Enantiomerically pure hydroxylated xanthine compounds to treat shock symptoms

There is disclosed compounds and pharmaceutical compositions that are a resolved R or S (preferably R) enantiomer of an .omega.-1 alcohol of a straight chain alkyl (C.sub.5-8) substituted at the 1-position of 3,7-disubstituted xanthine. The inventive compounds are effective in modulating cellular response to external or in situ primary stimuli, as well as to specific modes of administration of such compounds in effective amounts.

TECHNICAL FIELD OF THE INVENTION 
The invention relates to a discovery that an isomer of a 
hydroxy-substituted xanthine compound is an effective agent to modulate 
cellular responses to stimuli mediated through a stereo-specific cellular 
second messenger pathway. More specifically, the inventive compounds are 
an R or S (preferably R) enantiomer of an .omega.-1 alcohol of a straight 
chain alkyl (C.sub.5-8) substituted at the 1-position of 3,7-disubstituted 
xanthine. The inventive compounds are useful antagonists to control 
intracellular levels of specific sn-2 unsaturated phosphatidic acids and 
corresponding phosphatidic acid-derived diacylglycerols which occur in 
response to cellular proliferative stimuli and mediated through a 
phosphotidic acid (PA) pathway. 
BACKGROUND ART 
Pentoxifylline (1-(5-oxohexyl)-3,7-dimethylxanthine), abbreviated PTX, is a 
xanthine derivative which has seen widespread medical use for the increase 
of blood flow. PTX is disclosed in U.S. Pat. Nos. 3,422,307 and 3,737,433. 
Metabolites of PTX were summarized in Davis et al., Applied Environment 
Microbiol. 48:327, 1984. A metabolite of PTX is 
1-(5-hydroxyhexyl)-3,7-dimethylxanthine, designated M1 and as a racemic 
mixture. M1 (racemic mixture) was also disclosed as increasing cerebral 
blood flow (as opposed to just increasing blood flow) in U.S. Pat. Nos. 
4,515,795 and 4,576,947. In addition, U.S. Pat. Nos. 4,833,146 and 
5,039,666 disclose use of shorter chain tertiary alcohol analogs of 
xanthine for enhancing cerebral blood flow. In subsequent metabolism 
studies, PTX was found to be metabolized to the S enantiomer. 
Furthermore, U.S. Pat. No. 4,636,507 describes an ability of PTX and M1 
(racemic mixture), to stimulate chemotaxis in polymorphonuclear leukocytes 
in response to a stimulator of cherootaxis. PTX and related tertiary 
alcohol substituted xanthines inhibit activity of certain cvtokines to 
affect chemotaxis (U.S. Pat. No. 4,965,271 and U.S. Pat. No. 5,096,906). 
Administration of PTX and GM-CSF decrease tumor necrosis factor (TNF) 
levels in patients undergoing allogeneic bone marrow transplant (Bianco et 
al., Blood 76: Supplement 1 (522A), 1990). Reduction in assayable levels 
of TNF was accompanied by reduction in bone marrow transplant-related 
complications. However, in normal volunteers, TNF levels were higher among 
PTX recipients. Therefore, elevated levels of TNF are not the primary 
cause of such complications. 
It is common practice to market a drug with a chiral center as a racemate. 
The M1 metabolite has only been disclosed exclusive of its chirality. In 
fact, M1 appears to be made (metabolically in humans) only as the S 
isomer. The approach of manufacturing and dosing drugs as racemic mixtures 
means that each dose of a drug is contaminated with an equal weight of an 
isomer, which usually has no therapeutic value and has the potential to 
cause unsuspected side effects. For example, the sedative thalidomide was 
marketed as a racemate. The desired sedative activity resided in the 
R-isomer, but the contaminating S-isomer is a teratogen, causing the birth 
defects in babies born to mothers using this drug. The R,R-enantiomer of 
the tuberculostatic ethambutol can cause blindness. The lethal side 
effects associated with the nonsteroidal anti inflammatory drug 
benoxaprofen (Oraflex) might have been avoided had the drug been sold as a 
pure enantiomer. 
The issue of enantiomeric purity is not limited to the field of 
pharmaceuticals. For example, ASANA (.sup.i Pr=isopropyl) is a synthetic 
pyrethroid insecticide which contains two asymetric centers. The potent 
insecticidal activity resides overwhelmingly in just one of four possible 
stereoisomers. Moreover, the three non-insecticidal stereoisomers exhibit 
cytotoxicity toward certain plant species. Therefore, ASANA can only be 
sold as a single stereoisomer because the mixed stereoisomers would not be 
suitable. 
Therefore, there is a need in the art to discover effective therapeutic 
compounds that are safe and effective for human or animal administration 
and that can maintain cellular homeostasis in the face of a variety of 
inflammatory stimuli, and that are enantiomerically pure to have activity 
residing in a single isomer. The present invention was made in a process 
of looking for such compounds. 
SUMMARY OF THE INVENTION 
We have found that the compounds described herein can be used to maintain 
homeostasis of a large variety of target cells in response to a variety of 
inflammatory and proliferative stimuli. In addition, the inventive 
compounds and pharmaceutical compositions are suitable for normal routes 
of therapeutic administration (e.g., oral, topical and parenteral) and 
permit effective dosages to be provided. 
The inventive compounds and pharmaceutical compositions are a resolved R or 
S (preferably R) enantiomer of an .omega.-1 alcohol of a straight chain 
alkyl (C.sub.5-8) substituted at the 1-position of 3,7-disubstituted 
xanthine. The inventive compounds are effective in modulating cellular 
response to external or in situ primary stimuli, as well as to specific 
modes of administration of such compounds in effective amounts. 
The inventive compounds comprise compounds and pharmaceutical compositions 
having a compound comprising a xanthine core of the formula: 
##STR1## 
wherein R.sub.1 is independently a resolved enantiomer .omega.-1 secondary 
alcohol-substituted alkyl (C.sub.5-8) substantially free of the other 
enantiomer, and wherein each of R.sub.2 and R.sub.3 is independently alkyl 
(C.sub.1-12) optionally containing one or two nonadjacent oxygen atoms in 
place of a carbon atom. Preferably R.sub.1 is a C.sub.6 alkyl with the 
hydroxyl group as the R enantiomer. 
