AU2007353320B2 - Radiation protection and treatment for exposure to gamma-radiation - Google Patents

Abstract

Disclosed are radioprotective agents and methods of use for decreasing cellular damage and cell death resulting from radiation exposure.

Description

WO 2008/140574 PCT/US2007/084875 Radiation Protection and Treatment for Exposure to Gamma-radiation Cross-Reference to Related Applications 5 [001] This application claims the benefit of priority of earlier-filed United States Provisional Patent Application Number 60/865,871, filed November 15, 2006. Statement of Government Rights 10 [002] This research was supported, in part, by a United States Public Health Service Grant, number HL61469. The United States Government may therefore have certain rights in this invention. Field of the Invention [003] The invention relates generally to radioprotective agents, and more 15 specifically to compositions that act as effectors of the LPA2 receptor and their use as radioprotective agents. Background of the Invention [004] The stem cells of the intestinal mucosa represent one of the most 20 radiation-vulnerable cell types in the mammalian body. The threat of radiation exposure, whether by acts of terrorism, through workplace exposure, or as the result of a nuclear accident, is a serious public health concern. Three major syndromes accompany significant radiation exposure: (1) the hematopoietic syndrome is the result of destruction of bone marrow, resulting in infection and hemorrhage; (2) the 25 gastrointestinal syndrome is the result of destructive changes in the gastrointestinal tract and bone marrow, resulting in infection, dehydration and electrolyte imbalance, with death usually occurring within 2 weeks; and (3) the cardiovascular/central nervous WO 20081140574 PCT/US2007/084875 system syndrome is the result of collapse of the circulatory system, increased fluid and pressure in the brain, vasculitis, and meningitis. Death from the cardiovascular/central nervous system syndrome usually occurs within 3 days. Certain free radical scavengers may ameliorate the central nervous system syndrome. Bone marrow transplant can treat 5 the hematopoietic syndrome, although in cases of exposure of large numbers of individuals, bone marrow transplant would obviously be difficult to provide to those individuals who have been exposed. Effectively treating the gastrointestinal syndrome and the subsequent disruption of the intestinal barrier due to radiation-induced apoptosis of the intestinal stem cells has proven to be even more difficult. 10 [005] Lysophosphatidic acid (1 -radyl-2-hydroxy-sn-glycero-3-phosphate, LPA) is a growth factor-like lipid mediator with anti-apoptotic actions elicited through a set of G protein-coupled receptors (GPCR). At least five LPA GPCRs (LPA 1 , LPA 2 , LPA 3 ,

LPA

4 , and LPA 5 ) have been identified thus far (Ishii I, et al. Lysophospholipid receptors: signaling and biology. Annu Rev Biochem 2004;73:321-54.). The mouse intestine 15 expresses the LPA 1 and LPA2 receptor subtypes (Li C, et al. Lysophosphatidic acid inhibits cholera toxin-induced secretory diarrhea through CFTR-dependent protein interactions. J Exp Med 2005;202:975-86; Yun CC, et al. LPA2 receptor mediates mitogenic signals in human colon cancer cells. Am J Physiol Cell Physiol 2005;289:C2 11), and the LPA2 receptor is targeted to the apical surface of the enterocyte (Li C, et al. 20 Lysophosphatidic acid inhibits cholera toxin-induced secretory diarrhea through CFTR dependent protein interactions. J Exp Med 2005;202:975-86). [006] High levels of phospholipids have been detected in the colonic mucosa of patients with inflammatory bowel disease, and LPA significantly reduces the degree of 2 WO 2008/140574 PCT/US2007/084875 inflammation and necrosis in a rat model of colitis (Sano T, et al. Multiple mechanisms linked to platelet activation result in lysophosphatidic acid and sphingosine 1-phosphate generation in blood. J Biol Chem 2002;277:21197-206). LPA has been shown to stimulate restitution of intestinal epithelia via pertussis toxin (PTX)-sensitive 5 mechanisms (Hines OJ, et al. Lysophosphatidic acid stimulates intestinal restitution via cytoskeletal activation and remodeling. J Surg Res 2000;92:23-8). LPA 2 receptors play an important attenuating role in bacterial toxin-induced secretory diarrhea via PDZ domain-mediated protein-protein interactions inhibiting the activation of the CFTR Cl channel (Li C, et al. Lysophosphatidic acid inhibits cholera toxin-induced secretory 10 diarrhea through CFTR-dependent protein interactions. J Exp Med 2005;202:975-86). [007] Lysophosphatidic acid (LPA) as a potent anti-apoptotic agent for the intestinal epithelium (Gastroenterology, 123:206-216). Apoptosis in the intestinal epithelium is the primary pathological factor that leads to chemotherapy- or radiation induced gastrointestinal damage (Potten CS. Radiation, the ideal cytotoxic agent for 15 studying the cell biology of tissues such as the small intestine. Radiat Res 2004;161:123 36; Hall PA, et al. Regulation of cell number in the mannalian gastrointestinal tract: the importance of apoptosis. J Cell Sci 1994; 107 ( Pt 12):3569-77; Pritchard DM, Watson AJ. Apoptosis and gastrointestinal pharmacology. Pharmacol Ther 1996;72:149-69). However, exogenous LPA is rapidly metabolized in the gastrointestinal tract. Because 20 complex lipids, including phospholipids, are broken down to non-polar intermediates that traverse the plasma membrane, LPA's action is terminated by phospholipase- and lipase mediated deacylation or (lipid)phosphatase-mediated dephosphorylation, since the products of these enzymatic reactions can no longer activate LPA receptors. While this 3 mechanism rapidly renders F PA. inactive, it also li-mil5: LPA's efbt on the rcceptors present (in Ohla Wumirial slirfaC oV, "110 cpithii., 1ma.1king EwPA Lrx. unlikely ia.ididdc Ior use w, An eFr1cclivc radioF'rotcetaht in hmi rannasc-.<coed to 'y-radiation. [0081 There are a number ways by which a~n intdividuait mnay be eWosed to 5 signficawit .aloWi(; of radiation that ma,,,Y result in. fadiatki~u da'Mage to cclls and 6-;,u.CS an~d potentially to iradiatlion sickiicss Mid de.,ah. FbAingi effective radioprotcctivce agents is 331 important public health concern, and would 'henet'ii. those whlo, for, example, undergo .radiationh therapy, work with radioactive. ccllpouifJs at s:gnmficant levels, vwork in the field u(' nwi~cau energy, or may bi- the. targc~ps of f'e aso of radioacl"tivity into tile enrviron Ile It through) 1. 0 radiation terrorism. [008a] Throagfioul the dcsc.riptkon And. claijms of' theo sprecifcglioun, tboe word "comrine"' and variations oflte word, sudi as comprisingn" and %~ounpd.st~a~ ii; not. intended to exclude odlhr aodditivcz, coLYP011cnIit", ilg r r teps. 15 [008b] A reference herein to a patent document or otber matter. which -is give as prior art is not to he. taken as an adi-ission or a sunceto httat uunitw*,kono hi the. 'Information it contains was paft Of tile COIoil oncn.crai knowledge a .t hc proiy date of ally of the clainm. [008c] The preseni invcntion; provides a. method for decreasing damaget'. to and increb!sijig s~i4l of c l§ Of the se~tjoci ygtemrA prior to or s-ubserj( iib t rai-&-tion exposure, the met-hod comprisig admiris4tcm-ing to a hunmn or animal Lhiat 1iaF beert exposed to radiation a thkputc .-ftc Inearoulut IIa Compound as ill formula f(I). 4 X1, (I)7 wVhere in, at lett One of X', X and N! is (HO)2PS-Z-, or (HOg 2 PO-ZP(N)S2Z' and X 'inked togetvern -O-PS{OH) -O-, or X' and X 3 are liIAed rcgptogh ett -- ~PSi(OH)I -NH--; at lear one of X', X2, and X 3 is R'-Y'-A- widh each boiig the-same ot diffeect whe1 two of X' X 2 and X 3 Xm R-yi VA, or X2 and X3 at iineu together' as -C(0) -N(R')-; optionally, one of X", X 2 , and X3 ia l A is eiwr a directfink, (C)*A wit .rk being an integer from 0 to 30; or Y -(Oi-H witk I being al integer frm I to :0, -0-, -8-, or Z is -CH2).- -CFr-, -CF2CHt--. or -CH2,- wih "I bing an iteger frtr I to 50, -C(Rt)H-, -NH-, -O or -S-.; 4a

Z

2 is --(CH2)-- r -O(CHifiith h bitg an integer from I to- 50 or --- ; Q nd Q 2 are independently Hz, =Nf; =0, or a combination of.Rand -NRWR R", for each of X', X-% or X1, is independently hydrogen, a straight.or brnched chain CI to C30 alkyl. a straigbt or branched-dhaiII C2 to C30 alkenyl; an mromatic or heteroaromiat i ring with or wihhut mono-, di-, or ri-substons of the ring, In cy inAuding a Cl to (30 alkyl or an aromadc or heteroaomnatic ring, an nryhilkyl including strMight or brapched-chain C1 to C30 alkyj, an arytoxyatkyl including straight or branched--chain Cf to C3 alky L R R? -, Nf R NR0 R~ R 3 , P, 1R R~ R& ad are independently hydrogen, a straight or branched chain C I to C30 alkyl, a sraglit or branched-chani C24 (C30 al~enyl, anratic-or hwroarom c ig with ot w.it1out mrno-, db, Or tr Isabitutioof of the raig. an acyi including t CI tw 30 alkyl or aromie or 1eteroaroma1i ring, an rylalkyI including. tight or branched-chin CI to C30 alky, or an aryloxyalkyl including straight or branched-chabin C to C30.alkyj. 1009] T.he jnvention also provides compositions Lt use in methods for decreasing d "age to and iicx rasing Suvial of cell, of the gatrintetihal sVsRtI fIlowilg Tadiation 4b q,cposurp. the method cornpvising ad0ministerilig to ah~utnanW O.r anmlthat has been exposed to 5 wherei, 4c WO 2008/140574 PCT/US2007/084875 at least one of X 1 , X 2 , and X 3 is (HO) 2 PS-Z'-, or (HO) 2

