Ar.sub.4 and the other substituents are as described above. 
Alternatively, in these more preferred ICE inhibitors, R.sub.3 is --C(O)--H 
and the other substituents are as described above. 
Alternatively, in these more preferred ICE inhibitors, R.sub.3 is 
--CO--CH.sub.2 --T.sub.1 --R.sub.11 and R.sub.11 is --Ar.sub.4 and the 
other substituents are as described above. 
Most preferably, in these more preferred ICE inhibitors: 
m is 1; 
T.sub.1 is O or S; 
R.sub.13 is H or a --C.sub.1-4 straight or branched alkyl group optionally 
substituted with --Ar.sub.3, --OH, --OR.sub.9, or --CO.sub.2 H, wherein 
the R.sub.9 is a --C.sub.1-4 branched or straight alkyl group, wherein 
Ar.sub.3 is morpholinyl or phenyl, wherein the phenyl is optionally 
substituted with Q.sub.1 ; 
R.sub.21 is --H or --CH.sub.3 ; 
R.sub.51 is a C.sub.1-6 straight or branched alkyl group optionally 
substituted with Ar.sub.3, wherein Ar.sub.3 is phenyl, optionally 
substituted by --Q.sub.1 ; 
Ar.sub.2 is (hh); 
Y is O, and 
each Ar.sub.3 cyclic group is independently selected from the set 
consisting of phenyl, naphthyl, thienyl, quinolinyl, isoquinolinyl, 
pyrazolyl, thiazcolyl, isoxazolyl, benzotriazolyl, benzimidazolyl, 
thienothienyl, imidazolyl, thiadiazolyl, benzo[b]thiophenyl, pyridyl, 
benzofuranyl, and indolyl, and said cyclic group optionally being singly 
or multiply substituted by --Q.sub.1 ; 
each Ar.sub.4 cyclic group is independently selected from the set 
consisting of phenyl, tetrazolyl, pyridinyl, oxazolyl, naphthyl, 
pyrimidinyl, or thienyl, said cyclic group being singly or multiply 
substituted by --Q.sub.1 ; 
each Q.sub.1 is independently selected from the group consisting of 
--NH.sub.2, --Cl, --F, --Br, --OH, --R.sub.9, --NH--R.sub.5 wherein 
R.sub.5 is --C(O)--R.sub.10 or --S(O).sub.2 --R.sub.9, --OR.sub.5 wherein 
R.sub.5 is --C(O)--R.sub.10, --OR.sub.9, --NHR.sub.9, and 
##STR14## 
wherein each R.sub.9 and R.sub.10 are independently a --C.sub.1-6 
straight or branched alkyl group optionally substituted with Ar.sub.3 
wherein Ar.sub.3 is phenyl; 
provided that when --Ar.sub.3 is substituted with a --Q.sub.1 group which 
comprises one or more additional --Ar.sub.3 groups, said additional 
--Ar.sub.3 groups are not substituted with another --Ar.sub.3. 
The compounds of this invention may be modified by appending appropriate 
functionalities to enhance selective biological properties. Such 
modifications are known in the art and include those which increase 
biological penetration into a given biological compartment (e.g., blood, 
lymphatic system, central nervous system), increase oral availability, 
increase solubility to allow administration by injection, alter metabolism 
and alter rate of excretion. 
In order that this invention be more fully understood, the following 
examples are set forth. These examples are for the purposes of 
illustration only and are not to be construed as limiting the scope of the 
invention in any way. 
EXAMPLE 1 
ICE Cleaves and Activates pro-IGIF 
ICE and ICE Homolog Expression Plasmids 
A 0.6 kb cDNA encoding full length murine pro-IGIF (H. Okamura et al., 
Nature, 378, p. 88 (1995) was ligated into the mammalian expression vector 
pCDLSR.alpha. (Y. Takebe et al., Mol. Cell Biol., 8, p. 466 (1988)). 
