Keywords: psychedelic, asthma, 5-HT_2A receptor, inflammation, anti-inflammatory, chemical structure

(R)-DOI Prevents the Developmentof SymptomsAssociated with Asthma in Rats

In order to test several ofour compounds (seeFigure1 for complete library) without the confounding rapid metabolicissues present in mice,¹⁴ we used rats.Unfortunately, little work has been done by others with adult ratsas a model for allergic asthma. Therefore, we needed to develop andvalidate an appropriate rat model. On the basis of our prior workwith a BALB/c mouse model,¹¹ and certainprotocols in the literature for rats,^25,26 we developeda treatment paradigm as described in the “Methods” section (Figure2) that produces robust symptoms of allergicasthma in the Brown Norway rat.^28,29 The validation of ourmodel involved demonstrating similar physiological responses to OVAtreatment with respect to the mouse model we previously used, wheretreatment with low doses of (R)-DOI prevents thedevelopment of asthma-related symptoms. As anticipated, rats challengedwith OVA develop pronounced AHR in response to methacholine (MeCh),similar to what was observed in our mouse model (Figure3A). Pretreatment with verylow doses of nose-only (R)-DOI (0.01 mg/kg) alsocompletely prevents increases in enhanced pause (PenH) (Figure3B).

Lung Remains Responsiveto Other Asthma Treatment Modalitiesafter Exposure to (R)-DOI

To determine whetherthe lung remains responsive to a conventional treatment for asthmafollowing (R)-DOI administration, we assessed theability of a β2-receptor agonist, isoproterenol, to lower PenHfollowing administration of an approximate EC₅₀ dose of(R)-DOI and 32 mg/mL methacholine. As expected, isoproterenollowers PenH values to nearly those of naïve levels that wereequivalent between experimental treatment groups (Figure3C). These results suggest that5-HT_2A-receptor-mediated therapy for asthma in the clinicmay be compatible with traditional types of rescue therapy like bronchodilators.

(R)-DOI Treatment Prevents OVA Exposure-MediatedPulmonary Inflammation and Mucus Overproduction

Histologicalanalysis of the lungs reveals significant OVA-induced pulmonary inflammation(Figure4B,E,M) andmucus overproduction (Figure4H,K,N) in the OVA-alone treated animals. Pretreatment with(R)-DOI (0.5 mg/kg; nose-only) prevents OVA-inducedpulmonary inflammation (Figure4C,F,M) and mucus over production (Figure4I,L,N).

(R)-DOI Prevents OVA-InducedProinflammatoryRelated Gene Expression in the Lung

We validated our ratmodel at the gene expression level by assessing the pulmonary expressionof certain genes associated with the pathology of asthma by qRT-PCRanalysis. These included examining mRNA levels ofIL-1β,IL-4,IL-5,IL-6,IL-13,Muc5ac,TNFα, andGm-csf from whole lung homogenates. Similarto our earlier results with the acute OVA mouse model, (R)-DOI pretreatment prevented the expression of OVA-induced increasesin the mRNAs forIL-5,IL-6,IL-13,Gm-csf, andMuc5ac in the lung (Figure5A–F). Also similar to our previous results from the acuteOVA mouse model, (R)-DOI does not affect the expressionof mRNAs for other cytokines such asIL-4 (Figure5G), indicating thatas in the mouse (R)-DOI is not acting as a generalimmunosuppressant and is only affecting a subset of proinflammatory/asthma-relatedgenes.

Determination of Potency and Efficacy by Different Routes ofAdministration for (R)-DOI

To determinethe potency of (R)-DOI to prevent the symptoms ofallergic asthma, we performed dose–response experiments. Wealso compared different routes of administration to determine EC₅₀ values for both nebulized and intraperitoneal drug administration.The use of whole-body plethysmography (WBP) as a proxy to generatedose–response curves and test the effects of biological alterationsto the bronchoconstriction response has been described elsewhere.^30,31 Dose–response curves were generated for the effects of differentconcentrations of (R)-DOI (0.0001–1.0 mg/kg)plotted against the highest dose of methacholine administered (32mg/mL). We determined the EC₅₀ dose of (R)-DOI to be 0.008 mg/kg when delivered via nose-only (Figure6A) and 0.003 mg/kg when deliveredvia intraperitoneal (i.p.) injection (Figure6B). These doses are roughly 30-fold lessthan the minimal threshold dose for behavioral effects in rats whenadministered by i.p. injection (0.1 mg/kg)³²⁻³⁵ and are consistent with our earlierstudies in mice demonstrating therapeutic efficacy at levels far belowthose necessary to produce behaviors. The nearly equivalent potencyof noninhaled drug may at first seem surprising, but this may be dueto the lung serving as a depot for drugs of this class. In rats, [¹³¹I]-labeled DOI shows the highest tissue concentrations inthe lung after systemic injection.³⁶ Rapidlocalization to the lung is also observed in full body scans in humansfor the closely related compound 2,5-dimethoxy-4-bromoamphetamine(DOB) after systemic administration of radiolabeled DOB.³⁷ Furthermore, in rats given oral and subcutaneousdoses of DOB, the highest concentration of DOB is found in the lungs.³⁸ Therefore, we predict that i.p.-injected (R)-DOI is rapidly accumulating in the lung to provide effectsequivalent to those of the nebulized drug. Together, our data indicatethat the potential anti-inflammatory therapeutic levels for diseaseslike asthma in the clinic with drugs like (R)-DOIwould be sub-behavioral and potentially orally available.