PO-Z

2 -P(OH)S-Z'-, X' and X 2 are linked together as -O-PS(OH) -0-, or X 1 and X 3 are linked together as -0-PS(OH) -NH-; at least one of X', X 2 , and X 3 is R 1 -Y'-A- with each being the same or different 5 when two of X 1 , X 2 , and X 3 are R1-Y'-A-, or X 2 and X 3 are linked together as -N(H) -C(O) -N(R)-; optionally, one of X 1 , X 2 , and X 3 is H; A is either a direct link, (CH2)k with k being an integer from 0 to 30, or 0; Y' is -(CH 2 )1- with I being an integer from 1 to 30, -0-, -S-, 10C or -NR 2_ Z1, is -(CH2)m-, -CF2-, -CF 2

(CH

2 )m-, or -O(CH 2 )m- with m being an integer from 1 to 50, -C(R 3 )H-, -NH-, -0-, or -S-;

Z

2 is -(CH 2 )n- or -O(CH 2 )n- with n being an integer from 1 to 50 or -0-; 15 Q 1 and Q 2 are independently H 2 , =NR 4 , =0, or a combination of H and -NR 5

R

6 ; R1, for each of X1, X2, or X3, is independently hydrogen, a straight or branched chain C1 to C30 alkyl, a straight or branched-chain C2 to C30 alkenyl, an aromatic or heteroaromatic ring with or without mono-, di-, or tri-substitutions of the ring, an acyl including a C1 to C30 alkyl or an aromatic or heteroaromatic ring, an arylalkyl including 20 straight or branched-chain C1 to C30 alkyl, an aryloxyalkyl including straight or branched-chain C1 to C30 alkyl, 5 WO 2008/140574 PCT/US2007/084875 N N CHR N N C-NH-R 7 || NI, , R 8 , R 8 , NR 8 C- NH-R 7 C- NH-R 7 C-0- R 7 0 S 0 ;and 2 3 4 5 6 7 8 5 R , R3, R4, R , R', R', and R8 are independently hydrogen, a straight or branched chain C1 to C30 alkyl, a straight or branched-chain C2 to C30 alkenyl, an aromatic or heteroaromatic ring with or without mono-, di-, or tri-substitutions of the ring, an acyl including a C1 to C30 alkyl or aromatic or heteroaromatic ring, an arylalkyl including straight or branched-chain C1 to C30 alky, or an aryloxyalkyl including straight or 10 branched-chain C1 to C30 alkyl. [010] In some embodiments, a composition as in formula (I) may comprise a compound wherein: X' is R-Y-A-;

X

2 is -Z 1 -P(S)(OH)2; 15 X 3 is hydrogen; A is a direct link or (CH 2 ) with I being an integer from 1-30; Y' is (CH 2 ) with 1 being an integer from 1-30; Z is oxygen;

Q

1 and Q 2 are independently selected from the group consisting of H, =NR 4 , =0, 20 or -NR 5 R ; 6 WO 2008/140574 PCT/US2007/084875 R is independently a straight or branched-chain C 1 to C 3 0 alkyl, a straight or branched-chain C 2 to C 3 o alkenyl, an aromatic or heteroaromatic ring (optionally substituted), an acyl including a C 1 to C 30 alkyl, an aromatic or heteroaromatic ring, an arylalkyl including straight or branched-chain C 1 to C 30 alkyl, an aryloxyalkyl including 5 straight or branched-chain C 1 to C 3 o alkyl, N N CH R _R _ N N C-NH-R 7 || NH R8 , NR 8

C-NH-R

7

C-NH-R

7 C-0-R 7 0 , S , ;and 10 R 2, R3, R , R , R', R , and R 8 are independently hydrogen, a straight or branched chain C 1 to C 30 alkyl, a straight or branched-chain C 2 to C 3 0 alkenyl, an aromatic or heteroaromatic ring with or without mono-, di-, or tri-substitutions of the ring, an acyl including a C 1 to C 3 o alkyl or aromatic or heteroaromatic ring, an arylalkyl including straight or branched-chain C 1 to C 3 o alky, or an aryloxyalkyl including straight or 15 branched-chain C 1 to C 30 alkyl. [011] The step of administering to a human or animal that has been exposed to radiation a therapeutically-effective amount of a compound as in formula (I) may be performed by oral administration for the prevention of cellular damage, increasing cellular survival, and treating radiation sickness resulting from radiation exposure to the 7 WO 2008/140574 PCT/US2007/084875 gastrointestinal system, including, for example, cells of the small intestine, large intestine, or both. [012] The invention also provides methods for the use of compositions of formula (I) as described above for decreasing damage to and increasing survival of cells 5 of the hematopoietic system prior to or subsequent to radiation exposure, the method comprising administering to a human or animal that has been exposed to radiation a therapeutically-effective amount of a compound as in formula (I). [013] Administering to a human or animal that has been exposed to radiation a therapeutically-effective amount of a compound as in formula (I) for the prevention of 10 cell damage, cell death, and/or radiation sickness resulting from the hematopoietic syndrome may be performed by subcutaneous administration, by intramuscular administration, by intravenous administration, or other similar means, and may be more easily performed by subcutaneous administration or intramuscular administration via injection if self-administration is necessary. 15 [014] In conjunction with the methods of decreasing cellular damage and increasing cellular survival by use of the compositions of formula (I), the invention also provides kits for preventing or treating radiation sickness, the kit comprising a therapeutically-effective amount of a compound as in formula (I) in an orally administrable form and a form chosen from the group consisting of subcutaneous, 20 intramuscular, intravenous, and intraperitoneal administration. In one embodiment, such a kit may comprise an orally-administrable form of thiophosphoric acid O-octadec-9-enyl ester and an injectable form of thiophosphoric acid O-octadec-9-enyl ester for the 8 WO 2008/140574 PCT/US2007/084875 treatment of the gastrointestinal syndrome and the hematopoietic syndrome of radiation sickness. Brief Description of the Drawings 5 [015] Fig. 1. Chemical structures of LPA and OTP (Panel A). Molecular models of OTP docked into the ligand binding pocket of LPA receptors (Panel B). Ca 2 + transients elicited by OTP and LPA in RH7777 cells stably expressing the individual EDG family LPA receptors (Panel C). Wild type RH7777 cells show no Ca2+ transients in response to LPA up to concentrations as high as 10 [M.. 10 [016] Fig. 2. Activation of prosurvival signaling pathways by LPA and OTP in IEC-6 cells. IEC-6 cells were pretreated with pertussis toxin (PTX, 50 ng/ml) overnight, PD98059 (20 pM) for 1 h, or LY294002 (10 pM) for 30 min, followed by the addition of 10 OM OTP or LPA. Activation of ERK1/2 (Panel A), P38 MAPK (Panel B), and PKB/AKT (Panel C) was evaluated by Western blot 5 min after treatment with 10 pM 15 OTP or LPA. A total of 20 pg lysate protein was loaded for each lane. (Panel D) IEC-6 cells were exposed to 25 Gy y-irradiation 15 min after OTP or LPA treatment (10 pM). DNA fragmentation was evaluated 18 h postirradiation. *p < 0.05 compared to irradiation alone. **p < 0.01 compared to irradiation alone. Data shown are means ± SD of at least three experiments. 20 [017] Fig. 3. OTP selectively protects LPA 2 transfectants against TNF-a induced apoptosis. RH7777 cells were transfected with empty pCDNA3.1 vector or

