CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U. S. Provisional Application No. 62/571 ,335 filed October 12, 2017. The content of these earlier filed applications is hereby incorporated by reference herein in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under R01 NS082746, R01 DE26139, F31NS083358 and awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND

It is well known that women are at an increased risk for pain disorders or pain intensity. However, there is a large gap in therapeutics since analgesic drugs are not indicated for preferentially treating women in pain. It is has been shown that serotonin (5-HT) significantly enhances capsaicin-induced activation of pain neurons in tissue biopsies collected from female humans but not male humans. 5HT is involved in many pain disorders including migraine, other headaches, and visceral and skeletomuscular pain. Capsaicin activates the transient receptor potential subtype VI (TRPV1), which is a maj or pain transducing receptor expressed on a maj or class of pain-detecting sensory neurons (also referred to as nociceptors). Currently, there are no analgesics that have an indication for treatment of pain in women. Thus, useful compositions and methods to treat women in pain are needed.

SUMMARY

Disclosed herein are methods for treating pain in a female subj ect, the method comprising: (a) identifying a female subject in need of treatment; and (b) administering to the female subject a therapeutically effective amount of a pharmaceutical composition comprising a C3a receptor inhibitor, a C5a receptor inhibitor and a pharmaceutically acceptable carrier.

Disclosed herein are methods for treating pain in a female subj ect, the method comprising: (a) identifying a female subject in need of treatment; and (b) administering to the female subject a therapeutically effective amount of a pharmaceutical composition comprising an anti-C3a antibody or a C3a aptamer, an anti-C5a antibody or a C5a aptamer and a pharmaceutically acceptable carrier.

Disclosed herein are methods of reducing pain in a female subject susceptible to or having pain, the method comprising administering to the female subject a therapeutically effective amount of a pharmaceutical composition comprising a C3a receptor inhibitor, a C5a receptor inhibitor and a pharmaceutically acceptable carrier.

Disclosed herein are methods of reducing pain in a female subject susceptible to or having pain, the method comprising: administering to the female subject a therapeutically effective amount of a pharmaceutical composition comprising an anti-C3a antibody or a C3a aptamer, an anti-C5a antibody or a C5a aptamer and a pharmaceutically acceptable carrier.

Disclosed herein are pharmaceutical compositions comprising C3a receptor inhibitors, a C5a receptor inhibitors and pharmaceutically acceptable carriers.

Other features and advantages of the present compositions and methods are illustrated in the description below, the drawings, and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic of ICAP experiments.

FIG. 2 shows the evaluation of a soluble factor released from human dental pulp.

FIG. 3 shows the effects of pre-treatment with a combination of 5-HT antagonists (all 1 μΜ) to 5-HT2 (ritanserin), 5-HT4 (GR113808), 5-HT6 (SB258585) and 5-HT7

(SB269970) on 100 μΜ 5-HT-evoked release of CGRP from female human dental pulp. ANOVA: F=4.98; P<0.01; N=24-33/group.

FIG. 4 shows that the soluble factor is a protein and not a lipid.

FIG. 5 shows the results of proteomic analysis using 2D DIGE (differential in-gel electrophoresis) comparing supernatant from 5-HT-treated female and male human dental pulps.

FIG. 6 shows the results of the evaluation of complement C3a (C3a) receptor antagonist (SB290157) and anti-C3a-antibody on blocking excitatory factor released from female human dental pulp.

FIG. 7 shows the results of cultured female dental pulp.

FIG. 8 depicts confocal micrographs showing expression of C3a (first column) and C3a receptor (C3aR; second column) in normal (top row) and painful (symptomatic irreversible pulpitis; bottom row) dental pulp specimens from females. Expression of both is increased in intrinsic cells in pain specimen (arrows), whereas C3aR expression is also present in neurofilament heavy (NFH; third column)-identified nerve fibers in both normal and inflamed conditions (arrowheads).

FIG. 9 depicts the Western blot analysis and quantitative densitometry post-siRNA (Accell siRNA at 1 μΜ) treatment of female dental pulp cultures for 72 hours.

FIG. 10 is a schematic diagram illustrating proposed signaling pathways in which activation of ϋβγ from C3aR or C5aR leads to increased TRPVl activities by either increased channel opening or trafficking (Adapted from Meents et al., Trends Mol. Med. 16: 153, 2010; Cabrera-Vera et al, Endoc. Rev. 24:765, 2006, Qiagen Signaling Pathways, and Wang and Woolf, Neuron 46:9, 2005).

FIG. 11 shows the evaluation of a soluble factor released from human dental pulp.

FIGS. 12A-B show the effects of synthetic C3a and C5a on human peptidergic neurons. FIG. 12A shows the percent effect of complement in females. FIG. 12B shows the percent effect of complement in males.

DETAILED DESCRIPTION

The present disclosure can be understood more readily by reference to the following detailed description of the invention, the figures and the examples included herein.

Before the present compositions and methods are disclosed and described, it is to be understood that they are not limited to specific synthetic methods unless otherwise specified, or to particular reagents unless otherwise specified, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, example methods and materials are now described.

Moreover, it is to be understood that unless otherwise expressly stated, it is in no way intended that any method set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not actually recite an order to be followed by its steps or it is not otherwise specifically stated in the claims or descriptions that the steps are to be limited to a specific order, it is in no way intended that an order be inferred, in any respect. This holds for any possible non-express basis for interpretation, including matters of logic with respect to arrangement of steps or operational flow, plain meaning derived from grammatical organization or punctuation, and the number or type of aspects described in the specification.

All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided herein can be different from the actual publication dates, which can require independent confirmation.

As used in the specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise.

The word "or" as used herein means any one member of a particular list and also includes any combination of members of that list.

Throughout the description and claims of this specification, the word "comprise" and variations of the word, such as "comprising" and "comprises," means "including but not limited to," and is not intended to exclude, for example, other additives, components, integers or steps. In particular, in methods stated as comprising one or more steps or operations it is specifically contemplated that each step comprises what is listed (unless that step includes a limiting term such as "consisting of), meaning that each step is not intended to exclude, for example, other additives, components, integers or steps that are not listed in the step.

Ranges can be expressed herein as from "about" or "approximately" one particular value, and/or to "about" or "approximately" another particular value. When such a range is expressed, a further aspect includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about," or "approximately," it will be understood that the particular value forms a further aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint and independently of the other endpoint. It is also understood that there are a number of values disclosed herein and that each value is also herein disclosed as "about" that particular value in addition to the value itself. For example, if the value " 10" is disclosed, then "about 10" is also disclosed. It is also understood that each unit between two particular units is also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

As used herein, the terms "optional" or "optionally" mean that the subsequently described event or circumstance may or may not occur and that the description includes instances where said event or circumstance occurs and instances where it does not.

As used herein, the term "subject" refers to the target of administration, e.g., a human. Thus, the subject of the disclosed methods can be a vertebrate, such as a mammal, a fish, a bird, a reptile, or an amphibian. The term "subject" also includes domesticated animals (e.g., cats, dogs, etc.), livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), and laboratory animals (e.g., mouse, rabbit, rat, guinea pig, fruit fly, etc.). In one aspect, a subject is a mammal. In another aspect, the subject is a human. The term does not denote a particular age or sex. Thus, adult, child, adolescent and newborn subjects, as well as fetuses, whether male or female, are intended to be covered.

As used herein, the term "patient" refers to a subject afflicted with pain or a disease, disorder or condition associated with pain. The term "patient" includes human and veterinary subjects. In some aspects of the disclosed methods, the "patient" has been diagnosed with a need for treatment for pain, such as, for example, prior to the administering step.

As used herein, the term "treating" refers to partially or completely alleviating, ameliorating, relieving, delaying onset of, inhibiting or slowing progression of, reducing severity of, and/or reducing incidence of one or more symptoms or features of a particular disease, disorder, and/or condition. Treatment can be administered to a subject who does not exhibit signs of a disease, disorder, and/or condition and/or to a subject who exhibits only early signs of a disease, disorder, and/or condition for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and/or condition. For example, the disease, disorder, and/or condition can be pain or associated with pain.

As used herein, "treating" can also refer to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, condition, or disorder. In various aspects, the term covers any treatment of a subject, including a mammal (e.g., a human), and includes: (i) preventing, for example, pain from occurring in a subject that can be predisposed to pain but has not yet been diagnosed as having it; (ii) inhibiting pain, i.e., arresting its development; or (iii) relieving pain, i.e., causing regression of the pain.

As used herein, the terms "inhibit" or "inhibiting" mean decreasing or reducing pain that would occur without treatment and/or causing one or more symptoms of pain to decrease.

The terms "administering" and "administration" refer to any method of providing a pharmaceutical preparation to a subject. Such methods are well known to those skilled in the art and include, but are not limited to, oral administration, sublingual administration, trans- buccal mucosa administration, transdermal administration, administration by inhalation, nasal administration, topical administration, intravaginal administration, ophthalmic administration, intraaural administration, intracerebral administration, intrathecal administration, rectal administration, intraperitoneal administration, and parenteral administration, including injectable such as intravenous administration, intra-arterial administration, intramuscular administration, intradermal administration, and subcutaneous administration. Ophthalmic administration can include topical administration, subconjunctival administration, sub- Tenon's administration, epibulbar administration, retrobulbar administration, intra-orbital administration, and intraocular administration, which includes intra-vitreal administration. Administration can be continuous or intermittent. In various aspects, a preparation can be administered therapeutically; that is, administered to treat an existing disease or condition. In further various aspects, a preparation can be administered prophylactically; that is, administered for prevention of a disease or condition.

As used herein, the term "prevent" or "preventing" refers to precluding, averting, obviating, forestalling, stopping, or hindering something from happening, especially by advance action. It is understood that where reduce, inhibit or prevent are used herein, unless specifically indicated otherwise, the use of the other two words is also expressly disclosed. As used herein, the term "inhibitor" of a C3a receptor or a C5a receptor can function in a competitive or non-competitive manner. Inhibitors of the C3a receptor or a C5a receptor include any chemical or biological entity that, upon treatment of a cell, results in inhibition of a biological activity caused by activation of the C3a receptor or a C5a receptor in response to binding of its natural ligand.

The "C3a receptor" is also known as complement or complement component 3a receptor 1 (C3AR1), and is a G protein-coupled receptor protein involved in the complement system. The C3a receptor binds to complement component C3a. The C3a receptor can modulate immunity, arthritis, diet-induced obesity and cancers.

The "C5a receptor" is also known as complement or complement component 5a receptor 1 (C3AR1), and is a G protein-coupled receptor protein for C5a involved in the complement system.

Numerous studies indicate that women and men differ in prevalence of pain disorders or pain intensity (Berkley, 1997; Frankel et al, 1999; Stovner et al., 2006; Cairns and Gazerani, 2009; Fillingim et al, 2009; Greenspan et al., 2011; Greenspan and Traub, 2013), possibly due to sexually dimorphic differences in detection, processing or responses to noxious stimuli (Jensen et al, 1994; Bereiter, 2001; Cairns et al, 2001 ; You et al., 2006; Loyd et al., 2012b). The importance of this complex problem has been emphasized by recent NIH policies. Disclosed herein are studies that assess peripheral sexually dimorphic pain mechanisms in humans.

As disclosed in the Examples, it was tested whether 5-HT releases a "soluble factor" from female human biopsies but not male biopsies that sensitizes capsaicin-sensitive peripheral nerve fibers. Dental pulp from females and males participants were treated with 5- HT and a combination of 5-HT antagonists was added to the conditioned media (to prevent effects of residual 5-HT) and then applied to trigeminal neurons. Patch clamp

electrophysiology was used to determine the effects the conditioned media (CM) on rat trigeminal neurons and 2D gel electrophoresis/mass spectrometry and ELISA to identify the soluble factor released. The results show that CM from female dental pulp treated with 5-HT sensitized ICAP responses; the soluble factor is a peptide and not a lipid; proteomics revealed that the soluble factor is complement C3a (C3a); and C3a receptor antagonists and an anti- C3a antibody block CM-induced sensitization of ICAP. Collectively, these data suggest that 5- HT triggers a peripheral sexually dimorphic pain mechanism in women via release of complement peptides that leads to enhanced activity of capsaicin-sensitive trigeminal nociceptors.