Structure–ActivityRelationship Analysis

Wetested the efficacy of a panel of several known ligands with differentstructural features in our rat allergic asthma OVA model to determinehow structural modifications alter responses. All compounds were initiallytested at the same screening dose of 0.5 mg/kg delivered via nose-onlyroute and dissolved in sterile saline. Our rationale for this dosechoice was that it should provide for complete normalization of PenHand inflammation based on our dose–response study with (R)-DOI and be high enough so as to be a fully efficaciousdose for drugs that may have significantly lower potency than (R)-DOI. Therefore, in this study we are comparing only efficacy(E_MAX) and not potency (EC₅₀) of the compounds tested to reduce the magnitude of OVA-inducedAHR in response to MeCh challenge. Our results were grouped into threecategories based on their ability to rescue/normalize MeCh-inducedPenH: fully efficacious, partially efficacious, and no significanteffect. For a few drugs, like psilocin, we performed additional testingat different doses to address potency issues. The rationale for examiningonly efficacy at a high dose of test compound as the primary comparatorwas that given our throughput of one test compound/dose per week,it simply was not feasible to generate full dose–response curvesfor 20+ different drugs, which would have required nearly three yearsof continual weekly testing.Figure7 shows the PenH curves for phenylalkylamines (Figure7A–N), ergolines(Figure7O–Q),and tryptamines (Figure7R–V) screened.

Phenylalkylamines

Our primary focus in this study wason the phenylalkylamine chemotype. Phenylalkylamines are not onlymore selective for 5-HT₂ receptors but also better suitedfor drug development because they are generally more chemically stablethan ergolines and tryptamines. Knowledge of the structure–activityrelationships (SAR) of this class of drug with respect to anti-inflammatoryactivity will enable potential development of next generation 5-HT_2A receptor agonist therapeutics with less behavioral liability.Starting with (R)-DOI as the prototype anti-inflammatoryagonist, neither removal of the α-carbon from the side chainor replacing the 4-iodo with a bromine had a measurable effect onactivity, as 2C-B had full efficacy (Figure7A).

Modification of the side chainby rigidifying it (TCB-2) resulted in reduced efficacy (Figure7B). It should be noted thateach of these drugs that demonstrate reduced efficacy in our systemare known to be highly potent and efficacious drugs for canonicalsignaling and inducing mouse head twitch behaviors, with nearly thesame pharmacological properties as (R)-DOI. Therefore,we propose that a 2-carbon side chain ending in a primary amine isnecessary and sufficient, as well as able to tolerate the additionof an alpha-methyl, but any other more extreme types of modificationare not well tolerated for full anti-inflammatory efficacy.

The 2- and 5-methoxy substituents for phenethylamines are hypothesizedto serve as hydrogen bond acceptors to orient the compound in thecorrect orientation for proper receptor interaction.³⁹ Therefore, both moieties must be taken into account whenconsidering any modification to the basic DOx or2C structure. 2C-B-Fly, which tethers the 2- and 5-oxygens into adihydrofuran structure, has affinity for the 5-HT_2A receptorand agonist activity at canonical pathways nearly equal to (R)-DOI. However, 2C-B-Fly has significantly reduced efficacycompared to (R)-DOI for reducing AHR (Figure7C). Loss of the 5-methoxy (2,4-DMA)is also detrimental to efficacy (Figure7D), as is substitution of the 5-methoxy withan isopropoxy moiety (5-iPro-2C-E) (Figure7E). Together, these data indicate that conformationalrestraint of the methoxys at the 2- and 5- position is detrimental,and at least a methoxy at the 5-position is necessary for full anti-inflammatoryefficacy.

