US 20100016280A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2010/0016280 A1 Nichols et al. (43) Pub. Date: Jan. 21, 2010

(54) LOW DOSAGE SEROTONIN 5-HT2A Publication Classification RECEPTORAGONST TO SUPPRESS (51) Int. Cl. NFLAMMATION A 6LX 3L/397 (2006.01) A63L/38 (2006.01) A63L/48 (2006.01) (76) Inventors: Charles D. Nichols, New Orleans, A6IP II/06 (2006.01) LA (US); Bangning Yu, New A6IP 9/02 (2006.01) A6IP3/10 (2006.01) Orleans, LA (US) A6IP 25/28 (2006.01) (52) U.S. Cl...... 514/210.21; 514/646; 514/647: Correspondence Address: 514/288 PATENT DEPARTMENT (57) ABSTRACT TAYLOR, PORTER, BROOKS & PHILLIPS, Activation of 5-HT, receptors using agonists at Surprisingly LLP low concentrations was shown to potently inhibit TNF-C.- P.O. BOX 2471 induced inflammation in multiple cell types. Significantly, BATON ROUGE, LA 70821-2471 (US) proinflammatory markers were also inhibited by the agonist, (R)-DOI, even many hours after treatment with TNF-C. With the exception of a few natural toxins, no current drugs or Small molecule therapeutics demonstrate a comparable (21) Appl. No.: 12/501,105 potency for any physiological effect. TNF-C. and TNF-C. receptor mediated inflammatory pathways have been strongly implicated in a number of diseases, including ath (22) Filed: Jul. 10, 2009 erosclerosis, asthma, rheumatoid arthritis, psoriasis, type II diabetes, depression, Schizophrenia, and Alzheimer's dis ease. Importantly, because (R)-DOI can significantly inhibit Related U.S. Application Data the effects of TNF-C. many hours after the administration of TNF-C., potential therapies could be aimed not only at pre (60) Provisional application No. 61/079,576, filed on Jul. venting inflammation, but also treating inflammatory injury 10, 2008. that has already occurred or is ongoing. Patent Application Publication Jan. 21, 2010 Sheet 1 of 14 US 2010/0016280 A1

2 ICAIf

100 90 8 70 S 5 EC50 a $9.5 pM

3.

6 - 5 4 3 2 ------log (R-DOM Fig. 1A

VCAM-1

8 70 S. S. EC50 a 12.0 pA. A. 30 2

6 - 5 - 4 - 3 - - - - -8 - 6 iog R-DOM Fig. 1B Patent Application Publication Jan. 21, 2010 Sheet 2 of 14 US 2010/0016280 A1

IL-6

5 EC50 s 0.3 pM

S 4 3 2 at . 9 8 7 6 log (R-DOM Fig. 1C

2 ICAM-1 (-FBS)

O

EC50 c 360 pM

-16 -15 i4 (3 -12 - O 9 8 7 6 og ROOM Fig. 1D Patent Application Publication Jan. 21, 2010 Sheet 3 of 14 US 2010/0016280 A1

Control NF-ct. GO IDOOTNF f++ RS+D+ S8--D-T

Treatment Fig. 2A

WCA-f to Control TNF-ct. GO IO--TNF V+D+T RS-D S3+)-T

Treatent Fig. 2B

I6 Control 500 TNF-ct EO C DOE+TNF 2M+D+ SNRS+D+ 234. SB+)--T 10 O

O Treatment Fig. 2C Patent Application Publication Jan. 21, 2010 Sheet 4 of 14 US 2010/0016280 A1

OControl NF-o.

1nM DO+TNF-ct KX2C-BCB+TNF-ct, LA-SS-Az+ NF :: LSD+TNF 8 S. 8SS: 8.

:8

12345678OOOOOOOO900 OOOOOOOOO 1 nM Agonist 50 nM Agonist Fig. 3 Patent Application Publication Jan. 21, 2010 Sheet 5 of 14 US 2010/0016280 A1

|||||||| C+D+E GO--D--§ PNA-T F-22+ T Fig. 4 Patent Application Publication Jan. 21, 2010 Sheet 6 of 14 US 2010/0016280 A1

120 ICAM-1 Expression 110 100

50 = 237 in

O.O. 10 30 100 316 1000 Time (R)-DOi added after TNF-o. (minutes, log scale) Fig. 5 Patent Application Publication Jan. 21, 2010 Sheet 7 of 14 US 2010/0016280 A1

900 m

800

700

600

500

400

300

200 100 -

Control TNF-0 DO-T Che+D+T Fig. 6 Patent Application Publication Jan. 21, 2010 Sheet 8 of 14 US 2010/0016280 A1

:

Control TNF-O TNF-o + (R) - D01

Fig. 7A

Patent Application Publication Jan. 21, 2010 Sheet 10 of 14 US 2010/0016280 A1

IC50 F ~ 0.8 nanomolar

150

EC50 = 0.72nM

5027 55 O -15 - 14 - 13 - 12 - 11 - 10 -9 -8 - 7 -6 -5 DOM Fig. 8 Patent Application Publication Jan. 21, 2010 Sheet 11 of 14 US 2010/0016280 A1

DOI (1nM) 350 LA-SS-Az (1 nM) N DO (50 nM) ELA-SS-Az (50 nM) 250

2OO

150

100

50 3. 2

Fig. 9 Patent Application Publication Jan. 21, 2010 Sheet 12 of 14 US 2010/0016280 A1

VACM-1 120 : 7 O EC50 = 19.5 pM

6 O

-1 -16 - 15 - 14 -13 - 12 - 11 - 10 -9 -8 -7 -6 log (R-DOIM Fig. 10 Patent Application Publication Jan. 21, 2010 Sheet 13 of 14 US 2010/0016280 A1

L6 1200

900

300

Control TNF 10 pMDO Fig. 11A

VCAM1 16OO

1400

1200

1000

800

600

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200

Control TNF 10 pMDOl Patent Application Publication Jan. 21, 2010 Sheet 14 of 14 US 2010/0016280 A1

1 ug LPS 1 OOO 900 - 800 700 600 500 400 300 200 100 a

Fig. 12 US 2010/0016280 A1 Jan. 21, 2010

LOW DOSAGE SEROTONIN 5-HT2A 0007. Two other studies reported that DOI acting at 5-HT, RECEPTORAGONST TO SUPPRESS receptors partially blocked LPS and cytokine stimulated NFLAMMATION nitrite accumulation using a cocktail of TNF-C. and TNF-Y in C6 glioma cells, and reported an IC50 value of 8+3 nM. (Miller et al., 1997: Miller and Gonzalez, 1998). In earlier 0001. The benefit of the 10 Jul. 2008 filing date of U.S. reports, the synthesis of TNF-C. in response to LPS stimula provisional patent application 61/079,576 is claimed under tion of toll-like receptors was reported to be inhibited by 35 U.S.C. S 119(e). 5-HT through 5-HT, receptors in monocytes (Arzt et al., 0002 This invention was made with United States govern 1991). The use of ketanserin as the antagonist to block 5-HT ment support under Grant Nos. P20RR018766 and receptor activation in many of these studies to indicate HL072889 awarded by the National Institutes of Health. The 5-HT2 receptor involvement is problematic, since ketanserin United States government has certain rights in this invention. has only weak selectivity (-20-fold) for 5-HT, receptors 0003. This invention relates to the use of serotonin 5-HT over 5-HT, receptors, and has a high affinity for C.-adren receptor agonists at low dosages no greater than about 5 nM. ergic receptors, and significantly, is equipotent at blocking more preferably no greater about 1 nM, to treat tumor necro histamine H receptors, which are known to regulate inflam sis factor-alpha (TNF-C) related inflammation and inflamma matory processes. Ketanserin cannot be used to discriminate tion-related diseases and conditions. reliably between the effects of agonists acting at 5-HT, or 0004 Serotonin 5-hydroxytryptamine (5-HT) receptors 5-HT, receptors. and agonists. Serotonin, 5-hydroxytryptamine (5-HT), is a 0008. The presence of serotonin itself has been demon Small monoamine molecule primarily known for its role as a strated to be necessary for expression of the inflammatory neurotransmitter. Within the brain, 5-HT modulates a variety markers IL-6 and TNF-C., with lower serotonin levels induc of behaviors including cognition, mood, aggression, mating, ing, and higher levels decreasing expression of these markers feeding, and sleep (Nichols and Nichols, 2008). These behav (Kubera et al., 2005). This inverted-U shaped response iors are mediated through interactions at seven different clearly indicates that in vivo serotonin plays an important role receptor families (5-HT) comprised of fourteen distinct in modulating molecular components of the inflammatory subtypes (Nichols and Nichols, 2008). Each of these are process. The identity of the serotonin receptor mediating G-protein coupled receptors, with the exception of the 5-HT these processes has not been reliably established. receptor, which is a ligand-gated ion channel. Of all the sero 0009 Primary cultures of rat aortic smooth muscle tonin receptors, the 5-HT2 receptor, which is known to pri (RASM) area well established system to study inflammatory marily couple to the God effectorpathway (Roth et al., 1986), processes. Aortic Smooth muscle cells normally form the has been the one most closely linked to complex behaviors. media of the aorta, and serve to provide and regulate vascular There is a high level of expression of 5-HT, receptors within tone of the artery. Significantly, the pathophysiological status the frontal cortex, with significant localization to the apical of vascular Smooth muscle cells is a crucial determinant of dendrites of cortical pyramidal cells (Willins et al., 1997), and vascular disease (Hansson et al., 2006). During the develop further expression at lower levels throughout the brain ment of atherosclerosis, which is believed to have a major (Nichols and Nichols, 2008). These receptors have been inflammatory component, levels of cytokines such as TNF-C. shown to participate in processes such as cognition and work are elevated, leading to increased expression of genes and ing memory, have been implicated in affective disorders such proteins, e.g., ICAM-1 and VCAM-1, in aortic smooth as Schizophrenia, and have been shown to mediate the pri muscle (Blankenberg et al., 2003; Hansson et al., 2006). mary effects of hallucinogenic drugs (Nichols, 2004). ICAM-1 (CD 54) belongs to the immunoglobulin (IgG) 0005. In addition, many peripheral tissues express 5-HT Superfamily and is expressed in many cell types, including receptors. Within the vasculature, 5-HT, receptors are vascular endothelial cells, epithelial cells, fibroblasts, and known to modulate vasoconstriction (Nagatomo et al., 2004). macrophages (Hughes et al., 2000). Cytokine-mediated Its role in other tissues such as mesangial cells of the kidney, increased levels of intracellular adhesion molecules like fibroblasts, liver, and lymphocytes remains less defined, but ICAM-1 in aortic Smooth muscle serve as an important com has been linked to cellular proliferation and differentiation. ponent of atherosclerotic plaque formation. 0006. The presence of 5-HT, receptor mRNA (along 0010. The Immune Response System in Mammals. There with the mRNAs of other serotonin receptor subtypes) has are two immune response systems in mammals. One is an been found in tissues involved in the immune response (Ste innate immunity, and is the system whereby the organism fuljet al., 2000). The role of 5-HT, receptors in inflamma recognizes acute pathogens like invading bacteria. There are tory processes, however, is unclear, with only a few published specific receptors on cells within the organism known as and inconsistent reports. For example, blockage of 5-HT, “Toll-like receptors’ (TLRs) that are pattern recognition receptor function with the selective antagonist Sarpogrelate receptors that recognize antigens present on pathogenic has been reported to decrease expression of proinflammatory micro-organisms. When a cell encounters these pathogens, markers (Marconi et al., 2003: Akiyoshi et al., 2006), and TLRs are activated by antigens on the pathogen. This activa conversely reported to increased expression of proinflamma tion induces an inflammatory response which partially tory markers (Ito et al., 2000). Using extremely high non involves the activation of Nuclear Factor k-B (NFkB), and pharmacologically relevant drug doses (lowest dose was 25 transcription of proinflammatory genes including cytokines uM), a 5-HT, receptor specific agonist, 1-(2,5-dimethoxy-4- and cell adhesion molecules. Activation of TLRs, primarily iodophenyl)-2-aminopropane (DOI: the racemic form), was on monocytes and macrophage cells, with bacterially derived alleged to repress IL-1B expression and production of TNF-C. agents like lipopolysaccharide (LPS) induces the production due to lipopolysaccharide stimulation through 5-HT2 recep and release of large amounts of tumor necrosis factor-alpha tor activation (Cloez-Tayarani et al., 2003). (TNF-C.) from the monocyte and macrophage cells. US 2010/0016280 A1 Jan. 21, 2010