The present invention further provides a pharmaceutical composition 
comprising an inventive compound and a pharmaceutically acceptable 
excipient, wherein the pharmaceutical composition is formulated for oral, 
parenteral or topical administration to a patient. 
The present invention further provides a method for treating an individual 
having a variety of diseases, wherein the disease is characterized by or 
can be treated by inhibiting an immune response or a cellular response to 
external or in situ primary stimuli, wherein the cellular response is 
mediated through a specific phospholipid-based second messenger acting 
adjacent to the inner leaflet of the cell membrane of a cell. The second 
messenger pathway is activated in response to various noxious or 
proliferative stimuli characteristic of a variety of disease states and 
the biochemistry of this second messenger pathway is described herein. 
More specifically, the invention is directed to methods to treat or 
prevent clinical symptoms of various disease states or reduce toxicity's 
of other treatments by inhibiting cellular signaling through the second 
messenger pathway described herein. The disease states or 
treatment-induced toxicity's are selected from the group consisting of 
proliferation of tumor cells in response to an activated oncogene; 
hematocytopenia caused by cytoreductive therapies or caused by an 
infection of a microbial agent; autoimmune. diseases caused by a T cell 
response or a B cell response and antibody production; septic shock; 
resistance of mesenchymal cells to tumor necrosis factor (TNF); 
disregulation of cell activation or disregulated cell growth, such as 
proliferation of smooth muscle cells, endothelial cells, fibroblasts and 
other cell types in response to growth factors, such as PDGF-AA, BB, FGF, 
EGF, etc. (i.e., atherosclerosis, restenosis, stroke, and coronary artery 
disease); human immunodeficiency virus infection (AIDS and AIDS related 
complex); proliferation of kidney mesangial cells in response to IL-1, 
mip-1.alpha., PDGF or FGF resulting in various inflammatory renal 
deseases; inflammation; kidney glomerular or tubular toxicity in response 
to cyclosporin A or amphotericin B treatment; organ toxicity (e.g., 
gastrointestinal or pulmonary epithelial) in response to a cytoreductive 
therapy (e.g., cytotoxic drugs or radiation); enhancing antitumor effects 
of nonalkylating antitumor agents; allergies in response to inflammatory 
stimuli (e.g., TNF, IL-1 and the like) characterized by production of cell 
surface metalloproteases or by degranulation of mast cells and basophils 
in response to IgE, bone diseases caused by overproduction of 
osteoclast-activating factor (OAF) by osteoclasts, CNS diseases caused by 
reduced signal transduction of the neurotransmitters epinephrine and 
acetylcholine, and combinations thereof. 
When a cell is stimulated to express a particular cytokine or to 
proliferate in response to a proliferative or noxious stimuli, this 
process is mediated through a specific phospholipid-based second messenger 
signaling pathway. This second messenger pathway produces elevated levels 
of a subset of phosphatidic acid (PA) containing sn-2 non-arachidonate 
unsaturation that is rapidly converted to its corresponding diacylglycerol 
(DAG). The inventive compounds and pharmaceutical compositions specificly 
inhibit the stereo-specific enzymes involved in this second messenger 
pathway without affecting other second messenger pathways that are 
involved in normal house-keeping functions of a cell, such as the 
phosphatidyl inositol (PI) pathway. The result of inhibiting one or 
several enzymes involved in the second messenger pathway described herein 
is "modulation" of the response of a target cell to a stimulus, 
particulary a noxious stimulus. This biochemical event (i.e., inhibiting 
activity of a second messenger pathway enzyme that responds to a primary 
stimulus, such as a cytokine) effects cellular signaling and results in an 
effect upon many diverse disease states that are the result of abnormal, 
inflammatory or noxious cellular signaling mechanisms.

The invention is illustrated by the following examples which should not be 
regarded as limiting the invention in any way. In these examples PTX means 
pentoxifylline. 
EXAMPLE 1 
This example illustrates a synthesis for CT1501R by resolution of racemic M 
1. To ether saturated with M 1 in a reaction vial was added 3:0 equivalent 
of pyridine (freshly distilled from calcium hydride) and 3 equivalents of 
the acid chloride of R-(+)-1-methoxy-1-trifluoromethyl-phenylacetic acid 
((+)-MTFPA). The reaction vial was sealed, 30 warmed at fifty degrees C. 
for 1 hour, placed under a stream of nitrogen until dry, and then 
reconstituted in 75% MeOH/H2O. Separation was achieved using an isocratic 
system of 90% (75% MeOH/water), 3% acetonitrile (AcN), 7% water at a flow 
rate of 3.0 ml/min through a 250.times.10 mm Ultremex 5 C-18 column 
(Phenomenex, Torrance, CA 90501). Samples were prepared as 10% solutions 
of MTFPA-M1 in 75% MeOH/water, 100 .mu.l aliquots were injected every 7 
min for 21 min (4 injections). Compounds were eluted at 29.8 rain (S-M 
1)-MTFPA, and 31.4 min (R-M1)-MTFPA with 95% separation. Assignment of 
absolute configuration was based on NMR, as described by Dale et at. (J. 
Org. Chem. 34:2543, 1969). Collected fractions were combined and reduced 
in volume with a rotovap. Initially, samples were evaporated only to 
remove MeOH and AcN after which they were extracted with chloroform. The 
solvent was dried and removed in vacuo. Later, the extraction step was 
omitted after studies showed that the ester was stable to the minor 
elevation of temperature required to remove water. 
Hydrolysis of (+)-MTFPA-M1 Stcreoisomers: 
For monitoring the progress of hydrolysis of MTFPA esters, it was necessary 
to develop an assay that would quantkate M1 directly as well as the total 
ester present. A gradient program was used changing the mobile phase from 
40% (75% MeOH/water) 10% AcN 50% water to 40% (75% MeOH/water) 60% AcN 
from 1.0-8.0 min after injection. The final conditions were maintained for 
3 min after which the column was reequilibrated to starting conditions. 
Eluent was monitored at 280 nm and retention times were 6.5 min and 12.3 
min for free and derivatized MI, respectively. Using this system there was 
no separation of stereoisomers of defivatized M1 and so it could be used 
as an assay for monitoring of hydrolysis of MTFPA esters. 