Generally, plasmids (3 .mu.g) encoding active ICE (above), or the three 
ICE-related enzymes TX, CPP32, and CMH-1 in the pCDLSR.alpha. expression 
vector (C. Faucheu et al., EMBO, 14, p. 1914 (1995); Y. Gu et al., EMBO, 
14, p. 1923 (1995); J. A. Lippke et al., J. Biol. Chem., 271, p. 1825 
(1996)), were transfected into subconfluent monolayers of Cos cells in 
35-mm dishes using the DEAE-dextran method (Y. Gu et al., EMBO J., 14, p. 
1923 (1995)). Twenty-four hours later, cells were lysed and the lysates 
subjected to SDS-PAGE and immunoblotting using an antiserum specific for 
IGIF (H. Okamura et al., Nature, 378, p. 88 (1995). 
Polymerase chain reaction was used to introduce Nde I sites at the 5' and 
3' ends of the murine pro-IGIF cDNA using the following primers: 
GGAATTCCATATGGCTGCCATGTCAGAAGAC (forward) (SEQ ID No. 4) and 
GGTTAACCATATGCTAACTTTGATGTAAGTTAGTGAG (reverse) (SEQ ID No. 5). The 
resulting NdeI fragment was ligated into E. coli expression vector 
pET-15B(Novagen) at the NdeI site to create a plasmid that directs the 
synthesis of a polypeptide of 213 amino acids consisting of a 21-residue 
peptide (MGSSHHHHHHSSGLVPRGSHM (SEQ ID No. 6), where LVPRGS (SEQ ID No. 7) 
represents a thrombin cleavage site) fused in-frame to the N-terminus of 
pro-IGIF at Ala2, as confirmed by DNA sequencing of the plasmid and by 
N-terminal sequencing of the expressed proteins. E. coli strain BL21(DE3) 
carrying the plasmid was induced with 0.8 mM 
isopropyl-1-thio-.beta.-D-galactopyranoside for 1.5 hours at 37.degree. 
C., harvested, and lysed by microfluidization (Microfluidic, Watertown, 
Mass.) in Buffer A (20 mM sodium phosphate, pH 7.0, 300 mM NaCl, 2 mM 
dithiothreitol, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, and 2.5 
.mu.g/ml leupeptin). Lysates were cleared by centrifugation at 
100,000.times.g for 30 min. (His)6-tagged pro-IGIF protein was then 
purified from the supernatant by Ni-NTA-agarose (Qiagen) chromatography 
under conditions recommended by the manufacturer. 
In Vitro pro-IGIF Cleavaae Reactions 
In vitro cleavage reactions (30 .mu.l) contained 2 .mu.g of purified 
pro-IGIF and various concentrations of the purified proteases in a buffer 
containing 20 mM Hepes, pH 7.2, 0.1% Triton X-100, 2 mM DTT, 1 mM PMSF and 
2.5 .mu.g/ml leupeptin and were incubated for 1 hour at 37.degree. C. 
Conditions for cleavage by granzyme B were as described previously (Y. Gu 
et al., J. Biol, Chem., 271, p. 10816 (1996)). Cleavage products were 
analyzed by SDS-PAGE on 16% gels and Coomassie Blue staining, and were 
subjected to N-terminal amino acid sequencing using an ABI automated 
peptide sequencer under conditions recommended by the manufacturer. 
Kinetic Parameters of IGIF Cleavage by ICE 
The kinetic parameters (k.sub.cat /K.sub.M, K.sub.M, and k.sub.cat) for 
IGIF cleavage by ICE were determined as follows. .sup.35 
S-methionine-labeled pro-IGIF (3000 cpm, prepared by in vitro 
transcription and translation using, the TNT T7-coupled reticulocyte 
lysate system (Promega) and pro-IGIF cDNA in a pSP73 vector as template) 
were incubated in reaction mixtures of 60 .mu.l containing 0.1 to 1 nM 
recombinant ICE and 190 nM to 12 .mu.M of unlabeled pro-IGIF for 8-10 min 
at 37.degree. C. Cleavage product concentrations were determined by 
SDS-PAGE and PhosphoImager analyses. The kinetic parameters were 
calculated by nonlinear regression fitting of the rate vs. concentration 
data to the Michaelis-Menten equation using the program Enzfitter 
(Biosoft). 