Described herein are the results of studies showing that stimulation of female human tissues, including application of serotonin to female human dental pulp, releases soluble factors capable of significantly enhancing the activities of the sensory neurons, and, in particular, a capsaicin-sensitive class of nociceptors. Also described herein is the finding that 3C receptor antagonists block a sexually dimorphic pain mechanism by blocking the actions of complement proteins, and, in particular, complement 3Ca. In some aspects, complement 3Ca and complement 5Ca can be antagonized by the administration of antibodies, aptamers, solubilized receptors or other compounds capable of binding or neutralizing the actions of complement 3Ca and complement 5Ca.

METHODS OF TREATMENT

Disclosed herein, are methods of treating pain in a female subject. Disclosed herein, are methods comprising: (a) identifying a female subject in need of treatment; and (b) administering to the female subject a therapeutically effective amount of pharmaceutical composition comprising a C3a receptor inhibitor, a C5a receptor inhibitor and a

pharmaceutically acceptable carrier. In some aspects, disclosed herein, are methods comprising: (a) identifying a female subject in need of treatment; and (b) administering to the female subject a therapeutically effective amount of pharmaceutical composition comprising an anti-C3a antibody or a C3a aptamer, an anti-C5a antibody or a C5a aptamer and a pharmaceutically acceptable carrier.

Also, disclosed herein, are methods of reducing pain in a female subject susceptible to or having pain. Disclosed herein, are methods comprising: administering to the female subject a therapeutically effective amount of a pharmaceutical composition comprising a C3a receptor inhibitor, a C5a receptor inhibitor and a pharmaceutically acceptable carrier.

Disclosed herein, are methods comprising: (a) identifying a female subject in need of treatment; and (b) administering to the female subject a therapeutically effective amount of pharmaceutical composition comprising an anti-C3a antibody or a C3a aptamer, an anti-C5a antibody or a C5a aptamer and a pharmaceutically acceptable carrier.

Also, disclosed herein, are methods of reducing or neutralizing complement 3Ca and complement 5Ca actions in a female subject susceptible to or having pain. Disclosed herein, are methods comprising: administering to the female subject a therapeutically effective amount of a pharmaceutical composition comprising a C3a receptor inhibitor, a C5a receptor inhibitor and a pharmaceutically acceptable carrier. In some aspects, the methods can comprise: (a) identifying a female subject in need of treatment; and (b) administering to the female subject a therapeutically effective amount of pharmaceutical composition comprising an anti-C3a antibody or a C3a aptamer, an anti-C5a antibody or a C5a aptamer and a pharmaceutically acceptable carrier. In an aspect, the reduction or neutralizing effect of complement 3Ca and complement 5Ca actions in a female subject susceptible to or having pain can be compared to a female subject or a population of female subjects susceptible to or having pain but not undergoing any of the treatments as disclosed herein. In an aspect, the reduction or neutralizing effect of complement 3Ca and complement 5Ca actions in a female subject susceptible to or having pain can be compared to a the same female subject susceptible to or having pain before the administration of any of the treatments as disclosed herein.

In some aspects, the female subject in need of treatment can be identified by determining the level of expression of C3a and/or C5a. In some aspects, the level of expression of C3a and/or C5a can be determined in leukocytes or in human dental pulp fibroblasts.

In an aspect, the C3a receptor inhibitor can be SB290157. Examples of C3a receptor inhibitors include but are not limited to FLTChaAR and compound 4. Other examples of C3a receptor inhibitors include but are not limited to SB 290157, C3a-receptor antagonist (C3aRA), and TLQP-21.

In an aspect, the C5a receptor inhibitor can be CHIPS. Examples of C3a receptor inhibitors include but are not limited to W54011, NDT9520492, avacopan, DF2593A, AcPhe-Orn-Pro-D-Cha-Trp-Arg, A8delta71-73, PMX205, PMX53, N-methyl-Phe-Lys-Pro- D-Cha-Trp-D-Arg-Co2H, JPE1375, C089, RPR121154, and L-156,602.

The compositions described herein can be formulated to include a therapeutically effective amount of a C3a receptor inhibitor and a C5a receptor inhibitor; or an anti-C3a antibody or a C3a aptamer and an anti-C5a antibody or a C5a aptamer described herein. In some aspects, the compositions described herein can be formulated in include a

therapeutically effective amount of a C3a receptor inhibitor with an anti-C5a antibody or a C5a aptamer or a C5a receptor inhibitor with an anti-C3a antibody or a C3a aptamer described herein. In some aspects, therapeutic administration encompasses prophylactic applications. Based on genetic testing and other prognostic methods, a physician in consultation with their patient can choose a prophylactic administration where the patient has a clinically determined predisposition or increased susceptibility (in some cases, a greatly increased susceptibility) to a type of pain.

The compositions described herein can be formulated in a variety of combinations. The particular combination of a C3a receptor inhibitor with a C5a receptor inhibitor, an anti- C5a antibody or a C5a aptamer or a C5a receptor inhibitor with an anti-C3a antibody or a C3a aptamer can vary according to many factors, for example, the particular the type and severity of the pain.

The compositions described herein can be administered to the female subject (e.g., a human patient) in an amount sufficient to delay, reduce, or preferably prevent the onset of pain. Accordingly, in some aspects, the patient is a human patient. In some aspects, the patient is a female patient. In therapeutic applications, compositions can be administered to a female subject (e.g., a human patient) already with or diagnosed with pain or a condition associated with pain in an amount sufficient to at least partially improve a sign or symptom or to inhibit the progression of (and preferably arrest) the symptoms of the condition, its complications, and consequences. An amount adequate to accomplish this is defined as a "therapeutically effective amount." A therapeutically effective amount of a composition (e.g., a pharmaceutical composition) can be an amount that achieves a cure, but that outcome is only one among several that can be achieved. As noted, a therapeutically effective amount includes amounts that provide a treatment in which the onset or progression of the pain or condition or disorder is delayed, hindered, or prevented, or the pain or condition or disorder or a symptom of the pain or condition or disorder is ameliorated. One or more of the symptoms can be less severe. Recovery can be accelerated in an individual who has been treated.

In some aspects, the pain, condition or disorder can be one or more of orofacial pain, temporomandibular dysfunction, migraine, fibromyalgia, musculoskeletal pain, piriformis syndrome, vestibulodynia, trigeminal neuralgia, pain associated with labor, delivery and/or the recovery from labor and/or delivery or chronic pelvic pain. In some aspects, chronic pelvic pain can be due to conditions or disorders including but not limited to endometriosis, fibroids, irritable bowel syndrome, painful bladder, interstitial cystitis or pelvic congestion syndrome. Disclosed herein, are methods of treating a female subject with pain. The pain can be any pain or associated with any condition or disorder. In some aspects, the pain can be orofacial pain, headache pain, muscloskeletal pain, pelvic pain, joint pain, nerve pain or bladder pain. In some aspects, the female subject has been diagnosed with a condition or disorder associated with pain prior to the administering step. In an aspect, the pain can be associated with orofacial pain, temporomandibular dysfunction, migraine, fibromyalgia, musculoskeletal pain, piriformis syndrome, vestibulodynia, trigeminal neuralgia, pain associated with labor, delivery and/or the recovery from labor and/or delivery or chronic pelvic pain. In some aspects, chronic pelvic pain can be due to conditions or disorders including but not limited to due to conditions or disorders such as endometriosis, fibroids, irritable bowel syndrome, painful bladder, interstitial cystitis or pelvic congestion syndrome.

Pain can be described as a symptom or an indication of an underlying condition, disease or disorder. Pain itself can be considered a diagnosis or a condition.

The methods and compositions described herein can be used to treat acute and/or chronic pain or any etiology associated with orofacial pain, temporomandibular dysfunction, migraine, fibromyalgia, musculoskeletal pain, piriformis syndrome, vestibulodynia, trigeminal neuralgia, pain associated with labor, delivery and/or the recovery from labor and/or delivery or chronic pelvic pain due to conditions or disorders such as endometriosis, fibroids, irritable bowel syndrome, painful bladder, interstitial cystitis or pelvic congestion syndrome.

The compositions described herein can be formulated to include a therapeutically effective amount. In an aspect, compositions disclosed herein can be contained within a pharmaceutical formulation. In an aspect, the pharmaceutical formulation can be a unit dosage formulation.

The therapeutically effective amount or dosage of any of the C3a receptor inhibitors, the C5a receptor inhibitors, the anti-C3a antibodies or the C3a aptamers, and an anti-C5a antibodies or the C5a aptamers used in the methods as disclosed herein applied to mammals (e.g., humans) can be determined by one of ordinary skill in the art with consideration of individual differences in age, weight, sex, other drugs administered and the judgment of the attending clinician. Variations in the needed dosage may be expected. Variations in dosage levels can be adjusted using standard empirical routes for optimization. The particular dosage of a pharmaceutical composition to be administered to the patient will depend on a variety of considerations (e.g., the severity of the pain symptoms), the age and physical characteristics of the subject and other considerations known to those of ordinary skill in the art. Dosages can be established using clinical approaches known to one of ordinary skill in the art.

The duration of treatment with any composition provided herein can be any length of time from as short as one day to as long as the life span of the host (e.g., many years). For example, the compositions can be administered once a week (for, for example, 4 weeks to many months or years); once a month (for, for example, three to twelve months or for many years); or once a year for a period of 5 years, ten years, or longer. It is also noted that the frequency of treatment can be variable. For example, the present compositions can be administered once (or twice, three times, etc.) daily, weekly, monthly, or yearly.

Dosages of any of the C3a receptor inhibitors, the C5a receptor inhibitors, the anti- C3a antibodies or the C3a aptamers, and the anti-C5a antibodies or the C5a aptamers can be in the range of 0.1 μg to 1 g/kg. The dosage can be computed by one of ordinary skill in the art. In an aspect, the therapeutically effective dose of a C3a receptor inhibitor may be less when combined with a C5a receptor inhibitor, an anti-C5a antibody or a C5a aptamer disclosed herein. In an aspect, the therapeutically effective dose of a C5a receptor inhibitor may be less when combined with a C3a receptor inhibitor, an anti-C3a antibody or a C3a aptamer disclosed herein.

The total effective amount of the compositions as disclosed herein can be

administered to a subject as a single dose, either as a bolus or by infusion over a relatively short period of time, or can be administered using a fractionated treatment protocol in which multiple doses are administered over a more prolonged period of time. Alternatively, continuous intravenous infusions sufficient to maintain therapeutically effective

concentrations in the blood are also within the scope of the present disclosure.

In an aspect, the compositions or any of the combinations of compositions disclosed herein can be orally administered. In some aspects, the compositions or any of the combinations of compositions disclosed herein can be administered in a table or capsule dosage form one or more times a day.

The compositions described herein can be administered in conjunction with other therapeutic modalities to a subject in need of therapy. The present compounds can be given to prior to, simultaneously with or after treatment with other agents or regimes. For example, any of the compounds and combinations of compounds disclosed herein can be administered in conjunction with standard therapies used to treat pain or conditions or disorders associated with pain. In an aspect, any of the compounds or compositions described herein can be administered or used together with one or more analgesics. Suitable analgesics include, but are not limited to acetaminophen and acetaminophen-containing compounds and nonsteroidal anti-inflammatory (NSAID) drugs and NSAID-containing compounds such as, for example, salicylates, propionic acid derivatives, acetic acid derivatives, enolic acid derivatives, anthranilic acid derivatives, selective COX-2 inhibitors, sulfonanilides and LOX inhibitors.

In an aspect, a C3a receptor inhibitor and a C5a receptor inhibitor can be co- formulated. In an aspect, a C3a receptor inhibitor and an anti-C5 antibody or a C5 (or a C5a) aptamer can be co-formulated. In an aspect, a C5a receptor inhibitor and an anti-C3 antibody or a C3 (or a C3a) aptamer can be co-formulated.

Any of the compounds or compositions described herein can be administered as a term "combination." It is to be understood that, for example, a C3a receptor inhibitor can be provided to the subject in need, either prior to administration of a C5a receptor inhibitor, an anti-C5 antibody or a C5 (or a C5a) aptamer, concomitant with administration of said a C5a receptor inhibitor, an anti-C5 antibody or a C5 (or a C5a) aptamer (co-administration) or shortly thereafter. It is also to be understood that, for example, a C5a receptor inhibitor can be provided to the subject in need, either prior to administration of a C3a receptor inhibitor, an anti-C3 antibody or a C3 (or a C3a) aptamer, concomitant with administration of said a C3a receptor inhibitor, an anti-C3 antibody or a C3 (or a C3a) aptamer (co-administration) or shortly thereafter.