To 30 mg of pure MTFPA-M1 ester (R or S derivative) in 4.0 ml EtOH was 
added ca. 40 mg sodium borohydride (NaBH) and this mixture was heated in 
an oil bath to 70.degree. C. Additional sodium borohydride (20 mg) was 
added every 4 hours during the day.. After 54 hours the reaction was 
approximately 90% complete (via HPLC) with no loss of the total peak area 
of M1+ M1-ester. The reaction was terminated with monobasic sodium 
phosphate/water, adjusted to pH 3.5 with HCl, the EtOH was removed in 
vacuo, and unreacted ester and M1 extracted into chloroform. The 
chloroform was dried over sodium sulfate and removed in vacuo. The crude 
product was purified using preparative chromatography with the solvent 
gradient outlined above for M1 using a flow rate of 3.0 ml/min with the 
250.times.10 mm column. Collected fractions were reduced to dryness in 
vacuo, triturated with ether, and analyzed for enantiomeric excess (&gt;95% 
for both isomers). Ether was removed under nitrogen and the resulting 
crystals dissolved in a minimal volume of normal saline. Dilutions were 
made of this standardized solution with normal saline to produce 10 mM 
solutions of each enantiomer. Enantiomeric identity was confirmed by HPLC 
of reformed MTFPA esters. 
EXAMPLE 2 
This example illustrates a process for preparing (R) 
1-(5-hydroxyhexyl)-3,7-dimethylxanthine) (CT1501R) using R pinanediol as a 
chiral director on a laboratory scale. Triethylsilane (83.49 g, 0.718 
moles) and 4-bromo-1-butene (97 g, 0.718 moles) were mixed in a 500 ml 
round bottomed flask and cooled to -78.degree. C. In a one liter round 
bottomed flask, boron trichloride (84.13 g, 0.718 moles) gas was condensed 
at -78.degree. C. and 400 ml of penlane were added. The silane/butene 
mixture was added to the boron trichloride solution dropwise via a canula 
with stirring, under argon, while maintaining an internal temperature of 
-78.degree. C. When the addition was completed, three equivalents of 
methanol were added dropwise to the. The solution was then warmed to room 
temperature, and the pentane, HCI, and excess methanol were distilled off 
under argon at atmospheric pressure. The residue was vacuum distilled to 
give dimethyl 4-bromobutyl boronate; (bp 70.degree.-79.degree. C. at 0.9 
torr, yield 127.5 g, 85% yield). 
In a 500 ml round bottomed flask, (R)-pinanediol (62 g; 0.365 moles) and 
dimethyl 4-bromobutylboronate (75 g; 0.359 moles) were stirred with 200 ml 
of diethyl ether. After 30 min the ether was removed undervacuum and the 
residue was distilled to yield (R)-pinanediol-4-bromobutyl boronate; (bp 
134.degree.-141.degree. C. at 1.6-1.9 torr, 110.85 g, 98% yield). 
To perform the homologation reaction, methylene chloride (31.47 g; 0.370 
moles) and 500 ml of anhydrous TI-EF were cooled to -100.degree. C. under 
argon with stirring in a one liter round bottomed flask with a side arm. 
To the cooled solution, 212 ml of n-butyllithium (1.4 N in hexanes) were 
added dropwise down the side of the flask over 45 min with stirring under 
argon while maintaining the internal temperature at -100.degree. C. The 
solution was allowed to stir 20 rain after addition was complete. 
Pinanediol 4-bromobutylboronate (77.82 g; 0.247 moles) was mixed with 100 
ml anhydrous THF, cooled to -78.degree. C., and then added to the lithium 
methyl dichloride solution dropwise while keeping the internal temperature 
at 100.degree. C. Upon completion of the addition, rigorously dried zinc 
chloride (30.29 g; 0.222 moles) was added. The solution was stirred under 
argon for 10 hr and warmed to room temperature. The solvents were removed 
under vacuum. To the. residue was added 500 ml petroleum ether and 300 ml 
saturated aqueous ammonium chloride. The organic phase was separated and 
washed with saturated ammonium chloride (2.times.250 ml). The aqueous 
phases were combined and washed with petroleum ether (2.times.250 ml). The 
organic phases were combined and dried with sodium sulfate, filtered and 
evaporated to give crude pinanediol (R) bromopentylboronate, crude wt. 
92.13 g (102%). 
In a 500 ml round bottomed flask, 300 ml of anhydrous THF and the crude 
pinanediol (R)-1-chloro-5-bromopentylboronate from the previous reaction 
(assuming 89 g; 0.247 moles) were mixed and cooled to -78.degree. C. with 
stirring under argon. To the solution was added methylmagnesium bromide 
(3.26N, 79.6 ml). The solution was warmed to room temperature overnight. 
Petroleum ether (500 ml) and saturated ammonium chloride (250 ml) were 
added, forming an emulsion. The aqueous phases were separated. The organic 
phase was washed with saturated ammonium chloride (2.times.250 ml), 
causing the emulsion to disappear. The combined aqueous phases were washed 
with petroleum ether (2.times.250 ml). The combined organic phases were 
dried over sodium sulfate, filtered and evaporated under vacuum to yield 
85.85 g of crude pinanediol (R)-5-bromo-1-methylpentylboronate. 
In a 5 liter flask, 2 liters of DMSO and theobromine (44.51 g, 0.247 moles) 
were combined with stirring under argon. Sodium hydride (9.9 g; 0.247 
moles) was added in two aliquots to the solution and allowed to stir until 
the theobromine was dissolved. After 3 hr, the pinanediol 
(R)-5-bromo-l-methylpentylboronate from the previous reaction (84.75 g; 
0.247 moles).was added neat to the solution dropwise and allowed to stir 
for 12 hr. 
The DMSO was distilled from the solution (it may be recycled). The residue 
was treated with 500 ml of methylene chloride and 500 ml water. The 
aqueous phase was removed and the organic phase was washed (3.times.750 
ml) with water. The aqueous phases were combined and extracted with 
2.times.500 ml of methylene chloride. The organic phases were combined, 
dried with sodium sulfate, filtered and the solvents removed under vacuum 
to yield 88.14 g, 80% yield) of pinanediol 
(R)-5-(3,7-dimethylxanthine)-1-methylpentyl boronate. 