LPA

1 , LPA 2 , or LPA 3 and pre-incubated with 10 pM OTP or LPA for 15 in, followed by TNF-a (20 ng/mil) plus CHX (10 pg/ml) exposure to induce apoptosis (Panel A). 9 WO 2008/140574 PCT/US2007/084875 Whereas LPA reduced TNF-aCHX-induced DNA fragmentation in all three transfectants, OTP was effective only in LPA 2 cells. Both ligands elicited Ca 2 ' transients in these same cell lines (cf Fig. 1C). LPA 1 and LPA2 transfectants (Panel B) were pretreated with pertussis toxin overnight (PTX, 50 ng/ml), the MEK inhibitor PD98059 5 (20 tM) for 1 h, or the P13K inhibitor LY294002 (10 ptM) for 30 min, followed by the addition of 10 iM OTP or LPA for 15 min and challenged with TNF-a (20 ng/ml) plus CHX (10 pg/ml). DNA fragmentation was evaluated 6 h after TNF-aCHX addition. The reduction in DNA fragmentation was partially sensitive to PTX in LPA 2 cells and was abolished in LPA 1 cells. *p < 0.05, **p < 0.001 compared with TNF-aCHX alone. Data 10 shown represent means ± SD of three experiments. [018] Fig. 4. OTP inhibits apoptosis in small intestinal epithelia of mice following y-irradiation. Wild type C57BL6 (black bars) and LPA 1 (grey bars) or LPA 2 KO mice (open bars) were given vehicle orally (100 pl 200 pM BSA control), LPA (200 tM) or OTP (200 pM) 2 h before subjecting them to 15 Gy whole body y-irradiation. 15 Animals were sacrificed 4 h after irradiation to evaluate apoptosis in the small intestine by H&E staining. (Panel A) The mean number of apoptotic cells per crypt-villus unit. Both LPA and OTP caused a statistically significant decrease in the number of apoptotic bodies compared to the vehicle-treated animal group (p < 0.01). Furthernore, OTP was significantly more effective than LPA (*p < 0.05). The number of apoptotic bodies was 20 significantly higher in the LPA 2 KO mice compared to wild type or LPA 1 mice (*p < 0.01). (Panel B) Correlation between apoptotic cell index (percentage of apoptotic cells at a given cell position) and their position along the crypt-villus axis. Cells 1-2 are Paneth to WO 2008/140574 PCT/US2007/084875 cells in the graph. n = 6 animals in each group, and a minimum of 100 crypt-villus units were scored in each group. [019] Fig. 5. OTP and LPA reduced caspase 3 activation and activated prosurvival pathways in vivo. C57/BL6 mice were pretreated with 2 mg/kg LPA or OTP 5 for 2 i and subjected to 15 Gy radiation exposure. Mice were sacrificed 4 h after radiation. (Panel A) Quantification of active caspase 3 immunoreactive cells. Paraffin embedded jejunum sections from animals treated with radiation and pretreated with either LPA or OTP were stained with a rabbit polyclonal active caspase 3 antibody and fluorescein-labeled secondary antibody using indirect immunofluorescence as described 10 under Methods, and the sections were counterstained with DAPI. Active caspase 3 immunoreactive cells were counted in a minimum of 100 crypt-villus units in groups of four animals in the groups. Both agents significantly reduced the number of activated caspase 3 positive cells (p< 0.05) when compared with vehicle-treated controls. (Panel B) Caspase 3 activity was determined in epithelial cell lysates prepared from the same 15 animals whose jejunum segments were used for activated caspase 3 immunostaining in panel A. LPA and OTP both significantly reduced caspase 3 activity in the tissue lysates, a finding in agreement with the reduced number of active caspase 3 cells. (Panels C-D) Jejunum tissue from mice orally treated with vehicle, 2 mg/kg LPA, or OTP for the indicated times were homogenized and lysates analyzed using Western blotting with anti 20 phospho-ERKI/2 (C) or anti-phospho-AKT antibodies (7D) and appropriate antibodies to the nonphosphorylated forms of the kinases to monitor equal loading. Note that the activation of both kinases was more robust and longer lasting to OTP compared to LPA. 1I WO 2008/140574 PCT/US2007/084875 [020] Fig. 6. Immunohistochemical localization of antiapoptotic Bcl-XL protein in vehicle (200 [M BSA), LPA- and OTP-pretreated (2 mg/kg each), irradiated small intestinal sections of C57/BL6 mice. Sections of vehicle-treated mice (A) showed less Bcl-XL expression compared to sections obtained from mice treated with LPA (B) or 5 OTP (C). Calibration bar is 200 un. The patterns shown represent sections obtained from all mice in the corresponding group of four mice per treatment. [021] Fig. 7. Clonogenic regeneration assays reveal increased intestinal crypt survival in OTP- and LPA-treated mice following irradiation. (Panel A) Wild type C57/BL6 mice were given either vehicle (BSA 200 pM), LPA, or OTP (0 to 2 mg/kg) 10 orally 2 h before being subjected to 15 Gy whole body y-irradiation. (Panel B) Wild type C57/BL6 (black bars), LPA 1 KO (grey bars) or LPA 2 KO mice (open bars) were orally given vehicle, LPA, or OTP, 2 mg/kg, 2 h before 15 Gy y-irradiation, and the animals were sacrificed 4 days after irradiation. Crypt survival was evaluated by H&E staining combined with BrdU immunostaining. The number of animals was n = 6 in each group. 15 Data are expressed as means ± SD of surviving crypts per cross-section. The level of significance based on Student's t-test was *p < 0.05, or **p < 0.01, between the designated groups, and was #p < 0.01 compared to irradiation alone and between the mean crypt survival in wild type and LPA 1 and LPA 2 KO mice. [022] Fig. 8. Graphs illustrating the effects of oleyl thiophosphate (OTP) in 20 ameliorating radiation-induced acute hematopoietic syndrome. Left panel: OTP increases peripheral WBC counts, as indicated. Right panel: OTP increases peripheral platelet counts, as indicated. Mice were randomized into control and OTP treatment groups, and were then subjected to a single dose of 6 Gy whole-body irradiation. A 12 WO 2008/140574 PCT/US2007/084875 single dose of OTP (2.5 mg/kg) or vehicle was administered by subcutaneous injection 6 hrs after radiation exposure. Peripheral blood was collected at day 6, 12 and 18 after irradiation exposure. Total white blood cells (WBC) and platelets (PLT) were counted using a Z 1 cell coulter (Beckman, FL). 5 Detailed Description [023] The inventors had previously reported the discovery of a novel composition for use as a gastrointestinal radioprotectant when administered prior to radiation exposure. Here they report the use of the composition as a gastrointestinal 10 radioprotectant when administered after radiation exposure, being shown by the inventors to be effective if provided even multiple hours after radiation exposure, and the use of the composition as a radioprotectant for preventing or treating the hematopoietic syndrome associated with radiation exposure, if administered either prior to or multiple hours after radiation exposure. 15 [024] Also provided by the invention are radioprotectant kits for protecting and treating humans and animals exposed to T-radiation, the kits comprising a combination of a compound of formula (1) in a form for oral administration for the protection and treatment of cells of the gastrointestinal system and a compound of formula (I) for subcutaneous or intravenous administration for the protection and treatment of cells in the 20 hematopoietic system. Subcutaneous, intravenous, intraperitoneal, intramuscular, or other means of administration may be included in the use of the composition for protection and treatment of the hematopoietic system, intraperitoneal and other means of administration may be included in the use of the composition for protecting the cells and 13 WO 2008/140574 PCT/US2007/084875 tissues of the gastrointestinal system, and subcutaneous, intravenous, intraperitoneal or other means of achieving distribution of the composition outside the gastrointestinal system may also be included in the use of the composition for the protection of the cardiovascular and central nervous systems. Tablets, capsules, gelcaps, syrups, liquids, 5 and other orally-administrable formulations may be provided for the oral administration of a compound of formula (I), while syringes, single injection devices, and other injection means may be provided for the subcutaneous administration of a compound of formula (I). In one embodiment of the invention, for example, orally-administrable tablets, capsules, or other easy-to-administer formulations comprising the compound 10 thiophosphoric acid O-octadec-9-enyl ester (oleyl thiophosphate; OTP) may be provided in bottles, vials, blister packs, or other containers in a kit with a subcutaneously injectable form of OTP such as a combination of one or more vials of OTP and at least one syringe, or one or more auto-injectable single unit combinations of OTP and injection device. [025] Orally-administrable OTP may also be provided separately, while a kit 15 may comprise a subcutaneously-injectable formulation of OTP in a form such as, for example, a vial of OTP and a syringe, an autoinjectable single unit combination of OTP and injection device, or other combination of OTP and means for subcutaneous or intramuscular injection of OTP. Useful dosages of the compounds of formula (I) can be determined utilizing the information provided herein and by comparing their in vitro 20 activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to those of skill in the art. Formulations of the composition may be presented in a single dose form or as divided 14 WO 2008/140574 PCT/US2007/084875 doses for administration at appropriate intervals such as, for example, as two, three, four or more sub-doses per day. [026] Compounds according to formula (I) were previously shown by the inventors to be effective as LPA receptor agonists and antagonists. Among these 5 compounds are LPA 2 receptor agonists that demonstrate effectiveness in decreasing apoptosis, cell destruction, and tissue destruction associated with exposure to y-radiation. CQ1 CH CQ 2 I(II Xi X2 wherein, at least one of X1, X2, and X' is (HO) 2 PS-Z -, or (HO) 2

PO-Z

2

-P(OH)S-Z

1 -, X 1 10 and X 2 are linked together as -O-PS(OH) -0-, or X 1 and X3 are linked together as -0-PS(OH) -NH-; at least one of X 1 , X 2 , and X 3 is R 1 -Y-A- with each being the same or different when two of X 1 , X 2 , and X 3 are R 1 -Y -A-, or X 2 and X 3 are linked together as -N(H) -C(O) -N(R)-; 15 optionally, one of X 1 , X 2 , and X 3 is H; A is either a direct link, (CH2)k with k being an integer from 0 to 30, or 0; Y' is -(CH 2 )1- with I being an integer from 1 to 30, -0-, -S-, 0 or -NR2_ 15 WO 2008/140574 PCT/US2007/084875

Z

1 , is -(CH 2 )m-, -CF 2 -, -CF 2

(CH

2 )m,-, or -O(CH 2 )m 1 ,- with m being an integer from I to 50, -C(R 3 )H-, -NH-, -0-, or -S-; Z2 is -(CH 2 )n- or -O(CH 2 )n- with n being an integer from 1 to 50 or -0-; Q' and Q 2 are independently H 2 , =NR 4 , =0, or a combination of H and -NR 5

R

6 ; 5 R 1 , for each of X 1 , X 2 , or X 3 , is independently hydrogen, a straight or branched chain C1 to C30 alkyl, a straight or branched-chain C2 to C30 alkenyl, an aromatic or heteroaromatic ring with or without mono-, di-, or tri-substitutions of the ring, an acyl including a C1 to C30 alkyl or an aromatic or heteroaromatic ring, an arylalkyl including straight or branched-chain C1 to C30 alkyl, an aryloxyalkyl including straight or 10 branched-chain C1 to C30 alkyl, N N CH R7 R'7 CH/ _ N N C-NH-R 7 || NH, ,IR NR,

C-NH-R

7

C-NH-R

7 C-0-R 7 || || || 0 , S , 0 ;and 2 3 4 5 6 7 8 15 R , R , R , R , R', R , and R8 are independently hydrogen, a straight or branched chain C1 to C30 alkyl, a straight or branched-chain C2 to C30 alkenyl, an aromatic or heteroaronatic ring with or without mono-, di-, or tri-substitutions of the ring, an acyl including a C1 to C30 alkyl or aromatic or heteroaromatic ring, an arylalkyl including straight or branched-chain C1 to C30 alky, or an aryloxyalkyl including straight or 20 branched-chain C1 to C30 alkyl. 16 WO 2008/140574 PCT/US2007/084875 [027] One especially effective composition, thiophosphoric acid O-octadec-9 enyl ester (oleyl thiophosphate; OTP), is one of a class of compositions of formula (1) wherein: X' is R'-Y'-A-; 5 X 2 is -Z 1

-P(S)(OH)

2 ;

X

3 is hydrogen; A is a direct link or (CH 2 )1 with I being an integer from 1-30; Y' is (CH 2 ) with 1 being an integer from 1-30; Z' is oxygen; 10 Q1 and Q2 are independently selected from the group consisting of H, =NR, =0, or -NR 5

R

6 ;

R

1 is independently a straight or branched-chain C 1 to C 3 0 alkyl, a straight or branched-chain C 2 to C 30 alkenyl, an aromatic or heteroaromatic ring (optionally substituted), an acyl including a C 1 to C 30 alkyl, an aromatic or heteroaromatic ring, an 15 arylalkyl including straight or branched-chain C 1 to C 30 alkyl, an aryloxyalkyl including straight or branched-chain C 1 to C 3 o alkyl, N N CHRR N N C- NH-R 7 || NH, R 8 8 , NR ,