0011 Tumour necrosis factor-alpha (TNF-C.) is an inflam monocytes and macrophage cells. In contrast, therapeutic matory cytokine produced by circulating monocytes and resi strategies that are aimed at blocking the TNF-C. receptor dent macrophages during acute inflammation. Macrophages induced signal transduction pathways by 5-HT2 receptor are, upon stimulation, the main producers of TNF-C. as well as activity would aim at any tissue cell expressing TNF-C. recep they are also the primary infiltrating cells at the site of inflam tors, which include most cell types in the body (e.g. Smooth mation. After TNF-C. is released from these cells, one action muscle cells, neurons, skin cells). is to bind to its specific receptor proteins (TNFR1 and TNFR2) present on the surface of almost every cell type. DISCLOSURE OF INVENTION TNF-C. receptors (TNFRs) are members of a different class of receptor proteins than TLRs, and recognize the endogenously I0016 We have shown that activation of 5-HT, receptors produced cytokine TNF-C. Whereas TNF-C. is predominantly using agonists at concentrations no greater than 5 nm, more produced and released from circulating monocytes and resi preferably no greater than 1 nM. potently inhibits TNF-C.- dent macrophages at the site of infection or injury, TNF-C. induced inflammation in multiple cell types, including rat receptors are expressed in nearly every cell. Through a view aortic Smooth muscle cells, human neuroblastoma cells, rat different signaling pathway from that used by TLR receptors, glioma cells, rat aortic epithelial cells, and rat bronchoaveolar a pathway that involves completely different proteins, TNF-C. macrophage cells. We have shown that 5-HT, receptor receptor stimulation can lead to activation and nuclear trans stimulation with the agonist (R)-DOI rapidly inhibits a vari location of NF-kB, and transcription of proinflammatory ety of proinflammatory markers mediated by TNF-C. acting at genes including cytokines and cell adhesion molecules. its receptors, including ICAM-1, VCAM-1, and IL-6 gene expression, NOS activity, and nuclear translocation of NF 0012. When TNF-C. binds to its specific receptor, a diverse KB, with ICs values of only 10-20 pM. Significantly, proin range of signal transduction events are initiated from the flammatory markers also can be inhibited by (R)-DOI many activated receptor that lead to cellular responses, which hours after treatment with TNF-C. With the exception of a few include responses like inflammation, necrosis, apoptosis, cell natural toxins, no current drugs or Small molecule therapeu Survival, and cell migration. During acute inflammation. tics demonstrate a comparable potency for any physiological TNF-C. overproduction is crucial in the induction of inflam effect. TNF-C.-mediated inflammatory pathways have been matory genes, cell death, endothelial up-regulation, and in the strongly implicated in a number of diseases, including ath recruitment and activation of immune cells. TNF is a “mas erosclerosis, rheumatoid arthritis, asthma, psoriasis, type II ter” cytokine in inflammatory diseases. Anti-TNF agents diabetes, depression, Schizophrenia, and Alzheimer's dis have been very effective in chronic inflammatory conditions ease. Our results indicate that activation of 5-HT, receptors in patients such as rheumatoid arthritis, even in the absence of represents an extraordinarily potent, potential therapeutic other anti-cytokine agents. (Crisafulli et al 2009). avenue for the treatment of disorders involving TNF-C.-me 0013 Whereas production and release of TNF-C. from diated inflammation. Importantly, because (R)-DOI can sig monocytes and macrophages can be induced by TLRS during nificantly inhibit the effects of TNF-C. many hours after the the process of infection, there are many other mechanisms administration of TNF-C., potential therapies could be aimed independent of TLRs that can regulate TNF-C. concentra not only at preventing inflammation, but also treating inflam tions. For example, oxidative stress in cells can lead to matory injury that has already occurred or is ongoing. elevated levels of TNF-C., as well as direct activation of NFkB, to produce inflammation. Oxidative stress can be caused by pathological conditions that include diabetes, BRIEF DESCRIPTION OF DRAWINGS metabolic disorder, and neurological disorders, and the (0017 FIG. 1A illustrates the effect of 5-HT, receptor resulting increase in inflammation can contribute to the devel activation with the agonist (R)-DOI on the expression of opment and progression of diseases like atherosclerosis, ICAM-1 in primary rat aortic Smooth muscle cells (passage arthritis, and asthma. 4). The Y-axis represents percent of TNF-C. control induction 0014 TNF-C. and TNF-C. receptor-mediated inflamma for the dose of (R)-DOI indicated on the X-axis. The ICs for tory pathways have been strongly implicated in a number of proinflammatory gene expression inhibition for ICAM-1 is diseases including atherosclerosis, rheumatoid arthritis, pso 19.5 pM. riasis, type II diabetes, irritable bowel syndrome, Crohn's (0018 FIG. 1B illustrates the effect of 5-HT, receptor disease, and septicemia (e.g., Reimold, 2002; Popa et al., activation with the agonist (R)-DOI on the expression of 2007: Williams et al., 2007). Significantly, TNF-C. and other VCAM-1 in primary rat aortic Smooth muscle cells (passage cytokine induced inflammatory pathways also have been 4). The Y-axis represents percent of TNF-C. control induction linked to psychiatric conditions such as depression and bipo for the dose of (R)-DOI indicated on the X-axis. The ICs for lar disorder (Dunn et al., 2005; Kim et al., 2007), as well as proinflammatory gene expression inhibition for VCAM-1 is schizophrenia (Saetre et al., 2007), and neurodegenerative 12.0 pM. diseases like Alzheimer's and Parkinson's disease and stroke (0019 FIG. 1C illustrates the effect of 5-HT, receptor (Tweedie et al., 2007). As such, inhibitors of TNF-C.-medi activation with the agonist (R)-DOI on the expression of IL-6 ated proinflammatory pathways represent potential therapeu in primary rat aortic smooth muscle cells (passage 4). The tics for each of these conditions. Currently, the only available Y-axis represents percent of TNF-C. control induction for the therapeutic inhibitors of TNF-C. pathways are monoclonal dose of (R)-DOI indicated on the X-axis. The ICs for proin antibodies against TNF-C. (infliximab and adalimumab) and flammatory gene expression inhibition for IL-6 is 10.3 pM. soluble TNF-C. receptor (etanercept), and the development of 0020 FIG. 1D illustrates the effect of 5-HT, receptor small molecules for this purpose is highly desirable (Tracey et activation with the agonist (R)-DOI on the expression of al., 2007). ICAM-1 in primary rat aortic Smooth muscle cells (passage 4) 00.15 Potential therapeutic strategies aimed at blocking in serum-free media. The Y-axis represents percent of TNF-C. TNF-C. synthesis and release would primarily target the control induction for the dose of (R)-DOI indicated on the US 2010/0016280 A1 Jan. 21, 2010