The boronate was then dissolved in THF/water (300 ml each) and the mixture 
cooled to 0.degree. C. with stirring under argon. While maintaining the 
internal temperature at 0.degree. C., 95 ml of 3N potassium hydroxide was 
added dropwise followed by dropwise addition of 32.3 ml of 30% hydrogen 
peroxide. The mixture was stirred for 2 hr and the solids were filtered 
off. Water and methylene chloride (300 ml each) were added. The phases 
were separated and the aqueous phase was washed 4.times.100 ml with 
methylene chloride. The combined organic phases were dried over sodium 
sulfate, filtered and evaporated. The residue was recrystallized with a 
minimal mount of methylene chloride and a larger amount of ether. The 
yield was 46.3 g (87%) of (R)-1-(5-hydroxyhexyl)-3,7-dimethyl xanthine 
m.p. 105.degree.-108.degree. C., [.alpha.]D=-5.63, ca. 96% ee. 
EXAMPLE 3 
This example illustrates a process for preparing of 
(R)-1-(5-hydroxyhexyl)-3,7-dimethylxanthine (CT1501R) using DICHED as a 
chiral director: The pinanediol chiral director described in example 2 can 
be replaced with (1S,2S)-(1,2)-dicyclohexylethanediol (DICHED). This 
chiral director is more easily recovered (95%) than the pinanediol. DICHED 
can be prepared according to a procedure described in Sharpless et at.,J. 
Org. Chem. 57:2768, 1992. Dimethyl-4-bromobutylboronate (10.1 g, 48.06 
mmol), prepared as described above was mixed with 10.54 g (46.6 mmol) of 
(S,S)-DICHED in 100 ml of ether. After 30 min, the ether was removed and 
the residue put through a short column of 10 g of silica gel and eluted 
with petroleum ether/ether (9:1), yielding 17.8 g of the (S,S) DICHED 
analog 4-bromobutylboronate. 
The homologation reaction was performed as described in example 2 with the 
following amounts: 17.8 g of DICHED 4-bromobutylboronate; 6.1.1 g of 
methylene chloride; 41.2 ml of 1.4N n-butyllithium; 100 ml of THF and 5.89 
g of anhydrous zinc chloride. 
The Grignard reaction was performed, as in example 2, with the following 
amounts: DICHED (R)-1-chloro-5-bromopentylboronate, 20.04 g; 3.0 N 
methylmagnesium bromide, 16.7 ml; 150 ml of THF. The oxidation of the 
DICED boronate to give (R)-6-bromohexan-2-ol was performed by dissolving 
the DICHED 5-bromo-1-methylpentylboronate in THF (10 mI) in a 50 ml flask. 
Water (10 ml) was added and the solution. The solution was cooled to 
0.degree. C. while stirring under argon. Sodium carbonate (16.5 ml of a 3M 
solution) was added, followed by 8 ml of 30% hydrogen peroxide. The 
solution was filtered, 20 ml of pentane added, and filtered again. The 
aqueous phase was separated and washed with pentane (2.times.10 ml). The 
organic phase was dried over sodium sulfate, filtered and the solvent 
evaporated to give (R)-6-bromo-hexan-2-ol in a crude yield of 8.9 g. 
Vacuum distillation provided a yield of 7.1 g (84%) of pure material with 
a rotation of -13.89 [.alpha.].sub.549. 
The (R)-bromoalcohol was added to theobromine. A mixture of theobromine 
(2.02 g, 11.2 mmol) was stirred in DMSO (30 ml), and 282 mg of sodium 
hydride (11.8 mmol) was added. The reaction mixture was vigorously stirred 
for 80 minutes. The bromdalcohol (2.03 g, 11.2 mmol) was added dropwise 
and the stirring continued for 21 hr. DMSO was distilled off under full 
pump vacuum. Water (100 ml) was added. The mixture was extracted with 25% 
ethanol/methylene chloride (3.times.50 ml) and the combined extracts were 
dried over mag-nesium sulfate and evaporated. The residue was taken up in 
20 ml of methylene chloride and 150 ml of ether was added. Beige crystals 
formed of (R)-1-(5-hydroxyhexyl)-3,7dimethylxanthine (1.5 g, 5.36 mmol, 
47.8% yield). Another 500 mg of the product crystallized over the next 24 
hr to give a total yield of 2 g (64% overall) with greater than 94% ee by 
chiral chromatography. 
EXAMPLE 4 
This example illustrates a process for synthesis of (R or 
S)-1-(6-hydroxyheptyl)-3,7-dimethyl-10 xanthine or (R or 
S)-7-hydroxyoctyl)-3,7-dimethylxanthine. These compounds were prepared 
using the appropriate pinanediol and DICHED procedures described above by 
using as starting materials the longer chain bromoolefins, 
5-bromo-1-pentene and 6-bromo-1-hexene, 15 respectively. 
EXAMPLE 5 
This example illustrates a mixed lymphocyte reaction of CT1501R and PTX. 
The mixed lymphocyte reaction shows a proliferative response of PBMC 
(peripheral blood mononuclear cells) to allogeneic stimulation determined 
in atwo-way mixed lymphocyte reaction. Both CT 1501R and PTX showed 
activity in this immune modulating activity assay procedure as shown in 
FIG. 1. 
EXAMPLE 6 
This example shows the effects of CT1501R on inhibition of murine B-cell 
proliferation stimulated by anti-mu antibody crosslinked and/or 
interleukin-4 (IL-4). FIG. 2 shows that CT1501R inhibited B-cell 
proliferation caused by the indicated proliferative signals. 
FIG. 3 shows the effects of CT1501R inhibiting proliferation caused by 
Concanavalin A (ConA) and interleukin-1 alpha (IL-1.alpha.) or 
interleukin-2 (IL-2). CT1501R was added to the cells at the doses 
indicated two hours prior to activation with ConA and IL-1.alpha. or IL-2. 
CT1501R inhibited thymocyte proliferation in a dose-response manner as is 
shown in FIG. 3. Background counts wereless than 200 cpm. 
FIG. 4 shows the effects of CT1501R and PTX on inhibition of smooth muscle 
proliferation stimulated by PDGF (platelet derived growth factor) and 
IL-1. CT1501R and PTX were separately added to the cells two hours prior 
to activation with PDGF and IL-1. Both drugs inhibited smooth muscle cell 
proliferation at the higher doses tested as shown in FIG. 4 with CT1501R 
being more active than PTX. 
