Endogenous Metabolite

Oxytocin is a pleiotropic, peptide hormone with broad implications for general health, adaptation, development, reproduction, and social behavior. Endogenous oxytocin and stimulation of the oxytocin receptor support patterns of growth, resilience, and healing. Oxytocin can function as a stress-coping molecule, an anti-inflammatory, and an antioxidant, with protective effects especially in the face of adversity or trauma. Oxytocin influences the autonomic nervous system and the immune system. These properties of oxytocin may help explain the benefits of positive social experiences and have drawn attention to this molecule as a possible therapeutic in a host of disorders. However, as detailed here, the unique chemical properties of oxytocin, including active disulfide bonds, and its capacity to shift chemical forms and bind to other molecules make this molecule difficult to work with and to measure. The effects of oxytocin also are context-dependent, sexually dimorphic, and altered by experience. In part, this is because many of the actions of oxytocin rely on its capacity to interact with the more ancient peptide molecule, vasopressin, and the vasopressin receptors. In addition, oxytocin receptor(s) are epigenetically tuned by experience, especially in early life. Stimulation of G-protein-coupled receptors triggers subcellular cascades allowing these neuropeptides to have multiple functions. The adaptive properties of oxytocin make this ancient molecule of special importance to human evolution as well as modern medicine and health; these same characteristics also present challenges to the use of oxytocin-like molecules as drugs that are only now being recognized. SIGNIFICANCE STATEMENT: Oxytocin is an ancient molecule with a major role in mammalian behavior and health. Although oxytocin has the capacity to act as a "natural medicine" protecting against stress and illness, the unique characteristics of the oxytocin molecule and its receptors and its relationship to a related hormone, vasopressin, have created challenges for its use as a therapeutic drug [2].

The pituitary neuropeptide oxytocin promotes social behavior, and is a potential adjunct therapy for social deficits in schizophrenia and autism. Oxytocin may mediate pro-social effects by modulating monoamine release in limbic and cortical areas, which was investigated herein using in vivo microdialysis, after establishing a dose that did not produce accompanying sedative or thermoregulatory effects that could concomitantly influence behavior. The effects of oxytocin (0.03-0.3 mg/kg subcutaneous) on locomotor activity, core body temperature, and social behavior (social interaction and ultrasonic vocalizations) were examined in adult male Lister-hooded rats, using selective antagonists to determine the role of oxytocin and vasopressin V1a receptors. Dopamine and serotonin efflux in the prefrontal cortex and nucleus accumbens of conscious rats were assessed using microdialysis. 0.3 mg/kg oxytocin modestly reduced activity and caused hypothermia but only the latter was attenuated by the V1a receptor antagonist, SR49059 (1 mg/kg intraperitoneal). Oxytocin at 0.1 mg/kg, which did not alter activity and had little effect on temperature, significantly attenuated phencyclidine-induced hyperactivity and increased social interaction between unfamiliar rats without altering the number or pattern of ultrasonic vocalizations. In the same rats, oxytocin (0.1 mg/kg) selectively elevated dopamine overflow in the nucleus accumbens, but not prefrontal cortex, without influencing serotonin efflux. Systemic oxytocin administration attenuated phencyclidine-induced hyperactivity and increased pro-social behavior without decreasing core body temperature and selectively enhanced nucleus accumbens dopamine release, consistent with activation of mesocorticolimbic circuits regulating associative/reward behavior being involved. This highlights the therapeutic potential of oxytocin to treat social behavioral deficits seen in psychiatric disorders such as schizophrenia [1].

Analysis of monoamines[1]
Microdialysis samples were analyzed using High Performance Liquid Chromatography with electrochemical detection as described previously. Defrosted samples were kept on ice before injection (15 µl) into a Targa C18 3 µM column (100 × 1.0 m) using a Perkin Elmer Series 200 autosampler. Dopamine, 5-HT and their major metabolites; 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 5-hydroxyindoleacetic acid (5-HIAA) were detected using a mobile phase (20 mM potassium dihydrogen phosphate, 20 mM sodium acetate, 0.1 mM ethylenediaminetetraacetic acid, 0.15 mM octanesulfonic acid, and 10% methanol, pH 3.9) at 0.4 ml/min (Dionex P680 pump), and measured against standards with a DECADE II SDC Detector I and Clarity software using a potential of + 0.75 V. The percentage change from baseline for every microdialysate molecule was calculated for each individual rat. PFC samples were excluded from one rat due to incorrect probe placement and two others because of flow disruption. In one rat NAc dopamine was below the detection limit; n = 6/7 per group in the PFC, and n = 7/8 in the NAc.