IFN-.gamma. Induction Assays 
A.E7 Th1 cells (H. Quill and R. H. Schwartz, J. Immunol., 138, p. 3704 
(1987)) (1.3.times.10.sup.5 cells in 0.15 ml Click's medium supplemented 
with 10% FBS, 50 .mu.M 2-mercaptoethanol and 50 units/ml IL-2) in 96-well 
plates were treated with IGIF for 18-20 hours and the culture supernatant 
were assayed for IFN-.gamma. by ELISA (Endogen, Cambridge, Mass.). 
EXAMPLE 2 
Processing of pro-IGIF by ICE In Cos Cells 
Cos cells were transfected with various expression plasmid combinations as 
described in Example 1. Transfected Cos cells (3.5.times.10.sup.5 cells in 
a 35-mm dish) were labeled for 7 hours with 1 ml of methionine-free DMEM 
containing 2.5% normal DMEM, 1% dialyzed fetal bovine serum and 300 
.mu.Ci/ml .sup.35 S-methionine (.sup.35 S-Express Protein Labeling-Mix, 
New England Nuclear). Cell lysates (prepared in 20 mM Hepes, pH 7.2, 150 
mM NaCl, 0.1% Triton X-100, 5 mM N-ethylmaleimide, 1 mM PMSF, 2.5 .mu.g/ml 
leupeptine) or conditioned medium were immunoprecipitated with an antiIGIF 
antibody that recognizes both the precursor and the mature forms of IGIF 
(H. Okamura et al., Nature, 378, p. 88 (1995)). Immunoprecipitated 
proteins were analyzed by SDS-PAGE (polyacrylamide gel electrophoresis) 
and fluorography (FIG. 2A). 
We also measured the presence of IFN-.gamma. inducing activity in the cell 
lysates and the conditioned media of transfected cells (FIG. 2B). 
Transfected Cos cells (3.5.times.10.sup.5 cells in a 35-mm dish) were 
grown in 1 ml medium for 18 hours. Media was harvested and used at 1:10 
final dilution in the IFN-.gamma. induction assay (Example 1). Cos cell 
pellets from the same transfection were lysed in 100 .mu.l of 20 mM Hepes, 
pH 7.0, by freeze-thawing 3 times. Lysates were cleared by centrifugation 
as described above and were used at a 1:10 dilution in the assay. 
EXAMPLE 3 
IGIF is a Physiological Substrate of ICE 
Wild type (ICE+/+) and ICE-/- mice were primed with heat-inactivated P. 
acnes, and Kupffer cells were isolated from these mice 7 days after 
priming and were then challenged with 1 .mu.g/nl LPS for 3 hours. The 
amounts of IGIF in the conditioned media were measured by ELISA. 
Wild type or ICE-deficient mice were injected intraperitoneally with 
heat-killed p. acnes as described (H. Okamura et al., Infection and 
Immunity, 63, p. 3966 (1995)). Kupffer cells were prepared seven days 
later according to Tsutsui et al. (H. Tsutsui et al., 
Hepato-Gastroenterol., 39, p. 553 (1992)) except a nycodenz gradient was 
used instead of metrizamide. For each experiment, Kupffer cells from 2-3 
animals were pooled and cultured in RPMI 1640 supplemented with 10% fetal 
calf serum and 1 .mu.g/ml LPS. Cell lysates and conditioned medium were 
prepared 3 hours later. 