X-axis. The ICso for proinflammatory gene expression inhi (R)-DOI (1 nM) prior to TNF-C. (Go--D--T); pretreatment bition for ICAM-1 in serum-free media is 360 pM. with a PKC activator PMA (100 nM) prior to TNF-C. (PMA+ 0021 FIG. 2A illustrates the effect of 5-HT, receptor T); and pretreatment with a PKA inhibitor F-22 amide (100 activation on the expression of ICAM-1 in primary rat aortic nM) for 30 minutes prior to the addition of (R)-DOI (1 nM) Smooth muscle cells (passage 4) under various conditions: prior to TNF-C. (F-22+D+T). (*=p<0.01 vs control: ANOVA control, TNF-C. treatment alone (10 ng/ml) (TNF), pre-treat with Tukey post hoc). ment with (R)-DOI (1 nM) prior to TNF-C. (DOI+TNF), 0026 FIG. 5 illustrates a time course study examining pretreatment with the 5-HT, receptor selective antagonist 5-HT, receptor simulation by (R)-DOI (1 nM) and its effect M100907 (100 nM) 30 minutes prior to (R)-DOI (M+D+T), in inhibiting TNF-C. induced ICAM1 expression when given pretreatment with 100 nM of the 5-HT, receptor selective at various time intervals after addition of TNF-C. (10 ng/ml) antagonist SB20474130 minutes prior to (R)-DOI (S+D+T), (in minutes) in primary rat aortic Smooth muscle cells (pas or pretreatment with 100 nM of the 5-HT, receptor selective Sage 4). antagonist RS102221 30 minutes prior to (R)-DOI (R+D+T). (0027 FIG. 6 illustrates the effect of 5-HT, receptor acti (#p-0.01 vs control: *p-0.01 vs TNF-C. alone: ANOVA with vation on the levels of nitrite (an indicator of NOS activity) in Tukey post hoc) primary rat aortic Smooth muscle cells (passage 4) under 0022 FIG. 2B illustrates the effect of 5-HT, receptor various conditions: control; TNF-C. treatment alone (10 activation on the expression of VCAM-1 in primary rat aortic ng/ml) (TNF-C.); pretreatment with (R)-DOI (1 nM) prior to Smooth muscle cells (passage 4) under various conditions: TNF-C. (DOI+T), and pretreatment with the pan-PKC iso control, TNF-C. treatment alone (10 ng/ml) (TNF), pre-treat form inhibitor chelerythrine (100 nM) 30 mins prior to pre ment with (R)-DOI (1 nM) prior to TNF-C. (DOI+TNF), treatment with (R)-DOI (1 nM) prior to TNF-C. (Che+D+T) pretreatment with the 5-HT, receptor selective antagonist (*=p<0.01 vs. TNF-C. ANOVA with Tukey post hoc). M100907 (100 nM) 30 minutes prior to (R)-DOI (M+D+T), (0028 FIG. 7A illustrates both the p65 localization as visu pretreatment with 100 nM of the 5-HT, receptor selective alized with Alexafluor 48 conjugated secondary antibody (the antagonist SB20474130 minutes prior to (R)-DOI (S+D+T), top row), and the position of the nuclei as visualized with or pretreatment with 100 nM of the 5-HT, receptor selective DAPI (the bottom row) in primary rat aortic smooth muscle antagonist RS102221 30 minutes prior to (R)-DOI (R+D+T). cells (passage 4) under various conditions: control; TNF-C. (#p-0.01 vs control: *p-0.01 vs TNF-C. alone: ANOVA with treatment alone (10 ng/ml) (TNF-C.); and pretreatment with Tukey post hoc) (R)-DOI (1 nM) prior to TNF-C. (TNF-C+(R)-DOI). 0023 FIG. 2C illustrates the effect of 5-HT, receptor 0029 FIG. 7B illustrates the percentage of cells within a activation on the expression of IL-6 in primary rat aortic given field that predominantly showed p65 located in the Smooth muscle cells (passage 4) under various conditions: nucleus under various conditions: control; (R)-DOI alone (1 control, TNF-C. treatment alone (10 ng/ml) (TNF), pre-treat nM); TNF-C. treatment alone (10 ng/ml) (TNF-C.); and pre ment with (R)-DOI (1 nM) prior to TNF-C. (DOI+TNF), treatment with (R)-DOI (1 nM) prior to TNF-C. ((R)-DOI+ pretreatment with the 5-HT, receptor selective antagonist TNF-C.). (Average of three fields for each treatment; p<0.01 M100907 (100 nM) 30 minutes prior to (R)-DOI (M+D+T), vs. control, hip 0.01 vs TNF-C. ANOVA with Tukey posthoc). pretreatment with 100 nM of the 5-HT, receptor selective 0030 FIG. 8 illustrates the effect of 5-HT, receptor acti antagonist SB20474130 minutes prior to (R)-DOI (S+D+T), vation with the agonist ()-DOI on the expression of ICAM-1 orpretreatment with 100 nM of the 5-HT, receptor selective in human neuroblastoma cells. The Y-axis represents percent antagonist RS102221 30 minutes prior to (R)-DOI (R+D+T). of TNF-C. control induction for the dose of (R)-DOI indicated (#p-0.01 vs control: *p-0.01 vs TNF-C. alone: ANOVA with on the X-axis. The ICs for proinflammatory gene expression Tukey post hoc) inhibition for ICAM-1 is about 0.8 nM. 0024 FIG.3 illustrates the effect of 5-HT, receptor acti 0031 FIG. 9 illustrates the effect of 5-HT, receptor acti vation on the expression o ICAM-1 in primary rat aortic vation on the expression of ICAM-1 in rat C6 glioma cells Smooth muscle cells (passage 4) under various conditions: under various conditions: control; TNF-C. treatmentalone (10 control; TNF-C. treatment alone (10 ng/ml) (TNF-C.); pre ng/ml) (TNF); pretreatment with (R)-DOI (1 nM and 50 nM, treatment with (R)-DOI (1 nM, a 5-HT, receptor agonist) a 5-HT, receptor agonist) prior to TNF-C. (DOI), and pre prior to TNF-C. (DOI+TNF-C.), pretreatment with the phen treatment with the indolealkylamine LA-SS-AZ (1 nM and 50 ethylamine 2C-BCB (1 nM and 50 nM, a 5-HT, receptor nM, a 5-HT, receptor agonist) prior to TNF-C. (LA-SS-AZ). agonist) prior to TNF-C. (2C-BCB+TNF-C.); pretreatment 0032 FIG. 10 illustrates the effect of 5-HT, receptor with the indolealkylamine LA-SS-AZ (1 nM and 50 nM, a activation with the agonist ( )-DOI on the expression of 5-HT, receptor agonist) prior to TNF-C. (LA-SS-AZ+TNF VCAM-1 in primary rat bronchoaveolar macrophage cells. C.); and pretreatment with the indolealkylamine LSD (1 nM The Y-axis represents percent of TNF-C. control induction for and 50 nM, a 5-HT, receptoragonist) prior to TNF-C. (LSD+ the dose of (R)-DOI indicated on the X-axis. The ICs for TNF-o). proinflammatory gene expression inhibition for VCAM-1 is 0025 FIG. 4 illustrates the effect of 5-HT, receptor acti about 20 pM. vation on the expression o ICAM-1 in primary rat aortic 0033 FIG. 11A illustrates the effect of 5-HT, receptor Smooth muscle cells (passage 4) under various conditions: activation on the expression of IL-6 in rat primary aortic control; TNF-C. treatment alone (10 ng/ml) (TNF-C.); pre endothelial cells under various conditions: control; TNF-C. treatment with (R)-DOI (1 nM, a 5-HT, receptor agonist) treatment alone (10 ng/ml) (TNF); and pretreatment with prior to TNF-C. (D+T), pretreatment with the pan-PKC iso (R)-DOI (10pM, a 5-HT, receptoragonist) 60 minutes prior form inhibitor chelerythrine (100 nM) for 30 minutes prior to to TNF-o. (DOI). the addition of (R)-DOI (1 nM) prior to TNF-C. (C+D+T): 0034 FIG. 11B illustrates the effect of 5-HT, receptor pretreatment with the classical PKC isoform inhibitor activation on the expression of VCAM-1 in rat primary aortic Gö6976 (100 nM) for 30 minutes prior to the addition of endothelial cells under various conditions: control; TNF-C. US 2010/0016280 A1 Jan. 21, 2010 treatment alone (10 ng/ml) (TNF); and pretreatment with hydrochloride), HT receptor antagonist SB204741 (N-(1- (R)-DOI (10pM, a 5-HT, receptoragonist) 60 minutes prior Methyl-1H-indolyl-5-yl)-N'-(3-methyl-5-isothiazolyl)urea, to TNF-o. (DOI). PKC inhibitor Gö6976 (5,6,7,13-Tetrahydro-13-methyl-5- 0035 FIG. 12 illustrates the effect of 5-HT, receptor oxo-12H-indolo2,3-alpyrrolo3.4-ccarbazole-12-pro activation on the expression of ICAM-1 due to stimulation panenitrile), and PKC inhibitor chelerythrine (1.2- with lipopolysaccharide (LPS, 100ng and 1 Lig) in primary rat Dimethoxy-12-methyl 1.3 benzodioxolo.5,6-cphenanth aortic Smooth muscle cells (passage 4) under various condi ridinium chloride) were purchased from Tocris (Ellisville, tions: control; LPS treatment alone (100 ng and 1 g); pre Mo.). PKC activator PMA (Phorbol 12-myristate 13-acetate), treatment with (R)-DOI (100 nM, a 5-HT, receptoragonist) and PKA inhibitor fragment 6-22 amide (F-22) were pur prior to LPS treatment (LPS+DOI). chased from Sigma (St. Louis, Mo.). (R)-DOI ((R)-1-(2,5- 0036 Current anti-inflammatory agents directly target dimethoxy-4-iodophenyl)-2-aminopropane) (greater than other pathways, for example, the inhibition of prostaglandin 95% Renantiomer), LA-SS-AZ (2'S,4'S)-(+)-9,10-Didehy synthesis or production of cytokines. Most published reports dro-6-methylergoline-8f3-(trans-2,4-dimethylazetidide), probing the role of serotonin 5-HT, receptors in inflammation 2C-BCB (4-Bromo-3,6-dimethoxybenzocyclobuten-1-yl) have primarily focused on the use of antagonists (blockers) as methylamine, and MDL100907 (R(+)-a-(2,3-dimeth-ox anti-inflammatory agents. We have shown that the use of yphenyl)-1-2-(4-fluorophenylethyl)-4-pipeddine-metha serotonin 5-HT, receptor agonists (activators) at extremely nol) were obtained from Purdue University. Lysergic acid low concentration, e.g., (R)-DOI, inhibits inflammatory diethylamide (LSD) was provided by the National Institute on marker gene expression with a potency at least 100 times Drug Abuse. greater than any current drug on the market. This allows for 0039 RASM cells and treatment. Rat aortic smooth the treatment of inflammatory diseases and conditions with muscle (RASM) cells were isolated from adult 180 g male small doses of drug, which would decrease the likelihood of Sprague-Dawley rats and provided by the Cell and Molecular undesirable side effects. At the doses of drug found effective Core Facility in the Department of Pharmacology at LSU in this technology, inflammation processes could be targeted HSC-NO. Isolated RASM cells were grown in M-199 media very specifically with little to no undesirable side effects due containing 10% fetal bovine serum (Gibco), 100 units/mL to off target receptor activation. penicillin, and 100 g/mL streptomycin, and incubated at 37° 0037 We have discovered that activation of 5-HT, recep C. in 5% CO. Cells for all assays were used between pas tors in primary aortic Smooth muscle cells provides a previ sages 3 and 5. Prior to treatments, cells were grown in M-199 ously unknown and extremely potent inhibition of TNF-C.- media with 10% fetal bovine serum (FBS) until 30-50% mediated inflammation. 5-HT, receptor stimulation with the confluent. For most assays, cells were treated with (R)-DOI, agonist (R)-DOI rapidly inhibits a variety of proinflammatory or other drugs as indicated, at specific concentrations for 24 markers mediated by TNF-C. acting at its receptors, including hours, followed by TNF-C. (10 ng/ml) and (R)-DOI at the ICAM-1, VCAM-1, and IL-6 gene expression, NOS activity, same pretreatment concentrations in fresh M-199 media. and nuclear translocation of NF-KB, with ICs values of only After another 24 hours, the cells were scraped, pelleted, and 10-20 pM. Significantly, proinflammatory markers also can processed for RNA. Pretreatment and treatment times varied be inhibited by (R)-DOI many hours after treatment with with assays as indicated in the results section. TNF-C. With the exception of a few natural toxins, no current 0040. RNA isolation and Quantitative Realtime-PCR. drugs or Small molecule therapeutics demonstrate a compa RNA was extracted from cells using Illustra RNAspin Mini rable potency for any physiological effect. TNF-C.-mediated kits from GE Healthcare Life Sciences (Piscataway, N.J.) inflammatory pathways have been strongly implicated in a following protocols supplied by the manufacturer. First number of diseases, including atherosclerosis, asthma, rheu strand cDNA was generated using the ImProm-II clNA syn matoid arthritis, psoriasis, type II diabetes, depression, thesis kit (Promega) following the manufacturers protocols. Schizophrenia, and Alzheimer's disease. Our results indicate Q-RT-PCR was performed using the Probel library system that activation of 5-HT, receptors represents an extraordi from Roche (Indianapolis, Ind.) in combination with the Hot narily potent, potential therapeutic avenue for the treatment Start-IT probe qpcr master mix from USB Biological (Cleve of disorders involving TNF-C.-mediated inflammation. land, Ohio) following manufactures protocols. Importantly, because (R)-DOI can significantly inhibit the 0041. The sequences of primers used are: 5-HTR (5-hy effects of TNF-C. many hours after the administration of TNF droxytryptamine 2A receptor): forward primer 5'-TGATGT C., potential therapies could be aimed not only at preventing CACTTGCCATAGCTG-3 (SEQID NO: 1), reverse primer, inflammation, but also treating inflammatory injury that has 5TCGCACAGAGCTTGCTAGG-3 (SEQ ID NO: 2): already occurred or is ongoing. ICAM-1 (intracellular adhesion molecule 1): forward primer, 5'-TTCTGCCACCATCACTGTGT-3' (SEQ ID NO: 3, EXAMPLE1 reverse primer, 5'-AGCGCAGGATGAGGTTCTT-3' (SEQ Material and Methods ID NO: 4); VCAM-1 (vascular adhesion molecule 1): forward primer, 5'-CAAATGGAGTCTGAACCCAAA-3' (SEQ ID 0038 Reagents and Chemicals. Standard cell culture NO: 5), reverse primer, 5'-GGTTCTTTCGGAGCAACG-3' media (M-199) was provided by the Molecular and Cell core (SEQ ID NO: 6); IL-6 (interleukin-6): forward primer, at Louisiana State University Health Science Center in New 5'-CCTGGAGTTTGTGAA GAACAACT-3' (SEQ ID NO: Orleans (LSUHSC-NO). Supplements to the media were 7), reverse primer, 5'-GGAAGTTGGGGTAGGAAGGA-3' from Invitrogen (Carlsbad, Calif.). TNF-C. (rat and human) (SEQ ID NO: 8); and cyclophillin B (control amplicon): was purchased from Peprotech, Inc. (Rocky Hill, N.J.). The forward primer, 5'-ACGTGGTTTTCGGCAAAGT-3' (SEQ 5-HT, receptor antagonist RS102221 (8-5-(2,4- ID NO: 9), reverse primer, 5'-CTTGGTGTTCTCCACCT Dimethoxy-5-(4-trifluoromethylphenylsulphonamido)phe TCC-3' (SEQ ID NO: 10). Primers were synthesized by IDT nyl-5-oxopentyl-1,3,8-triazaspiro4.5 decane-2,4-dione (Coralville, Iowa). Probel library probes from Roche were: US 2010/0016280 A1 Jan. 21, 2010