C-NH-R

7

C-NH-R

7 C-0-R 7 0 , S , ;and 20 17 WO 2008/140574 PCT/US2007/084875 R 2, R', R4, R', R', R , and R 8 are independently hydrogen, a straight or branched chain C 1 to C 30 alkyl, a straight or branched-chain C 2 to C 30 alkenyl, an aromatic or heteroaromatic ring with or without mono-, di-, or tri-substitutions of the ring, an acyl including a C 1 to C 3 o alkyl or aromatic or heteroaromatic ring, an arylalkyl including 5 straight or branched-chain C 1 to Cqo alky, or an aryloxyalkyl including straight or branched-chain C 1 to C 30 alkyl. [028] The inventors had previously demonstrated that LPA demonstrates radioprotective effects and more recently had discovered that their compound, thiophosphoric acid O-octadec-9-enyl ester (oleyl thiophosphate; OTP) S HO P O- C 1

H

35 10 OH is significantly more effective as a radioprotectant than is LPA. LPA consists of a glycerol backbone with a hydroxyl group, a phosphate group, and a fatty acid or fatty alcohol chain. Modeling studies done by the inventors, while identifying the absolute requirement for a negatively charged headgroup and an aliphatic tail, also revealed that 15 the glycerol backbone was not necessary for ligand binding and receptor activation. Therefore, they began to develop LPA analogs-compounds with agonist or antagonist effects on LPA receptors, similar to the effects demonstrated by LPA-and discovered OTP, a compound that could provide a radioprotective effect if administered prior to or subsequent to significant exposure to 7-radiation. OTP has demonstrated effectiveness 20 in the inventors' experimental in decreasing cell damage and increasing cell survival in the gastrointestinal system, while LPA is rapidly metabolized in the GI system. Only small amounts of orally administered OTP enter the bloodstream, so the discovery that 18 WO 2008/140574 PCT/US2007/084875 the LPA 2 receptor is the target of OTP's radioprotective effects allows the inventors and others to further develop agents that have the radioprotective effect of OTP, but may be orally administered to treat not only the gastrointestinal syndrome caused by radiation exposure, but also the hematopoietic syndrome and the cardiovascular/central nervous 5 system syndrome. Should a radiation accident or an incident involving radiation terrorism occur, orally-administered radioprotectant compounds would be especially beneficial for saving the lives of significant numbers of people and animals. [029] Orally administered radioprotectant compounds for use following radiation exposure could also be provided in food or food supplement products, which 10 might have special benefit for the protection and treatment of animals, particularly pets and agricultural animals to whom their human owners may administer a radioprotectant coiposition. [030] The inventors have demonstrated that OTP protects intestinal epithelial cells from apoptosis both in vitro and in vivo and was significantly stronger in reducing 15 caspase 3 activation and DNA fragmentation compared to LPA. Unlike LPA-elicited protection, OTP-elicited protection was partially sensitive to PTX. OTP-induced reduction of DNA fragmentation and of caspase 3 activation required MEK-ERK1/2 and PI3K-AKT. Whereas LPA prevented tumor necrosis factor-a plus cycloheximide (TNF a/CHX)-induced DNA fragmentation through any of the three EDG family LPA 20 receptors inserted into RH7777 cells, OTP protected only cells expressing LPA 2 . Following radiation exposure, the rate of apoptosis in the stem cell region of the crypts of

LPA

2 knockout mice was significantly higher, and crypt survival was significantly lower compared to that in wild type mice. Both LPA and OTP reduced apoptosis and caspase 3 19 WO 2008/140574 PCT/US2007/084875 activation and increased crypt survival; however, both were ineffective in reducing apoptosis and increasing crypt survival in LPA 2 knockout mice. [031] The invention may be further described by means of the following non limiting examples. 5 Examples Materials and Methods [032] Reagents. LPA (oleoyl) was purchased from Avanti Polar Lipids (Alabaster, AL). LPA and OTP (synthesized as described in Durgam GG, et al. Synthesis, structure-activity relationships, and biological evaluation of fatty alcohol 10 phosphates as lysophosphatidic acid receptor ligands, activators of PPARgamma, and inhibitors of autotaxin. J Med Chem 2005; 48: 4919-30) were applied to cells complexed with fatty acid-free BSA (Sigma, St. Louis, MO) as previously described Virag T, et al. (Fatty Alcohol Phosphates are Subtype-Selective Agonists and Antagonists of LPA Receptors. Mol Pharmacol 2003;63:1032-1042). Camptothecin and cycloheximide 15 (CHX) were purchased from Sigma. Recombinant rat TNF-c was purchased from BD Pharmingen (San Diego, CA). PD98059 and PD158780 were purchased from Calbiochem (San Diego, CA). Pertussis toxin (PTX), AG1296, AG1487, N-acetyl-Asp Glu-His-Asp-p-nitroanilide (Ac-DEVD-pNA) and N-acetyl-Leu-Glu-His-Asp-p nitroanilide (Ac-LEHD-pNA) colorimetric caspase substrates were from Biomol 20 Laboratories Inc., (Plymouth Meeting, PA). The following antibodies and sources were used: rabbit anti-caspase 3 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), rabbit anti active caspase 3 (Abcam, Inc., Cambridge, MA), mouse monoclonal anti-JNK1 (BD Pharmingen), and mouse monoclonal anti-phospho (Thr 83Tyr' )-JNK; rabbit anti 20 WO 2008/140574 PCT/US2007/084875 ERK1/2 and rabbit anti-phospho-(Tyr2 0 2 /Tyr 2 0 4 ) ERK1/2, rabbit anti-AKT, and rabbit anti -phospho-(Ser 47 3 )-AKT, rabbit anti-Bcl-2, monoclonal mouse anti-Bcl-XL (Cell Signaling, Inc., Beverly, MA), monoclonal mouse anti-phospho-(ThrIOTyr 12)-P38 (Promega, Madison, WI), and mouse monoclonal anti-actin (Calbiochem). Horseradish 5 peroxidase-conj ugated anti-rabbit and anti-mouse secondary antibody used for Western blotting was purchased from Sigma. FITC-labeled goat anti-rabbit IgG was purchased from Molecular Probe (Eugene, OR). Normal goat serum and VECTASHIELD* Mounting Medium with DAPI were purchased from Vector Laboratories, Inc.(Burlingame, CA). 10 [033] Computational Modeling. The detailed methods used to develop computational models of LPA 1 , LPA 2 , and LPA 3 have been published previously (Wang D, et al. A Single Amino Acid Determines Ligand Specificity of the S lPl (EDG1) and LPA1 (EDG2) Phospholipid Growth Factor Receptors. J. Biol. Chem. 2001; 276: 49213 15 49220; Sardar VM, et al. Molecular basis for lysophosphatidic acid receptor antagonist selectivity. Biochim Biophys Acta 2002; 1582: 309-17; Fujiwara Y, et al. Identification of residues responsible for ligand recognition and regioisomeric selectivity of LPA receptors expressed in mammalian cells. J Biol Cheim 2005; 280: 35038-50). Briefly, the inventors' validated model of the SIPi receptor (Parrill AL, et al. Identification of Edg1 20 receptor residues that recognize sphingosine 1-phosphate. J Biol Chem 2000; 275: 39379-84; Wang D, et al. A Single Amino Acid Determines Ligand Specificity of the SIP 1 (EDG 1) and LPA 1 (EDG2) Phospholipid Growth Factor Receptors. J. Biol. Chem. 2001; 276: 49213-49220; Inagaki Y, et al. Sphingosine 1-phosphate analogue recognition and selectivity at S 1P4 within the endothelial differentiation gene family of 21 WO 2008/140574 PCT/US2007/084875 receptors. Biochem J 2005; 389: 187-95) was used as a template for generating LPA receptor models. Homology model development was performed using the automated algorithm implemented in the MOE software program (MOE. (2002) 03 ed. Montreal: Chemical Computing Group). The best model was geometry optimized using the 5 MMFF94 (Halgrei TA. Merck Molecular Force Field. I. Basis, Form, Scope, Parameterization, and Performance of MMFF94*. J. Comp. Chem. 1996; 17: 490-519) forcefield to a root mean square gradient of 0.1 kcal/mol-A. The individual receptor models were used in docking studies with OTP bearing a total charge of -2. Docking calculations were performed using Autodock 3.0 software (Morris GM, et al. Automated 10 Docking Using a Lanmarckian Genetic Algorithm and an Empirical Binding Free Energy Function. J. Comput. Chem. 1998; 19: 1639-1662) with default values for all parameters except the number of runs (10), energy evaluations (9.0 x 1010), generations (60,000), and local search iterations (3000). The complex chosen as the best geometry from each docking calculation was that with the lowest final docked energy value. Results are 15 described using a numbering system that facilitates comparisons among homologous positions in G protein-coupled receptors as described by Ballesteros and Weinstein (Ballesteros JA, Weinstein H. Chapter 19. In: Conn PM, Sealfon SC, eds. Methods in Neurosciences. Volume 25. San Diego: Academic Press, 1995: 366-428). In this system, each amino acid in the transmembrane domain is given a number in the format X.YY 20 where X indicates the number of the helix where the amino acid is found and YY indicates the position relative to the most conserved amino acid in that helix at reference position 50. 22 WO 2008/140574 PCT/US2007/084875 [034] Radiolabeling of OTP. Tritiated OTP was synthesized in the inventors' laboratory. Briefly, Pyridinium chlorochromate-mediated oxidation of oleyl alcohol gave oleyl aldehyde, which was subjected to reduction with NaB 3