Kupffer cells from wild type and ICE-/- mice were metabolically labeled 
with .sup.35 S-methionine as for Cos cells (described above in Example 2) 
except that methionine-free RPMI 1640 was used in place of DMEM. IGIF 
immunoprecipitation experiments were performed on cell lysates and 
conditioned media and immunoprecipitates were analyzed by SDS-PAGE and 
fluorography as described in Example 1. See FIG. 3. 
EXAMPLE 4 
Induction of IFN-.gamma. Production In Vivo 
LPS mixed with 0.5% carboxymethyl cellulose in PBS, pH 7.4, was 
administered to mice by intraperitoneal injection (30 mg/kg LPS) in a dose 
volume of 10 ml/kg. Blood was collected every 3 h for 24 h from groups of 
three ICE-deficient or wild type mice. Serum IFN-.gamma. levels were 
determined by ELISA (Endogen). 
EXAMPLE 5 
Human PBMC Assays 
Buffy coat cells were obtained from blood donors and peripheral blood 
mononuclear cells (PBMC) were isolated by centrifugation in LeukoPrep 
tubes (Becton-Dickinson, Lincoln Park, N.J.). PBMC were added 
(3.times.10.sup.6 /well) to 24 well Corning tissue culture plates and 
after 1 hr incubation at 37.degree. C., non-adherent cells were removed by 
gently washing. Adherent mononuclear cells were stimulated with LPS (1 
.mu.g/ml) with or without ICE inhibitor in 2 ml RPMI-1640-10% FBS. After 
16-18 hr incubation at 37.degree. C., IGIF and IFN-.gamma. were 
quantitated in culture supernatants by ELISA. 
EXAMPLE 6 
ICE Inhibition Assays 
We obtained inhibition constants (K.sub.i) and IC.sub.50 values for 
compounds of this invention using the three methods described below: 
1. Enzyme Assay with UV-visible Substrate 
This assay was run using an Succinyl-Tyr-Val-Ala-Asp-pNitroanilide 
substrate (SEQ ID No. 8). Synthesis of analogous substrates is described 
by L. A. Reiter (Int. J. Peptide Protein Res. 43, 87-96 (1994)). The assay 
mixture contained: 
65 .mu.l buffer (10 mM Tris, 1 mM DTT, 0.1% CHEPS @pH 8.1) 
10 .mu.l ICE (50 nM final concentration to give a rate of .about.1 mOD/min) 
5 .mu.l DMSO/Inhibitor mixture 
20 .mu.l 400 .mu.M Substrate (80 .mu.M final concentration) 
100 .mu.l total reaction volume 
The visible ICE assay was run in a 96-well microtiter plate. Buffer, ICE 
and DMSO (if inhibitor is present) were added to the wells in the order 
listed. The components were left to incubate at room temperature for 15 
minutes starting at the time that all components were present in all 
wells. The microtiter plate reader was set to incubate at 37.degree. C. 
After the 15 minute incubation, substrate was added directly to the wells 
and the reaction monitored by following the release of the chromophore 
(pNA) at 405-603 nm at 37.degree. C. for 20 minutes. A linear fit of the 
data was performed and the rate calculated in mOD/min. DMSO was only 
present during experiments involving inhibitors, buffer was used to make 
up the volume to 100 .mu.l in the other experiments. 
2. Enzyme Assay with Fluorescent Substrate 
This assay was run essentially according to Thornberry et al. (Nature 356: 
768-774 (1992)), using substrate 17 referenced in that article. The 
substrate was: Acetyl-Tyr-Val-Ala-Asp-amino-4-methylcoumarin (AMC) (SEQ ID 
No. 9). The following components were mixed: 
65 .mu.l buffer(10 mM Tris, 1 mM DTT, 0.1% CHAPS (@pH8.1) 
10 .mu.l ICE (2-10 nM final concentration) 
5 .mu.l DMSO/inhibitor solution 
20 .mu.l 150 .mu.M Substrate (30 .mu.M final) 
100 .mu.l total reaction volume 
The assay was run in a 96-well microtiter plate. Buffer and ICE were added 
to the wells. The components were left to incubate at 37.degree. C. for 15 
minutes in a temperature-controlled wellplate. After the 15 minute 
incubation, the reaction was started by adding substrate directly to the 
wells and the reaction monitored at 37.degree. C. for 30 minutes by 
following the release of the AMC fluorophore using an excitation 
wavelength for 380 nm and an emission wavelength of 460 nm. A linear fit 
of the data for each well was performed and a rate determined in 
fluorescence units per second. 