The nature of the 4-position substituent of phenethylaminepsychedelicshas been previously linked to 5-HT₂ receptor selectivityas well as agonist properties at 5-HT₂ receptors.⁴⁰ Analysis of the 4-position demonstrated thatthe identity of the moiety at this position was rather flexible. Fullyefficacious substitutions at the 4-position included the halogensiodine and bromine (R)-DOI (Figure3), 2C-B (Figure7A), methoxy (TMA-2) (Figure7G), short-chain hydrocarbons (R)-DOM (Figure7H),(R)-DOET) (Figure7I), and a branched hydrocarbon (DOiBu) (Figure7J). An exception was 2C-T-33(Figure7K), whichhas a large hydrophobic 2-methoxythiophenyl at the 4′-positionthat demonstrated reduced efficacy. That may be due to the overallbulk at the 4-position or to the thiol. Further testing of additional2C-T compounds is necessary to answer this issue.

Although allof these 4-substituted compounds, with the exceptionof 2C-T-33, demonstrated full efficacy at 0.5 mg/kg inhaled, potenciesmay differ. To examine this possibility, we tested (R)-2,5-DMA (Figure7L) at 0.03 mg/kg, a fully efficacious dose for (R)-DOI, and found that (R)-2,5-DMA was only partiallyefficacious at this lower dose (Figure7M). Conversely, for drugs that only demonstrated partialefficacy at 0.5/mg/kg inhaled, their efficacy may be due to not providingsufficient levels to achieve full efficacy. To test for this possibility,we doubled the dose of 5-iPrO-2C-E to 1.0 mg/kg (Figure7F). No increase in efficacywas found between the 0.5 and the 1.0 mg/kg doses. Together, theseresults indicate that aside from absolute potencies the 0.5 mg/kgdose we chose for screening was likely a maximally efficacious doseacross the panel of compounds tested and valid for meaningful comparisonsof efficacy between drugs.

Remarkably, we found that a substitutionat the 4-position wasnot even necessary to provide full efficacy. The compounds 2,5-DMA(Figure7L) and 2C-H(Figure7N) were bothfully efficacious at 0.5 mg/kg. Interestingly, Dr. Alexander Shulginhas reported no behavioral effects of 2,5-DMA in humans up to a veryhigh dose.⁴¹ Whether or not the lack ofbehavioral effects in humans and rodents is due to potential rapidmetabolism from first-pass metabolism after oral or i.p. administrationthat is not present following direct application to the lung remainsto be tested. With all of our data taken together, we propose that2C-H represents the pharmacophore anti-inflammatory structure forthe phenylethylamine class of psychedelic. Modifications of the alkylside chain, with the exception of the addition of an alpha-methyl,are not well tolerated. Furthermore, modifications or substitutionsof the 2- and 5-methoxys are not well tolerated. However, substitutionsat the 4-position are well-tolerated and can maintain anti-inflammatoryactivity, but perhaps only to a point where steric bulk exceeds somecritical value.

Ergolines

LSD and ETH-LAD, whichhave significantlyhigher affinities than (R)-DOI for the 5-HT_2A receptor and are more potent for eliciting HTR in mice, demonstrateonly partial normalization of PenH (Figure7O,P). Lisuride, which has been proposed tobe a nonhallucinogenic agonist at the 5-HT_2A receptor hadno ability to normalize PenH at the tested dose (Figure7Q). LSD is only a partial agonistwith respect to canonical signaling pathways at the 5-HT_2A receptor. The closely related drug ETH-LAD (Figure7P) has the same affinity for the receptoras LSD, however, is a full agonist at canonical signaling pathways.Because both LSD and ETH-LAD had only partial efficacy to preventthe development of allergic asthma symptoms, the degree of efficacyat the receptor by ergolines by canonical signaling is likely notthe primary driver for efficacy in the asthma model. Together, ourresults indicate that ergolines as a class may simply be less efficaciousat engaging anti-inflammatory pathways than phenethylamines, althoughseveral more would need to be tested to make this claim definitively.