H

4 to form the tritiated oleyl alcohol. Alcohol 3 was phosphorylated as reported earlier (Durgam GG, et al. Synthesis, 5 structure-activity relationships, and biological evaluation of fatty alcohol phosphates as lysophosphatidic acid receptor ligands, activators of PPARgamma, and inhibitors of autotaxin. J Med Chem 2005;48:4919-30) to give protected oleyl thiophosphate ester. Treatment of oleyl thiophosphate ester with methanolic KOH, followed by acidification, yielded [ 3 H]-OTP. The specific activity of the product was 10.8 nCi/Innol. 10 [035] Determining OTP absorption from the gut. To determine oral absorption of OTP, female 8- to 10-week-old C57BL/6 mice (average body weight 20 g) were used. Mice were maintained on a 12:12-h light-dark cycle and fed standard laboratory mouse chow and water ad libitum. Conscious mice were administered through oral gavage 1.5 mg/kg OTP in 1 mM BSA in PBS including 106 dpm OTP. Groups of four mice were 15 sacrificed after 30 min, 90 min, and 180 min, and blood samples were collected through cardiac puncture using 0.2% EDTA anti-coagulant. Ten microliters whole-blood samples from each mouse were mixed with Ecolume (Packard, Boston, MA) liquid scintillation cocktail and counted after 24 h of equilibration in a liquid scintillation counter. [036] Assaying OTP metabolism by pancreatic lipase and lipid phosphate 20 phosphatase 1 (LPP1). To determine the enzymatic stability of OTP by phospholipases, [3H]-OTP (5.2 x 106 dpn) mixed with 0.03 mM\4 cold OTP was subjected to enzymatic hydrolysis for 24 h by using bovine pancreatic lipase (Sigma) followed by thin-layer chromatography (TLC) separation of the products using a previously established protocol 23 WO 2008/140574 PCT/US2007/084875 (Kates M. Techniques of Lipidology. Elsevier, 1988). Lipid phosphate phosphatase was obtained from mouse embryonic fibroblasts derived from mice with transgenic overexpression of the enzyme. Membrane fractions (300 ig/reaction) prepared by centrifugation at 10 4 x g from transgenic LPP1 fibroblasts were added to [ 3 H]-OTP 5 (1.5x 106 dpm) mixed with 8 iiole cold OTP and subjected to LPP- 1 hydrolysis for 8 h using a previously established protocol (Yue J, et al. Mice with transgenic overexpression of lipid phosphate phosphatase- I display multiple organotypic deficits without alteration in circulating lysophosphatidate level. Cell Signal 2004; 16: 385-99). The reaction mixture was dried in vacuo and the residue was acidified with 100 l 1 N HCl and 10 extracted four times with 0.5 ml ethyl acetate. The extracts were combined and the solvent was evaporated. The extracted reaction products were taken up in 2 ml ethyl acetate and a 20 [d aliquot was applied to TLC using imethanol:ether solvent (2:98. V/V) as described (Kates M. Techniques of Lipidology. Elsevier, 1988). [037] Cell culture and induction of apoptosis in vitro. IEC-6 cells were obtained 15 from the American Type Culture Collection (Manassas, VA) at passage 13 and were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, insulin (10 pig/ml), and gentamicin sulfate (50 pg/ml) at 37 'C in a humidified 90% air/10% CO 2 atmosphere. RH7777 cells, stably expressing LPA 2 receptors, were provided by Dr. Fumikazu Okajima (Gunma University, Japan). RH7777 cells stably 20 expressing LPAI or LPA 3 receptors were generated by the inventors' group and characterized elsewhere (Fischer DJ, et al. Short-chain phosphatidates are subtype selective antagonists of lysophosphatidic acid receptors. Mol Pharmacol 2001; 60: 776 84). Wild type and stably transfected RH7777 cells were grown in Dulbecco's modified 24 WO 2008/140574 PCT/US2007/084875 Eagle's medium with 10% fetal bovine serum and 2 mM glutamine containing 250 ptg/ml G418 for the stable transfectants. Apoptosis in IEC-6 cells was induced by exposing them to 20 pM camptothecin or a 25-Gy Cs137 source y-irradiation (Mark I model 25 Gamma Irradiator, J. L. Shepherd & Associates, San Fernando, CA) at a rate of 4.80 5 Gy/min. DNA fragmentation and caspase 3 activity were measured 6 h after camptothecin treatment or 18 h postirradiation. Apoptosis in RH7777 cells was induced by 10 ng/ml TNF-a plus 10 Og/ml CHX and evaluated 6h later. [038] Pharmacological characterization of OTP. The ligand properties of OTP were evaluated using RH7777 cells stably transfected with each LPA receptor of the 10 EDG family exactly as described in the inventors' previous report (Durgam GG, et al. Synthesis, structure-activity relationships, and biological evaluation of fatty alcohol phosphates as lysophosphatidic acid receptor ligands, activators of PPAR-gamma, and inhibitors of autotaxin. J Med Chem 2005; 48: 4919-30). RH7777 cells lack endogenous Ca 2 ' responses to LPA applied as high as 30 piM, the highest concentration tested but 15 acquire these responses upon transfection of any of the LPA receptors (Virag T, et al. Fatty Alcohol Phosphates are Subtype-Selective Agonists and Antagonists of LPA Receptors. Mol Pharnacol 2003;63: 1032-1042; Durgam GG, et al. Synthesis, structure activity relationships, and biological evaluation of fatty alcohol phosphates as lysophosphatidic acid receptor ligands, activators of PPARgamma, and inhibitors of 20 autotaxin. J Med Chem 2005; 48: 4919-30; Fischer DJ, et al. Short-chain phosphatidates are subtype-selective antagonists of lysophosphatidic acid receptors. Mol Pharmacol 2001;60:776-84). 25 WO 2008/140574 PCT/US2007/084875 [039] RT-PCR. RNA was extracted using Trizol® (Invitrogen, Carlsbad, CA) from wild type and KO mice using four 0.5-cm segments spaced equally along the jejunum. Gene-specific primers for LPA 1 , LPA 2 , LPA 3 , LPA 4 , LPA 5 , and $-actin were used. Reverse-transcription and PCR was done using the Superscript III kit (Invitrogen) 5 and a total of 31 cycles were performed; the products were applied in full to agarose gels and stained with ethidium bromide. [040] Evaluating apoptosis by DNA fragmentation and caspase activity. DNA fragmentation was measured by ELISA following the procedure provided with the Cell Death Detection kit from Roche, Inc.(Indianapolis, IN) as described previously (Deng W, 10 et al. Lysophosphatidic acid protects and rescues intestinal epithelial cells from radiation and chemotherapy-induced apoptosis. Gastroenterology 2002; 123: 206-16; Deng W, et al. LPA protects intestinal epithelial cells from apoptosis by inhibiting the mitochondrial pathway. Am J Physiol Gastrointest Liver Physiol 2003; 284: G821-9) and was expressed as absorbance units (at 405 nm) per microgram protein per minute. Caspase 3 15 and caspase 9 activity in IEC-6 cells was measured by ELISA by using the specific Ac DEVD-pNA chromogenic substrate (for caspase 3) and Ac-LEHD-pNA for caspase 9 as described previously. Small intestine samples were washed thoroughly with PBS, scraped off the muscle layer, mixed with lysis buffer, homogenized, and centrifuged. The supernatants were collected for evaluating caspase 3 activity as described above. Caspase 20 activity was expressed as picomole pNA cleaved per minute per microgram protein. [041] Western blotting. Caspase 3 was measured in IEC-6 cells or intestinal epithelial lysates prepared for caspase 3 activity as described in the inventors' previous reports. ERK1/2 and AKT were measured in total cellular protein lysates by using a 26 WO 2008/140574 PCT/US2007/084875 previously described procedure. Briefly, IEC-6 cells were lysed in 62.5 mM TRIS-HCl (pH 6.8), 2% SDS, 25% glycerol, 1 mM NaF, 1 mM orthovanadate, and protease inhibitor cocktail (Sigma). The lysates were cleared by centrifugation (104 x g) for 15 min at 4'C, and supernatants were collected. Protein concentrations were determined 5 using the BCA reagent kit (Pierce Biotechnology, Inc., Rockford, IL). For ERK1/2 10 jig, for AKT 40 ig, for p 3 8 20 pg, and for JNK 50 pg of the cell lysate was fractionated by SDS-PAGE and transferred to PVDF membranes, blocked with 5% nonfat milk, and incubated with various primary antibodies. Blots were reacted with the appropriate HRP conjugated secondary antibodies and developed using the SuperSignal 10 chemiluminescence reagent (Pierce Biotechnology, Inc.). [042] Animal treatment and irradiation. The whole-body irradiation protocol was