For determination of enzyme inhibition constants (K.sub.i) or the mode of 
inhibition (competitive, uncompetitive or noncompetitive), the rate data 
determined in the enzyme assays at varying inhibitor concentrations were 
computer-fit to standard enzyme kinetic equations (see I. H. Segel, Enzyme 
Kinetics, Wiley-Interscience, 1975). 
The determination of second order rate constants for irreversible 
inhibitors was performed by fitting the fluorescence vs time data to the 
progress equations of Morrison. Morrison, J. F., Mol. Cell. Biophys., 2, 
pp. 347-368 (1985). Thornberry et al. have published a description of 
these methods for measurement of rate constants of irreversible inhibitors 
of ICE. Thornberry, N. A., et al. Biochemistry, 33, pp. 3923-3940 (1994). 
For compounds where no prior complex formation can be observed 
kinetically, the second order rate constants (k.sub.inact) were derived 
directly from the slope of the linear plots of k.sub.obs vs. [I]. For 
compounds where prior complex formation to the enzyme could be detected, 
the hyperbolic plots of k.sub.obs vs. [I] were fit to the equation for 
saturation kinetics to first generate K.sub.i and k'. The second order 
rate constant k.sub.inact is then given by k'/K.sub.i. 
3. PBMC Cell Assay 
IL-1.beta. or IGIF Assay with a Mixed Population of Human Peripheral Blood 
Mononuclear Cells (PBMC) or Enriched Adherent Mononuclear Cells 
Processing of pre-IL-1.beta. or pro-IGIF by ICE can be measured in cell 
culture using a variety of cell sources. Human PBMC obtained from healthy 
donors provides a mixed population of lymphocyte subtypes and mononuclear 
cells that produce a spectrum of interleukins and cytokines in response to 
many classes of physiological stimulators. Adherent mononuclear cells from 
PBMC provide an enriched source of normal monocytes for selective studies 
of cytokine production by activated cells. 
Experimental Procedure: 
An initial dilution series of test compound in DMSO or ethanol was 
prepared, with a subsequent dilution into RPMI-10% FBS media (containing 2 
mM L-glutamine, 10 mM HEPES, 50 U and 50 .mu.g/ml pen/strep) respectively 
to yield drugs at 4.times. the final test concentration containing 0.4% 
DMSO or 0.4% ethanol. The final concentration of DMSO was 0.1% for all 
drug dilutions. A concentration titration which brackets the apparent 
K.sub.i for a test compound determined in an ICE inhibition assay was 
generally used for the primary compound screen. 
Generally 5-6 compound dilutions were tested and the cellular component of 
the assay was performed in duplicate, with duplicate ELISA determinations 
on each cell culture supernatant. 
PBMC Isolation and IL-1 or IGIF Assay: 
Buffy coat cells isolated from one pint of human blood (yielding 40-45 ml 
final volume plasma plus cells) were diluted with media to 80 ml and 
LeukoPREP separation tubes (Becton Dickinson) and each were overlaid with 
10 ml of cell suspension. After 15 min centrifugation at 
1500-1800.times.g, the plasma/media layer was aspirated and then the 
mononuclear cell layer was collected with a Pasteur pipette and 
transferred to a 15 ml conical centrifuge tube (Corning). Media was added 
to bring the volume to 15 ml, the cells gently mixed by inversion and 
centrifuged at 300.times.g for 15 min. The PBMC pellet was resuspended in 
a small volume of media, cells counted and adjusted to 6.times.10.sup.6 
cells/ml. 