Tryptamines

Psilocin, which has significantly weakeraffinity for the 5-HT_2A receptor and less potency than(R)-DOI at activating canonical signaling pathways,is potent at normalizing PenH (Figure7R), and a low sub-behavioral dose of 0.01 mg/kg (Figure7S) inhaled is sufficientto completely prevent OVA-induced AHR to methacholine. The closelyrelated drugN,N-dimethyl tryptamine(N,N-DMT) had no measurable effect(Figure7T), nor did5-MeO–DMT (Figure7U). However, 4-OH-DiPT (Figure7V) does possess full efficacy to normalize PenH at0.5 mg/kg. These results indicate that perhaps for tryptamines a 4-OHgroup is necessary for therapeutic efficacy and also that the requirementsfor the identity ofN′-R groups are less stringent.Further testing of additional compounds will be necessary, however,to test this hypothesis fully. Our results withN,N-DMT having no efficacy in our asthma model, andby extrapolation no anti-inflammatory activity, are at odds with thework of others who have proposed it does have anti-inflammatory effects.Specifically,N,N-DMT has been proposedto act on the sigma-1 receptor on cells of the immune system to preventinflammation.⁴² That we did not observeany measurable effects in our model system suggests sigma-1-receptor-mediatedanti-inflammatory mechanisms may not be relevant for therapeutic efficacyin pulmonary inflammation, and/or that the anti-inflammatory activityofN,N-DMT is dependent on specifictissues that may or may not express sufficient sigma-1 receptor protein.⁴³

Activation of Canonical Signaling Is NotCorrelated with Efficacyto Prevent OVA-Induced AHR

Several, but not all, drugs weexamined have known affinities for the 5-HT_2A receptorand known behavioral potencies.^8,41,44 In order to attempt to correlate canonical signaling downstreamof the 5-HT_2A receptor to anti-asthma efficacy, we measuredefficacy and potencies for each of the drugs used for calcium flux,which is induced by activation of the Gα_q canonicalsignaling pathway, in heterologously expressed human 5-HT_2A receptors in HEK cells. In a comparison of PenH-AUC values determinedfor each drug as a proxy measure of anti-inflammatory efficacy (Figure8A) to either EC₅₀ orE_Max for calcium mobilizationdownstream of 5-HT_2A receptor activation (Table1), we found no correlation betweencanonical signaling and anti-asthma efficacy (Figure8B,C). As expected, our prototypical drug(R)-DOI, which is fully anti-inflammatory, was apotent and full activator of calcium mobilization. Drugs with similarreceptor potencies such as 2C-B-Fly, however, were only partiallyefficacious as anti-inflammatories (Table1;Figure8A). These results are consistent with noncanonicalsignaling pathways linking anti-inflammatory activity to 5-HT_2A receptor activation that remain to be elucidated in futureexperiments. This uncoupling of traditional pharmacological propertiesof drug–receptor interaction and therapeutic effect is notunprecedented.⁴⁵ Therefore, it is likelythat noncanonical signaling pathways yet to be identified underliethe anti-inflammatory/asthma effects of psychedelics.

5-HT_2B and 5-HT_2C Receptors Are Not Involvedin the Effects of (R)-DOI

Because (R)-DOI, as well as all other psychedelic drugs tested, alsohas significant affinity for and are agonists at 5-HT_2B and 5-HT_2C receptors, we sought to determine any potentialinvolvement in the therapeutic effects to normalize PenH. We tested0.5 mg/kg of a selective 5-HT_2C agonist and 5-HT_2B inverse agonist (1-methyl psilocin) (Figure9A), and two mixed 5-HT_2B/2C agonists(Ro 60–0175 fumarate and BW 723C86) (Figure9B,C). There were no significant effects onPenH hyperresponsiveness to MeCh in the OVA treated animals for anyof these three drugs. These results indicate that activity at 5-HT_2B/2C receptors is not involved in the therapeutic effectsof (R)-DOI to prevent allergic asthma in our model.Although these results are highly indicative of activity at the 5-HT_2A receptor being necessary and sufficient for anti-inflammatory/asthmaactivity, we performed a further experiment examining the abilityof (R)-DOI to prevent allergic asthma inHTR2A^–/– knockout mice lackingexpression of the 5-HT_2A receptor (Figure S1). We followed protocols as previously describedfor our mouse model of acute OVA allergic asthma¹¹ and administered 0.5 mg/kg (R)-DOI noseonly prior to each OVA exposure. We found that there was no effectof (R)-DOI to prevent the development of AHR. Utilizingour models, we have demonstrated that agonism at serotonin 5-HT_2B and 5-HT_2C receptors does not have therapeuticefficacy. Combined with our data demonstrating no anti-asthma effectsin theHTR2A^–/– mouse, weconclude that the activation of 5-HT_2A receptors by selectpsychedelics is necessary and sufficient to confer anti-inflammatoryactivity and to prevent the symptoms of asthma in our model. Thisfinding is in agreement with our previousin vitro(9) andin vivo studies.¹⁰⁻¹² Regardless, a possibility we cannot rule out without further testingis that some of these drugs may be interacting with off-target receptorsto produce their effects. The most common off-target receptors forpsychedelics include 5-HT_1A receptors (tryptamines andergolines) and α2 adrenergic receptors (phenakylamines).⁸ We do not, however, believe that this is thecase. There is little to no evidence in the literature of 5-HT_1A receptors having any anti-inflammatory or therapeutic effectsin the lung relevant to asthma, and in human clinical studies testingthe effects of the α2 agonist clonidine on methacholine-inducedairway reactivity, no significant differences between placebo andclonidine groups were measured.⁴⁶