PHARMACEUTICAL COMPOSITIONS

As disclosed herein, are pharmaceutical compositions, comprising a C3a receptor inhibitor and a C5a receptor inhibitor and a pharmaceutical acceptable carrier. Also disclosed herein, are pharmaceutical compositions, comprising a C3a receptor inhibitor and anti-C5a antibody or a C5a aptamer and a pharmaceutical acceptable carrier. Also disclosed herein, are pharmaceutical compositions, comprising a C5a receptor inhibitor and anti-C3a antibody or a C3a aptamer and a pharmaceutical acceptable carrier. In some aspects, compositions disclosed herein can be formulated for oral or parental administration. In an aspect, the parental administration is intravenous, subcutaneous, intramuscular or direct injection. The compositions can be formulated for administration by any of a variety of routes of administration, and can include one or more physiologically acceptable excipients, which can vary depending on the route of administration. As used herein, the term "excipient" means any compound or substance, including those that can also be referred to as "carriers" or "diluents." Preparing pharmaceutical and physiologically acceptable compositions is considered routine in the art, and thus, one of ordinary skill in the art can consult numerous authorities for guidance if needed.

The compositions disclosed herein can be administered directly to a subject.

Generally, the compositions can be suspended in a pharmaceutically acceptable carrier (e.g., physiological saline or a buffered saline solution) to facilitate their delivery. Encapsulation of the compositions in a suitable delivery vehicle (e.g., polymeric microparticles or implantable devices) may increase the efficiency of delivery.

The compositions disclosed herein can be formulated in various ways for parenteral or nonparenteral administration. Where suitable, oral formulations can take the form of tablets, pills, capsules, liquids or powders, which may be enterically coated or otherwise protected. Sustained release formulations, suspensions, elixirs, aerosols, and the like can also be used. In some aspects, the pharmaceutical compositions disclosed herein can be in the form of a liquid, foam, cream, ointment, gel, tablet or capsule.

Pharmaceutically acceptable carriers and excipients can be incorporated (e.g., water, saline, aqueous dextrose, and glycols, oils (including those of petroleum, animal, vegetable or synthetic origin), starch, cellulose, talc, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, glycerol monosterate, sodium chloride, dried skim milk, glycerol, propylene glycol, ethanol, and the like). The compositions may be subjected to conventional pharmaceutical expedients such as sterilization and may contain conventional pharmaceutical additives such as preservatives, stabilizing agents, wetting or emulsifying agents, salts for adjusting osmotic pressure, buffers, and the like. Suitable pharmaceutical carriers and their formulations are described in "Remington's Pharmaceutical Sciences" by E.W. Martin, which is herein incorporated by reference. Such compositions will, in any event, contain an effective amount of the compositions together with a suitable amount of carrier so as to prepare the proper dosage form for proper administration to the patient.

The pharmaceutical compositions as disclosed herein can be prepared for oral or parenteral administration. Pharmaceutical compositions prepared for parenteral administration include those prepared for intravenous (or intra-arterial), intramuscular, subcutaneous, intraperitoneal, transmucosal (e.g., intranasal, intravaginal, or rectal), transdermal (e.g., topical), epidural, buccal, intrathecal or sublingual administration. Aerosol inhalation can also be used. Thus, compositions can be prepared for parenteral administration that includes any of C3a receptor inhibitors, C5a receptor inhibitors, anti-C5 antibodies or C5 (or C5a) aptamers or an anti-C3 antibodies or C3 (or C3a) aptamers dissolved or suspended in an acceptable carrier, including but not limited to an aqueous carrier, such as water, buffered water, saline, buffered saline (e.g., PBS), and the like. One or more of the excipients included can help approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, detergents, and the like. Where the compositions include a solid component (as they may for oral administration), one or more of the excipients can act as a binder or filler (e.g., for the formulation of a tablet, a capsule, and the like).

The pharmaceutical compositions can be sterile and sterilized by conventional sterilization techniques or sterile filtered. Aqueous solutions can be packaged for use as is, or lyophilized, the lyophilized preparation, which is encompassed by the present disclosure, can be combined with a sterile aqueous carrier prior to administration. The pH of the

pharmaceutical compositions typically will be between 3 and 11 (e.g., between about 5 and 9) or between 6 and 8 (e.g., between about 7 and 8). The resulting compositions in solid form can be packaged in multiple single dose units, each containing a fixed amount of the above- mentioned agent or agents, such as in a sealed package of tablets or capsules.

ARTICLES OF MANUFACTURE

The composition described herein can be packaged in a suitable container labeled, for example, for use as a therapy to treat pain or pain associated with a condition or disorder or any of the methods disclosed herein. Accordingly, packaged products (e.g., sterile containers containing the composition described herein and packaged for storage, shipment, or sale at concentrated or ready-to-use concentrations) and kits, including any of the compounds or compositions as described herein and instructions for use, are also within the scope of the disclosure. A product can include a container (e.g., a vial, jar, bottle, bag, or the like) containing the composition described herein. In addition, an article of manufacture further may include, for example, packaging materials, instructions for use, syringes, buffers or other control reagents for treating or monitoring the condition for which prophylaxis or treatment is required. The product may also include a legend (e.g., a printed label or insert or other medium describing the product's use (e.g., an audio- or videotape)). The legend can be associated with the container (e.g., affixed to the container) and can describe the manner in which the compound therein should be administered (e.g., the frequency and route of administration), indications therefor, and other uses. The compounds can be ready for administration (e.g., present in dose-appropriate units), and may include a pharmaceutically acceptable adjuvant, carrier or other diluent. Alternatively, the compounds can be provided in a concentrated form with a diluent and instructions for dilution.

In an aspect, C3a receptor inhibitor and a C5a receptor inhibitor can be co-packaged. In an aspect, a C3a receptor inhibitor and a C5a antibody or a C5a aptamer can be co- packaged. In an aspect, a C5a receptor inhibitor and a C3a antibody or a C3a aptamer can be co-packaged. Any of the compositions described herein can also be co-packaged with one or more analgesics disclosed herein.

EXAMPLES

Example 1: Regulation of TRPVl Activities by a Sexually Dimorphic

Mechanism

Experiments were designed to further understand sexually dimorphic pain mechanisms. The objective is to determine the effects of serotonin (5-HT), applied to dental pulp biopsies from women versus men, on activation of capsaicin-sensitive nociceptors, and the mechanisms mediating this response. This study tests whether 5-HT preferentially releases complement peptides C3a or C5a from peripheral tissues of women compared to men, leading to a sexually dimorphic increase in TRPVl activities in trigeminal (TG) sensory neurons. Data previously collected demonstrated that 5-HT produced a sexually dimorphic difference in capsaicin activation of human peptidergic fibers. Described herein are findings showing that 5-HT released a "soluble factor" from female human tissue biopsies, but not male human biopsies (Fig. 2; and Fig. 11), that this factor significantly increased ICAP in cultured rat TG neurons (Fig. 2; and Fig. 11), that the 5-HT effect was mediated by 5-HTRs since antagonists blocked the effect (Fig. 3), that the soluble factor was a protein and not a lipid (Fig. 4), and that this soluble factor included complement C3a, as identified by 2D gel electrophoresis/mass spectrometry (Fig. 5) and confirmed using C3a receptor antagonists and anti-C3a antibody (Fig. 6). Collectively, these data suggest that 5-HT triggers a peripheral sexually dimorphic pain mechanism in women via release of complement peptides that leads to enhanced activity of capsaicin-sensitive trigeminal nociceptors. However, there is a large gap in knowledge conceming unknown cell types and mechanism(s) mediating 5-HT-evoked release of C3a and C5a, and the mechanism(s) for complement peptide sensitization of TRPV1 is not understood. Described herein are experiments and results addressing this gap.

Study #1 : Determine the effects of sex and inflammation on cellular expression patterns and release of C3a and C5a peptides in human dental pulp. Immunohistochemical methods will be used to determine the co-expression of complement peptides with markers of fibroblasts, afferent neurons, immune cells, and endothelium. Additional studies will investigate the effects of sex and inflammation on cellular expression patterns of the receptors C3aR and C5aR. Finally, the effects of sex and inflammation on tissue release of C3a and C5a will be determined by superfusion of isolated human dental pulp.

Study #2: Determine the 5-HT receptor subtype(s) and G-protein and effector signaling pathways mediating 5-HT-evoked release of C3a and C5a from female and male human pulp. The results disclosed herein demonstrate that 5-HT applied to female human dental pulp evokes the release of C3a and subsequent increase in TRPV1 activities. Given that 5-HTR antagonists block the evoked release of this excitatory factor (Fig. 3), pharmacological and molecular (knockdown) methods will be used to determine the 5-HTR subtypes and signaling mechanisms regulating peripheral release of complement peptides.

Study #3: Determine the receptor and cellular signaling mechanisms mediating C3a- and C5a-evoked increase in activities of capsaicin-sensitive trigeminal neurons.

Pharmacological antagonists and cultured TG neurons from C3aR and C5aR knockout animals will be employed to determine the cellular mechanisms by which C3a and C5a increase ICAP.

These studies will provide a new model for sexually dimorphic pain mechanisms with therapeutic implications. Human dental pulp will be used as a model tissue, in part due to the significance of orofacial pain as a health care problem (Gibbs J, Hargreaves KM (2013) In: Wall and Melzack's Textbook of Pain, 6 Edition (McMahon S, Koltzenberg MT, I., Tracey I, Turk D, eds), pp 803-214. Philadelphia, PA: Elsevier), including the prevalence of females experiencing orofacial pain (Greenspan J, Traub R (2013) In: Wall and Melzack's Textbook of Pain, 6 Edition (McMahon S, Koltzenberg MT, I., Turk D, eds), pp 221-231. Philadelphia, PA: Elsevier) The results from these studies on this model tissue may generalize to other human tissues and therefore shed light on other sexually dimorphic pain disorders. The NIH has recently recognized sex as a significant biological variable for research (NOT OD 15-102). This focus applies to the field of pain research as numerous studies have documented sex and gender differences in pain (Berkley KJ (1997) Behav Brain Sci 20:371- 380; discussion 435-513; Frankel S, Eachus J, Pearson N, Greenwood R, Chan P, Peters TJ, Donovan J, Smith GD, Dieppe P (1999) Lancet 353: 1304-1309; Stovner LJ, Zwart JA, Hagen K, Terwindt GM, Pascual J (2006) Eur J Neurol 13:333-345; Cairns BE, Gazerani P (2009) Maturitas 63:292-296; Fillingim RB, King CD, Ribeiro-Dasilva MC, Rahim-Williams B, Riley JL, 3rd (2009) J Pain 10:447-485; and Greenspan JD, Slade GD, Bair E, Dubner R, Fillingim RB, Ohrbach R, Knott C, Mulkey F, Rothwell R, Maixner W (2011) J Pain 12:T61- 74; Greenspan J, Traub R (2013) In: Wall and Melzack's Textbook of Pain, 6 Edition (McMahon S, Koltzenberg MT, I., Turk D, eds), pp 221-231. Philadelphia, PA: Elsevier). Previous results (Loyd DR, Sun XX, Locke EE, Salas MM, Hargreaves KM (2012) Pain 153:2061-2067) have demonstrated that sexually dimorphic responses to serotonin, in which 5-HT significantly increases capsaicin-evoked CGRP release from normal (un-inflamed) female human dental pulp, with no effect on male dental pulp. This effect is not due to sex differences in TRPV1 expression or CGRP content, as control experiments demonstrated no differences when capsaicin was applied without 5-HT pretreatment. Thus, human dental pulp displays a significant, sex-dependent difference in serotoninergic modulation of capsaicin- sensitive peptidergic fibers (Loyd et al, 2012, Pain 153:2061-2067).