For the cellular assay, 1.0 ml of the cell suspension was added to each 
well of a 24-well flat bottom tissue culture plate (Corning), 0.5 ml test 
compound dilution and 0.5 ml LPS solution (Sigma #L-3012; 20 ng/ml 
solution prepared in complete RPMI media; final LPS concentration 5 
ng/ml). The 0.5 ml additions of test compound and LPS are usually 
sufficient to mix the contents of the wells. Three control mixtures were 
run per experiment, with either LPS alone, solvent vehicle control, and/or 
additional media to adjust the final culture volume to 2.0 ml. The cell 
cultures were incubated for 16-18 hr at 37.degree. C. in the presence of 
5% CO.sub.2. 
At the end of the incubation period, cells were harvested and transferred 
to 15 ml conical centrifuge tubes. After centrifugation for 10 min at 
200.times.g, supernatants were harvested and transferred to 1.5 ml 
Eppendorf tubes. It may be noted that the cell pellet may be utilized for 
a biochemical evaluation of pre-IL-1.beta. or pro-IGIF and/or mature 
IL-1.beta. or IGIF content in cytosol extracts by Western blotting or 
ELISA with pre-IL-1.beta. and/or IGIF-specific antisera. 
Isolation of Adherent Mononuclear cells: 
PBMC were isolated and prepared as described above. Media (1.0 ml) was 
first added to wells followed by 0.5 ml of the PBMC suspension. After a 
one hour incubation, plates were gently shaken and nonadherent cells 
aspirated from each well. Wells were then gently washed three times with 
1.0 ml of media and finally resuspended in 1.0 ml media. The enrichment 
for adherent cells generally yields 2.5-3.0.times.10.sup.5 cells per well. 
The addition of test compounds, LPS, cell incubation conditions and 
processing of supernatants proceeds were as described above. 
ELISA: 
We have used Quantikine kits (R&D Systems) for measurement of mature 
IL-1.beta. or IGIF. Assays were performed according to the manufacturer's 
directions. Mature IL-1.beta. levels of about 1-3 ng/ml in both PBMC and 
adherent mononuclear cell positive controls were observed. ELISA assays 
were performed on 1:5, 1:10 and 1:20 dilutions of supernatants from 
LPS-positive controls to select the optimal dilution for supernatants in 
the test panel. 
The inhibitory potency of the compounds can be represented by an IC.sub.50 
value, which is the concentration of inhibitor at which 50% of mature 
IL-1.beta. or IGIF is detected in the supernatant as compared to the 
positive controls. 
For example, we obtained the following data for compound 412 of this 
invention using the methods described. (The structure of compound 412 is 
shown further below). 
______________________________________ 
Cell PBMC 
Compound UV-Visible Ki (nM) avg. IC50 (nM) 
______________________________________ 
412 1.3 580 
______________________________________ 
The preparation of compound 412 is described below. Other ICE inhibitors 
may be prepared in a similar manner. The preparation of ICE inhibitors may 
also be found in the references cited and incorporated by reference 
herein. 