Summary

Our previousin vitro workdemonstrated that different 5-HT_2A receptor agonists havedisparate anti-inflammatory properties.⁹ We hypothesized that structural differences between ligands gaverise to differential anti-inflammatory potencies and efficacy throughfunctionally selective (ligand biased) mechanisms. Here, we examinedthe structure–activity relationships between agonist and anti-inflammatory/asthmaefficacy in a new rat model of allergic asthma. We first validatedour asthma model, and then used it to examine structure–activityrelationships. During this process, we demonstrated the utility ofour rat model as anin vivo platform that is robustand long-lasting for the analysis and/or screening of compounds fortherapeutic efficacy to treat symptoms associated with pulmonary inflammationand asthma. Parameters measured were more consistent between individualrats than we have found for mice, and once sensitized to OVA, therats maintain a stable hyper-allergic state and have a robust testingperiod of ∼1 year, compared to only ∼6 months in BALB/cmice. Results showing equipotency for nebulized compared to intraperitonealinjection for (R)-DOI indicate that potential therapiesin the clinic need not be limited to inhaled formulations. SAR analysisindicates that 2C-H represents the pharmacophore for phenalkylamineanti-inflammatory efficacy, and it also highlights certain modificationsthat are permissive or detrimental to efficacy for phenalkylaminesand tryptamines and that ergolines as a class may not be fully efficacious.In vitro data with regard to potency and efficacy at canonicalsignaling pathways indicate that relevant anti-inflammatory pathwaysdo not correlate with canonical signaling pathways. These results,together with no apparent correlation between behavioral effects,support the notion that anti-inflammatory and behavioral effects maybe separable through functionally selective mechanisms in drug discoveryefforts to identify nonbehavioral 5-HT_2A receptor agonistswith anti-inflammatory activity for the treatment of inflammation-relateddiseases like asthma in the clinic.

The data in theSupporting Information further support our findingsin rats that activation of 5-HT_2A receptors is necessaryand sufficient for the anti-asthma effects of psychedelics. In summary,treatment with a high dose of the 5-HT₂ receptor agonist(R)-DOI does not reduce or affect OVA-induced AHRinHTR2A^–/– knockout mice.

Drugs and Reagents

(R)-DOI was synthesizedand provided by Dr. Bruce Blough at Research Triangle Institute (ResearchTriangle Park, NC) and by Eleusis Therapeutics (New York, NY). LSD;ETH-LAD, 2,4-DMA, 5-iPrO-2CE, 2C-B, TCB-2, (R)-DOB,(R)-DOM, (R)-DOiBu, (S)-DOI, (R)-DOET, 5-MeO-DMT, psilocin, and mescalinewere synthesized and provided by Dr. David Nichols (Purdue University).2C-T-33 was synthesized and provided by Dr. Daniel Traschel (ReseaChemGmbh, Burgdorf, CH). 2C-H, 2C-I, 4-OH-DIPT, 2,5-DMA, and TMA-2 werepurchased from Cayman Chemical (MI). Lisuride; 1-methylpsilocin; Ro-0175fumarate; and BW723C86 were purchased from Tocris Bioscience (Bristol,UK).N,N-DMT, 5-HT, ovalbumin (OVA),methacholine (MeCh), anddl-isoproterenol hydrochloride werepurchased from Sigma-Aldrich (St. Louis, MO). SeeFigure1 for the structures of eachcompound.

Animals

For all experiments described, respiratory-pathogen-freeBrown Norway (RijHsd-BN) rats were obtained from Envigo (Somerset,NJ) and Charles River (Raleigh, NC). All animals used in these studieswere male and 6–8 weeks of age on arrival with a body weightof 170–220 g. Animals were allowed to acclimate at least 1week prior to initiation of OVA sensitization. For the duration ofthe study, rats were housed singly in the animal care facility atthe Louisiana State University Health Sciences Center (New Orleans,LA) in ventilated cages housed in a pathogen-free animal facilitywithad libitum access to food and water on a 12h/12 h light/dark cycle. All protocols were prepared in accordancewith theGuide for the Care and Use of Laboratory Animals(340) and approved by the InstitutionalAnimal Care and Use Committee at Louisiana State University HealthSciences Center.