Dose–response and antagonist studies with oxytocin on core body temperature and locomotor activity[1]
To establish a suitable dose of oxytocin, which would not suppress locomotor activity (LMA) or produce hypothermia during microdialysis studies, rats (n = 12) were tested using a within-subjects design on four occasions at weekly intervals following injection of vehicle and each dose of oxytocin (0.03, 0.1, or 0.3 mg/kg s.c.) in a pseudo-random order to serve as their own control. This range was selected from previous reports showing that oxytocin doses above 0.3 mg/kg s.c. or i.p. suppress spontaneous locomotion in other rat strains so we included lower doses to identify those devoid of this unwanted effect. To establish the relative contribution of oxytocin and vasopressin V1a receptors to hypothermia produced by the highest dose, a further 12 rats received vehicle or oxytocin (0.3 mg/kg s.c.) in the presence and absence of the non-peptide selective V1a receptor antagonist SR49059 (1 mg/kg i.p.) or the selective oxytocin antagonist L-368,899 (2 mg/kg i.p.), on six occasions at weekly intervals (within-subjects design). Although original peptide antagonists for these receptors showed poor stability and selectivity the development of non-peptide antagonists greatly improved pharmacokinetic properties. The current non-peptide antagonists (SR49059 and L-368,889) were selected because they possess the best overall profile of commercially available oxytocin and V1a antagonists; having high affinity, relative selectivity, good BBB penetration, and plasma half-life and are devoid of partial agonist activity. Doses of these brain penetrant antagonists were selected from previous studies showing < 15 min onset and 2–4 h duration in rodents. SR49059 prevented oxytocin-induced pro-social behavior and hypothermia, whereas L-368,899 (which has brain penetration demonstrated by PET studies), prevented anxiolytic effects of oxytocin in the open field, reduced conditioned disgust behavior during social interaction and attenuated sexual motivation in male rats]. The cross-over repeat within-subject design for dose–response and antagonist studies greatly reduced the number of rats required and the inter-individual variation of measurements made in line with the 3 R’s principle.

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Doses and routes of administration:
Compounds were dissolved in 0.154 M saline (vehicle also containing 5% dimethyl sulfoxide for the antagonists) and administered at volume of 1 ml/kg subcutaneous (s.c.) (oxytocin)

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Effect of oxytocin on PCP-induced hyperactivity, social interaction, and PFC and NAc dopamine and 5-HT efflux[1]
Oxytocin at 0.03 and 0.1 mg/kg were selected for further investigation, as these doses did not produce confounding effects on ambulation and body temperature in dose–response studies described above. A separate group of rats (n = 32, Figure S1) was used to examine the effect of these two doses on PCP-induced hyperactivity, and on the basis of these findings 0.1 mg/kg oxytocin was administered 7 days later, prior to assessment of social interaction and USVs. The following week rats underwent stereotaxic surgery to implant microdialysis probes into the PFC and NAc and after 7 days recovery the effects of oxytocin on dopamine efflux from these brain regions was assessed. One week was left between each of the three protocols (Figure S1) to ensure complete drug wash-out and minimize any carry over effects from the previous procedure.

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Locomotor activity[1]
LMA was assessed on a single occasion as described above. Animals received oxytocin or vehicle after 30 min arena habituation, and vehicle or PCP (5.6 mg/kg i.p.; an established dose to examine “antipsychotic-like” activity) 30 min later, resulting in four treatment combinations: vehicle + vehicle, PCP + vehicle, PCP + 0.03 mg/kg oxytocin, PCP + 0.1 mg/kg (n = 8/group; between-subjects design).

Absorption, Distribution and Excretion
Oxytocin is administered parenterally and is fully bioavailable. It takes approximately 40 minutes for oxytocin to reach steady-state concentrations in the plasma after parenteral administration.
The enzyme oxytocinase is largely responsible for the metabolism and regulation of oxytocin levels in pregnancy; only a small percentage of the neurohormone is excreted in the urine unchanged.
In a study that observed 10 women who were given oxytocin to induce labor, the mean metabolic clearance rate was 7.87 mL/min.
Oxytocin is destroyed by chymotrypsin in the GI tract. Uterine response occurs almost immediately and subsides within 1 hour following iv administration of oxytocin. Following im injection of the drug, uterine response occurs within 3-5 minutes and persists for 2-3 hours. Following intranasal application of 10-20 units of oxytocin (nasal preparations are no longer commercially available in the US), contractions of myoepithelial tissue surrounding the alveoli of the breasts begin within a few minutes and continue for 20 minutes; iv oxytocin produces the same effect with a dose of 100-200 milliunits.
Like vasopressin, oxytocin is distributed throughout the extracellular fluid. Small amounts of oxytocin probably reach the fetal circulation.
It is not known whether this drug is excreted in human milk.
Its rapid removal from plasma is accomplished largely by the kidney and the liver. Only small amounts oxytocin are excreted in the urine unchanged.
For more Absorption, Distribution and Excretion (Complete) data for Oxytocin (7 total), please visit the HSDB record page.