Another recent study demonstrated that 5-HT increases capsaicin-evoked CGRP release from female human tissue, and also releases a soluble factor that significantly increases capsaicin-evoked inward currents when applied to cultured rat TG neurons (ICAP; Fig. 2). This soluble factor has no effect on inducing a significant current by itself

(conditioned media alone generates no inward current); instead, it enhances responses to capsaicin. Human dental pulp was collected from female, (left; N=8) or male (right; N=9) subjects and treated with either vehicle or 5-HT (100 μΜ). The conditioned media from pulp was collected, 5-HTR antagonists were added (see, Fig. 3) and the mixture was then applied to cultured rat TG neurons with measurement of ICAP (100 nM) by whole cell patch clamp (**P<0.001 ANOVA). Thus, the studies described herein are significant for revealing a sexually dimorphic pain mechanism involving TRPV1. This is a significant finding because of sex differences in pain, but also because the use of isolated human tissue permits studies on cellular mechanisms. Described herein is an experimental approach in which soluble factors are collected from isolated human dental pulp and then applied onto cultured rat trigeminal neurons (e.g., Figs. 2, 4, 6, 11). Fig. 1 provides a schematic illustration of the experimental designed used in Figs. 2 and 4. The combination of the methods used herein provides a powerful experimental approach wherein parallel studies can focus on 5-HT receptors and signaling pathways evoking release of the soluble factor from dental pulp, and additionally determine receptors and signaling pathways by which complement 3Ca and C5a increases ICAP in cultured TG neurons. These studies could not be conducted in an earlier model of superfused dental pulp since it is not possible to measure ICAP, and interventions such as pathway inhibitors would simultaneously affect both the pulpal tissue and the neuronal terminals, confounding interpretation of the results. Thus, the experimental approach described herein permits a focused study on receptors and signaling pathways that mediate both the release of C3a and C5a, as well as increases in ICAP. An additional benefit of this approach is based on the knowledge that serotonin has multiple functions both in the CNS and in the periphery. Moreover, serotonin has been implicated in many sexually dimorphic pain conditions including migraine, fibromyalgia, vestibulodynia and trigeminal neuralgia, and

polymorphisms of serotonin receptors, transporters or enzymes are associated with an increased risk of pain (Nicolodi M, Sicuteri F (1996) Advances in experimental medicine and biology 398:373-379; Aoki J, Hayashida M, Tagami M, Nagashima M, Fukuda K, Nishizawa D, Ogai Y, Kasai S, Ikeda K, Iwahashi K (2010) Neurosci Lett 479:40-43; Cui W, Yu X, Zhang H (2014) The j ournal of headache and pain 15 :42; and Heddini U, Bohm-Starke N, Gronbladh A, Nyberg F, Nilsson KW, Johannesson U (2014) The j ournal of sexual medicine 11 :3064-3071). Thus, it is important to use an experimental approach that isolates one aspect of serotonin physiology to avoid confounding effects of engaging both central and peripheral 5-HT receptors. The experimental designs employed in the studies described herein address this issue and permit focused evaluation of the cellular mechanisms by which peripheral serotonin evokes C3a and C5a release and enhances the activation of TRPV1 in women. Although degranulating platelets are one likely source of peripheral 5-HT release, it is worth noting that two enzymes involved in 5-HT synthesis, namely tyrosine hydroxylase and aromatic L-amino acid decarboxylase, are both expressed in peripheral tissues, including cells of the dental pulp (Nomura T, Inoue K, Creveling CR, Komatsu F, Ohta N, Chino T, Karasawa N, Nagatsu I (1996) Brain research 735:314-316; and O'Sullivan M, Tipton KF, McDevitt WE (2002) Archives of oral biology 47:399-406).

The studies described herein are focused on peripheral mechanisms for sexually dimorphic differences, namely 5-HT-evoked release of C3a and C5a and enhancement of capsaicin-sensitive nociceptors. Prior studies have implicated estrogens in modulating both complement peptide and 5-HTR expression. For example, estradiol increases expression of C3 as well as 5-HTR-1D, and -2B in cultures of human dental pulp (Inaba T, Kobayashi T, Tsutsui TW, Ogawa M, Uchida M, Tsutsui T (2013) Archives of oral biology 58:943-950). Moreover, the C3a gene has an estrogen response element (Vik DP, Amiguet P, Moffat GJ, Fey M, Amiguet-Barras F, Wetsel RA, Tack BF (1991) Biochemistry 30: 1080-1085) and estradiol drives both C3a- and C5a-evoked activities (Sundstrom SA, Komm BS, Ponce-de- Leon H, Yi Z, Teuscher C, Lyttle CR (1989)The Journal of biological chemistry 264: 16941- 16947; Harrington WR, Sheng S, Barnett DH, Petz LN, Katzenellenbogen JA,

Katzenellenbogen BS (2003) Mol Cell Endocrinol 206: 13-22; Wanda GJ, Starcke S, Zierau O, Njamen D, Richter T, Vollmer G (2007) Planta Med 73:512-518; Farkas I, Varju P, Szabo E, Hrabovszky E, OkadaN, Okada H, Liposits Z (2008) Neurochemistry international 52:846-856; and Lee YL, Cheong AW, Chow WN, Lee KF, Yeung WS (2009) Mol Reprod Dev 76:301-308). Several preclinical studies have implicated complement proteins, including C3a and C5a, in both neuropathic and inflammatory pain models (Twining CM, Sloane EM, Schoeniger DK, Milligan ED, Martin D, Marsh H, Maier SF, Watkins LR (2005) J Pain 6: 174-183; Clark JD, Qiao Y, Li X, Shi X, Angst MS, Yeomans DC (2006) Anesthesiology 104: 1274-1282; Griffin RS, Costigan M, Brenner GJ, Ma CH, Scholz J, Moss A, Allchorne AJ, Stahl GL, Woolf CJ (2007) The Journal of neuroscience : the official j oumal of the Society for Neuroscience 27:8699-8708; Li M, Peake PW, Charlesworth JA, Tracey DJ, Moalem-Taylor G (2007) The European journal of neuroscience 26:3486-3500; Levin ME, Jin JG, Ji RR, Tong J, Pomonis JD, Lavery DJ, Miller SW, Chiang LW (2008) Pain 137: 182- 201; Ting E, Guerrero AT, Cunha TM, Verri WA, Jr., Taylor SM, Woodruff TM, Cunha FQ, Ferreira SH (2008) Br J Pharmacol 153: 1043-1053; Jang JH, Clark JD, Li X, Yorek MS, Usachev YM, Brennan TJ (2010) Pain 148:343-352; Ren K, Dubner R (2010) Nat Med 16: 1267-1276; Jang JH, Liang D, Kido K, Sun Y, Clark DJ, Brennan TJ (2011) Journal of neuroinflammation 8:80; LaCroix-Fralish ML, Austin JS, Zheng FY, Levitin DJ, Mogil JS (2011) Pain 152: 1888-1898; Liang DY, Li X, Shi X, Sun Y, Sahbaie P, Li WW, Clark JD (2012) Pain 153:366-372; and Moriconi A et al. (2014) Proceedings of the National Academy of Sciences of the United States of America 111 : 16937-16942). However, no studies have focused on the complement proteins in human pain mechanisms; nor have any of these preclinical studies revealed a sexually dimorphic pain mechanism.

Prior studies have established that 5-HT has direct effects on TG neurons Loyd DR, Weiss G, Henry MA, Hargreaves KM (2011) Pain 152:2267-2276; and Loyd DR, Henry MA, Hargreaves KM (2013) Seminars in cell & developmental biology 24:51-57). Therefore, it is important to add 5-HTR antagonists to the collected media before neuronal exposure in order to ensure that the measured response is due to release of a soluble factor, rather than the effect of residual 5-HT. Fig. 3 indicates that the ability of 5-HT to enhance capsaicin-induced release of CGRP is abolished by adding a mixture of antagonists to the 5-HT2, 5-HT4, 5-HT6 and 5-HT7 receptor subtypes (100 nM of ritanserin, GR113808, SB258585 and SB269970). This finding is replicated in the patch clamp studies (Fig. 2 and Fig. 11), where the same 5- HTR antagonists were added to the conditioned media before performing electrophysiologic recordings. Fig. 2 (and Fig. 11) indicates that 5-HT applied to female dental pulp increases La with no effect observed when 5-HT is applied to male dental pulp; if the effect were due to residual 5-HT, then the 5-HT/male group would have had similar effects as the 5- HT/female group. Experiments in Study 2 will determine which of the 5-HTR subtypes mediate serotonin-evoked release of this soluble factor.

It is possible that this soluble factor is either a protein or a lipid. To distinguish between these two alternatives, 5-HT-conditioned media from female dental pulp was incubated with either proteinase K, a broad-spectrum serine protease (Kraus E, Femfert U (1976) PHoppe-Seyler's Zeitschrift fur physiologische Chemie 357:937-947), or

Cleanascite™, a non-ionic polymeric matrix that selectively removes lipids without affecting proteins (Antunes RF, Brandao C, Maia M, Arosa FA (2011) Immunology and cell biology 89: 111-121). As indicated in Fig. 4, the 5-HT-evoked soluble factor is a protein as its activity was abolished by incubation with proteinase K, with no effect of Cleanascite™. For this study, the determination of protein versus lipid structure for substance released from female dental pulp. Dental pulp from female participants was treated with vehicle or 5-HT (100 nM). The 5-HT group underwent either no further treatment or was incubated with proteinase K (1 mg/ml X 2 hr; terminated by addition of mini proteinase inhibitior table (Roche) or had removal of lipids by additional of Cleanascite™ (Biotech Support Group, LLC)). Samples were then applied to cultured TG neurons with measurement of 100 nM ICAP (**p<0.01 ; ANOVA: F=6.9; P<0.005; N=9/group). The TG neurons in this experiment and in Fig. 2 were maintained in whole cell patch for 15 minutes before recording ICAP to ensure no inward currents due to cultured media in subsequent patch experiments (Figs. 6, 7). ICAP responses were obtained immediately after entering whole cell configuration.

Next, quantitative 2D DIGE (Difference Gel Electrophoresis) was combined with mass spectrometry for a proteomics analysis of conditioned media collected after 5-HT treatment to female and male human pulp. 2D DIGE has the advantages of simultaneously analyzing two samples in one gel by labeling proteins with one of two fluorescent dyes, and contains internal standards permitting quantitative measurements of protein levels (Lilley KS, Friedman DB (2004) Expert review of proteomics 1 :401-409; Rukmangadachar LA, Kataria J, Hariprasad G, Samantaray JC, Srinivasan A (201 1) Clinical proteomics 8:4; May C, Brosseron F, Chartowski P, Meyer HE, Marcus K (2012) Methods Mol Biol 893 :75-82; and Bi F, Huang C, Tong J, Qiu G, Huang B, Wu Q, Li F, Xu Z, Bowser R, Xia XG, Zhou H (2013) Proceedings of the National Academy of Sciences of the United States of America 110:4069-4074). Fig. 5 presents the results of the 2D DIGE analysis. Dental pulp was treated with 5-HT (100 nM) and two pools were made from N=6 female and N=6 male participants. Primary amino groups on proteins were labeled with CyDye DIGE fluors coupled to N-hydroxy succinimidyl esters, subjected to DIGE and regions of interest (ROI) were visualized with a Typhoon Scanner. ROI that were >2-fold greater in female samples were collected with an Ettan automated spot picker and identified by Mass

Spectrometry /MALDI TOF. Mass spectrometry data was then performed on all regions of interest (ROI) with >2-fold (p<0.05) increased levels in the female versus male samples. A literature search was conducted on all identified proteins and a single publication was identified that was associated with pain: complement C3a. Interestingly, prior studies in rats demonstrate that C3a (and C5a) activated and sensitized nociceptors (Jang JH, Clark JD, Li X, Yorek MS, Usachev YM, Brennan TJ (2010) Pain 148:343-352; and Jang JH, Liang D, Kido K, Sun Y, Clark DJ, Brennan TJ (2011) Journal of neuroinflammation 8: 80). Although the 2D DIGE proteomic analysis detected C3a, both C3a and C5a will be studied since both peptides activate nociceptors and inflammation increases the expression and release of both peptides (Fosse E, Mollnes TE, Ingvaldsen B (1987) J Thorac Cardiovasc Surg 93: 860-866; Zhang J, Koh J, Lu J, Thiel S, Leong BS, Sethi S, He CY, Ho B, Ding JL (2009) PLoS Pathog 5:el000282; and Chimenti MS, Perricone C, Graceffa D, Di Muzio G, Ballanti E, Guarino MD, Conigliaro P, Greco E, Kroegler B, Perricone R (2012) Clin Exp Rheumatol 30:23-30). While the liver is a primary site for synthesis of complement proteins, many other cell types including fibroblasts, macrophages, and endothelial cells are now recognized to secrete various complement-related proteins (Li K, Sacks SH, Zhou W (2007) Mol Immunol 44:3866-3874; and Gene-Cards (2015) Complement C3. http://www.genecards.org/cgi- bin/carddisp.pl?gene=C3&keywords=complement#protein_expr ession). Moreover, the C3a gene has an estrogen response element (Vik DP, Amiguet P, Moffat GJ, Fey M, Amiguet- Barras F, Wetsel RA, Tack BF (1991) Biochemistry 30: 1080-1085) and estradiol drives both C3a- and C5a-evoked activities (Sundstrom SA, Komm BS, Ponce-de-Leon H, Yi Z, Teuscher C, Lyttle CR (1989) The Journal of biological chemistry 264: 16941-16947;

Harrington WR, Sheng S, Bamett DH, Petz LN, Katzenellenbogen JA, Katzenellenbogen BS (2003) Mol Cell Endocrinol 206: 13-22; Wanda GJ, Starcke S, Zierau O, Njamen D, Richter T, Vollmer G (2007) Planta Med 73:512-518; Farkas I, Varju P, Szabo E, Hrabovszky E, Okada N, Okada H, Liposits Z (2008) Neurochemistry international 52:846-856; and Lee YL, Cheong AW, Chow WN, Lee KF, Yeung WS (2009) Mol Reprod Dev 76:301-308), suggesting that these peptides function in a sexually dimorphic fashion. Experiments described in Study 1 will identify the cell types expressing C3a and C5a.