reviewed and approved by the University of Tennessee Health Science Center Animal Care and Use Committee. Eight-(8) to 10-week-old C57/BL6 mice were purchased from Harlan (Indianapolis, IN) and maintained on a 12:12-h light-dark cycle 15 and fed standard laboratory mouse chow and water ad libitum. LPA 1 and LPA 2 KO mice generously provided by Dr. Jerold Chun (Scripps Institute, La Jolla, CA) were bred in house and used between the ages of 8 and 12 weeks. Mice were fasted overnight before whole-body 7-irradiation (Cs 1 37 source at a rate of 4.80 Gy/min). LPA or OTP was administered by oral gavage 2 h before irradiation. Mice were killed either 4 h after 20 irradiation by isoflurane inhalation for analysis of apoptosis or 4 days later for the clonogenic assay. Four segments of the jejunum and the ileuin were fixed in 10% neutralized formaldehyde (pH 7.4) buffer and processed for histological evaluation. Mice used in the clonogenic regeneration assay received bromo-deoxy uridine (BrdU, 120 27 WO 2008/140574 PCT/US2007/084875 mg/kg) and 5-fluoro-2'-deoxyuridine (12 mg/kg) intraperitoneally 2 h before death to label the S-phase regenerating cells in intestine. [043] Assessing apoptosis and crypt survival in intestine. Paraffin cross-sections were cut perpendicular to the long axis of the small intestine and stained with 5 heiatoxylin and eosin (H&E) or inuuno stained with a monoclonal auti-BrdU antibody. Epithelial cell apoptosis and crypt survival were analyzed in four sections per segment as previously described (Deng W, et al. Lysophosphatidic acid protects and rescues intestinal epithelial cells from radiation- and chemotherapy-induced apoptosis. Gastroenterology 2002; 123: 206-16). To detect apoptosis, a minimum of 100 half crypt 10 villus units from each experimental group were scored, and the number of apoptotic bodies per intestinal circumference was counted. For the clonogenic assay, the number of surviving crypts per jejunal circumference was counted in the sections from the different segments. A surviving crypt was defined as a regenerative crypt that contained a cluster of 10 or more H&E-stained cells. The viability of surviving crypts was confirmed by 15 positive immunostaining for BrdU of incorporation into five or more crypt cells. [044] Iinmunohistological staining. To identify the BrdU-labeled S-phase cells in the crypts, the inventors used a staining protocol described by the inventors (Balazs L, et al. Topical application of the phospholipid growth factor lysophosphatidic acid promotes wound healing in vivo. Am J Physiol Regul Integr Comp Physiol 2001; 280: 20 R466-72). For activated caspase 3 staining, formalin-fixed paraffin-embedded tissue slides were used with heat-mediated antigen retrieval for 15 min. After three 5-min washes in TRIS-buffered saline (TBS, 25 mM TRIS, 150 mM NaCl pH 7.6), slides were blocked with 10% normal goat serum in TBS/0. 1% Tween for 2 h at room temperature. 28 WO 2008/140574 PCT/US2007/084875 Rabbit active caspase 3 antibody was diluted 1:50 in 1% BSA/IBS/0. 1% Tween and incubated at 4 0 C overnight. FITC-labeled goat anti-rabbit IgG was applied for 2 h after washes, the slides were mounted with VECTASHIELD* with DAPI (Vector Laboratories) and visualized by Nikon Eclipse 80i fluorescence microscope. At least 100 5 villi per animal were counted for the presence of activated caspase 3 positive cells. [045] Immunohistochenical staining for Bcl-XL was done using 5-pm thick sections cut from formalin-fixed, paraffin-embedded blocks of jejunum and ileum taken 4 h after irradiation from a total of six mice. Upon antigen unmasking, inmunostaining with Bcl-XL antibody (1:25 dilution) was performed using the rabbit Vectastain ABC 10 Elite kit (Vector Laboratories) following the manufacturer's instructions. Vector 3,3' diaminobenzidine peroxidase substrate (Vector Laboratories) was used for color development. Stained sections were then dehydrated and counterstained with Vector Hematoxylin QS (Vector Laboratories), and immunoreactivity was assessed by at least two investigators. 15 [046] Statistical analysis. Data are expressed as means ± SD or SEM. Each in vitro experiment was repeated at least three times. For animal studies, each experimental group consisted of at least six mice; a minimum of eight KO mice were used per group. Student's t-test was used for comparing control and treatment groups. A p value of <0.05 was considered significant. 20 Example 2 - Hematopoietic Syndrome [047] Mice were randomized into control and OTP treatment groups, and were then subjected to a single dose of 6 Gy whole-body irradiation. A single dose of OTP 29 WO 2008/140574 PCT/US2007/084875 (2.5 mg/kg) or vehicle was administered by subcutaneous injection 6 hrs after radiation exposure. Peripheral blood was collected at day 6, 12 and 18 after irradiation exposure. Total white blood cells (WBC) and platelets (PLT) were counted using a Z1 cell coulter (Beckman, FL). 5 [048] As illustrated in Fig. 8, OTP ameliorates radiation-induced heinatopoietic syndrome by increasing white blood cell production and/or promoting their release from central hematopoietic system; and (2) stimulating platelet production. Results [049] Molecular modeling and pharmacological characterization of OTP. 10 Complexes of OTP with the LPA receptor models are shown in Figure 1B. The LPA 1 complex shows a strong ionic interaction between the thiophosphate group and R3.28 (2.0 A P-O:H-N distance). No interaction occurs involving Q3.29, a residue required for LPA binding and LPA-induced LPA 1 receptor activation (Wang D, et al. A Single Amino Acid Determines Ligand Specificity of the S 1PI (EDG1) and LPAI (EDG2) 15 Phospholipid Growth Factor Receptors. J. Biol. Chem. 2001;276:49213-49220). Similar to the LPA 1 complex, the LPA 3 complex fails to show a strong hydrogen bonding interaction with Q3.29 due to the unfavorably nonlinear angle fonned by the amide N-H and the hydrogen bond acceptor (1120). However, a greater number of strong ionic interactions are observed in the LPA 3 complex involving not only R3.28 but also K7.35 20 (2.2 A P-O:H-N and 2.4 A P-S:H-N distances, respectively). In contrast to the inventors 36 previous studies demonstrating a role for R5.38 in LPA-induced LPA 3 activation 3, OTP failed to interact with R5.38. The failure of OTP to interact with residues known to be required for LPA-induced activation of LPA 1 and LPA 3 is consistent with the observation 30 WO 2008/140574 PCT/US2007/084875 of only partial agonism at these receptors (Fig. IC). OTP shows both strong ionic interaction with R3.28 (1.6 A P-O:H-N distance) and moderate hydrogen bonding interaction with Q3.29 (1420). This hydrogen bonding angle is within the most highly populated cluster of angles observed in crystal structures. The LPA 2 receptor lacks 5 cationic amino acid residues at the top of TM7, so these two interactions comprise the required headgroup interactions for full agonism. [050] LPA and OTP activate LPA receptors stably expressed in RH7777 cells, providing a simple assay platform to study the pharmacological properties of these receptors as described in many previous reports. OTP was compared in wild type, LPA 1 , 10 LPA 2 , and LPA 3 transfected RH7777 cells using Ca' mobilization as a measure of receptor activation (Fig. 1C). OTP was inactive in wild type RH7777 cells up to 30 pM, the highest concentration tested. However, it activated all three EDG-family LPA receptors with varying potency and efficacy. OTP was always less potent than oleoyl LPA at all three receptors and was less efficacious at LPA 1 and LPA 3 receptors. At the 15 LPA 2 receptor subtype OTP was a full agonist and showed an apparent EC 50 of 90 nM compared 1 nM for oleoyl LPA. [051] Metabolic resistance of OTP to pancreatic lipase and lipid phosphate phosphatase 1. The two major pathways of LPA breakdown include (phospho)lipase mediated deacylation and lipid phosphate phosphatase-mediated dephosphorylation 20 (Brindley DN, et al. Lipid phosphate phosphatases regulate signal transduction through glycerolipids and sphingolipids. Biochim Biophys Acta 2002;1582:33-44). To determine the enzymatic stability of OTP by pancreatic lipase and LPP1 the inventors synthesized