EXAMPLE 7 
This example illustrates in vitro effects of CT1501R, including inhibition 
of cytokine release and cellular adhesion. We determined the effects of 
CT1501R on endotoxin, TNF-.alpha. or IL-1.alpha.-stimulated cytokine 
release and adhesion to activated human umbilical endothelial cells. 
Murine peritoneal exudate cell macrophages (PEC) were isolated by perfusion 
of mouse peritoneum and plated into 96-well trays at 5.times.10.sup.5 
cells per well. Cells were stimulated with 5 .mu.g/ml Salmonella abortus 
equi -derived endotoxin (LPS; Sigma Chemical Co., L-1887) or 50 ng/ml 
IL-1.alpha. (Genzyme; Cambridge, Mass.) with or without the addition of 
CT1501R added to the cultures one hour prior to addition of the stimulus. 
At various times thereafter, supernatants were removed and levels of 
either TNF-.alpha. or IL-1.alpha. were assayed using commercial 96-well 
microtiler immunoassay kits (Genzyme; TNF; Endogen, Boston, Mass.; 
IL-1.alpha.) according to manufacturer's specifications. For the adhesion 
studies, early passage human umbilical vein endothelial cells (HUVEC) were 
obtained. from commercial suppliers (Clonetics, San Diego, Calif. or Cell 
Systems, Seattle, Wash.) and cultured in defined, Hepes buffered, serum 
free medium (Cell Systems, cat 301-180) supplemented with acidic FGF (Cell 
Systems cat 401-111). 4000 cells were plated into each well of a 96-well 
microtiter plate and allowed to incubate for 72 hrs at 37.degree. C. The 
histiocytic leukemia cell line U937 was stimulated in RPMI 1640 medium 
supplemented with 10% fetal calf serum. Either the HUVEC or U937 cells 
were stimulated with either LPS, IL-1.alpha. or TNF-.alpha. for 12 hrs. 
For the adhesion assay, U937 cells were labeled with the fluorescent 
viability stain, 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfiuorescein 
acetoxymethyl ester (BCECF). Briefly, the cells were labeled with 10 
.mu.g/ml BCECF in RPMI-1640 media plus 10% fetal calf serum for 30 min at 
37.degree. C. The cells were washed once with full media and 50,000 
cells/well added to the HUVEC cultures. The samples were incubated at 
37.degree. C. for 30 min, inverted and centrifuged for 10 min at 
400.times.g. After addition of 100 ml Hanks Balanced Salt Solution (HBSS), 
the microtiter plate was then analyzed on a Millipore Cytofluor 
fluorescent plate reader (excitation 488 nm; emission 525 nm). 
Fluorescence of serial dilutions of cells showed a linear concentration 
relationship over wide ranges of cell concentrations from 20 to 100,000 
cells per well (data not shown). All experiments were repeated at least in 
duplicate with all results confirming the findings of those shown. 
CT1501R inhibited TNF-.alpha. release from LPS stimulated PEC cells. Cells 
were pretreated with 250 mM CT1501R and supernatants assayed by ELISA for 
released TNF-.alpha. as a function of time following LPS stimulation. 
CT1501R significantly inhibited TNF-.alpha. release at all time points 
measured from approximately 50% of LPS induced levels at 6-24 hrs to 
approximately 70% at 48 hr. A dose response of the effects of CT1501R on 
released TNF is shown in FIG. 18. PEC cells were stimulated with LPS 1 
hour following treatment with various concentrations of CT1501R. The 
concentration for CT 1501R 50% inhibition was approximately 30 .mu.M. At 
500 .mu.M the inhibitory effects of CT1501R were approximately 40% of the 
maximal LPS stimulation (1428 pg/ml). 
LPS stimulates IL-1.alpha. release in peritoneal macrophages, increasing to 
levels of 24 pg/ml by 12 hours following LPS stimulation, and 31 pg/ml by 
48 hrs (FIG. 19). Addition of CT1501R significantly attenuated IL-1.alpha. 
release at 12-48 hrs, with a maximum inhibition of approximately 50% at 24 
hours. If the peritoneal macrophages were stimulated with 50 ng/ml 
IL-1.alpha. rather than LPS (FIG. 20), CT1501R also inhibited release of 
TNF-.alpha. (FIG. 20). Addition of CT1501R resulted in a 50% -70% 
inhibition of induced levels of TNF-.alpha. at the tested time points. 
Cells of the immune lineage leave the blood by recognizing and binding to 
vascular endothelial cells. Thereafter immune cells migrate between 
endothelial cells and surrounding tissue. Models of immune cell adhesion 
to endothelial cells and extravasation provide predictive models for in 
vivo manipulation an inflammatory and an atherogenic response. Activation 
of endothelial cells with TNF-.alpha., IL-1.alpha. or LPS up-regulates 
certain adhesion molecules and increases their adhesiveness for 
lymphocytes, monocytes, eosinophils and neutrophils. The cell line U937 
was used to test the effects of CT1501R on inhibition of adhesion to 
activated HUVEC. U937 cells express many of the phenotypic characteristics 
of monocytes including expression of the integrin VLA-4, which mediates 
monocyte attachment to endothelial cells via vascular adhesion molecule 1 
(VCAM), a member of the immunoglobulin superfamily which is expressed on 
endothelial cells. HUVEC were treated overnight with an activating agent 
selected from IL-1.alpha., TNF-.alpha. or LPS with or without the addition 
of 250 .mu.M CT1501R, added 1 hour prior to addition of the activating 
agent. As shown in FIG. 21, there was no significant effect on background 
attachment of U937 cells to HUVEC by the addition of CT1501R. However, 
there was a marked suppression in the relative adhesiveness of the 
activated HUVEC pretreated with CT1501R. This inhibition by CT1501R was 
observed with stimulation of the HUVEC with either TNF-.alpha., 
IL-1.alpha. or LPS. 
Conversely, activating U937 cells with IL-1-.alpha. also increased their 
relative adhesiveness to non-stimulated HUVEC (FIG. 22). Pre-treatment of 
U937 cells with 250 .mu.M CT1501R effectively blocked the IL-1.alpha. 
mediated increase in adhesion to HUVEC to near background levels. 