U3, U74, U13, U106, U79 for 5-HTR, ICAM-1, VCAM-1, gets, whereas steroidal anti-inflammatory drugs typically IL-6, and cyclophillin B, respectively. Quantitative determi have ICso values in the low nanomolar range (Huntjens et al., nation of gene expression levels using a 2-step cycling pro 2005). The ICs values in the low picomolar range for (R)- tocol was performed on a MyIQ-5 Cycler (Bio-Rad, Hercules DOI to block proinflammatory markers show that (R)-DOI Calif.). Relative gene expression levels were calculated using activation of 5-HT, receptors is ~300-fold more potent than the 2^' method. Levels of all targets from the test the more effective current anti-inflamatory agents. With the samples were normalized to rat cyclophillin B expression. exception of a few natural toxins (e.g. botulinum toxin), no 0042. Nitric Oxide Synthetase (NOS) Activity. NOS activ current drugs or Small molecule therapeutics demonstrate a ity was determined by detection of nitrite levels in the cell comparable potency for any physiological effect. culture medium following (R)-DOI/TNF-C. treatments utiliz ing the Nitrate Detection kit from Assay Designs (Ann Arbor, EXAMPLE 3 Mich.) following the manufacturers protocols. Absorbances were detected at 539 nm on a Molecular Dynamics Spectra Inhibition of Proinflammatory Markers Mediated Max M2 plate reader. Through 5-HT, Receptor Activation 0043. Nuclear Translocation of p65. RASM cells were 0048 Because (R)-DOI is an agonist at all three 5-HT grown in M-199 medium+10% FBS until 50% confluent in 8 receptor isoforms, receptor selective antagonists were well chamber slides. Cells were treated with 1 nM (R)-DOI selected to determine which receptor was mediating the (R)- for various times as indicated, and TNF-C. (10 ng/ml; tumor DOI effect. RASM cells were treated with a control solution necrosis factor alpha) added. Thirty minutes after TNF-C. (Control), TNF-C. alone (TNF-C), (R)-DOI alone (DOI), pre treatment, cells were fixed and processed with rabbit antip65 treatment with (R)-DOI (1 nM) prior to TNF-C. (DOI+TNF), primary and Alexafluor 488-conjugated goat secondary anti pretreatment with the 5-HT, receptor selective antagonist bodies as described in Zerfaoui et al. (2008) (Zerfaoui et al., M100907 (100 nM) 30 minutes prior to (R)-DOI and TNF-C. 2008). Fluorescent signal was visualized on a Leica DMRA2 (M+D+T), pretreatment with 100 nM of the 5-HT, receptor fluorescent microscope. selective antagonist SB204741 prior to (R)-DOI and TNF-C. (S+D+T), and pretreatment with 100 nM of the 5-HT, recep EXAMPLE 2 tor selective antagonist SB204741 prior to (R)-DOI and TNF-C. (S+D+T). (R)-DOI Super-Potently Inhibits TNF-C. Induced 0049. The results are shown in FIGS. 2A, 2B, and 2C for Expression of Proinflammatory Genes the induced expression of ICAM-1, VCAM-1, and IL-6, 0044 Primary rat aortic smooth muscle (RASM) cells respectively. TNF-C. treatment alone (10 ng/ml) (TNF-C.) were verified to express 5-HT, mRNA by QRT-PCR using induced expression of ICAM-1, VCAM-1, and IL-6. (R)-DOI primer sequences and probe as described in the methods at 1 nM alone had no effect on expression of any of the three section of Example 1 (data not shown). To examine the effects genes (DOI). Pre-treatment with (R)-DOI (1 nM) prior to of 5-HT, receptor activation on TNF-C. mediated proinflam TNF-C. completely blocked the increase in proinflammatory matory gene expression, RASM cells were pretreated with gene expression by TNF-C. (DOI+TNF). Pretreatment with (R)-DOI, a selective 5-HT, receptor agonist, as described the 5-HT, receptor selective antagonist M100907 (100 nM) above. Dose-response curves were determined for the effects 30 minutes prior to (R)-DOI blocked the effects of (R)-DOI on ICAM-1, VCAM-1, and IL-6 gene expression for twelve (M+D+T). Pretreatment with 100 nM of the 5-HT, receptor different concentrations of (R)-DOI ranging from 0.1 pM to selective antagonist SB204741 (S+D+T), or the 5-HT, 100 nM, with each experiment repeated in triplicate. The receptor selective antagonist RS102221 (R+D+T) did not results are shown in FIGS. 1A, 1B, and 1C.. In FIGS. 1A-1C, block the effects of (R)-DOI. In FIGS. 2A, 2B, and 2C, the the Y-axis represents a percent of TNF-C. for the dose of symbols represent the following: if p<0.01 vs control; (R)-DOI indicated on the X-axis. The ICs for proinflamma *=p<0.01 vs TNF-C. alone as analyzed with an ANOVA with tory gene expression inhibition is between 10-20 pM for all Tukey post hoc) three (ICAM-1=19.5 pM; VCAM-1=12.0 pM; IL-6=10.3 0050. As shown above, pretreatment of the cells with 100 pM). This indicates that (R)-DOI is a surprisingly super nM of either the 5-HT, receptor selective antagonist potent inhibitor of each of these genes with an ICso in the RS102221, or the 5-HT, receptor selective antagonist picomolar range. SB204741 for 30 minutes prior to the addition of 1 nM (R)- 0045. The experiments were repeated with fresh dilutions DOI (a dose that completely inhibits TNF-C. induced proin of drug and different batches of RASM cells, with the same flammatory gene expression) had no effect. Pretreatment with results: a super-potent effect. TNF-C. alone consistently 100 nM of the 5-HT, receptor selective antagonist increased baseline mRNA expression of these genes eight-ten MDL100907 for 30 minutes, however, completely blocked fold, whereas (R)-DOI had no effect by itself (Data not the inhibitory effects of 1 nM (R)-DOI on TNF-C. mediated shown). ICAM-1, VCAM-1, and IL-6 gene expression changes 0046 Because complete media with FBS contains seroto (FIGS. 2A, 2B, and 2C, respectively), indicating that the nin, which may affect the results, the effects of (R)-DOI on effects of (R)-DOI on these processes are being mediated TNF-C-induced ICAM1 expression were examined in cells exclusively through the 5-HT, receptors. serum starved for 8 hours. FIG. 1D shows an ICAM1 expres sion dose response curve in serum-free media. The ICso EXAMPLE 4 increased slightly to 360 pM over that seen in the experiments with FBS. All experiments were performed in RASM cells at Additional 5-HT Receptor Agonists Inhibit Proin passage 4. (FIG. 1D). flammatory Marker Expression 0047. Non-steroidal anti-inflammatories (NSAIDS) typi 0051) To examine if these effects were exclusive for (R)- cally have ICso values in the micromolar range for their tar DOI acting at 5-HT2 receptors, other 5-HT agonists were US 2010/0016280 A1 Jan. 21, 2010