##STR15## 
______________________________________ 
compound R.sup.1 
______________________________________ 
502z, 412 
2 
______________________________________ 
[ 3S(1S,9S)] t-Butyl 
3-[6,10-dioxo-9-(isoquinolin-1-oylamino)-1,2,3,4,7,8,9,10-octahydro-6H-pyr 
idazino[1,2-a][1,2]-diazepine-1-carboxamido]-4-oxobutanoate semicarbazone 
(502z), (3S)-3-(1-Fluorenylmethyloxycarbonylamino)-4-oxobutyric acid 
tert-butyl ester semicarbazone (210 mg, 0.4 mol, Prepared in a similar 
manner to the benzyloxycarbonyl analog in Graybill et al., Int. J. Protein 
Res., 44, pp. 173-82 (1994).) was dissolved in 10 ml of DMF and 2 ml of 
diethylamine and stirred for 2 h. The reaction was concentrated in vacuo 
to give (3S)-3-amino-4-oxobutyric acid tert-butyl ester semicarbazone. The 
0.degree. C. solution of the above residue and the acid corresponding to 
502z (200 mg, 0.42 mmol) in 5 ml of DMF and 5 ml of CH.sub.2 Cl.sub.2 was 
treated with 1-hydroxybenzotriazole (57 mg, 0.42 mmol) and 
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (98 mg, 0.51 
mmol). The reaction was stirred at RT for 18 h, poured onto EtOAc (75 ml) 
and washed with aq. 0.3 N KHSO.sub.4, sat. aq. NaHCO.sub.3 and sat. aq. 
NaCl, dried over NaSO.sub.4 and concentrated in vacuo to afford a pale 
yellow solid (90%): mp. 142-145.degree. C.; [.alpha.].sub.D.sup.24 
-136.5.degree. (c 0.06, CH.sub.2 Cl.sub.2); .sup.1 H NMR (CDCl.sub.3) 
.delta. 9.51-9.46 (1H, m), 9.11 (1H, s), 8.83 (1H, d, J=7.8), 8.53 (1H, d, 
J=5.5), 7.89-7.83 (2H, m), 7.77-7.65 (2H, m), 7.55 (1H, d, J=7.2), 7.18 
(1H, d, J=2.7), 5.26-5.12 (2H, m), 4.87 (1H, m), 4.59 (1H, m), 3.25-3.12 
(2H, m), 2.95-2.76 (2H, m), 2.59-2.38, 2.18-1.94, 1.70 (5H, 3m), 1.44 (9H, 
s). 
[3S(1S,9S)] 
3-[6,10-Dioxo-9-(isoquinolin-1-oylamino)-1,2,3,4,7,8,9,10-octahydro-6H-pyr 
idazino[1,2-a][1,2]-diazepine-1-carboxamido]-4-oxobutanoic acid (412). 412 
was stirred with 10 ml of 33% TFA/H.sub.2 O for 4 h and concentrated in 
vacuo. The residue was dissolved in 7 ml of MeOH/acetic acid/37% aq. 
formaldehyde (5:1:1) and stirred for 18 h to afford a white glassy solid 
(69%): mp. 138-141.degree. C.; [.alpha.].sub.D.sup.23 -105.5.degree. (c 
0.5, CH.sub.2 Cl.sub.2); IR (KBr) 3375, 1787, 1659, 1515, 1421, 1278, 
1256; .sup.1 H NMR (CDCl.sub.3) .delta. 9.32 (1H, m), 8.79 (1H, m), 8.47 
(1H, m), 7.86-7.64 (4H, m), 5.31, 5.18, 4.59, 4.37 (4 or 5H, m), 
3.55-2.76, 2.49-2.39, 2.05, 1.65 (11H, 4m). Anal. Calcd for C.sub.24 
H.sub.25 N.sub.5 O.sub.7.1.5H.sub.2 O: C, 55.17; H, 5.40; N, 13.40. Found: 
C, 54.87; H, 5.22; N, 13.15. MS (ES.sup.+, m/z) 494 (M.sup.+ -1, 100%). 