CT 1501R inhibited LPS, TNF-.alpha. and IL-1.alpha.-mediated inflammatory 
signaling pathways and concomitant cellular responses in peritoneal 
macrophages, the human U937 histiocytic leukemia cell line, and in human 
umbilical vein endothelial cells. CT1501R significantly decreased release 
of the pro-inflammatory cytokines TNF-.alpha. and IL-1.alpha. from 
peritoneal macrophages stimulated with LPS. The IC50 for TNF-.alpha. 
inhibition using 10 .mu.g/ml LPS stimulation was approximately 30 .mu.M. 
CT1501R blocked TNF-.alpha. release from IL-1.alpha. activated PEC. 
CT1501R inhibited the increase in adhesion of U937 cells to TNF-.alpha., 
IL-1.alpha. or LPS activated HUVEC. Finally, CT1501R inhibited 
IL-1.alpha.-induced activation and increased adhesiveness of U937 cells to 
non-stimulated HUVEC. 
EXAMPLE 8 
This example illustrates nude mouse hair studies involving topical 
application of CT1501R. Six to eight week old, female, nu/nu mice from 
Charles River Laboratories were housed at Biosupport Research Support 
Services (Seattle, Wash.) in autoclaved micro isolator units with 
hyperchlorinated autoclaved water, irradiated rodent chow and kept under a 
laminar flow hood. Caging was changed weekly, and water was changed twice 
weekly. The mice were acclimated for 5 days before beginning each study. 
The first topical formulation was prepared by adding CT1501R to a heated 
hydrophilic ointment (USP) at a 1% concentration, and allowed to solidify. 
Nude mice were painted twice daily for 16 days with the first topical 
formulation of CT1501R on the left flank and another compound in the same 
base on the right flank with sterile applicators. Mice were handled under 
the laminar flow hood with applicator wearing face mask and sterile 
gloves. After 16 days, one mouse was sacrificed by cervical dislocation 
and skin biopsies were taken of the treated areas of the shoulder/flank 
and from the non-treated area of the dorsal pelvis (rump). Specimens were 
placed in 10% buffered formalin solution. Biopsies were sent to a 
veterinary dermopathologist for histopathology. Six weeks following 
treatment, a second mouse was euthanatized and biopsied the same as the 
first. Samples were sent for histopathology. 
The results from the first experiment show that treated sections had 
significantly more normal appearing hair follicles than the non treated 
sections. Numerous hair shafts were seen exiting the follicles in the 
treated sections vs. none in the non treated sections for both CT1501R and 
the other compound. 
In a second experiment, topical application of CT1501R was performed along 
with a commercial topical minoxidil preparation (Rogaine.RTM., Upjohn) 
that is approved for a hair growth indication. Five to six week old, 
female, nu/nu mice from Bantin and Kingman Universal were housed at 
Biosupport Research Support Services (Seattle, Wash.) in autoclaved micro 
isolator units with hyper chlorinated autoclaved water, irradiated rodent 
chow and kept under a laminar flow hood. Caging was changed weekly, and 
water was changed twice weekly. Mice were acclimated for 7 days before 
beginning study. 
CT1501R, and two other compounds were prepared in a topical formulation of 
60% ETOH, 10% water and 30% PEG along with vehicle only. Minoxidil was 
purchased as the commercial preparation Rogaine.RTM. from a local 
pharmacy. Rogaine.RTM. is sold in the same formulation base. Mice were 
identified by unique tail markings specific for each test article. Two 
mice per group were treated with CT1501R and another compound. One mouse 
was treated with each of vehicle and minoxidil. The mice were handled 
under the laminar flow hood with the applicator wearing face mask and 
sterile gloves. Each mouse was treated twice daily with a separate sterile 
applicator to avoid contamination of the solutions or the mice. All mice 
were applied a strip of appropriate test article along the center line of 
the back from the base of the skull to the base of the tail, approximately 
1.5 cm wide. After 34 days all mice were euthanatized by overdose of 
halothane anesthesia. Skin biopsies were taken from each of the 7 mice 
over the nape of the neck between the scapulas (approximate size was 
1.times.1.5 cm). All specimens were blotted on gauze to remove blood and 
fluids, affixed to a cut piece of wooden tongue depressor, and placed 
specimen side down in separately marked containers with 10% buffered 
formalin. Specimens were shipped to a veterinary dermatopathologist for 
qualitative histopathological determinations. 
Subjective examination revealed that the vehicle treated mouse (control) 
had the least well developed hair follicles. A fair response to treatment 
was evident in the minoxidil treated mouse. CT1501R and the other compound 
treated mice had the greatest concentration of hair folicles. Further, 
visual inspection of the mice revealed that the CT1501R mice were the most 
"hairy" of a group consisting of CT1501R, minoxidil and control. 
Therefore, CT1501R is at least as good at promoting hair growth in this 
model as minoxidil. Accordingly, a topical formulation of 1-4% CT15001R 
(by weight) is an effective therapeutic composition for treating baldness 
and promoting hair growth. 
EXAMPLE 9 
This example illustrates the effect of CT1501R on survival in mice given a 
lethal dose of endotoxin. We determined if CT1501R protects against 
endotoxin induced lethality in a murine model. Septic shock was modeled by 
endotoxin injection of 6-8 week old female Balb/c mice similar to 
previously published reports (Ashkenazi et al. Proc. Natl. Acad. Sci. 
U.S.A. 88: 10535-10539, 1991.) under protocols approved by the Animal Use 
Committee of the Biomembrane Institute, Seattle, Wash. Animals were 
injected intravaneously (i.v.) with an approximate LD 100 dose (10 
.mu.g/g) of Salmonella abortus equi-derived endotoxin (Sigma Chemical Co., 
L-1887) in phosphate buffered saline (PBS). CT1501R at a dose of 100 
.mu.g/kg was injected intrapedtoneally (IP) 3 times per day (100. 
ml/injection), Control mice were injected at the same times with a similar 
volume of vehicle control (PBS). Survival was followed for at least 72 
hours. 
For ELISA measurements of cytokine levels in plasma, blood was collected by 
retro-orbital or cardiac puncture of anesthetized mice, immediately 
centrifuged and the EDTA plasma stored at -70.degree. C. Particulate free 
plasma was thawed on ice and cytokine levels determined utilizing 
commercial ELISA assays with normal mouse plasma used to generate standard 
curves. Murine tumor necrosis factor-co and intefieukin-2.alpha. kits were 
purchased from Genzyme (Cambridge, Mass.) and Endogen (Boston, Mass.), 
respectively. Each data point was an average of two ELISA measurements 
made from EDTA serum pooled from three mice. The data summarized in Table 
3 were compiled from two independent experiments; data from Table 4 and 5 
were from single experiments of three mice per dam point. 