tested for ability to inhibit proinflammatory marker expres nM) for 30 minutes prior to the addition of (R)-DOI (1 nM) Sion. These included an additional phenethylamine, blocked the effects of TNF-C-induced gene expression for 4-Bromo-3,6-dimethoxybenzocyclobuten-1-yl) methy ICAM1 (C+D+T). Pretreatment with the classical PKC iso lamine (2C-BCB), and two indolealkylamines, (2S,4'S)-(+)- form inhibitor Gö6976 (100 nM) (Go--D+T) only blocked 9,10-Didehydro-6-methylergoline-8f3-(trans-2,4-dimethy about 50% of the effects of (R)-DOI (1 nM) on TNF-C- lazetidide) (LA-SS-AZ) and lysergic acid diethylamide induced proinflammatory gene expression, indicating that (LSD). All three have high affinity for rat 5-HT, receptors more than one PKC isoform, at least one from each class, is (Ki of 2C-BCB=0.73 nM; LA-SS-AZ-8.3 nM; LSD=3.5 nM) mediating the anti-inflammatory effects of 5-HT2 receptor as well as high potency for activating PI turnover (ECso of stimulation. Activation of PKC with PMA (100 nM) in the 2C-BCB-36 nM; LA-SS-AZ-19 nM; LSD=15 nM) (Nichols absence of (R)-DOI also blocks the effects of TNF-C. (PMA+ et al., 2002; McLean et al., 2006). T). Inhibition of PKA with F-22 amide (100 nM) had no effect 0052. The phenethylamine 2C-BCB, and the indolealky (F-22+D+T). In FIG. 4, the symbol * represent *=p-0.01 vs lamines LA-SS-AZ and LSD, were tested for their ability to control; as analyzed with ANOVA with Tukey post hoc. The block TNF-C.-mediated increases in proinflammatory gene effects of these PKC inhibitors were the same for VCAM1 expression at both 1 nM and 50 nM concentrations. The and IL6 gene expression (Data not shown). Together, these results with ICAM1 are shown in FIG. 3. Results for VCAM1 data indicate that the 5-HT, receptor-mediated inhibitory and IL6 were identical (Data not shown). Whereas (R)-DOI effects on proinflammatory gene expression are mediated blocked gene expression at 1 nM, 2C-BCB and LSD only had through stimulation of PKC. weak to moderate effects at 1 nM. LA-SS-AZ was moderately 0056. Thus, a pan-PKC isoform inhibitor, chelerythrine effective at this dose and blocked about 50% of induced gene (100 nM), completely inhibited the effects of 1 nM (R)-DOI expression. At the higher concentration of 50 nM, 2C-BCB on TNF-C-induced ICAM-1 gene expression (FIG. 4), indi and LA-SS-AZ effectively blocked TNF-C. induced gene cating that PKC plays a critical role in the mechanism of expression. However, LSD only blocked about 85% of the action of 5-HT, receptor-mediated inhibition of proinflam effect. matory markers. To further delineate the role of specific iso 0053. Thus, the effects at both 1 nM and 50 nM pretreat forms of PKC in this process, RASM cells were pretreated ment on gene expression of other 5-HT2 receptor ligands with the conventional isoform inhibitor Gö6976 (100 nM) show that they also may have potent effects (FIG.3). Whereas and tested the ability of (R)-DOI to inhibit TNF-C-induced the effects are potent (predicted ICso values in the low nano proinflammatory marker expression. Only 50% of the effect molar range), they are not extraordinarily potent as is the case of (R)-DOI was blocked, indicating that 5-HT, receptor for (R)-DOI. Based upon the affinity (a measure of how stimulated anti-inflammatory effects are mediated through at tightly a particular molecule attaches to its protein target) of least two isoforms of PKC: one conventional, and one non (R)-DOI for the receptor (Affinity—Ki-0.5 nM), and potency conventional (FIG. 4). Finally, the ability of exogenous acti (a term relating to how much of a drug is necessary to produce vation of PKC was examined using Phorbol 12-myristate a half maximal response) of (R)-DOI in standard pharmaco 13-acetate (PMA) in RASM cells to block the effects of logical assays that measure activity of the 5-HT, receptor TNF-C. on proinflammatory marker expression. In the (e.g. phosphoinositide turnover; ECso -10-20 nM), which absence of (R)-DOI, PMA (100 nM) was found to completely are similar to the affinity and potency values of other known block TNF-C. mediated expression of ICAM1, VCAM1, and 5-HT, receptor agonists including 2C-BCB and LSD, the IL6 (FIG. 4, results for ICAM1. Data are not shown for extreme potency of (R)-DOI in blocking TNF-C. mediated VCAM1 and IL6). If the effects are completely mediated inflammation with an ICs of ~20 picomolar, about 1000 through PKC, inhibiting other GPCR initiated effector path times less than is necessary to produce a half maximal effect ways like PKA would have no effect. Indeed, when the effects in standard pharmacological measures of 5-HT, receptor of blocking PKA with the inhibitor F-22 amide (100 nM) was activity, is unpredicted and unexpected. tested on the ability of (R)-DOI to block TNF-C. on proin flammatory marker expression, no effect was observed (FIG. EXAMPLE 5 4). 5-HT, Receptor Mediated Inhibition of TNF-C. Induced Proinflammatory Marker Expression EXAMPLE 6 Involves Protein Kinase C (PKC) Effects of (R)-DOI at Blocking Proinflammatory 0054. It has been well established that ICAM-1 gene Gene Expression is Rapid, and Present Many Hours expression can be induced through pathways involving pro after Addition of TNF-C. tein kinase C (PKC) (Roebuck and Finnegan, 1999). Further more, PKC can be activated through 5-HT, receptor stimu 0057 The initial dose response experiments examined the lation (Rothet al., 1986). To delineate the role of PKC, RASM effects of 24 hour pretreatment with (R)-DOI. To determine cells were pretreated with several chemicals prior to treat whether this length of pretreatment was necessary to block ment with (R)-DOI (1 nM) and TNF-C., and then measured for the effects of TNF-C., a time course analysis was performed to ICAM-1, VCAM-1, and IL-6 gene expression. The results examine the ability of (R)-DOI (1 nM) to block the effects of were identical for all three genes, and the results for ICAM-1 TNF-C. (10 ng/ml) on ICAM-1 gene expression with 24 hour are shown in FIG. 4. The results for the other two genes are not and one hour pretreatments, simultaneous treatment, and shown. various time points after treatment with TNF-C. The 24 hour 0055. The ICAM-1 gene expression was first measured and one hour pretreatments, and simultaneous treatment with using a control solution (control), only TNF-C. (TNF-C.), and (R)-DOI completely blocked the effects of TNF-C. (Data not pretreatment with (R)-DOI prior to TNF-C. (D+T). Pretreat shown). These data indicated that (R)-DOI very rapidly ment with the pan-PKC isoform inhibitor chelerythrine (100 blocks proinflammatory marker expression. US 2010/0016280 A1 Jan. 21, 2010