[

3 H]-OTP for monitoring the enzymatic hydrolysis followed by TLC separation of the 31 WO 2008/140574 PCT/US2007/084875 products using previously established protocols. TLC analysis of the pancreatic lipase mediated hydrolysis of [3H]-OTP for up to 24 h showed neither the breakdown nor formation of the expected [3H]-oleyl alcohol. In contrast, LPA was fully metabolized under these conditions. The lack of cleavage is consistent with the requirement for the 5 glycerol backbone that is absent in the OTP structure. LPP1 cleavage of [ 3 H]-OTP for up to 8 h showed no detectable dephosphorylation, indication that the thiophosphate moiety is considerably more resistant to cleavage by this enzyme than the phosphate moiety present in LPA. [052] Absorption of orally applied OTP into the blood. The absorption of orally 10 administered OTP into the bloodstream was determined by injecting 101 dpin [ 3 H]-OTP tracer mixed with cold OTP to yield 1.5 mg/kg into the stomach. Blood samples collected at 60, 90, and 180 min later revealed that as little as 0.67% ± 0.15, 0.61% 0.2, and 0.45% ± 0.15 of the applied radioactivity appeared in the blood compartment. Assuming that all of it was non-metabolized OTP, this would yield a maximal blood concentration 15 of 180 nM which could activate LPA receptors. However, TLC analysis of the radioactively labeled compounds extracted from blood revealed no more than 0.01% of the injected material at the position of the authentic [3H]-OTP standard, indicating that the little amount of radioactivity represents metabolites of the compound. The very low amount of [ 3 H]-OTP found in the blood after oral administration is reinforced by the lack 20 of metabolism by pancreatic lipase and LPP1 present in the intestinal lumen, since these enzymes would produce the neutral oleyl alcohol, which could more effectively be taken up than a charged compound like OTP. 32 WO 2008/140574 PCT/US2007/084875 [053] OTP surpasses the antiapoptotic activity of LPA in IEC-6 cells. The inventors previously showed that LPA protects intestinal epithelial cells from apoptosis induced by four different mechanisms (Deng W, et al. Lysophosphatidic acid protects and rescues intestinal epithelial cells from radiation- and chemotherapy-induced 5 apoptosis. Gastroenterology 2002; 123: 206-16; Deng W, et al.. LPA protects intestinal epithelial cells from apoptosis by inhibiting the mitochondrial pathway. Am J Physiol Gastrointest Liver Physiol 2003;284:G821-9). The inventors compared the antiapoptotic effect of OTP with that of LPA. Although both OTP and LPA dose-dependently protected IEC-6 cells from 7-irradiation-induced DNA damage, OTP at concentrations 10 greater than 1 pM conferred significantly higher protection (Fig. 2A). Both OTP and LPA applied at 10 pM significantly protected IEC-6 cells from either topoisomerase inhibitor camptothecin- or the proinflammatory cytokine TNF-a-induced DNA damage as compared to control (p<0.001), and at this concentration OTP showed stronger antiapoptotic activity than did LPA (p<0.05, Fig. 2B). OTP and LPA significantly 15 inhibited caspase 3 and caspase 9 activity induced with y-irradiation; again, the OTP induced inhibition was significantly higher than that of LPA (p<0.05, Fig. 2C). Furthermore, analysis of caspase 3 activation by Western blotting demonstrated that both OTP and LPA significantly inhibited the conversion of the 32-kDa procaspase into its active form, and OTP showed significantly higher inhibition compared to LPA (Fig. 2D. 20 p<0.05). These results together indicate that OTP mimics the antiapoptotic action of LPA in IEC-6 cells that express LPA 1 and LPA 2 receptors. [054] The antiapoptotic activity of OTP requires PTX-sensitive G protein, mitogen activated protein kinase and P13-kinase/AKT signaling. The inventors have 33 WO 2008/140574 PCT/US2007/084875 previously shown that in IEC-6 cells the antiapoptotic action of LPA requires PTX sensitive G protein, MEK, and P13-kinase signaling. The inventors found that OTP, just like LPA, activated ERK1/2 and p38 MAPK and AKT phosphorylation (Fig. 3A-C). The inventors did not detect JNK phosphorylation under identical conditions. To evaluate 5 whether these same signaling mechanisms are required for OTP-elicited anti-apoptotic activity, the inventors examined these pathways by using pharmacological inhibitors and exposing these cells to OTP or LPA with or without 25 Gy 7-irradiation. In nonirradiated cells, just like LPA, OTP treatment for 5 min induced a significant increase in ERK1/2, p 3 8, and AKT phosphorylation (Fig. 3). Blocking of ERK1/2 and AKT activation 10 upstream by the MEK inhibitor PD98059 and P13K inhibitor LY294002 completely abolished both OTP- and LPA-elicited activation, as well as protection (Fig. 3A, C, and D), indicating that the antiapoptotic activity of OTP required the activation of the ERK1/2 and AKT pathways. Pretreatment of IEC-6 cells with PTX completely abolished LPA-elicited ERKI/2 and AKT activation (Fig. 3A and C). In agreement with the 15 requirement for a PTX-sensitive G protein in the antiapoptotic mechanism, PTX cancelled LPA-elicited protection from 7-irradiation-induced DNA damage. In contrast, although PTX abolished OTP-induced AKT phosphorylation, it only partially attenuated ERK1/2 activation, indicating that OTP-stimulated ERK1I/2 activation was mediated through not only PTX-sensitive G proteins. In support of this result, PTX pretreatment 20 only partially reduced OTP-initiated protection, which remained statistically significant following treatment with this toxin (p < 0.05, Fig. 3D). These results indicate some similarities, as well as differences, between the signaling pathways used by OTP and LPA. 34 WO 2008/140574 PCT/US2007/084875 [055] OTP selectively protects LPA 2 transfectants from TNF-a-induced apoptosis. RH7777 cells do not endogenously express the EDG-family LPA receptor but express low amounts of LPA 5 transcripts. When LPA or OTP was applied to wild type RH7777 cells exposed to TNF-a plus cycloheximide to induce apoptosis, neither 5 compound applied at 10 pM attenuated DNA fragmentation (Fig. 4A). In contrast, in RH7777 cells individually transfected with LPA 1 , LPA 2 , or LPA 3 , DNA fragmentation was significantly reduced by LPA pretreatment (Fig. 4A). OTP applied at 10 pM showed no significant antiapoptotic activity in LPA 1 or LPA 3 stable transfectants but evoked highly significant protection in the LPA 2 transfectants, surpassing the effect of that of 10 10 pM LPA (Fig. 3A, p < 0.001). It is important to note that OTP and LPA both activate Ca 2 * transients in LPA 1 , LPA 2 , or LPA 3 transfectants (Fig. IC) but not in wild type RH7777 cells. Hence, it appears that the antiapoptotic effect of OTP is not merely linked to receptor activation and Ca2* mobilization but to the activation of a specific receptor subtype, which is LPA 2 . Just as in IEC-6 cells, the protective effect of OTP in RH7777 15 LPA 2 transfectants was only partially PTX-sensitive and was completely abolished only by blocking ERK1/2 and PI3K/AKT pathways (Fig. 4B). These results identify LPA 2 in this model system as the main target of OTP and pinpoint a mechanism that involves MEK/ERK1/2 and PI3K/AKT activation mediated through multiple PTX-insensitive and -sensitive G proteins. LPA 2 is known to be coupled to Gq and it mediates ERK1/2 20 activation in part, a finding consistent with the partial inhibitory effect of PTX on the apoptotic effect found in IEC6, as well as in LPA 2 transfected RH7777 cells. [056] OTP attenuates radiation-induced apoptosis in the intestine. The inventors have previously demonstrated that LPA given orally prevents apoptosis induced by y 35 WO 2008/140574 PCT/US2007/084875 irradiation in the stem cell region of the intestinal crypt (Deng W, et al. Lysophosphatidic acid protects and rescues intestinal epithelial cells from radiation- and chemotherapy induced apoptosis. Gastroenterology 2002; 123:206-16). The inventors further tested whether OTP exerted a similar effect and compared its anti-apoptotic efficacy with that 5 of LPA by quantifying apoptotic bodies in the jejunal epithelium 4 h after 15 Gy 7 irradiation (-LDioo 10 ). In agreement with the inventors' previous report using ICR mice, the number of apoptotic bodies in the jejunum of control non-irradiated animals was less than 0.5 per crypt-villus unit (Fig. 5A). In vehicle-treated (100 pL of 200 pM BSA in PBS) wild type C57/BL6 mice, the number of apoptotic bodies per crypt-villus unit 10 increased 6-fold 4 h following 7-irradiation. Oral pretreatment with LPA (100 pL of 200 pM into the stomach 2 h before irradiation) significantly reduced the number of apoptotic bodies per crypt-villus unit (p<0.01, Fig. 5A). Oral OTP pretreatment (same timing and concentration as LPA) reduced the number of apoptotic bodies by 50%, which was significantly higher than that elicited by LPA (p<0.05, Fig. 5A). Analysis of the 15 distribution of the apoptotic bodies along the crypt-villus unit showed that y-irradiation resulted in a high frequency of apoptotic cells over the stem cell zone of the crypt, four to seven cell positions from the base (Fig. 5B). Both LPA and OTP significantly reduced the number of apoptotic cells over the stem cell zone in the irradiated animals. [057] The inventors' in vitro data using the RH7777 heterologous over 20 expression model indicated that OTP elicited its anti-apoptotic action through the LPA 2 receptor subtype (Fig. 4). To evaluate which LPA receptor mediates the protective effect of OTP in vivo, the inventors performed the same treatments in LPA 1 and LPA 2 knockout mice. The inventors detected no difference in the basal count of apoptotic bodies between 36 WO 2008/140574 PCT/US2007/084875 wild type C57/BL6 mice and LPA 1 or LPA2 KO mice on this same genetic background. Surprisingly, y-irradiation induced a significantly higher rate of apoptosis per crypt-villus unit in LPA 2 knockouts compared to that in wild type mice, whereas there was no difference between wild type and LPA 1 mice (Fig. 5A). LPA and OTP had the same 5 protective effect in wild type and LPA 1 mice. In contrast, LPA and OTP failed to attenuate y-irradiation-induced apoptosis in LPA2 KO mice. In wild type animals the biggest radiation-induced increase in apoptotic bodies was found in the stem cell region of the crypt (Fig. 5B). LPA and OTP caused the biggest decrease in the number of apoptotic cells in this region of the crypt suggesting that the cellular targets of LPA and 10 OTP are the stem cells. RT-PCR analysis of the LPA receptors showed that all but LPA 4 transcripts were expressed in the tissue (Fig. 5C). The LPA 1 and LPA 2 KO animals showed no compensatory change in the expression of the other LPA receptors. Thus the in vivo data are consistent with the inventors' in vitro findings that LPA 2 is required for the anti-apoptotic effect of OTP. Nonetheless, these data differ from the in vitro findings 15 in the case of LPA and point to the essential role of LPA 2 in mediating the in vivo anti apoptotic effect of LPA. Furthermore, the increased rate of apoptosis in LPA 2 KO mice suggests that this receptor may play a physiological role in the radiation sensitivity of the small intestine. These results also point to the fundamentally different role of the two LPA receptor subtypes in radiation sensitivity and apoptotic protection. 20 [058] Next, the inventors sought to obtain direct evidence that LPA and OTP treatments activate the same signal transduction mechanisms the inventors have identified in vitro. First, the inventors examined whether LPA and OTP attenuate the activation of caspase 3, as the inventors have seen in IEC-6 cells in vitro (Fig. IC). Jejunum and ileum 37 WO 2008/140574 PCT/US2007/084875 sections were immunostained with an antibody specific for the activated form of caspase 3, and the number of positive cells was counted in a minimum of 100 crypt-villus units in slides prepared from groups of four animals. In the non-irradiated vehicle-treated group, the inventors could not detect caspase 3 positive cells, possibly due to the low amount of 5 antigen present in cells naturally undergoing apoptosis combined with their low incidence in the healthy small intestine. In contrast, in the vehicle-treated irradiated group, caspase 3 positive cells were readily detected. Both LPA and OTP treatments significantly reduced the number of activated caspase 3 positive cells by -60% (Fig. 6A, p<0.05). The inhibition of caspase 3 activation by LPA and OTP was also confirmed in intestinal 10 epithelial lysates by measuring enzymatic activity (Fig. 6B). These experiments revealed that, in control non-irradiated animals, a low level of caspase 3 substrate cleaving activity is present, which increased 4-fold after 15 Gy y-irradiation. LPA and OTP reduced caspase 3 activity significantly, although the inhibitory effect of OTP was greater. [059] The inventors' previous and present in vitro studies using IEC-6 and other 15 types of cells have established the requirement of ERK1/2 and AKT activation in the antiapoptotic response to LPA and OTP. They next determined whether these pathways were activated in intestinal cell homogenates prepared from animals that have been treated with the two ligands by oral gavage. As shown in Fig. 6 C-D, both agents activated ERK1/2 and AKT phosphorylation as early as 30 min after administration in 20 nonirradiated animals. However, the effect of OTP was considerable longer lasting in the tissue compared to the effect of LPA, which was absent in the samples collected 60 min after administration. 38 WO 2008/140574 PCT/US2007/084875 [060] In vitro studies using IEC-6 cells showed that LPA upregulated the expression of the antiapoptotic Bcl-2 and had no effect on Bcl -XL, BAD, and Bak. In jejunum homogenates prepared from LPA- and OTP-treated mice 4 h after irradiation (6h after treatment), the inventors could not detect a significant change in Bcl-2 expression. 5 However, innnunostaining with a Bcl-XL specific antibody revealed marked increases in LPA and OTP treated animals (Fig. 7) compared to jejunum and ileum (not shown) from the vehicle-treated animals. OTP treatment caused the most intense immunostaining (Fig. 7C), although LPA also elicited a substantial increase in the immunoreactivity (Fig. 7B). These results taken together establish strong similarities, as well as some differences, 10 between the prosurvival signals elicited by these two agents in cultured cells in vitro and the small intestinal tissue in vivo. [061] OTP and LPA enhance intestinal crypt survival after radiation injury. y irradiation induces apoptosis in crypt cells, which in turn reduces the regenerative or clonogenic potential leading to the disruption of the barrier and absorptive function in the 15 injured gut. The inventors examined the effect of LPA and OTP on crypt survival in irradiated mice using H&E staining and BrdU incorporation to monitor regenerating S phase enterocytes. Irradiation with a dose of 15 Gy caused a 90% reduction in the number of crypts in jejunum in wild type mice within 4 days (Figs. 8-9). Oral LPA or OTP pretreatment enhanced intestinal crypt survival in a dose-dependent manner (Fig. 8A). 20 OTP treatment of mice with doses above 0.2 mg/kg significantly enhanced crypt survival, while LPA treatment required a minimum dose of 1.5 mg/kg to cause a significant protection in crypt survival (p<0.05, Fig. 8A). OTP at doses above 1 mg/kg maintained a significantly greater number of crypts than did LPA (p<0.05, Fig. 8A). Following 15 Gy 39 WO 2008/140574 PCT/US2007/084875 7-irradiation, oral OTP applied at 2 mg/kg increased the number of surviving crypts in the jejunum from 10 crypts to an average of 27 crypts per circumference (Fig. 8A). [062] To further validate LPA 2 as the target of the antiapototic action of LPA and OTP, the inventors also examined crypt survival in LPA2 KO mice treated the same 5 way as their wild type counterparts described above. In contrast to irradiated wild type mice, LPA 1 and particularly LPA 2 KO mice showed lower crypt survival. However, in LPAi KO mice LPA and OTP both elicited significantly increased crypt survival, which was not significantly different from that seen in wild type mice treated the same manner.