Ten mice were treated each with PBS alone or CT1501R alone on the same 
schedule as the experimental mice. There were no adverse effects noted and 
survival was 100% throughout the course of the experiment (data not 
shown). Endotoxin survival data were summarized from a total of six (6) 
independent experiments. Summary Table 1 details the results of each of 
the six experiments. Cumulative percent survival is given in Table 2 and 
plotted as FIG. 5. In FIG. 5, each time point represents a minimum of 
three (3) experiments comprising at least twenty (20) mice per group. A 
probability analysis of the survival data using a Fisher's Exact One 
Tailed Test is given as Table 3. Significant protection was conferred if 
CT1501R was administered immediately after the LPS treatment. The 
cumulative percent survival at 72 hours post LPS treatment was 60% 
compared to 7% for the LPS-only treated animals (p=&lt;0.0005; Table 3). 
The effect of delaying the time of administration of CT1501R following the 
LPS treatment is also shown in FIG. 5. Animals were treated with LPS and 
given CT1501R either two or four hours after the LPS treatment. Again, 
CT1501R conferred significant protection. Survival of the CT1501R treated 
mice was 55% at 2 hrs and 37% at 4 hrs compared to 7% for the LPS-only 
treated mice (p=0.0005 and p=0.001; respectively; Table 3). 
Levels of TNF-.alpha., IL-10.alpha. and IL-6 were measured in the plasma of 
mice as a function of time following treatment with S. abortus endotoxin. 
These data were compared to animals treated with endotoxin followed 
immediately with a single i.p. injection of CT1501R. TNF-.alpha. levels 
peaked within 1 hour of treatment of endotoxin (Table 4 and FIG. 6). 
Treatment of the mice with CT1501R decreased the levels of TNF-.alpha. in 
the EDTA plasma of endotoxin treated mice at all time points measured. In 
particular, peak levels of TNF-.alpha. at 0.5 and 1 hour post endotoxin 
were decreased 2..5 and 2.6 fold respectively in the CT1501R treated mice. 
Plasma levels of IL-1.alpha. were also decreased by treatment with CT1501R 
immediately following the endotoxin (Table 5; FIG. 7). In particular, peak 
levels observed at 6, 12 and 18 hours post endotoxin were decreased 1.2, 
4.3 and 3.2-fold, respectively. Finally, IL-6 measurements were made in a 
similar manner (Table 6, FIG. 8). Peak plasma levels at 3, 6 and 12 hours 
were also decreased in the CT1501R treated animals (1.7, 2.0 and 4.1-fold 
decrease, respectively). 
CT1501R significantly enhanced survival in mice that received a dose of 
endotoxin that was lethal to 41 of 44 mice. Survival was improved compared 
to control when CT1501R was administered simultaneous with endotoxin or 
after 2 or 4 hours following endotoxin treatment. Administration of a 
single dose of CT 1501R immediately following the endotoxin significantly 
decreased peak plasma levels of TNF-.alpha., IL-1.alpha. and IL-6. 
TABLE 1 
______________________________________ 
Survival data from six independent mouse sepsis experiments. 
Time of Hours Post LPS 
CT-1501R 
(Number Surviving/Total) 
Expt. # Post LPS 24 Hr 48 Hr 72 Hr 
______________________________________ 
1 LPS only 2/4 0/4 0/4 
t = 0 Hr 4/5 4/5 4/5 
t = 2 Hr 4/5 3/5 3/5 
2 LPS only 0/5 0/5 0/5 
t = 0 Hr 5/5 5/5 5/5 
t = 2 Hr 5/5 5/5 5/5 
3 LPS only 1/5 1/5 1/5 
t = 4 Hr 5/5 3/5 3/5 
4 LPS only 1/10 1/10 1/10 
t = 4 Hr 7/10 5/10 5/10 
5 LPS only 0/10 0/10 0/10 
t = 0 Hr 9/10 7/10 3/10 
t = 2 Hr 5/10 3/10 3/10 
t = 4 Hr 7/10 4/10 2/10 
6 LPS only 3/10 1/10 1/10 
t = 4 Hr 3/10 3/10 3/10 
______________________________________ 
TABLE 2 
______________________________________ 
Cumulative mouse survival from data derived from Table 1. 
Time of Hours Post LPS 
CT-1501R 
(Number Surviving/Total) 
No. Expts. Post LPS 24 Hr 48 Hr 72 Hr 
______________________________________ 
n = 6 LPS only 7/44 3/44 3/44 
n = 3 t = 0 Hr 18/20 16/20 12/20 
n = 3 t = 2 Hr 14/20 11/20 11/20 
n = 4 t = 4 Hr 22/35 15/35 13/35 
______________________________________ 
TABLE 3 
______________________________________ 
Fisher's Exact P Values (one tailed) for the mouse sepsis survival 
data in Table 2. 
Time of P Value (Hours Post LPS) 
No. Expts. 
CT-1501R 24 Hr 48 Hr 72 Hr 
______________________________________ 
n = 6 LPS Only 
n = 3 t = 0 Hr &lt;0.0005 &lt;0.0005 &lt;0.0005 
n = 3 t = 2 Hr &lt;0.0005 0.0005 0.0005 
n = 4 t = 4 Hr &lt;0.0005 0.0002 0.001 
______________________________________ 
TABLE 4 
______________________________________ 
Averaged Data From Mouse Plasma TNF-.alpha. ELISA 
Measurements 
Hours Post Plasma TNF (pg/ml) 
LPS LPS Only LPS + CT-1501R 
______________________________________ 
0 0 0 
0.17 8 6 
0.5 990 390 
1 4480 1728 
2 1585 940 
4 655 400 
6 545 350 
______________________________________ 
TABLE 5 
______________________________________ 
Data From Mouse Plasma IL-1.alpha. ELISA Measurements 
Hours Post Plasma TNF (pg/ml) 
LPS LPS Only LPS + CT-1501R 
______________________________________ 
0 0 0 
0.17 0 0 
0.5 0.01 22 
1 2.25 10.8 
3 41.3 33.2 
6 166 133 
12 139 32.3 
18 150 46.7 
24 16.6 43.5 
36 all dead 18.8 
48 all dead 0 
______________________________________ 
TABLE 6 
______________________________________ 
Data From Mouse Plasma IL-6 ELISA Measurements. 