0058 FIG. 5 shows a time course study examining when this process. The ability of (R)-DOI to block translocation 5-HT, receptor stimulation is necessary to inhibit TNF-C.- when administered simultaneously with TNF-C., and not as a induced ICAM1 expression in RASM cells. (R)-DOI (1 nM) pretreatment, is in agreement with the above time course gene can block TNF-C-induced proinflammatory gene expression expression studies, and further supports that the effects of after addition of TNF-C. (10 ng/ml) with a half maximal effect 5-HT, receptor stimulation on inhibiting inflammatory pro at 4 hours. Significantly, the addition of (R)-DOI after TNF-C. cesses are very rapid. treatment substantially blocked ICAM-1 expression with a 50% effect at about 4 hours (FIG. 5). 0059. The activation of 5-HT, receptors by (R)-DOI EXAMPLE 9 blocks not only the effects of TNF-C. when added as a pre- or co-treatment, but also when added many hours after TNF-C. 5-HT, Receptor Activation Potently Blocks Proin treatment with a half maximal effect at 4 hours post-TNF-C. flammatory Marker Expression in Different Cell Types EXAMPLE 7 0063 Experiments were conducted to analyze for the Nitric Oxide Synthase (NOS) Activity is Inhibited by effect of 5-HT, receptor activation on proinflammatory 5-HT, Receptor Activation with (R)-DOI marker expression in other cell types. Human neuroblastoma 0060 A key participant in inflammatory processes is NOS cells (SHSY5Y) and rat glioma cells (C6) can be obtained activity (Guzik et al., 2003). NOS activity can regulate NF-kB from the American Type Culture Collection (Manassas, Va.). activation (Ckless et al., 2007), and cytokine pathway-acti but were provided Purdue University. The cells were grown in vated NF-kB can transcriptionally regulate iNOS gene DMEM+10% FBS. DMEM was made and provided by the expression (Guo et al., 2007). To examine the effects of Cell and Molecular Core of the Mentoring in Cardiovascular 5-HT2 receptor activation on this component of inflamma Biology COBRE center at LSUHSC-NO. FBS was pur tory mechanisms, primary RASM cells were used to examine chased from Invitrogen (Carlsbad, Calif.). Primary rat aortic the ability of 1 nM (R)-DOI to block TNF-C. mediated endothelial cells were prepared fresh and provided by the Cell increases in nitrite levels. and Molecular Core of the Mentoring in Cardiovascular Biol 0061. As an indicator of NOS activity, nitrite levels were ogy COBRE center at LSUHSC-NO. Cells were tested at measured in the cell culture media after treatments and are passages 3-4. Cells were grown in endothelial cell growth shown in FIG. 6 as % control. TNF-C. treatment (10 ng/ml) for medium (Cell Applications, Inc.). Primary rat bronchoaveo 24 hours significantly increased nitrite levels eight-fold, indi lar macrophage cells were obtained by lung lavage from rats cating increased NOS activity. Pretreatment with 1 nM (R)- and used at day 3 or 4 post plating in DMEM+10% FBS. DOI for 24 hours blocked TNF-C. mediated increases in nitrite 0064 All treatments with 5-HT, receptor agonists accumulation (DOI+T). Pretreatment with the pan-PKC iso occurred 1 hour prior to the addition of TNF-alpha (10 ng/ml) form inhibitor chelerythrine (100 nM) completely inhibited or LPS (100 ng or 1 mg) depending on the experiment. All the effects of (R)-DOI (Che+D+T) (*=p<0.01 vs. TNF-C.: RNA isolations, cDNA synthesis, QPCR gene expression ANOVA with Tukey post hoc). These results indicate that assays examining ICAM1, VCAM1, and IL6, and data analy PKC activation is upstream of NOS activity. sis were performed as described above in Example 1, except the primers were different for the human cell experiment. The EXAMPLE 8 human gene PCR primer sequences and universal probe num bers were as follows: cyclophillin (control amplicon; Probe Nuclear Translocation of the Activated NF-kB Sub U64): forward primer, 5'-CCCAGTTCTTCATCACGACA unit p65 is Blocked by 5-HT, Receptor Activation 3' (SEQ ID NO: 11), reverse primer, 5'-GTCTTGGT with (R)-DOI GCTCTCCACCTT-3' (SEQ ID NO: 12); and human 0062 Both ICAM-1 and VCAM-1 genetranscription dur ICAM-1 (Probe U71, intracellular adhesion molecule 1): for ing inflammatory processes is regulated by the transcription ward primer, 5'-CCTTCCTCACCGTGTACTGG-3 (SEQID factor NF-kB (Collins et al., 1995). During this process, NO: 13), reverse primer, 5'-AGCGTAGGGTAAGGTTCT NF-kB must be activated, and then translocated from the TGC-3' (SEQ ID NO: 14). cytoplasm to the nucleus, where it promotes gene transcrip 0065. As shown in FIG. 8, 5-HT, receptor activation with tion. To investigate whether 5-HT, receptor stimulated inhi (R)-DOI blocked ICAM-1 gene expression in human neuro bition of NF-kB activation and translocation might be a pos blastoma cells similar to results seen in RASM cells. As sible mechanism for blockade of proinflammatory gene shown in FIG. 9, 5-HT, receptor activation with (R)-DOI expression changes, a series of immunohistochemical experi and LA-SS-AZ in rat glioma cells blocked ICAM-1 gene ments were conducted examiningp65 translocation in RASM expression similar to results seen in RASM cells. cells. TNF-C. treatment for 30 minutes caused the expected 0066. As shown in FIG. 10, (R)-DOI potently blocked dramatic shift in localization of the p65 subunit of NF-kB VCAM-1 gene expression in response to TNF-C. in primary from the cytoplasm to the nucleus (FIG. 7A). Pretreatment rat bronchoaveolar macrophage cells similar to results seen in with 1 nM (R)-DOI for 24 hours (not shown), one hour, and RASM cells. The IC50 was about 20 pM. together (not shown) with TNF-C. (10 ng/ml) completely 0067. As shown in FIG. 11A, 5-HT, receptor stimulation blocked p65 nuclear translocation (FIG. 7B). FIG.7B shows with (R)-DOI (10 pM) administered 60 minutes prior to the the percentage of cells within a given field that predominantly addition of TNF-alpha (10 ng/ml) potently inhibited TNF-C.- had p65 located in the nucleus. Furthermore, pretreatment of induced IL-6 expression in rat primary aortic endothelial the 1 hour time point with the PKC inhibitor chelerythrine cells. However, the effect at inhibiting the TNF-alpha induced blocked the (R)-DOI induced inhibition of p85 translocation increase in VCAM-1 gene expression, was less potent (FIG. (not shown), indicating that PKC activation is upstream of 11B). US 2010/0016280 A1 Jan. 21, 2010

0068 Thus (R)-DOI was shown to inhibit proinflamma ingredient(s) from digestion by use of enteric coatings, cap tory gene expression in other types of cells, including human Sules or other methods known in the art. cells. 0074 Pharmaceutically acceptable carrier preparations include sterile, aqueous or non-aqueous solutions, Suspen EXAMPLE 10 sions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as 5-HT Receptor Activation on ICAM-1 Gene olive oil, and injectable organic esters such as ethyl oleate. Expression after Stimulation with Lipopolysaccha Aqueous carriers include water, alcoholic/aqueous Solutions, ride emulsions or Suspensions, including saline and buffered 0069. The effect of (R)-DOI (60 minute pretreatment) on media. Parenteral vehicles include sodium chloride solution, the expression of ICAM-1 in primary rat aortic smooth Ringer's dextrose, dextrose and sodium chloride, lactated muscle cells after stimulation with LPS was examined. As Ringer's, or fixed oils. The active therapeutic ingredient may shown in FIG. 12, at a low dose of LPS (100 ng) producing be mixed with excipients that are pharmaceutically accept only 2-fold induction of ICAM-1 gene expression, (R)-DOT able and are compatible with the active ingredient. Suitable was able to block the response. At a higher dose of LPS (1 lug), excipients include water, Saline, dextrose, glycerol and etha there was no effect of (R)-DOI on the increase in ICAM-1. nol, or combinations thereof. Intravenous vehicles include Together, these data indicate that whereas there may be a fluid and nutrient replenishers, electrolyte replenishers, such moderate effect on low levels of LPS stimulation, by and large as those based on Ringer's dextrose, and the like. Preserva there is little to no effect of 5-HT, receptor stimulation on tives and other additives may also be present Such as, for inflammation induced by LPS, indicating that the anti-in example, antimicrobials, anti-oxidants, chelating agents, flammatory effects of 5-HT, receptor activity are specific for inert gases, and the like. the TNF-C. receptor activated inflammatory mechanisms. 0075. The form may vary depending upon the route of 0070 These results indicate that activation of 5-HT administration. For example, compositions for injection may receptors by (R)-DOI, as well as additional 5-HT, receptor be provided in the form of an ampoule, each containing a unit agonists, represents an extremely potent, therapeutic avenue dose amount, or in the form of a container containing multiple to explore for the treatment of diseases and disorders involv doses. ing TNF-C.-mediated inflammation. TNF-C. and TNF-C. 0076. A compound in accordance with the present inven receptor mediated pathways are believed to be a major com tion may be formulated into therapeutic compositions as ponent of many inflammatory conditions that include athero pharmaceutically acceptable salts, for example a hydrochlo Sclerosis, rheumatoid arthritis, psoriasis, type II diabetes, ride salt (e.g., the (R)-DOI used in the above examples). asthma, Crohn's Disease, inflammatory bowel syndrome, These salts include acid addition salts formed with inorganic depression, Schizophrenia, and Alzheimer's disease. Notably, acids, for example hydrochloric or phosphoric acid, or 5-HT2 receptor expression has been detected in most, if not organic acids such as acetic, oxalic, or tartaric acid, and the all, of the tissues mediating the inflammatory conditions men like. Salts also include those formed from inorganic bases tioned above. Given the unprecedented and extremely high Such as, for example, Sodium, potassium, ammonium, cal potency of (R)-DOI to suppress multiple proinflammatory cium or ferric hydroxides, and organic bases such as isopro markers rapidly, ranging from NOS activity, through NF-KB pylamine, trimethylamine, histidine, procaine and the like. translocation, to gene expression of ICAM-1, VCAM-1 and 0077. A method for controlling the duration of action IL-6, the predicted therapeutic dose would be at least two comprises incorporating the active compound into particles orders of magnitude below that necessary to produce unde of a polymeric Substance such as a polyester, peptide, hydro sirable hallucinogenic side effects. Importantly, because (R)- gel, polylactide? glycolide copolymer, or ethylenevinylac DOI can significantly inhibit the effects of TNF-C. many etate copolymers. Alternatively, an active compound may be hours after the administration of TNF-C., potential therapies encapsulated in microcapsules prepared, for example, by could be aimed at not only preventing inflammation, but also coacervation techniques or by interfacial polymerization, for treating inflammation or injury that has already occurred or is example, by the use of hydroxymethylcellulose or gelatin ongoing. microcapsules or poly(methylmethacrylate) microcapsules, (0071 Miscellaneous respectively, or in a colloid drug delivery system. Colloidal 0072 Following successful completion of animal trials dispersion systems include macromolecule complexes, nano using common mammals, (R)-DOI and other 5-HT ago capsules, microspheres, beads, and lipid-based systems nists will be tested in human patients with symptoms or including oil-in-water emulsions, micelles, mixed micelles, diseases of enhanced immunological response in clinical tri and liposomes. als conducted in compliance with applicable laws and regu 0078. As used herein, the term “5-HT, agonists’ is any lations. compound that increases the activity of a 5-hydrox 0073 Specific 5-HT agonists used in the present inven ytryptamine 2A receptor. Examples of such agonists include tion may be administered to a patient by any suitable means, DOI (+)-1-(2,5-dimethoxyphenyl)-2-aminopropane hydro including oral, intravenous, parenteral, Subcutaneous, intra chloride; (R)-DOI ((R)-1-(2,5-dimethoxy-4-iodophenyl)-2- pulmonary, and intranasal administration. Parenteral infu aminopropane) (greater than 95% Renantiomer); LA-SS-AZ sions include intramuscular, intravenous, intraarterial, or (2'S,4'S)-(+)-9,10-Didehydro-6-methylergoline-8B-(trans-2, intraperitoneal administration. The compounds may also be 4-dimethylazetidide); 2C-BCB (4-Bromo-3,6-dimethoxy administered transdermally, for example in the form of a benzocyclobuten-1-yl) methylamine; and lysergic acid slow-release Subcutaneous implant. They may also be admin diethylamide (LSD). istered by inhalation. Although direct oral administration (0079. As used herein, an “therapeutically effective may cause some loss of anti-inflammatory activity, the ago amount of a compound is an amount, that when administered nists could be packaged in Such a way to protect the active to a patient, human or animal, (whether as a single dose or as US 2010/0016280 A1 Jan. 21, 2010