Next, whether the active factor released by addition of 5-HT to female dental pulp contains complement C3a was tested. First, the effect of 5-HT on released C3a was measured using ELISA assay (Abeam C3a Elisa Kit abl33037). 5-HT evoked a three-fold (p<0.01) increase in C3a released from female human dental pulp as compared to male dental pulp. Second, 5-HT conditioned media from female dental pulp was treated with either SB290157, a receptor antagonist to the C3aR (Ames RS, Nuthulaganti P, Kumar C (1996) FEBS Lett 395: 157-159), or with monoclonal anti-C3a antibody (BioLegend (2015) LEAF™ Purified anti-human C3a/C3a(desArg)/C3 Antibody. In.) and then the effect on La was evaluated in cultured TG neurons. Fig. 6 indicates that both interventions significantly blocked the effect of 5-HT. Additional control experiments (Bars #4 and #6 in Fig. 6) verified that these two interventions had no effect on I _cap by themselves. Since SB290157 has been reported to have agonist-like actions in some assays (Mathieu MC, Sawyer N, Greig GM, Hamel M, Kargman S, Ducharme Y, Lau CK, Friesen RW, O'Neill GP, Gervais FG, Therien AG (2005) Immunology letters 100: 139-145), it is important to note that both the antagonist and the monoclonal antibody produced maximal inhibitory effects. Taken together, these data indicate that 5-HT evokes the release of C3a from female human dental pulp and that C3a significantly increase I _cap in TG neurons under basal conditions. For these experiments, female human dental pulp (N=6-10/group) were treated with vehicle or 5-HT (100 nM), with subsequent addition of 5-HT receptor antagonists (see, Fig. 3) as well as SB290157 (2 μΜ) or an anti-C3a antibody (4 μg/ml at 2 h) and then applied to cultured TG neurons with measurement of 100 nM ICAP (ANOVA: F=7.5; P<0.0005).

Study 2 will focus on 5-HT receptors, G-proteins and cell signaling pathways mediating the release of C3a and C5a from female human tissues. Pharmacological and molecular approaches will be used for these studies. Acutely superfused tissue biopsies as well as primary cultures of dental pulp will be studied. Prior studies have shown that cultured human dental pulp expresses transcripts for C3, C5 as well all 7 5-HTR subtypes and that estradiol up-regulates C3, C5, as well as 5HT-1D and -2B (Inaba T, Kobayashi T, Tsutsui TW, Ogawa M, Uchida M, Tsutsui T (2013) Archives of oral biology 58:943-950).

Accordingly, it will determined whether 5HT evokes the release of a sensitizing factor from cultured female human dental pulp. Female human dental pulp was cultured for one week and then treated with either vehicle or 5-HT (100 nM). Dental pulp was collected from a female participant, cultured for 1 week and then conditioned media was collected after treatment with vehicle (Veh), or 5-HT 100 nM. The 5-HT group was then incubated with normal goat serum or anti-C3a antibody with subsequent addition of 5-HT receptor antagonists. Samples were then applied to cultured TG neurons with measurement of 100 nM ICAP. "Media" refers to culture media alone (no exposure to dental pulp; ANOVA: F=7.12; P<0.0001). As seen in Fig. 7, 5-HT evokes the release of a soluble factor from cultured dental pulp and its effects on leap are significantly reduced with anti-C3a immunoneutralization. In addition, ELISA indicates that 5-HT evokes a 4-fold increase in C3a from cultured female dental pulp

(p<0.01) and western immunoblots reveal that these human dental pulp cultures express C3 protein (Fig. 9). Collectively, these studies provide independent and converging lines of evidence indicating that 5-HT evokes the release of complement peptides from female human tissues that then increases TRPV1 activities.

As described herein, these studies evaluate whether 5-HT preferentially releases complement peptides C3a or C5a from peripheral tissues of women compared to men, leading to a sexually dimorphic increase in TRPV1 activities in trigeminal (TG) sensory neurons.

Study #1 will determine the effects of sex and inflammation on cellular expression patterns and release of C3a and C5a peptides in human dental pulp. Given that the C3a gene has an estrogen response element (Vik DP, Amiguet P, Moffat GJ, Fey M, Amiguet-Barras F, Wetsel RA, Tack BF (1991) Biochemistry 30: 1080-1085) and estradiol drives C3a- and C5a- evoked activities (Sundstrom SA, Komm BS, Ponce-de-Leon H, Yi Z, Teuscher C, Lyttle CR (1989) The Journal of biological chemistry 264: 16941-16947; Harrington WR, Sheng S, Barnett DH, Petz LN, Katzenellenbogen JA, Katzenellenbogen BS (2003) Mol Cell

Endocrinol 206: 13-22; Wanda GJ, Starcke S, Zierau O, Njamen D, Richter T, Vollmer G (2007) Planta Med 73:512-518; Farkas I, Varju P, Szabo E, Hrabovszky E, Okada N, Okada H, Liposits Z (2008) Neurochemistry international 52:846-856; and Lee YL, Cheong AW, Chow WN, Lee KF, Yeung WS (2009) Mol Reprod Dev 76:301-308) as well as expression in human dental pulp (Inaba T, Kobayashi T, Tsutsui TW, Ogawa M, Uchida M, Tsutsui T (2013) Archives of oral biology 58:943-950), Studies 1 and 2 will include secondary analyses to determine whether days since last menses is associated with alterations in experimental outcomes. Since complement peptides are upregulated in inflammatory conditions, these studies will also evaluate the effects of inflammation (irreversible pulpitis) on expression and release of C3a and C5a from these tissues.

Experiment 1.1: Determine effects of sex and inflammation on the cell types expressing the C3a and C5a peptides and C3aR and C5aR in human dental pulp.

Data disclosed herein identified complement C3a being released from female dental pulp by both 2D DIGE and ELISA. These experiments do not identify the cell source of complement peptides, leaving a gap in knowledge to understand this sexually dimorphic pain mechanism. To address this gap, double immunohistochemical labeling methods will be used to identify the co-expression of C3a, C5a, C3aR or C5aR, with cell type-specific markers. Again, C5a/C5aR will be included in these initial studies as this system activates nociceptors and is increased during inflammation (Twining CM, Sloane EM, Schoeniger DK, Milligan ED, Martin D, Marsh H, Maier SF, Watkins LR (2005) J Pain 6: 174-183; Jang JH, Clark JD, Li X, Yorek MS, Usachev YM, Brennan TJ (2010) Pain 148:343-352; and Jang JH, Liang D, Kido K, Sun Y, Clark DJ, Brennan TJ (2011) Journal of neuroinflammation 8:80). Fig. 8 provides support for this Study as C3a and C3aR are expressed in both normal and inflamed human female dental pulp. Importantly, these images also demonstrate that both C3a and C3aR are increased with inflammation, and that the C3aR was co-expressed with NFH on afferent neuronal terminals in female human dental pulp (Fig. 8).

Healthy female and male participants (age 18-45; mean age = 22.4) with normal or inflamed teeth will be used. Normal teeth will be fully developed third molars. Inflamed teeth will have a clinical diagnosis of symptomatic irreversible pulpitis. This bacterial-induced inflammatory condition is painful (Jena A, Shashirekha G (2013) J Conserv Dent 16: 171- 174; and Rogers BS, Botero TM, McDonald NJ, Gardner RJ, Peters MC (2014) J Endod 40:753-75) and is associated with a massive influx of immune cells (Ruparel S, Hargreaves KM, Eskander M, Rowan S, de Almeida JF, Roman L, Henry MA (2013) Pain 154:2363- 2371). Symptomatic irreversible pulpitis will be diagnosed using standard methods (Levin LG, Law AS, Holland GR, Abbott PV, Roda RS (2009) J Endod 35: 1645-1657). Dental pulp will be collected, fixed, sectioned and stained as described (Henry MA, Freking AR, Johnson LR, Levinson SR (2006) Pain 124:222-233; Alvarado LT, Perry GM, Hargreaves KM, Henry MA (2007) J Endod 33: 1167-1171 ; Luo S, Perry GM, Levinson SR, Henry MA (2008) Molecular pain 4: 16; Henry MA, Luo S, Foley BD, Rzasa RS, Johnson LR, Levinson SR (2009) J Pain 10:750-758; Luo S, Perry GM, Levinson SR, Henry MA (2010) Neuroscience 169: 1881-1887; and Henry MA, Luo S, Levinson SR (2012) BMC Neurosci 13:29). The confocal expression patterns present within these images will be quantified with objective measures of area and the intensity of the immunofluorescence signal as determined with NIH ImageJ image analysis software. The N=40 teeth, with n=10 each for the male and female groups under either normal or inflamed conditions.

Table 1. Antibody (all are anti -human)

*allows double label with C3aR

#C3a & C3aR Abs made in rabbit to be used in double-labeling experiments with cell specific antibodies made in mouse to determine expressions in specific cell types

The neurofilament heavy (NFH)/nerve fiber analysis will determine the average pixel intensity and percent nerve fiber area showing C3a, C3aR, C5a, and C5aR expression, whereas the use of cell specific antibodies will determine C3a/C3aR and C5a/C5aR expression in each cell type. Univariate statistics of the entire sample population (e.g., normal/inflamed; female/male) will be calculated with statistical analyses conducted. The "n" values equal the number of patients evaluated (1 tooth/patient) and a Bonferroni correction for multiple t-tests within any one experiment will be performed. Each pulp specimen typically generates 40 different 30 urn sections; a number that allows completion of all staining combinations outlined in Table 1.

This study establishes an important foundation for understanding the cellular origin of C3a and C5a peptides and their receptors in human tissues and sets the stage for research on mechanisms regulating their release. It is expected that inflammation will lead to an increase in the expression of C3a and C5a as they are expressed by leukocytes and human dental pulp fibroblasts (Chmilewsky F, Jeanneau C, Laurent P, About I (2014) Am J Pathol 184: 1991- 2000; and Arreola R, Becerril-Villanueva E, Cruz-Fuentes C, Velasco-Velazquez MA, Garces-Alvarez ME, Hurtado-Alvarado G, Quintero-Fabian S, Pavon L (2015) J Immunol Res 2015:354957). Moreover, as estrogens increase C3a and C5a activities (Harrington WR, Sheng S, Bamett DH, Petz LN, Katzenellenbogen JA, Katzenellenbogen BS (2003) Mol Cell Endocrinol 206: 13-22; Wanda GJ, Starcke S, Zierau O, Njamen D, Richter T, Vollmer G (2007) Planta Med 73:512-518; and Lee YL, Cheong AW, Chow WN, Lee KF, Yeung WS (2009) Mol Reprod Dev 76:301-308), it is expected that the level of C3a and C5a expression will be greater in female human tissues than male tissues.

Experiment 1.2: Determine the effects of sex and inflammation on 5-HT-evoked release of C3a and C5a. Prior clinical studies (and Fig. 8) have demonstrated increased expression of C3a and/or C5a in inflammatory conditions such as surgery, arthritis and bacterial infection (Fosse E, Mollnes TE, Ingvaldsen B (1987) J Thorac Cardiovasc Surg 93:860-866; Zhang J, Koh J, Lu J, Thiel S, Leong BS, Sethi S, He CY, Ho B, Ding JL (2009) PLoS Pathog 5:el000282; and Chimenti MS, Perricone C, Graceffa D, Di Muzio G, Ballanti E, Guarino MD, Conigliaro P, Greco E, Kroegler B, Perricone R (2012) Clin Exp Rheumatol 30:23-30). However, there is a gap in knowledge regarding whether 5-HT-evoked release of C3a and C5a are altered based on patient sex or a pre-existing painful inflammatory condition.