Hours Post Plasma TNF (pg/ml) 
LPS LPS Only LPS + CT-1501R 
______________________________________ 
0 0 0 
0.17 0.2 0.14 
0.5 3.86 1 
1 24 31.8 
3 317 183 
6 247 121 
12 119 29.6 
18 59 19.8 
24 31 25.6 
36 0 1.64 
48 2 0.4 
______________________________________ 
EXAMPLE 10 
This example illustrates the effect of CT1501R on 5-fluorouracil (5-FU) 
induced bone marrow suppression in mice. We determined if CT1501R 
influenced the time required for hematopoietic reconstitution following 
cytotoxic chemotherapy in a murine model. Female Balb/C mice (VAF, Charles 
River Laboratories, 6-8 wks of age approximately 17.3-18.5 g) were treated 
with 5-FU at a dose of 200 mg/kg intraperitoneally (i.p.) in experiment 1 
or 190 .mu.g/kg in experiment 2. CT1501R or vehicle control was given at a 
dose of 100 .mu.g/kg i.p. bid starting 1 day prior to 5-FU and was 
continued until the last mice were sacrificed on day 13 in experiment 1 
and day 15 in experiment 2. Controls included mice treated with CT1501R or 
vehicle without 5-FU. Mice (4 per group) were sacrificed starting 2 days 
after 5-FU by cardiac puncture under halothane anesthesia followed by 
cervical dislocation and had total white blood cell counts and 
differential counts performed. Platelet counts were performed using 
phase-contrast microscopy in duplicate. Femurs were harvested and the 
number of granulocyte-macrophage colony forming cells (CFU-GM) per femur 
were measured using a standard assay (Terry Fox Laboratories, Vancouver, 
British Columbia) using pokeweed mitogen spleen conditioned medium as the 
growth factor. Each femur was plated separately in triplicate and the mean 
and means for each experimental point was calculated. Standard deviations 
used for statistical analysis were deviations of the mean number of 
colonies measured for each femur. 
The mice treated with vehicle control or CT1501R had no apparent adverse 
effects. The mice treated with the vehicle Control alone had a rise in 
absolute neutrophil count (ANC) and white blood cell counts (WBC) with 
time which was not seen in the CT1501R treated mice. These differences 
were significantly different from day 0 values on days 4 and 8 (p=0.012 
and p=0.016, respectively; two-tailed student T test). When compared to 
mice receiving CT1501R, the control treated mice had significantly higher 
white blood cell counts on day 4 (p=0.009) and significantly higher 
neutrophil counts on day 12 (p=0.028). Control treated mice had a 
significant rise in granulocyte count on day 12 compared to the value on 
day 0 (p=0.028). The CT-1501R treated mice's WBC and ANC remained within 1 
SD of control values. 
The WBC's of 5-FU treated mice were significantly lower in vehicle treated 
controls than in CT1501R treated mice on days 6 and 10 (p=0.019 and 0.036, 
respectively) (FIG. 10). On differential blood counts all cells had the 
morphology of lymphocytes in both groups on days 6, and 8. Some monocytes 
and rare granulocytes were noted in the CT1501R treated animals on day 10. 
On day 13, CT1501R had a mean.+-.SD ANC of 440.+-.17/mm.sup.3 while the 
control mice had 180.+-.10/mm.sup.3 (P=0.05) (FIG. 10). 
An estimate of recovery of hematopoietic progenitor cells is provided by 
measurement of tile number of CFU-GM/femur. Following 5-FU treatment both 
vehicle control and CT1501R treated mice had suppression of CFU-GM to near 
unmeasurable levels until day 8 when recovery began (FIG. 13). By day 13, 
there was significant divergence. CT1501R treated mice had significantly 
more CFU-GM/femur on day 13 than either the vehicle control animals 
(p=0.024) and animals sacrificed before any treatment on day 0 (p=0.05) 
indicating that there was an overshoot of progenitor recovery. 
Experiment 2 was similar to experiment 1 with the following exceptions: (1) 
the dose of 5-FU was decreased to 185 mg/kg; (2) daily blood draws were 
performed between days 10 and 15; and (3) platelet counts were performed. 
Results from experiment 2 are displayed graphically in FIGS. 10-13. As in 
the first experiment, mice treated with CT1501R had significantly higher 
WBC's (FIG. 10) Neutrophil recovery was accelerated in the CT1501R treated 
mice (FIG. 12). The platelet count nadir and rate of recovery were also 
increased compared to vehicle control animals (FIG. 11) The number of 
cells per femur was increased during hematopoietic recovery compared to 
control animals as was the number of CFU-GM/femur (FIG. 13). 
In mice that received a highly marrow suppressive dose of 5-FU, CT1501R 
treatment increased the rate of rise in neutrophil counts. and the rate of 
marrow repopulation with committed myeloid progenitor cells. CT1501R 
inhibited 5-FU induced suppression of the total WBC at each time point 
measured. However, until day 10, the increase was in cells with the 
morphologic appearance of small lymphocytes. On day 13 in experiment 1 and 
on day 10 in experiment 2, the neutrophils in mice treated with CT1501R 
became significantly higher than in vehicle control treated mice. The 
stimulation of hematopoietic recovery by CT1501R also affected the 
megakaryocyte lineage. Test animals had a significantly higher platelet 
nadir than control animals and had a more rapid rise in platelet count and 
a greater overshoot than did vehicle controls. 
Stimulation of hematopoiesis was also evidenced by both the return in 
marrow cellularity and the quantification of marrow progenitor cells. 
CT1501R treated mice had an approximately two-fold increase in the number 
of CFU-GM/femur during hematopoietic recovery compared to control animals. 
In animals that did not receive 5-FU, treatment with CT1501R prevented both 
the rise in ANC and rise in CFU-GM/femur associated with the vehicle 
control. All CT1501R treated animals maintained ANCs within one standard 
deviation of non-injected controls. It appears that CT1501R prevented the 
stress-induced and probably cytokine-mediated response to twice daily i.p. 
injections. 
Overall, these data support a method of using CT1501R and the inventive 
compounds to accelerate the reconstitution of hematopoiesis following 
cytotoxic drugs or cytoreductive therapies.