a time course of treatment) inhibits or reduces the release of alpha in the regulation of inflammation in macrophases. proinflammatory compounds to a clinically significant Pharmacol. Res. Epub 2009 May 18. degree; or alternatively, to a statistically significant degree as I0087. Dunn A. J. Swiergiel A H and de Beaurepaire R compared to control. “Statistical significance' means signifi (2005) Cytokines as mediators of depression: what can we cance at the P-0.05 level, or such other measure of statistical learn from animal studies? Neurosci Biobehav Rev 29:891 significance as would be used by those of skill in the art of 909. biomedical statistics in the context of a particular type of I0088. Guo Z, Shao L, Du Q, Park K S and Geller DA treatment or prophylaxis. The term “therapeutically effective (2007) Identification of a classic cytokine-induced amount therefore includes, for example, an amount Suffi enhancer upstream in the human iNOS promoter. FasebJ cient to decrease the release of proinflammatory compounds 21:535-542. to a clinically relevant level that is statistically significant to I0089 Guzik TJ, Korbut R and Adamek-Guzik T (2003) decrease inflammation. The dosage ranges for the adminis Nitric oxide and Superoxide in inflammation and immune tration of a compound are those that produce the desired regulation.JPhysiol Pharmacol 54:469-487. effect, but an effective dose that would result in the tissue cells seeing no greater than abody fluid concentration of no greater 0090 Hansson G. K. Robertson A K and Soderberg-Nau than 5 nM, more preferable no greater than 1 nM, and most cler C (2006) Inflammation and atherosclerosis. Annu Rev preferably no greater than 0.5 nM. For example, a single dose Pathol 1:297-329. that would result in a 1 nM final body fluid concentration in a (0091 Hughes J. M. Arthur CA, Baracho S, Carlin S M, 60 kg human would be about 20 ug, or for (R)-DOI a single Hawker KM, Johnson PR and Armour CL (2000) Human dose that would result in a final body fluid concentration near eosinophil-airway Smooth muscle cell interactions. Media the IC50 of about 20 pM would be about 0.4 ug. This amount tors Inflamm 9:93-99. could be adjusted based on the size of the human or animal. 0092 Huntjens D R, Danhof M and Della Pasqua O E Generally, the dosage will vary with the age, weight, condi (2005) Pharmacokinetic-pharmacodynamic correlations tion, and the degree of the inflammation. A person of ordinary and biomarkers in the development of COX-2 inhibitors. skill in the art, given the teachings of the present specification, Rheumatology (Oxford) 44:846-859. may readily determine Suitable dosage ranges for the various (0093. Ito T, Ikeda U, Shimpo M, Yamamoto K and Shi sizes of different mammals. The dosage can be adjusted by mada K (2000) Serotonin increases interleukin-6 synthesis the individual physician or veterinarian in the event of any in human vascular Smooth muscle cells. Circulation 102: contraindications. In any event, the effectiveness of treatment 2522-2527. can be determined by monitoring the extent of release of (0094) KimYK, Jung HG, Myint AM, Kim Hand Park S known inflammatory parameters by methods well known to H (2007) Imbalance between pro-inflammatory and anti those in the field. inflammatory cytokines in bipolar disorder. JAffect Disord 104:91-95. REFERENCES 0095 Kubera M, Maes M. Kenis G, KimYK and Lason W 0080 Akiyoshi T. Zhang Q, Inoue F, Aramaki O, Hatano (2005) Effects of serotonin and serotonergic agonists and M. Shimazu M. Kitajima M, Shirasugi N and Niimi M antagonists on the production of tumor necrosis factor (2006) Induction of indefinite survival of fully mismatched alpha and interleukin-6. Psychiatry Res 134:251-258. cardiac allografts and generation of regulatory cells by (0096. Kurrasch-Orbaugh DM, Watts VJ, Barker E L and Sarpogrelate hydrochloride. Transplantation 82:1051 Nichols D E (2003) Serotonin 5-hydroxytryptamine 2A 1059. receptor-coupled phospholipase C and phospholipase A2 0081 Arzt E. Costas M. Finkielman S and Nahimod VE signaling pathways have different receptor reserves. J (1991) Serotonin inhibition of tumor necrosis factor-alpha Pharmacol Exp Ther 304:229-237. synthesis by human monocytes. Life Sci 48:2557-2562. (0097. Little KY, Elmer L. W. Zhong H. Scheys J O and I0082 Blankenberg S. Barbaux S and Tiret L (2003) Adhe Zhang L (2002) Cocaine induction of dopamine trans sion molecules and atherosclerosis. Atherosclerosis 170: porter trafficking to the plasma membrane. Mol Pharmacol 191-2O3. 61:436-445. 0083 Ckless K. van der Vliet A and Janssen-HeiningerY 0.098 Marconi A, Darquenne S. Boulmerka A. Mosnier M (2007). Oxidative-nitrosative stress and post-translational and D'Alessio P (2003) Naftidrofuryl-driven regulation of protein modifications: implications to lung structure-func endothelial ICAM-1 involves nitric oxide. Free Radic Biol tion relations. Arginase modulates NF-kappaB activity via Med 34:616-625. a nitric oxide-dependent mechanism. Am J Respir Cell Mol 0099 McLean T H. Parrish J C, Braden M R, Marona Biol. 36:645-653. Lewicka D. Gallardo-Godoy A and Nichols DE (2006) I0084 Cloez-Tayarani I, Petit-Bertron A F, Venters HD 1-Aminomethylbenzocycloalkanes: conformationally and Cavaillon J M (2003) Differential effect of serotonin restricted hallucinogenic phenethylamine analogues as on cytokine production in lipopolysaccharide-stimulated functionally selective 5-HT2A receptor agonists. J Med human peripheral blood mononuclear cells: involvement Chen 49:5794-5803. of 5-hydroxytryptamine2A receptors. Int Immunol 15:233 0100 Miller K J and Gonzalez H A (1998) Serotonin 240. 5-HT2A receptor activation inhibits cytokine-stimulated I0085 Collins T. Read MA, Neish A S, Whitley MZ, inducible nitric oxide synthase in C6 glioma cells. Ann NY Thanos D and Maniatis T (1995) Transcriptional regula AcadSci 861: 169-173. tion of endothelial cell adhesion molecules: NF-kappa B 0101 Miller KJ, Mariano C L and Cruz W R (1997) and cytokine-inducible enhancers. FasebJ 9:899-909. Serotonin 5HT2A receptor activation inhibits inducible I0086) Crisafulli C, Galuppo M, and CuZZocreaS (2009) nitric oxide synthase activity in C6 glioma cells. Life Sci Effects of genetic and pharmacological inhibition of TNF 61:1819-1827. US 2010/0016280 A1 Jan. 21, 2010 10