OVA-Induced Acute Allergic Airway Inflammation

Sensitization-challengeprotocols used in this study are summarized inFigure2. For sensitization, Brown Norway rats (7–9weeks old) were i.p. injected with (500 μL) of 2.0 mg of chickenOVA (Sigma-Aldrich) emulsified in 2.0 mL of Imject Alum [Al(OH)₃/Mg(OH)₂; Pierce, Rockford, IL] on days 0 and 7,as described by Elwood et al.²⁵ OVA exposuremethods were based on our previously described mouse model of acuteasthma.¹¹ OVA-alone treated rats were exposedto 3 times weekly exposure of 10.0 mg of OVA slowly dissolved in 10.0mL of 0.9% sterile saline solution (Baxter Healthcare Corp., Deerfield,IL) in a 15 L (38.00 × 19.05 × 19.7 cm) acrylic inductionchamber. No more than 6 animals were exposed in the chamber per challenge.OVA aerosol was generated using an ultrasonic nebulizer (Pari Pronebnebulizer, Midlothian, WA) in conjunction with a Pari Proneb pumpat a 1.0% OVA concentration for a total duration of 30 min, as describedin Palmans et al.²⁶

Drug Exposure

For nose-only exposures, rats were exposedin groups of 3–4 rats/group to the appropriate concentrationof drug dissolved in a total volume of 4.5 mL of sterile saline usingan inExpose nose-only inhalation system (SCIREQ, Montreal, QC, Canada)30 min prior to each OVA challenge. Each 4.5 mL of sample was aerosolizedusing a VixOne nebulizer (Westmed, Inc., Tucson, AZ) in conjunctionwith a Pari Proneb pump. Exposures lasted 15 min. All respiratoryparameters were measured 48 h after the final OVA exposure (Figure2). For i.p. administration,the appropriate drug was dissolved in sterile saline at a concentrationof 1.0 mg/kg and injected at the appropriate weight/volume ratio.

Noninvasive Whole-Body Plethysmography (WBP) and MethacholineChallenge

To minimize the impact of circadian influences,all respiratory recordings were performed between 10 am and 3 pm.⁴⁷⁻⁴⁹ For measurement of airway responsiveness to MeCh, a noninvasivebias flow ventilated whole body plethysmography system (EMKA Technologies,Falls Church, VA) was used in spontaneously breathing, unrestrainedrodents. The plethysmograph (PLY3215; diameter 10 in Buxco Electronics,Troy, NY) was ventilated by a continuous flow of 2.5 L/min (Bias FlowRegulator, Vent2, EMKA Technologies, Paris, FR).⁵⁰ A differential pressure transducer (EMKA Technologies,FR) was connected on one pole to the main chamber and on the secondpole to a reference chamber. The transducer measures pressure differencesbetween both chambers as caused by the respiratory cycle, mainly inhalationand exhalation.⁵¹ The IOX2 Software System(IOX 2.8.2.13; EMKA Technologies, FR) provides a breath-by-breathanalysis of pressure signals and transforms these pressure differencesvia computerized calculations to a dimensionless empirically establishedvalue, enhanced pause or PenH. Numerous experiments from our lab^11,12 and others^52,53 have shown PenH to be a reliableand sensitive measure of bronchoconstriction and a superior measurein assessing the degree of bronchoconstriction compared to other derivedparameters such as box pressure or box flow,⁵³ and it faithfully reproduces the results of forced respiratory techniquessuch as flexiVent (flexiVent, SCIREQ, Montreal, CA).¹¹ Despite these findings, it must be acknowledged that theuse of PenH as a measure of airway mechanics is a matter of debate.⁵⁴ A large portion of this controversy stems frominterpretation of pulmonary responsiveness using older plethysmographyequipment, in which fluctuations to any ambient parameter (i.e., temperatureand humidity) impact airways resistance calculations.⁵⁵ As such, the type of WBP is an important factor to consider.Although any data collected from a sealed chamber plethysmograph (PressureWBP)does not represent pulmonary resistance, data gathered from a WBPchamber with a pneumotachograph in its wall (FlowWBP) is correlativewith pulmonary resistance,⁵⁶ which is thetype of WBP performed in our studies. Accordingly, with appropriatemathematical analysis and a properly calibrated FlowWBP system, PenHdoes quantitatively measure fluctuations in airway resistance regardlessof variations in ambient parameters. Although a degree of cautionshould be exercised using PenH data from a properly calibrated FlowWBPsystem as a proxy for direct measurement using forced ventilationto measure pulmonary airways resistance (FlexiVent), FlexiVent isa terminal assay, whereas FlowWBP can be reliably repeated in thesame cohort of animals, necessitating its use for evaluating largenumbers of drugs and doses such as we have performed.