The dental pulp superfusion method (Fehrenbacher JC, Sun XX, Locke EE, Henry MA, Hargreaves KM (2009) Pain 144:253-261; and Loyd DR, Sun XX, Locke EE, Salas MM, Hargreaves KM (2012b) Pain 153:2061-2067) will be used to determine whether 5-HT- evoked release of C3a and C5a is increased in female versus male patients from control or inflamed human dental pulp. Control (non-inflamed, normal pain responsiveness) dental pulp will collected from fully developed third molars. Inflamed human dental pulp will be collected from teeth with a diagnosis of symptomatic irreversible pulpitis. The pulp will be isolated within 10 min of extraction and, following a 20 min washout, will be treated with either 5-HT (100 nM) or vehicle. To minimize loss of 5-HT activity, non-selective blockers of the serotonin-, norepinephrine- and dopamine-transporters (DOV216303 100 nM and DOV21947 300 nM) as well as a MAO inhibitor (pargyline 2uM) will be added to the buffer (Lengyel K, Pieschl R, Strong T, Molski T, Mattson G, Lodge NJ, Li YW (2008)

Neuropharmacology 55:63-70; and Vavilova T, Ostrovskaya I, Axenova L, Buneeva O, Medvedev A (2009) Med Sci Monit 15:BR289-292). C3a and C5a will be measured by ELISA (Abeam Elisa Kits, C3a: abl33037; C5a: abl93695) from the same superfusates.

Data will be analyzed by a three-way ANOVA: (Pulpitis) X (5-HT) X (Sex) with Bonferroni post-hoc tests. A sample size of n=24/group will generate an 80% power at 25% treatment effect (increased variance due to inflammatory state led to increased sample size compared to our prior studies on healthy control teeth (e.g., Fig. 2)). Although tissue levels of C3a and C5a are increased with inflammation, no study has determined whether this alters their release by 5-HT. It is expected that an inflammatory state will lead to increased 5-HT- evoked release of C3a and C5a. Since estrogens increase C3a and C5a activities (Harrington WR, Sheng S, Bamett DH, Petz LN, Katzenellenbogen JA, Katzenellenbogen BS (2003) Mol Cell Endocrinol 206: 13-22; Wanda GJ, Starcke S, Zierau O, Njamen D, Richter T, Vollmer G (2007) Planta Med 73:512-518; and Lee YL, Cheong AW, Chow WN, Lee KF, Yeung WS (2009) Mol Reprod Dev 76:301-308), it is also expected that the 5HT-evoked release of C3a and C5a will be greater in female human tissues than male tissues.

Male dental pulp will be included in these studies for comparative purposes and to explore whether inflammation alters the sexually dimorphic effect of 5-HT on evoked release of C3a and C5a. The concentration of 5-HT was selected to be >100 times its Kd in order to saturate 5-HT receptors (Kenakin T (1997) Pharmacologic analysis of drug-receptor interaction, 3 Edition. New York: Raven Press). Although the studies described herein have focused on C3a, due in part to the 2D DIGE findings, C5a will also be included in Study 1 due to the known upregulation of complement peptides during inflammation and to the well- recognized effects of both C3a and C5a on increasing primary afferent nociceptor activities.

Study #2will determine the 5-HT receptor subtype(s) and G-protein and effector signaling pathways that mediate 5-HT-evoked release of C3a and C5a from female and male human tissues.

Experiment 2.1: Determine the effects of sex and inflammation on cellular co- expression patterns of 5-HTR subtypes with C3a and C5a in human dental pulp.

Experiments 1.1 and 1.2 will characterize C3a and C5a expression in female and male human dental pulp under control and inflamed conditions. Immunohistochemical methods will be used to determine which 5-HTR subtypes are co-expressed with C3a and C5a peptides.

The methods and statistical analyses described in Experiment 1.1 will be used along with the anti-5-HTR antibodies listed in column 2 of Table 2. It is expected that

inflammation will lead to an increase in the co-expression of 5-HTR subtypes with C3a and C5a since they are expressed by leukocytes and human dental pulp fibroblasts (Chmilewsky F, Jeanneau C, Laurent P, About I (2014) Am J Pathol 184: 1991-2000; and Arreola R, Becerril-Villanueva E, Cruz-Fuentes C, Velasco-Velazquez MA, Garces-Alvarez ME, Hurtado-Alvarado G, Quintero-Fabian S, Pavon L (2015) J Immunol Res 2015:354957). Moreover, as estrogens increase C3a and C5a activities (Harrington WR, Sheng S, Barnett DH, Petz LN, Katzenellenbogen JA, Katzenellenbogen BS (2003) Mol Cell Endocrinol 206: 13-22; Wanda GJ, Starcke S, Zierau O, Njamen D, Richter T, Vollmer G (2007) Planta Med 73:512-518; Lee YL, Cheong AW, Chow WN, Lee KF, Yeung WS (2009) Mol Reprod Dev 76:301-308; and Inaba T, Kobayashi T, Tsutsui TW, Ogawa M, Uchida M, Tsutsui T (2013) Archives of oral biology 58:943-950) and increase expression of 5-HTR1D and 2B in cultured human dental pulp (Inaba T, Kobayashi T, Tsutsui TW, Ogawa M, Uchida M, Tsutsui T (2013) Archives of oral biology 58:943-950), it is expected that the 5-HTR and C3a and C5a co-expression will be greater in female tissues than male tissues.

Experiment 2.2: Determine 5-HT receptor subtypes mediating the evoked release of C3a and C5a by employing 5-HT receptor antagonists in primary cultures of female and male human dental pulp.

5-HT is known to activate at least 7 classes of receptors (Yun HM, Rhim H (2011) Experimental neurobiology 20: 159-168; and IUPHAR (2015) Guide to Pharmacology. 5- Hydroxytryptamine receptors.

http://www.guidetopharmacology.org/GRAC/FamilyDisplayForw ard?familyId=l. In.), and subtypes of all 7 classes of receptors have been identified in both human sensory neurons (Pierce PA, Xie GX, Meuser T, Peroutka SJ (1997) Neuroscience 81 :813-819; and

National. Ctr. Biotech. Inf. (2015) Gene Expression Omnibus.

http://www.ncbi. nlm.nih.gov/geo/geo2r/?acc=GSE44677. In.) and human dental pulp (Inaba T, Kobayashi T, Tsutsui TW, Ogawa M, Uchida M, Tsutsui T (2013) Archives of oral biology 58:943-950). Moreover, prior studies have demonstrated that cultured human dental pulp fibroblasts express C3a and C5a (Inaba T, Kobayashi T, Tsutsui TW, Ogawa M, Uchida M, Tsutsui T (2013) Archives of oral biology 58:943-950; Chmilewsky F, Jeanneau C, Laurent P, About I (2014) Am J Pathol 184: 1991-2000; and Chmilewsky F, Jeanneau C, Laurent P, About I (2015) J Dent Res 94: 166-174). For this experiment, selective 5HTR antagonists (Table 2) will be used to evaluate their effect on the EC50 of 5-HT for evoking release of C3a and C5a from primary cultures of dental pulp. All 5-HT receptor subtypes can be studies given their potential for complex interactions on 5-HT pharmacology when expressed in the same cells (Wada K, Hu L, Mores N, Navarro CE, Fuda H, Krsmanovic LZ, Catt KJ (2006) Molecular endocrinology 20: 125-135).

Primary cultures of female and male dental pulp will be used to determine whether antagonists to 5-HTR subtypes block the evoked release of C3a and C5a. Dental pulp will be isolated within 10 min of extraction, enzymatically dispersed and cultured in 24 well plates. One week later, cultures will be treated with either vehicle, 5-HT (0.1-1,000 nM) or a combination of 5-HT (0.1-1,000 nM) together with a selective antagonist (Table 2) to 5- HT1A, 5-HT1B, 5-HT1D, 5-HT2A, 5-HT2B, 5-HT2C, 5-HT3, 5-HT4, 5-HT6 and 5-HT7 receptors. Antagonists were selected based on pKi and known selectivities (IUPHAR (2015) Guide to Pharmacology. 5-Hydroxytryptamine receptors.

http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward ?familyId=l. In.) and will be used at a concentration of 30 X Ki (pKi values are presented in parentheses of column 4 in Table 2). To minimize loss of 5-HT activity, non-selective blockers of the serotonin-, norepinephrine- and dopamine-transporters (DOV216303, 100 nM and DOV21947 300, nM) as well as a MAO inhibitor (pargyline, 2uM) will be added to the buffer (Lengyel K, Pieschl R, Strong T, Molski T, Mattson G, Lodge NJ, Li YW (2008) Neuropharmacology 55:63-70; and Vavilova T, Ostrovskaya I, Axenova L, Buneeva O, Medvedev A (2009) Med Sci Monit 15:BR289-292). C3a and C5a will be measured by ELISA (Abcam.com).

Data will be analyzed by non-linear regression for determination of EC50 (GraphPad Prism). Control experiments will verify that the antagonists alone have no effect on C3a or C5a release. There will be a minimum of three replicates per group and three independent repeats of the experiment. A 5-HTR subtype mediates 5-HT-evoked release of C3a or C5a, if the EC50 for the 5-HT effect is increased significantly in the presence of a given antagonist. If more than one antagonist has a significant inhibitory effect on 5-HT-evoked release combinations of the antagonists can be used.

An important translational value of this antagonist study is that it identifies interventions that may have potential therapeutic benefit in treating sexually dimorphic pain disorders. To maximize translational implications, Experiment 2.3 will be carried out confirm these effects using a 5-HTR selective agonist + selective antagonist in acutely isolated superfused female dental pulp. Released C3a and C5a will be measured from the same cultures, which increases experimental efficiency.

Table 2. 5-HT Receptor Subtypes and Primary Signaling Pathways. Adapted from (Chang et al., Sci STKE 2000:pll ; Bockaert et al., 2006, Cell Tissue Res 326:553-572; Yun and Rhim, 2001,

Experimental neurobiology 20: 159-168; and IUPHAR, 2015, Guide to Pharmacology. 5- Hydroxytryptamine receptors.)

PKA (PKI 14- 22 amide, myristoylated) (Tocris, 2015)

Experiment 2.3: Determine the effects of sex and inflammation on 5-HT receptor subtypes mediating the evoked release of C3a and C5a from acutely isolated and superfused human dental pulp.

This experiment will determine whether the 5-HTR subtype identified as mediating C3a or C5a release under cell culture conditions also mediates 5-HT effects in acutely superfused female dental pulp Selective receptor agonists will be studied at a single fixed concentration (30 X Kd) alone, and in combination with its corresponding selective

antagonist at a fixed concentration (30 X Ki).

Dental pulp superfusion method (Fehrenbacher JC, Sun XX, Locke EE, Henry MA, Hargreaves KM (2009) Pain 144:253-261; and Loyd DR, Chen PB, Hargreaves KM (2012) Neuroscience 203:207-215) will be used to identify which 5HTR subtypes mediate the evoked release of C3a or C5a in female and male, normal or inflamed, dental pulp. The pulp will be isolated within 10 min of extraction and, following a 20 min washout, will be treated with either vehicle, 5-HT (100 nM), or 1 of the 11 selective 5-HTR agonists listed in column 3 of Table 2. Follow-up studies will focus on those agonists able to evoke C3a or C5a release; these studies will evaluate the effects of co-administration of the agonist together with the corresponding 5-HTR antagonist (Table 2, column 4). Complement C3a and C5a will be measured by ELISA from the same superfused samples.

Data will be analyzed by a three-way ANOVA (Sex X Inflammation X Drug) with Bonferroni's post-hoc test. Control experiments will verify that the antagonists alone have no effect on C3a or C5a release. A given 5-HTR subtype will be determined to mediate the 5-HT effect if the selective agonist significantly evokes release of C3a and the effects are blocked by co-administration of its corresponding antagonist; similar requirements will be applied for interpreting 5-HTR regulation of C5a release. These results will be compared to those obtained under cell culture conditions.

The results from superfused dental pulp (Experiment 2.3) will be compared to primary cultures of dental pulp (Experiment 2.2). If differences are observed, it is likely due to differences in cellular composition (cultures do not express neurons or immune cells). This possibility can then be evaluated using FACS of acutely dispersed human dental pulp cells to identify cell types mediating this effect.