LPA

2 KO mice revealed significantly higher radiation sensitivity. Whereas in wild type 10 mice exposed to a 15 Gy dose the mean crypt survival per circumference was 10, in LPA2 KO mice it was as little as 1 crypt/circumference (Fig. 8B). Neither LPA nor OTP applied at 2 ing/kg, the highest dose tested, showed any effect in enhancing intestinal crypt survival in the LPA 2 KO mice. [063] The inventors' data illustrate that OTP is a highly effective antiapoptotic 15 agent that engages prosurvival pathways similar to those elicited by LPA through the

LPA

2 receptor subtype. OTP shares a pharmacological profile similar to that of LPA in that it activated the three EDG family LPA receptors expressed heterologously in RH7777 cells. The rank order of OTP's ECo values was LPA 2 (90 nM) < LPA 3 < LPAi based on Ca 2 ' transients elicited in this heterologous expression system. These values 20 render OTP a considerably weaker ligand of the three LPA receptors compared to LPA 18:1. OTP when compared to LPA in three different apoptosis models, which included radiation- and camptothecin-elicited DNA damage-induced and TNF-c/CHX-elicited receptor-induced mechanisms, always surpassed the protective effect of LPA. In 40 WO 2008/140574 PCT/US2007/084875 agreement with the in vitro cellular models of apoptosis, OTP was more effective in reducing the number of apoptotic bodies, caspase 3 positive cells, and caspase 3 activity in C57/BL6 mice expose to an LD 100 15 dose of y-irradiation. [064] OTP was not cleaved by pancreatic lipase, the major lipase in the intestine. 5 It is not degraded by lipid phosphate phosphatase 1, which is the other major mechanism for the inactivation of LPA. Due to the lack of a glycerol backbone, unlike LPA, OTP cannot be acylated by lysophosphatidate transacetylases. Likely due to its polar character, OTP does not transverse the cell membrane readily, and the inventors could not detect radioactively labeled OTP in the blood of experimental animals following 10 application via oral gavage. These differences in its metabolism and bioavailability relative to LPA may represent some of the causes for its higher efficacy in the apoptosis assays. The lack of absorption of [ 3 H]-OTP into the systemic circulation indicates that it exerts its effect topically from within the intestinal lumen without appearing in a pharmacologically and biologically effective concentration in the bloodstream. 15 Radioactively labeled LPA has been shown to be taken up rapidly (within minutes) into cells where it is rapidly dephosphorylated and reacylated; nevertheless, -15% remains intact in the cytoplasm up to 30 miii after extracellular application. These differences in bioavailability could, at least in part, explain the differences in the in vivo efficacy of OTP compared to LPA. 20 [065] Another important difference was that the LPA 2 receptor subtype is sufficient and necessary for the antiapoptotic effect of OTP. In the receptor reconstitution experiments carried out in RH7777 cells stably transfected with the individual receptor subtypes, although OTP activated Ca' transients through each receptor subtype, only 41 WO 2008/140574 PCT/US2007/084875 cells expressing LPA 2 were protected against apoptosis. In agreement with the role of

LPA

2 in this model system, experiments conducted in LPA 2 KO mice confirmed that this receptor was absolutely necessary for the attenuation of apoptosis and increased crypt survival elicited by OTP. A third line of evidence, namely the increased radiation 5 sensitivity of LPA 2 KO gut tissue compared to wild type animals, suggests that this receptor subtype is uniquely involved in mediating pro-survival signals. Only if the inventors assign the pro-survival function to LPA2 can it explain the lack of protective effect in response to LPA administration observed in the LPA 2 KO mice, which continue to express LPA 1 and LPA 3 . RT-PCR analysis has identified only the LPA 1 and LPA 2 10 receptor subtypes in the mouse intestine, as well as in IEC-6 cells used in the present study. The partial sensitivity of the OTP-elicited antiapoptotic response to PTX is consistent with the Gq-coupling of LPA 2 . However, LPA also showed antiapoptotic protection in RH7777 cells expressing either LPA 1 or LPA 3 in the TNF-W/CHX induced apoptosis model. The RH7777 cells show extreme resistance to radiation that precluded 15 studying the radioprotective effect exerted by these receptors in this model. LPA 2 is distinct from the other two subtypes in that its C-terminus has been shown to interact with PDZ and LIM domain-containing proteins. LPA 2 can signal through specific protein-protein interactions in a non-G-protein-coupled manner and access triple LIM domain-containing proteins including TRIP6, zyxin, LPP, and Siva- 1. G protein-linked 20 activation of c-src by LPA 2 phosphorylates TRIP6, which in turn augments LPA-induced ERK activation and could lead to increased BAD phosphorylation and inhibition of procaspase 9. The potential interaction of LPA2 with Siva- 1 offers an exciting possibility, because DNA damage-induced activation of Siva- 1 scavenges the antiapoptotic Bcl-X. 42 WO 2008/140574 PCT/US2007/084875

LPA

2 may capture Siva- 1 and enable Bcl-XL to attenuate apoptosis triggered by DNA damage. The PDZ domain-mediated interactions including the PDZ-binding protein NHERF2 provide yet another link with antiapoptotic signaling, because stable knockdown of NHERF2 in CaCo-2 cells has been found to attenuate LPA2-induced 5 ERK1/2, AKT, and PLCD activation. [066] Orally administered OTP offers other uses that reach beyond the protection of the GI system. Experiments done with intraperitoneal and subcutaneous application of OTP in C57/BL6 mice at doses similar to those used in the present experiments also demonstrated a significant reduction in lethality. 10 15 43

Claims (4)

1. A method for doersing dariieq to and increaSing survival fl cells- of the hematopoietic system prior to or subsequent to radiation exposure, the method comprising administering to a hunan or animal that has been exposed to radiation a. thempeufcally tffective amount of ia compound as i1 fbriauta (1) X(3 whe rem, at least one -of X X2 and X 3 is (HO 2 P3-, or(H%.P~tP(OH)$A40, X' and X 2 &iie linked togiher as -IPS(OH) -O-, or X, and X 3r linked ogeber as -0-PSO) -NW oleast one of X1, X2I and X is R'-Y-A- with each being the sarn or different when two of X', U and X are R' -ilA, or X nd X are linked together as -N(H) -C(0) -N(R).-; optionally, one of Xk Xaxnd Xt is H; A is either a diret link; (O2)tvith k being an intege nm0 to 30 or O; Y1 izi -(CH2)r- widi I belog andateger from I to 30, -0-, -S-, or -NR Z is -(CIl- -CF--, -C2(CH2)c, or -O(C-Jz)mr- with m being an integer-from I to 50, -C(RW)-l-, -NH--, -O, or -S .4 Z 2 is -- (CHte),- or -P0-)with n being an integer from I to 50 or -0 Q1 tad Q" are independently fl, uNR 4 .0. or a conbination of H and -NR R Ri' fri ca of XN' X 2 , or X, is independently hydrogen, a straight or branched chain C! to C30-akyl, a straightor branched-chain C2 to C30 alkenyl, an aromatic or hetrarnmatki ring with oi without pono1 0 di, or tri- substitions of the ring, an acyl including a C I to C30 alkyI or marnmadc or heeromatic ring, an arylatkyl including. straight or branched-chai Ci to C30 alkyl, an aryoaxy-Ikl1 including st ight or ranched-chain Cl to, C30 alkyl, It N C-N--RR NN- C---NH -R O S and R R R R' R'. R ' d R 8 are independenly hydrogetn ai sbtrigbt or brnmhed-. chainrC1 to C3 alky], a straight or branched-chain C2 to C$0.alkeny imaic or hetemarmatic ring~ with or without w ono-, db-,or tr-substiturions of the ring, an acyl including a C1 to C30. alkyl or aromatic or letecaroaomatic ring, an arylalkyl inchiding straight ;.r'bauched-chain CI to C30 alky, 'or an aryjoKyalkyI inchdhig straight or branhed-chCi to C30 alkyl.

2. The method ofclaiPn i Wiherein in lhe chnpoimund of fornula (T): 45 X is RY--V-A A-; X( ia ZPS)Os X 3 la hydftogenf A is. a direct Iink or (CNkh with being an imeget hom i-30; 'is (CHI, with Ibeig ma integer from.1-3M Z is oxygen; (QP and Q trer nRw dently sdeced ftlqitinhe group Consisting of , =NP, -O, ur Rl i.q independundy . straight or brached-chain C, to Q lkyL v straight ir bric%-d-chuin C 2 to Can. keny, an brade or hieteraomatic ring (optonally Mbstitited ),n W.I a-yi includitg C.,tO C adky). n aromatic or heteoaromatic ing, an atryhdkyl inchiding straight or bned-chi C 1 io Ca alkyl anaryloxyajky including t oc bahed jehin C, to Cv akyt -7 R7 '-N H-R*? NHT, AS NR' . C $ ; anid R"R, R" mRad R'S are independently hydrogen),a sttaignt oe htmi'ched7 ciin C, to. Cg, alty, a sightorached chin C 2 to (,, alkeny, W rmtIiO Or hetrarubnuttik. fing with or without mon&, di-, or indabtitutionis of the ring, An tyl inacuding a C, tO Cor alkyl or aromafiu or beteroarirnatic ring, ari rydtkyIindijbig straight or branched-chain C 1 to Co alky, or n aryloxyaikyl inluding straight or branched'chain Ct to:C~ a kyl. 46 'I roel I licod of clan~ I or claim 2 vhbemin -te step~ of adriif-ring to a humani obr anirhga t-ha has beo! expic o -10radiation a th ue.noxmy-ffeiv if~ of a compound ag in formula (I) is pcrfbrm~d by subeutaticous admrnlnistruti on.

The nicihod of claim I or ii2 hei te p of administering ic hmno animal that has been -. xposcd. to radiation. aitxrpitcliyefuieaon of a conilound as in Forrmila (1) is perfbrmcd by intrarnusiar adntinigtration. 5, tho method of clajil 1, subs t .ntially as heroin described with rcfcrence to fuly of the Fxaipl d/o coiayigFgts

4.7