For theassay, the chamber pressure signal is calibrated by dynamic injectionof 5 mL of room air via syringe (BD; 10 mL syringe with luer-lok tip;Franklin Lakes, NJ).⁵⁰ Animals are thenplaced in the chamber, where baseline data are recorded for 5 minfollowing a 10 min habituation period in the plethysmograph.⁵³ After measurement of baseline PenH, either aerosolizedsaline (0.9% NaCl Solution) or an aqueous solution of MeCh in increasingconcentrations (4, 8, 16, 32 mg/mL) were nebulized through an inletof the plethysmography chamber for 3 min, followed by measurementsof PenH values for 3 min.^51,57 An Aeroneb Pro vibrating-meshnebulizer (Aerogen Ltd., Galway, Ireland) was used to generate aerosol.Following recordings, to prevent a MeCh gradient there was a wash-outperiod of 7 min in which the animal was provided with fresh air.⁵⁸⁻⁶⁰ For studies involving isoproterenol, the above protocol was utilized;however, following the 32 mg/mL MeCh challenge animals were exposedto 30 mg/mL isoproterenol for 3 min, and PenH values were measuredfor 3 min. The dosage of isoproterenol was extrapolated from Voginet al.⁶¹ and deemed by Kondo et al.⁶² not to have lasting myocardial effects. Datafrom the IOX2 software were analyzed using Datanalyst software (Datanalystv2.6.1.14; EMKA Technologies, France) and expressed as the mean SEMof maximal PenH values per group.

For testing, we maintainedsix cohorts of rats: two saline-onlycontrol groups (n = 6/group), and four OVA-sensitizedand exposed groups (n = 6/group). These cohorts werefurther divided into two sets, each set with one saline-only group,and two OVA-exposed and -sensitized groups. In any given testing round,we used one set of treatment groups with one OVA-treated group usedas an OVA-only control that was exposed only to OVA, another OVA-treatedgroup was used as our experimental group for drug pretreatment priorto OVA exposure, and one saline-only control group that was exposedonly to aerosolized saline. The following round we utilized the otherset with the same group designations. On the third round, we returnedto the first set; however, we utilized the previously drug-exposedOVA-treated group as the OVA-only group (to ensure resetting of theimmune response following drug treatments) and the previously usedOVA-only group as the OVA + drug group. This staggering allowed atleast 2 weeks between experimental manipulation on any given rat,so we could ensure the previously drug-treated animal’s responsesreturned to normal prior to another drug treatment and validated thatthere were no cumulative drug effects between trials to confound resultson an ongoing basis. At this rate we could test one experimental drug/doseper week, every week. The same cohorts of rats were used for testingdrugs over periods of about 3–9 months. During this time, themeasured PenH in the control groups, as well as the OVA-only treatedgroup measurements, did not significantly differ between trials, indicatingthat there were no age-related confounding effects on pulmonary responseto OVA over time and that repeated drug exposures did not affect subsequentresponses to OVA exposures and/or drug treatments. As a final test,the terminal round of testing for all rats after completion of experimentaltesting was to reassess the effects of inhaled (R)-DOI (0.1 mg/kg) to ensure animals were still equally responsiveat the end of testing as they were at the beginning several monthsearlier. The first cohort of experimental groups was used to developand validate the model using (R)-DOI only and togenerate lung tissue for gene expression analysis. The second cohortwas used to validate further the model with (R)-DOI,to collect BALF, and to generate lung tissues for histopathologicalanalysis. The third cohort was used for compound screening and theroute of administration experiments.

Histopathology

Lungs were isolated and prepared 48h after the final OVA exposure. Animals were humanely sacrificed byexsanguination under anesthesia (ketamine/xylazine mixture; 100 and10 mg/kg, respectively). A 14-guage angiocatheter (Excel Safelet Catheter,Excel International, Los Angeles, CA) was inserted into the trachea,and a 4–0 silk suture (Oasis, Mettawa, IL) was tied aroundthe trachea. The catheter was withdrawn, and the lungs were removeden bloc. A gentle infusion of 10 mL of Zinc Formal-Fixx Concentrate(Thermo Scientific, Shandon, Inc., Pittsburgh, PA) was used to inflation-fixthe lungs, which were immersed in Zinc Formal-Fixx at room temperatureovernight and dehydrated in a graded series of ethanol solutions.Fixed lungs were embedded in paraffin and sectioned at 4 μmthickness. Each lung section was stained with periodic acid–Schiff(PAS) for examination of mucus cell metaplasia and evaluated as previouslydescribed.⁶³ Adjacent sections were stainedwith hematoxylin and eosin (H&E) to assess severity of interstitialand peribronchial inflammation. Five trained individuals blinded tothe background of the samples assigned a total inflammation scorefor each lung section as follows: 0, normal; 1, inflammatory infiltrationcomprises less than 25% of entire section; 2, inflammatory infiltrate25–50% entire section; and 3, more than 50% of entire sectionexhibits inflammatory infiltration.