Experiment 2.4: Determine 5-HT receptor subtypes mediating the evoked release of C3a and C5a by molecular suppression (knockdown) of 5-HTR subtypes in primary cultures of dental pulp.

Experiments 2.2 and 2.3 will use pharmacological methods to explore 5-HTR subtypes mediating the evoked release of C3a or C5a. While this approach has considerable translational significance, siNRA knockdown, which provides an independent and selective intervention for will be used to confirm these findings. Accordingly, siRNA knockdown will be employed to more precisely identify which 5-HTR subtypes mediate the evoked release of C3a or C5a. This experiment will follow Experiments 2.2 and 2.3 to be able to focus on those receptor subtypes implicated in mediating the effect.

The focus of this experiment is directed to those 5-HTR subtypes which were implicated in Experiments 2.2 and 2.3. If all 5-HTR receptor antagonists are active in Experiment 2.2 and Experiment 2.3, then this would include 11 siRNAs against 5-HT1A, 5- HT1B, 5-HT1D, 5-HT1F, 5-HT2A, 5-HT2B, 5-HT2C, 5-HT3, 5-HT4, 5-HT6 and 5-HT7 as well as a scrambled siRNA control. In brief, Dharmicon Accell™ siRNA that employs passive diffusion as a delivery method and excludes the use of lipid- or viral-based delivery systems (Dharmacon TS (2010) Thermo Scientific Dharmacon Accell siRNA Delivery Protocol. http://dharmacon.gelifesciences.com/uploadedFiles/Resources/ accell-delivery- protocol.pdf. In) will be used. Accell™ siRNA exhibits high knockdown efficiency in primary cultures (Larsen HO, Roug AS, Nielsen K, Sondergaard CS, Hokland P (2011) Experimental hematology 39: 1081-1089), without triggering expression of innate immune proteins such as complement (Strezoska Z, Yamada C (2010) Accell™ siRNA Reagents: Achieving Long-term Gene Silencing.

http://dharmacon.gelifesciences.com/uploadedFiles/Resourc es/accell-app-gene-silencing- appnote.pdf. In). Accell siRNA (1 μΜ) targeted to each human 5-HTR subtype (Raymond J, Turner J, Gelasco A, Ayiku H, Coaxum S, Arthur J, Garnovskaya M (2008) 5HT Receptor Signal Transduction Pathways. In: The Receptors: The Serotonin Receptors: From Molecular Pharmacology to Human Therapeutics (Roth B, ed), pp 143-206. Totowa, NJ: Humana Press) or scrambled control siRNA will be added to primary cultures of female or male dental pulp in the presence of Accell delivery media for 72-96hr (Strezoska Z, Yamada C (2010) Accell™ siRNA Reagents: Achieving Long-term Gene Silencing.

http://dharmacon.gelifesciences.com/uploadedFiles/Resourc es/accell-app-gene-silencing- appnote.pdf. In). Fig. 9 illustrates the effectiveness of the Accell siRNA approach and provides additional evidence for the expression of C3 protein in the primary cultures of female human dental pulp. Western blot analysis was done using C3 primary antibody (C3 antibody H-300; Santa Cruz, Dallas, TX) at 1 :5000 concentration. Densitometric analysis was done using ImageJ software (*p<0.05; ANOVA with Dunnett's post hoc comparisions; n=2/group). Using methods previously described (Loyd DR, Sun XX, Locke EE, Salas MM, Hargreaves KM (2012) Pain 153:2061-2067), WB of cell lysates will be used to document 5- HTR subtype expression in cultured female and male dental pulp using anti-5-HTR antibodies (column 2 of Table 2). Next, it will be evaluated whether 5-HTR knockdown alters the effects of 5-HT (100 nM) for evoking release of C3a and C5a as measured by ELISA. To minimize loss of 5-HT activity, non-selective blockers of the serotonin-, norepinephrine- and dopamine-transporters (DOV216303, 100 nM and DOV21947, 300 nM) as well as a MAO inhibitor (pargyline, 2μΜ) will be added to the buffer (Lengyel K, Pieschl R, Strong T, Molski T, Mattson G, Lodge NJ, Li YW (2008) Neuropharmacology 55:63-70; and Vavilova T, Ostrovskaya I, Axenova L, Buneeva O, Medvedev A (2009) Med Sci Monit 15:BR289- 292). Data will be analyzed by two-way ANOVA: (Sex) X (5HT) X (11 siRNA + 1 scrambled siRNA control) with Bonferroni post-hoc tests. There will be a minimum of three replicates per group and three independent repeats of the experiment. C3a and C5a release will be measured from the same cultures.

Selectivity of each knockdown target will be evaluated by conducting WB analyses using antibodies against all 5-HTR subtypes. If off-target effects are observed (e.g., suppression of 5-HT2A with siRNA designed against 5-HTR4), then alternative siRNA sequences will be tested. It is possible that the 5-HT effect might be mediated by more than one receptor. Therefore, if any knockdown partially reduces the 5-HT-evoked release of C3a, then combinations of effective siRNAs will be evaluated. NMale dental pulp will be included in these studies for both comparative purposes and to explore whether culturing conditions alters the sexually dimorphic effect of 5-HT on evoked release of C3a and C5a. Alternative approaches such as CRISPR/Cas9 or morpholino technologies can also be used. The Accell siRNA was selected due to its efficiency in primary cultures and absence of triggering upregulation of innate immune proteins such as complement. The combined use of siRNA knockdown (Experiment 2.4) and 5-HTR antagonists (Experiments 2.2 and 2.3) provide strong independent tests to determine which 5-HTR subtype(s) mediates the evoked release of C3a and C5a. If a disparity is observed (e.g., 5-HT2A antagonist blocks 5-HT in cultures, but the siRNA for 5-HT2A has no effect), then 1) additional antagonists and siRNA will be included; and 2) whether the antagonist retains ability to inhibit 5-HT in cultures previously treated with the corresponding siRNA will be evaluated. If the antagonist is still active under conditions where the receptor has reduced expression, then it will be concluded that the antagonist lacks sufficient selectivity.

Experiment 2.5: Identify the G-protein mediating 5-HT-evoked release of C3a and C5a from superfused dental pulp.

As recently reviewed (Raymond J, Turner J, Gelasco A, Ayiku H, Coaxum S, Arthur J, Garnovskaya M (2008) 5HT Receptor Signal Transduction Pathways. In: The Receptors: The Serotonin Receptors: From Molecular Pharmacology to Human Therapeutics (Roth B, ed), pp 143-206. Totowa, NJ: Humana Press) and summarized in Table 2, the 5-HTRs are differentially coupled to multiple G protein signaling systems. The G-proteins by which 5-HT evokes the release of C3a and C5a will be evaluated. Membrane permeable blocking peptides will be used to disrupt these signaling proteins; these peptides mimic protein-binding domains and therefore selectively disrupt specific protein-protein binding interactions. These membrane permeable blocking peptides have been applied to determine 5-HTR activated G- proteins in other cell types (Chang M, Zhang L, Tam JP, Sanders-Bush E (2000) Endogenous 5-HT(2C) receptors in choroid plexus epithelial cells. The Journal of biological chemistry 275:7021-7029; and Chang MS, Tam JP, Sanders-Bush E (2000) Sci STKE 2000:pll).

Acute superfused dental pulp from female and male participants will be used to determine 5-HTR signaling pathways in normal pulp tissue. In all experiments, the blocking compound will be pre-treated for 30 min (Chang MS, Tam JP, Sanders-Bush E (2000) Sci STKE 2000:pll) before the addition of 5-HT (100 nM) and collection of media 30 min later for C3a and C5a measurement (Abeam ELISA). Specific blocking peptides (Table 2) will be used to evaluate whether 5-HTR signals via God, Gaq, Gas or the ϋβγ subunit to trigger C3a and C5a release. The control groups will consist of pretreatment with MPS alone (i.e., the packaging sequence without the blocking peptide). To minimize loss of 5-HT activity, nonselective blockers of the serotonin-, norepinephrine- and dopamine-transporters

(DOV216303, 100 nM and DOV21947, 300 nM) as well as a MAO inhibitor (pargyline, 2μΜ) will be added to the buffer (Lengyel K, Pieschl R, Strong T, Molski T, Mattson G, Lodge NJ, Li YW (2008) Neuropharmacology 55:63-70; and Vavilova T, Ostrovskaya I, Axenova L, Buneeva O, Medvedev A (2009) Med Sci Monit 15:BR289-292).

Data will be analyzed by three-way ANOVA: (5-HT) X (4 MPS blocking peptides and 1 MPS alone) X (Sex) with Bonferroni post-hoc test. The dependent measure will be released C3a and C5a. Power calculations (80% power, two-sided p<0.05 using prior variance estimates) give n=16/group.

One advantage of using the membrane-permeable G-protein blocking peptides in this experiment is that it facilitates conducting studies in acutely superfused female and male dental pulp which closely resembles the clinical situation.

Experiment 2.6: Identify the effector signaling pathways mediating 5-HT release of C3a and C5a from dental pulp.

It is recognized that the 5-HTRs are fairly promiscuous in both binding to G-proteins and activating effector signaling pathways and tissue-dependent signaling has often been reported (Wada K, Hu L, Mores N, Navarro CE, Fuda H, Krsmanovic LZ, Cart KJ (2006) Molecular endocrinology 20: 125-135; and Raymond J, Turner J, Gelasco A, Ayiku H, Coaxum S, Arthur J, Gamovskaya M (2008) 5HT Receptor Signal Transduction Pathways. In: The Receptors: The Serotonin Receptors: From Molecular Pharmacology to Human Therapeutics (Roth B, ed), pp 143-206. Totowa, NJ: Humana Press). Thus, it is important to study both G-proteins and effector signaling pathways engaged by ligand binding to a 5- HTR. Membrane permeable blocking peptides will be used to disrupt these effector signaling pathways (Table 2).

This experiment will determine whether the 5-HTR signals via PLC i, PLC 2, PLA2 or PKA to evoke C3a and C5a release using MPS blocking peptides and two PLA2 inhibitors (Table 2), compared to the control groups consisting of either vehicle or MPS alone (i.e., the packaging sequence without the blocking peptide). Acute superfused dental pulp from female and male participants will be used to determine 5-HTR signaling pathways in native tissue. In all experiments, the blocking compound will be pre-treated for 30 min (Chang MS, Tarn JP, Sanders-Bush E (2000) Sci STKE 2000:pll) before the addition of 5-HT (100 nM) and collection of media 30 min later for C3a and C5a measurement (ELISA). To minimize loss of 5-HT activity, non-selective blockers of the serotonin-, norepinephrine- and dopamine- transporters (DOV216303, 100 nM and DOV21947, 300 nM) as well as a MAO inhibitor (pargyline, 2 μΜ) will be added to the buffer (Lengyel K, Pieschl R, Strong T, Molski T, Mattson G, Lodge NJ, Li YW (2008) Neuropharmacology 55:63-70; and Vavilova T, Ostrovskaya I, Axenova L, Buneeva O, Medvedev A (2009) Med Sci Monit 15:BR289-292).

Data will be analyzed by three-way ANOVA: (5-HT) X (Sex) X (4 MPS blocking peptides & 1 MPS alone & 2 PLA2 inhibitors) with Bonferroni post-hoc test. The dependent measure will be released C3a and C5a. Power calculations (80% power, 25% treatment effect, two-sided p<0.05 using prior variance estimates) give n=16/group. Follow-up experiments will confirm 5-HT activation of a signaling pathway by measuring cellular accumulation of [ ³ H]-IP2,3, of [ ³ H]-arachidonic acid and immunoreactive cAMP as described (Berg KA, Patwardhan AM, Sanchez TA, Silva YM, Hargreaves KM, Clarke WP (2007) The Journal of pharmacology and experimental therapeutics 321 :839-847).

Alternative sequences of cell penetrating peptides are available (Bechara C, Sagan S (2013) FEBS Lett 587: 1693-1702). The use of binding domain peptides as inhibitors of receptor signaling is well established and more selective than many traditional small molecule approaches. However, the concentration of these peptides is generally in the range of 5-100 μΜ and studies verifying that the MPS blocking peptides are active will be carried out by determining their effect on 5-HT-evoked increases in [ ³ H]-IP2,3, of [ ³ H]-arachidonic acid and cAMP. A membrane permeable blocking peptide for PLA2 isoforms is not available. Therefore, PLA2 involvement will be evaluated with two small molecule compounds (Table 2).