__________________________________________________________________________ 
# SEQUENCE LISTING 
- - - - (1) GENERAL INFORMATION: 
- - (iii) NUMBER OF SEQUENCES: 9 
- - - - (2) INFORMATION FOR SEQ ID NO:1: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 4 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (iii) HYPOTHETICAL: NO 
- - (iv) ANTI-SENSE: NO 
- - (ix) FEATURE: 
(A) NAME/KEY: Modified-sit - #e 
(B) LOCATION: 1 
(D) OTHER INFORMATION: - #/note= "tyrosine is acetylated" 
- - (ix) FEATURE: 
(A) NAME/KEY: Modified-sit - #e 
(B) LOCATION: 4 
(D) OTHER INFORMATION: - #/note= "aspartic acid carboxy 
terminus - #is reduced to an aldehyde" 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
- - Tyr Val Ala Asp 
1 
- - - - (2) INFORMATION FOR SEQ ID NO:2: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 4 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (iii) HYPOTHETICAL: NO 
- - (iv) ANTI-SENSE: NO 
- - (ix) FEATURE: 
(A) NAME/KEY: Modified-sit - #e 
(B) LOCATION: 1 
(D) OTHER INFORMATION: - #/note= "aspartic acid is 
acetylated" 
- - (ix) FEATURE: 
(A) NAME/KEY: Modified-sit - #e 
(B) LOCATION: 4 
(D) OTHER INFORMATION: - #/note= "aspartic acid carboxy 
terminus - #is reduced to an aldehyde" 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
- - Asp Glu Val Asp 
1 
- - - - (2) INFORMATION FOR SEQ ID NO:3: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 5 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (iii) HYPOTHETICAL: NO 
- - (iv) ANTI-SENSE: NO 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
- - Asn Phe Gly Arg Leu 
1 5 
- - - - (2) INFORMATION FOR SEQ ID NO:4: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 31 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc - #= "forward primer" 
- - (iii) HYPOTHETICAL: NO 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
- - GGAATTCCAT ATGGCTGCCA TGTCAGAAGA C - # - # 
31 
- - - - (2) INFORMATION FOR SEQ ID NO:5: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 37 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: cDNA 
- - (iii) HYPOTHETICAL: NO 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
- - GGTTAACCAT ATGCTAACTT TGATGTAAGT TAGTGAG - # 
- # 37 
- - - - (2) INFORMATION FOR SEQ ID NO:6: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (iii) HYPOTHETICAL: NO 
- - (iv) ANTI-SENSE: NO 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
- - Met Gly Ser Ser His His His His His His Se - #r Ser Gly Leu Val 
Pro 
1 5 - # 10 - # 15 
- - Arg Gly Ser His Met 
20 
- - - - (2) INFORMATION FOR SEQ ID NO:7: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (iii) HYPOTHETICAL: NO 
- - (iv) ANTI-SENSE: NO 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
- - Leu Val Pro Arg Gly Ser 
1 5 
- - - - (2) INFORMATION FOR SEQ ID NO:8: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 4 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (iii) HYPOTHETICAL: NO 
- - (iv) ANTI-SENSE: NO 
- - (ix) FEATURE: 
(A) NAME/KEY: Modified-sit - #e 
(B) LOCATION: 1 
(D) OTHER INFORMATION: - #/note= "tyrosine is succinylated" 
- - (ix) FEATURE: 
(A) NAME/KEY: Modified-sit - #e 
(B) LOCATION: 4 
(D) OTHER INFORMATION: - #/note= "aspartic acid terminal 
carboxy g - #roup is derivatized as a p-nitroanilide 
derivative" 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
- - Tyr Val Ala Asp 
1 
- - - - (2) INFORMATION FOR SEQ ID NO:9: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 4 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (iii) HYPOTHETICAL: NO 
- - (iv) ANTI-SENSE: NO 
- - (ix) FEATURE: 
(A) NAME/KEY: Modified-sit - #e 
(B) LOCATION: 1 
(D) OTHER INFORMATION: - #/note= "tyrosine is acetylated" 
- - (ix) FEATURE: 
(A) NAME/KEY: Modified-sit - #e 
(B) LOCATION: 4 
(D) OTHER INFORMATION: - #/note= "aspartic acid carboxy 
terminus - #is derivatized as an amino-4-methylco 
umarin de - #rivative" 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
- - Tyr Val Ala Asp 
__________________________________________________________________________