Cytokine and ChemokineAnalysis by qRT-PCR

Lungs wereharvested 48 h after the final OVA exposure and frozen until processingat −80 °C. Total RNA was extracted from the left lobeof each rat with TRIzol reagent, purchased from Life Technologies(Carlsbad, CA), following the manufacturer’s instruction. TheRNA pellet was resuspended in nuclease-free H₂O, and RNAwas quantified by spectrophotometer (NanoDrop ND-1000; NanoDrop Technologies,Inc., Wilmington, DE). RNA was quantified at A260 and A280, with noRNA utilized that was below a 1.6 260/280 nm ratio. Suitable RNA wasprocessed into first-strand cDNA using the ImProm-II cDNA synthesiskit (Promega, Madison, WI) following the manufacturer’s instructions.Gene expression levels of cytokines were determined using reversetranscription and quantitative real-time PCR (qRT-PCR). The inputcDNA for each reaction was 1000 ng of total RNA. Primers were designedto be compatible with the Universal ProbeLibrary system using theUniversal ProbeLibrary Assay Design Center (Roche Diagnostics, Indianapolis,IN) and synthesized by Integrated DNA Technologies (Coralville, IA).Primer sequences used in this study are listed inTable2. Probes utilized in this reportwere from the Universal ProbeLibrary (Roche Diagnostics, Indianapolis,IN) and are listed with the following universal probe numbers: U78,U21, U2, U106, U17, U124, U5, U68, U89, U15, and U42 forIL-1β,IL-4,IL-5,IL-6,IL-13,Gm-csf,MUC5AC,TNFα, andGusB. Triplicate amplification reactions usingthe first-strand cDNA sample from each rat were performed on a Roche480 LightCycler II LC (Roche Diagnostics) using the qPCR PerfeCTaFastMix II (Quanta Biosciences, Inc., Gaithersburg, MD) followingthe manufacturer’s directions. Amplification ofβ-glucuronidase (GUSB), a housekeeping gene consistently expressedamong all samples, was used as the reference standard. Relative expressionwas determined using the ΔΔ-Ct method. Significance ofexpression was measured using a one-way analysis of variance (ANOVA),with all genes marked as significant exhibiting at least ap < 0.05.

Calcium Flux Assay

Gα_q-mediated calciumflux downstream of 5-HT_2A receptor activation was determinedusing HEK293 cells stably expressing the human 5-HT_2A receptor.⁶⁵⁰ Cells were seeded in DMEM supplemented with10% fetal bovine serum, 100 units/mL penicillin, 100 mg/mL streptomycin,and 100 mg/mL Zeocin onto 96-well poly-d-lysine plates withclear bottoms (12 000 cells/well) and cultured at 37 °C.The following day, media was aspirated and replaced with serum-freeDMEM for 12 h. On the day of the experiment, the cells were washedonce with HBSS supplemented with 20 mM HEPES, loaded with 75 μLof 3 μM Fluo-2 AM HA (Ion Indicators, LLC) diluted in HBSS–HEPESbuffer, incubated for 1 h at 37 °C, washed again with HBSS–HEPES,and maintained in 50 μL HBSS–HEPES at 25 °C. Theplates of dye-loaded cells were placed into a FlexStation 3 microplatereader (Molecular Devices, LLC) to monitor fluorescence (excitation,485 nm; emission, 525 nm; cutoff, 515 nm). Plates were read for 20s (2 s interval) to establish baseline fluorescence and then challengedwith compounds diluted in a range of 10 pM to 10 μM or bufferand read for an additional 80 s. After obtaining a calcium mobilizationtrace for each sample, the mean baseline fluorescence was subtractedfrom peak fluorescence in each well. E_MAX values were determinedby normalization to the maximum 5-HT response (100%) on the same plate.The data were analyzed using nonlinear regression curve-fitting routinesin Graph-Pad Prism 8.0 (GraphPad Software, Inc.) to generate EC₅₀ values. These data for test compounds were then normalizedto 5-HT by calculating a ratio of mean EC₅₀ value acrossall test plates versus same plate response and multiplying it by individualtest compound EC₅₀ values.

Statistics

Allstatistical analysis was performed usingGraphPad Prism (GraphPad Software, La Jolla, CA).

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Supplementary Materials