Experiment #3 will determine the receptor and cellular signaling mechanisms mediating C3a- and C5a-evoked increased activities of capsaicin-sensitive trigeminal neurons. Pharmacological antagonists and cultured TG neurons from C3aR and C5aR knockout animals will be used to determine the cellular mechanisms by which C3a and C5a increase ICAP.

Experiment 3.1: Determine whether the 5-HT-evoked soluble factor increases neuronal ICAP via C3aR or C5aR.

Fig. 6 implicates C3aR in mediating increases in ICAP since the enhancement is blocked by pretreatment with the C3aR antagonist SB290157 (Ames RS, Lee D, Foley JJ, Jurewicz AJ, Tornetta MA, Bautsch W, Settmacher B, Klos A, Erhard KF, Cousins RD, Sulpizio AC, Hieble JP, McCafferty G, Ward KW, Adams JL, Bondinell WE, Underwood DC, Osborn RR, Badger AM, Sarau HM (2001) J Immunol 166:6341-6348). However, C3a can also bind to the C5 receptor, but with reduced activity (Ames RS, Nuthulaganti P, Kumar C (1996) FEBS Lett 395: 157-159). Moreover, it is possible that 5HT also evokes the release of C5a. These considerations indicate that follow-up experiments must be conducted.

Vehicle or 5-HT (100 nM) will be applied to acutely superfused female and male human dental pulp, 5-HTR antagonists will be added to the conditioned media (to block effects of residual 5-HT; Fig 3), and this mixture will be applied to primary cultures of trigeminal (TG) sensory neurons with measurement of ICAP (100 nM) by whole cell patch electrophysiology. Additional experimental groups will include vehicle alone and recombinant human C3a and C5a (10 nM). The primary TG cultures will be taken from wild type mice, C3aR ^_/" mice, or C5aR ^_/" mice from Jackson Labs (stock #005712 and #006845, respectively). Whole cell patch recordings will be performed as described previously (Akopian AN, Ruparel NB, Patwardhan A, Hargreaves KM (2008) The Journal of neuroscience : the official journal of the Society for Neuroscience 28: 1064-1075; Patwardhan AM, Scotland PE, Akopian AN, Hargreaves KM (2009) Proceedings of the National Academy of Sciences of the United States of America 106: 18820-18824; Patwardhan AM, Akopian AN, Ruparel NB, Diogenes A, Weintraub ST, Uhlson C, Murphy RC, Hargreaves KM (2010) J Clin Invest 120: 1617-1626; and Eskander MA, Ruparel S, Green DP, Chen PB, Por ED, Jeske NA, Gao X, Flores ER, Hargreaves KM (2015) The Journal of neuroscience: the official journal of the Society for Neuroscience 35: 8593-8603). Recordings will be made in whole-cell patch-clamp (holding potential of -60 mV) configuration at 22-24°C from the somata of small- to medium-sized neurons (15-40 pF) within 24h of culture.

Data will be analyzed by one-way ANOVA with Bonferroni's test. All experiments will conducted in triplicate and repeated in at least three independent experiments. Based onthe results of the study described herein with SB290157, it is expected that the increase in lea will be abolished in TG neurons from C3aR ^_/" mice.

If leap is reduced in C3aR ^_/" neurons or in C5aR ^_/" neurons, then this result will be interpreted that the relevant complement receptor contributed to 5HT-evoked increases in leap. It is possible that leap will be reduced in both knockout groups and this may be due to release of both C3a and C5a, or to possible activation of C5aR by C3a (Ames RS,

Nuthulaganti P, Kumar C (1996) FEBS Lett 395: 157-159). These possibilities will be interpreted based on the release of C3a and C5a from female dental pulp (Experiment 1.3). Note that Study 3 uses rodent TG cultures as a model tissue to evaluate the role of complement system in enhancing TRPV1 activities. This interpretation is in line with the results that demonstrated sexually dimorphic differences exist by measuring peripheral release of complement peptides. Although human sensory neurons are available (Huang JH, Zager EL, Zhang J, Groff RF, Pfister BJ, Cohen AS, Grady MS, Maloney-Wilensky E, Smith DH (2008) J Neurosurg 108:343-347; and Davidson S, Copits BA, Zhang J, Page G, Ghetti A, Gereau RWt (2014) Pain 155: 1861-1870), to date, access is limited and primarily restricted to DRG neurons and not TG neurons. Moreover, many neuronal properties are similar between human and rodent sensory neurons (Davidson S, Copits BA, Zhang J, Page G, Ghetti A, Gereau RWt (2014) Pain 155: 1861-1870). Accordingly, Study 3 will be conducted in rodent TG neurons and will verify findings in human TG neurons if they become available.

Experiment 3.2: Identify the signaling mechanisms by which C3a and C5a increases leap in sensory neurons.

The rapid enhancement of ICAP is interpreted as due to post-translational regulation of TRPV1 since the patch clamp recording is completed within seconds. The mechanisms for post-translational regulation of TRPV1 have been extensively studied and include increased phosphorylation (PKA, PKC, CamKII), trafficking to the plasma membrane, PIP2 metabolism and oxidative changes (Bhave G, Gereau RWt (2004) J Neurobiol 61 :88-106; Stucky CL, Dubin AE, Jeske NA, Malin SA, McKemy DD, Story GM (2009) Brain Res Rev 60:2-23; Kishimoto E, Naito Y, Handa O, Okada H, Mizushima K, Hirai Y, Nakabe N, Uchiyama K, Ishikawa T, Takagi T, Yagi N, Kokura S, Yoshida N, Yoshikawa T (2011) Am J Physiol Gastrointest Liver Physiol 301 :G230-238; and Planells-Cases R, Valente P, Ferrer- Montiel A, Qin F, Szallasi A (2011) Advances in experimental medicine and biology 704:491-515). Both C3aR and C5aR are GPCRs that bind to God (Zaidi AK, Ali H (2007) Advances in experimental medicine and biology 598: 126-140), as well as other G proteins since effects persist after pertussis toxin treatment (Amatruda TT, 3rd, Gerard NP, Gerard C, Simon MI (1993) The Journal of biological chemistry 268: 10139-10144; and Ahamed J, Venkatesha RT, Thangam EB, Ali H (2004) J Immunol 172:6961-6968). The C3aR and C5aR activates PLC , PKC, POkinase (PI3K), ERK1/2 as well as the RAS-MAP p42/44 kinases (Monsinjon T, Gasque P, Chan P, Ischenko A, Brady JJ, Fontaine MC (2003) FASEB J 17: 1003-1014; and Zaidi AK, Ali H (2007) Advances in experimental medicine and biology 598: 126-140). In addition, the C3aR cross-sensitizes the trkA tyrosine kinase and significantly enhances NGF effects by a mechanism resistant to pertussis toxin (Ahamed J, Venkatesha RT, Thangam EB, Ali H (2004) J Immunol 172: 6961 -6968). It is important to note that C3aR and C5aR activate these multiple signal pathways in a cell-specific fashion and that no study published to date has evaluated its signaling pathways in sensory neurons. Based on prior studies (Torti M, Crouch MF, Lapetina EG (1992) Biochem Biophys Res Commun 186:440-447; Zhu X, Birnbaumer L (1996) Proceedings of the National Academy of Sciences of the United States of America 93:2827-2831 ; Cabrera-Vera TM, Thomas TO, Vanhauwe J, Depree KM, Graber SG, Hamm HE (2002) Methods in enzymology 344:69-81 ; Wang H, Woolf CJ (2005) Neuron 46: 9-12; and Meents JE, Neeb L, Reuter U (2010) Trends in molecular medicine 16: 153-159) and known C3aR and C5aR signaling pathways

(Monsinj on T, Gasque P, Chan P, Ischenko A, Brady JJ, Fontaine MC (2003) FASEB J 17: 1003-1014; Zaidi AK, Ali H (2007) Advances in experimental medicine and biology 598: 126-140), it will be tested whether (Fig 10) increased TRPVl opening and trafficking are mediated by C3aR or C5aR activation of both the God and ϋβγ subunits, leading to signaling via PLC, SRC and PI3K.

The first experiment will determine whether C3a or C5a signals through God, Gaq, Gas or the ϋβγ subunit to trigger increased ICAP in cultured TG neurons. Cultures will be pretreated with the MPS blocking peptides (Table 2) against God, Gaq, Gas or the ϋβγ subunit. The control groups will consist of pretreatment with either vehicle or MPS alone (i.e., the packaging sequence without the blocking peptide). Cultures will be pretreated with blocking peptide (50 μΜ - 30 min) or vehicle and then stimulated with application of recombinant C3a or C5a (10 nM) followed by capsaicin (100 nM) under whole cell recording conditions. The dependent measure will be ICAP (pA/pF) and the data will be analyzed by one way ANOVA/Bonferroni's test. All experiments will conducted in triplicate and repeated in at least three independent experiments.

The second experiment will determine whether C3a or C5a signals via ΡΙ β, PKC, PI3K, ERK1/2 or the RAS-MAP p44 and p42 kinases to increase ICAP. Cultures will be pretreated with vehicle or compounds (Table 3) to inhibit ΡΙΧβ, PKC, PI3K, ERK1/2 or the RAS-MAP p44 and p42 kinases and SRC, and then stimulated by application of recombinant C3a or C5a (10 nM) followed by capsaicin (100 nM) under whole cell recording conditions. The dependent measure will be ICAP (pA/pF) and the data will be analyzed by one way ANOVA/Bonferroni's test. All experiments will be conducted in triplicate and repeated in at least three independent experiments.

Table 3. Inhibitors to Major C3aR and C5aR Signaling Pathways

Although it is expected that C3aR or C5aR enhancement of TRPV1 will be significantly reduced by pretreatment with cell permeable peptides that inhibit Gai or ϋβγ signaling, interventions designed to block Gaq and Gas signaling will also be included to ensure complete testing. It is expected that inhibitors to PLC, PKC, PI3K and SRC will significantly reduce the C3a- and C5aR-induced increases in ICAP. However, other known signaling pathways (Table 3) will also be tested. As the MPS blocking peptides are generally not available for the pathways studied in this experiment, two structurally distinct small molecule compounds will be used to independently evaluate activity in each pathway (Table 3).

The studies and results provide converging support for a sexually dimorphic pain mechanism in females. Experiments carried out and disclosed herein provide important and comprehensive testsMoreover, the findings that a C3aR receptor antagonist blocks this mechanism may provide a therapeutic target for treating women in pain.

Vertebrate Animals. Patch clamp studies using mouse TG neurons will be used as the results can be generalized to a large published data set of prior studies in male rodents.

Animals will be completely anesthetized (4% isoflurane) prior to decapitation for collection of TG neurons. C3aR ^_/" mice and C5aR ^/_ mice will be obtained from Jackson Labs (stock #005712 and #006845) and their recommended wild type strain will be used as a control group (BALB/cJ stock #000651).

Rats will be euthanized by isoflurane overdose prior to decapitation.

Human Subjects. This study will recruit 464 subj ects between the ages of 18-45 who seek treatment and have a clinical indication for extraction of either normal developed third molars or teeth with a diagnosis of painful irreversible pulpitis (Table 4). It is estimated that about 720-960 eligible patients will be seen over a five year period. Subjects will provide extracted teeth and non-PHI (pulpal diagnosis, pain on a visual analog scale, age, sex, ethnicity/race).

Table 4. Study design.

2.4 5HT Receptors 48 groups (Sex X 5HT X n=3 X 3 reps 36 siRNA* 12 siRNAs inc

scrambled)

2.5 5HT G-proteins 20 groups (Sex X 5HT X 16 320

5 MPS peptides inc MPS

alone)

2.6 5HT Signaling 28 Groups (Sex X 5HT 16 448

Pathways X 7 [MPS peptides/MPS

along/2 PLA2

inhibitors])

3.1 C3a IcAP C3aR & no human tissue 0

C5R KO

3.2a C3a G-protein no human tissue 0

3.3b C3a Signaling no human tissue 0

Pathways

Total # Teeth: 1854

Total # Participants: 464

Primary Culture Study (1 tooth ~ 12 wells)

Sources of data include the following: 1) Patient's report of the intensity of their pain using 100mm visual analog scale; 2) extracted teeth; and 3) limited demographic information (sex, age, race, ethnicity, diagnosis).

Patients who possess an indication for extraction of either developed third molars or a tooth with a diagnosis of symptomatic irreversible pulpitis and a willingness to provide the extracted teeth may be included in the study described herein.