0102 Nagatomo T. Rashid M. Abul Muntasir H and (O112 Saucier C. Morris SJ and Albert PR (1998) Endog Komiyama T (2004) Functions of 5-HT2A receptor and its enous serotonin-2A and -2C receptors in Balb/c-3T3 cells antagonists in the cardiovascular system. Pharmacol Ther revealed in serotonin-free medium: desensitization and 104:59-81. down-regulation by serotonin. Biochem Pharmacol (0103) Nichols D E (2004) Hallucinogens. Pharmacol 56:1347-1357. Ther. 101:131-181. 0113 Shulgin A and Shulgin A (1991) PiHKAL. Trans 0104. Nichols DE, Frescas S. Marona-Lewicka D and form Press, Berkeley. Kurrasch-Orbaugh DM (2002) Lysergamides of isomeric 0114 Steful J, Jerne B, Cicin-Sain L. Rinner I and 2,4-dimethylaZetidines map the binding orientation of the Schauenstein K (2000) mRNA expression of serotonin diethylamide moiety in the potent hallucinogenic agent receptors in cells of the immune tissues of the rat. Brain N,N-diethyllysergamide (LSD). J Med Chem 45:4344 Behav Immun 14:219-224. 4349. 0115 Tracey D. Klareskog L, Sasso E. H. Salfeld JG and 0105 Nichols D E and Nichols C D (2008) Serotonin Tak PP (2007) Tumor necrosis factor antagonist mecha Receptors. Chem. Rev 108:1614-1641. nisms of action: A comprehensive review. Pharmacol Ther. 0106 Popa C, Netea MG, van Riel P L van der Meer J W 0116 Tweedie D, Sambamurti K and Greig N H (2007) and Stalenhoef A F (2007) The role of TNF-alpha in TNF-alpha inhibition as a treatment strategy for neurode chronic inflammatory conditions, intermediary metabo generative disorders: new drug candidates and targets. lism, and cardiovascular risk. J Lipid Res 48:751-762. Curr Alzheimer Res 4:378-385. 0107 Reimold AM (2002) TNFalpha as therapeutic tar 0117 Urban J. D. Clarke W. P. von Zastrow M, Nichols D get: new drugs, more applications. Curr Drug Targets E, Kobilka B, Weinstein H, Javitch JA, Roth BL, Chris Inflamm Allergy 1:377–392. topoulos A, Sexton P. M. Miller K J. Spedding M and 0108 Roebuck KA and Finnegan A (1999) Regulation of Mailman R B (2007) Functional selectivity and classical intercellular adhesion molecule-1 (CD54) gene expres concepts of quantitative pharmacology. J Pharmacol Exp Sion. J. Leukoc Biod 66:876-888. Their 320:1-13. 0109 Roth B L and Chuang DM (1987) Multiple mecha 0118 Williams RO, Paleolog E and Feldmann M. (2007) nisms of serotonergic signal transduction. Life Sci Cytokine inhibitors in rheumatoid arthritis and other 41:1051-1064. autoimmune diseases. Curr Opin Pharmacol 7:412-417. 0110 Roth B L, Nakaki T, Chuang D M and Costa E 0119 Willins D L, Deutch A Y and Roth B L (1997) (1986) 5-Hydroxytryptamine2 receptors coupled to phos Serotonin 5-HT2A receptors are expressed on pyramidal pholipase C in rat aorta: modulation of phosphoinositide cells and interneurons in the rat cortex. Synapse 27:79. turnover by phorbolester.JPharmacol Exp. Ther 238:480 0120 Zerfacui M, Suzuki Y, Naura AS, Hans CP, Nichols 485. C and Boulares AH (2008) Nuclear translocation of p85 0111 Saetre P. Emilsson L, Axelsson E. Kreuger J. Lind NF-kappaB is sufficient for VCAM-1, but not ICAM-1, holm E and Jazin E (2007) Inflammation-related genes expression in TNF-stimulated smooth muscle cells: Differ up-regulated in schizophrenia brains. BMC Psychiatry ential requirement for PARP-1 expression and interaction. 7:46. Cell Signal 20:186-194.

SEQUENCE LISTING

<16 Oc NUMBER OF SEO ID NOS : 14

SEO ID NO 1 LENGTH: 21 TYPE: DNA ORGANISM: Artificial sequence FEATURE; OTHER INFORMATION: Synthetic <4 OO SEQUENCE: 1

tgatgtcact togccatagot g 21

SEO ID NO 2 LENGTH 19 TYPE: DNA ORGANISM: Artificial sequence FEATURE; OTHER INFORMATION: Synthetic

<4 OO SEQUENCE: 2

togcacagag cttgctagg 19

<210 SEQ ID NO 3 US 2010/0016280 A1 Jan. 21, 2010 11

- Continued

<211 LENGTH: 2O &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic <4 OO SEQUENCE: 3 ttctgccacc at cactgtgt

<210 SEQ ID NO 4 <211 LENGTH: 19 &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic <4 OO SEQUENCE: 4 agcgcaggat gaggttctt 19

<210 SEQ ID NO 5 <211 LENGTH: 21 &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic <4 OO SEQUENCE: 5 caaatggagt ctgaac ccaa a 21

<210 SEQ ID NO 6 <211 LENGTH: 18 &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic

<4 OO SEQUENCE: 6 ggttctitt.cg gagcaacg 18

<210 SEQ ID NO 7 <211 LENGTH: 23 &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic

<4 OO SEQUENCE: 7

Cctggagttt gtgaagaa.ca act 23

<210 SEQ ID NO 8 <211 LENGTH: 2O &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic

<4 OO SEQUENCE: 8 ggaagttggg gtaggaagga

<210 SEQ ID NO 9 <211 LENGTH: 19 &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: US 2010/0016280 A1 Jan. 21, 2010 12

- Continued OTHER INFORMATION: Synthetic

SEQUENCE: 9 acgtggittitt C9gcaaagt 19

SEQ ID NO 10 LENGTH: 2O TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Synthetic

SEQUENCE: 1.O cittggtgttc. tccacct tcc

SEQ ID NO 11 LENGTH: 2O TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Synthetic

SEQUENCE: 11 cc.cagttctt cat cacgaca

SEQ ID NO 12 LENGTH: 2O TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Synthetic

SEQUENCE: 12 gtc.ttggtgc tict coaccitt

SEQ ID NO 13 LENGTH: 2O TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Synthetic

SEQUENCE: 13 cct tcc toac cqtgtactgg

SEQ ID NO 14 LENGTH: 21 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Synthetic

SEQUENCE: 14 agcgtagggit aaggttcttg C 21 US 2010/0016280 A1 Jan. 21, 2010

We claim: dimethylazetidide)(LA-SS-AZ), lysergic acid diethylamide 1. A method for the treatment of an inflammatory disorder (LSD), and known anti-inflammatory drugs. in a mammal, said method comprising administering to a 9. A method to reduce the expression of the intracellular mammal in need of Such treatment an therapeutically effec adhesion molecule 1 (ICAM-1) gene associated with stimu tive amount of a 5-HT2 receptor agonist in an amount no lation of the tumor necrosis factor-alpha receptor in a mam mal, said method comprising administering to a mammal in greater than that required to result in a body fluid concentra need of such reduction antherapeutically effective amount of tion no greater than 5 nM in a pharmaceutically acceptable a 5-HT, receptor agonist in an amount no greater than that carrier. required to result in a body fluid concentration no greater than 2. The method of claim 1, wherein the 5-HT, receptor 5 nM in a pharmaceutically acceptable carrier. agonist is selected from the group consisting of DOI, (R)-1- 10. A pharmaceutical composition which comprises a dose (2,5-dimethoxy-4-iodophenyl)-2-aminopropane ((R)-DOI), of a 5-HT2 receptoragonist in an amount no greater than that (4-Bromo-3.6 dimethoxybenzocyclobuten-1-yl) methy required to result in a body fluid concentration no greater than lamine (2C-BCB), and (2'S,4'S)-(+)-9,10-Didehydro-6-me 5 nM in a pharmaceutically acceptable carrier. thylergoline-8f3-(trans-2,4-dimethylazetidide) (LA-SS-AZ) 11. The composition of claim 10, wherein the 5-HT, 3. The method of claim 1, wherein the 5-HT, receptor receptoragonist is selected from the group consisting of DOI. agonist is (R)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopro (R)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane ((R)- pane ((R)-DOI). DOI), (4-Bromo-3.6 dimethoxybenzocyclobuten-1-yl) 4. The method of claim3, wherein the ((R)-DOI) is admin methylamine (2C-BCB), and (2'S,4'S)-(+)-9,10-Didehydro istered in an amount in an amount no greater than that 6-methylergoline-8f3-(trans-2,4-dimethylazetidide) (LA-SS required to result in a body fluid concentration no greater than AZ) 12. The composition of claim 10, wherein the 5-HT, 1 nM. receptor agonist is (R)-1-(2,5-dimethoxy-4-iodophenyl)-2- 5. The method of claim3, wherein the ((R)-DOI) is admin aminopropane ((R)-DOI). istered in an amount in an amount no greater than that 13. The composition of claim 12, wherein the ((R)-DOI) is required to result in a body fluid concentration no greater than in an amount no greater than that required to result in a body 50 pM. fluid concentration no greater than 1 nM. 6. The method of claim3, wherein the (((R)-DOI) is admin 14. The composition of claim 12, wherein the ((R)-DOI) is istered in an amount in an amount no greater than that in an amount no greater than that required to result in a body required to result in a body fluid concentration no greater than fluid concentration no greater than 50 pM. 20 p.M. 15. The composition of claim 12, wherein the ((R)-DOI) is 7. The method of claim 1, wherein the inflammatory dis in an amount no greater than that required to result in a body order is associated with a disease selected from the group fluid concentration no greater than 20 pM. consisting of atherosclerosis, asthma, rheumatoid arthritis, 16. The composition of claim 9, additionally comprising psoriasis, type II diabetes, irritable bowel syndrome, Crohn's administering one or more compounds selected from the disease, septicemia, depression, Schizophrenia, and Alzhe group consisting of DOI, (R) — 1-(2,5-dimethoxy-4-io imer's disease. dophenyl)-2-aminopropane ((R)-DOI), (4-Bromo-3,6 8. The method of claim 1, additionally comprising admin dimethoxybenzocyclobuten-1-yl) methylamine (2C-BCB), istering one or more compounds selected from the group (2'S,4'S)-(+)-9,10-Didehydro-6-methylergoline-8B-(trans-2, consisting of DOI, (R)-1-(2,5-dimethoxy-4-iodophenyl)-2- 4-dimethylazetidide) (LA-SS-AZ), lysergic acid diethylamide aminopropane ((R)-DOI), (4-Bromo-3,6-dimethoxybenzocy (LSD), and known anti-inflammatory drugs. clobuten-1-yl) methylamine (2C-BCB), (2S,4'S)-(+)-9,10 Didehydro-6-methylergoline-8f3-(trans-2,4- c c c c c