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Metabolism / Metabolites
Oxytocin is rapidly removed from the plasma by the liver and kidney. The enzyme oxytocinase is largely responsible for the metabolism and regulation of oxytocin levels in pregnancy and only a small percentage of the neurohormone is excreted in the urine unchanged. Oxytocinase activity increases throughout pregnancy and peaks in the plasma, placenta and uterus near term. The placenta is a key source of oxytocinase during gestation and produces increasing amounts of the enzyme in response to increasing levels of oxytocin produced by the mother. Oxytocinase activity is also expressed in mammary glands, heart, kidney, and the small intestine. Lower levels of activity can be found in the brain, spleen, liver, skeletal muscle, testes, and colon. The level of oxytocin degradation is negligible in non-pregnant women, men, and cord blood.
Oxytocinase, a circulating enzyme produced early in pregnancy, is also capable of inactivating the polypeptide.
During pregnancy ... "oxytocinase" ... is capable of inactivating oxytocin by cleavage of the 1-cysteine to 2-tyrosine peptide bond.

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Biological Half-Life
The plasma half-life of oxytocin ranges from 1-6 minutes. The half-life is decreased in late pregnancy and during lactation.
Oxytocin has a plasma half-life of about 3 to 5 minutes.

Interactions
Severe hypertension has been reported when oxytocin was given 3-4 hours following prophylactic administration of a vasoconstrictor in conjunction with caudal block anesthesia.
Cyclopropane anesthesia may modify oxytocin's cardiovascular effects, producing less pronounced tachycardia but more severe hypotension than occurs with oxytocin alone; maternal sinus bradycardia with abnormal atrioventricular rhythms has been noted when oxytocin was used concomitantly with cyclopropane anesthesia.
Oxytocin reportedly has delayed induction of thiopental anesthesia by producing venous spasm that caused peripheral pooling of thiopental; however, this interaction has not been conclusively established.

[1]. Oxytocin attenuates phencyclidine hyperactivity and increases social interaction and nucleus accumben dopamine release in rats. Neuropsychopharmacology. 2019 Jan;44(2):295-305.

[2]. Is Oxytocin "Nature's Medicine"? Pharmacol Rev. 2020 Oct;72(4):829-861.

Therapeutic Uses
Oxytocin is indicated for the medical rather than the elective induction of labor. Available data and information are inadequate to define the benefits-to-risks considerations in the use of the drug product for elective induction. Elective induction of labor is defined as the initiation of labor for convenience in an individual with a term pregnancy who is free of medical indications. /Included in US product label/
Oxytocin is indicated for the initiation or improvement of uterine contractions, where this is desirable and considered suitable for reasons of fetal or maternal concern, in order to achieve early vaginal delivery. It is indicated for induction of labor in patients with a medical indication for the initiation of labor, such as Rh problems, maternal diabetes, preeclampsia at or near term, when delivery is in the best interests of mother and fetus or when membranes are prematurely ruptured and delivery is indicated... /Included in US product label/
Oxytocin is indicated for stimulation or reinforcement of labor, as in selected cases of uterine inertia... /Included in US product label/
Oxytocin is indicated as adjunctive therapy in the management of incomplete or inevitable abortion. In the first trimester, curettage is generally considered primary therapy. In second trimester abortion, oxytocin infusion will often be successful in emptying the uterus. Other means of therapy, however, may be required in such cases. /Included in US product label/
For more Therapeutic Uses (Complete) data for Oxytocin (11 total), please visit the HSDB record page.

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Drug Warnings
When oxytocin is administered in excessive dosage, with abortifacients or to sensitive patients, hyperstimulation of the uterus, with strong (hypertonic) and/or prolonged (tetanic) contractions, or a resting uterine tone of 15-20 mm H2O between contractions may occur, possibly resulting in uterine rupture, cervical and vaginal lacerations, postpartum hemorrhage, abruptio placentae, impaired uterine blood flow, amniotic fluid embolism, and fetal trauma including intracranial hemorrhage.
Increased uterine motility may cause adverse fetal effects, including sinus bradycardia, tachycardia, premature ventricular complexes and other arrhythmias, permanent CNS or brain damage, and death secondary to asphyxia. Excessive maternal dosage or administration of the drug to sensitive women also can cause uteroplacental hypoperfusion and variable deceleration of fetal heart rate, fetal hypoxia, perinatal hepatic necrosis, and fetal hypercapnia. Rare incidents of pelvic hematoma have been reported, but these were probably also related to the high incidence of operative vaginal deliveries in primiparas, the fragility of engorged pelvic veins (especially if varicosed), and faulty episiotomy repair.
When large amounts of oxytocin are administered, severe decreases in maternal systolic and diastolic blood pressure, increases in heart rate, systemic venous return and cardiac output, and arrhythmia may occur; these effects may be particularly hazardous to patients with valvular heart disease and those receiving spinal and epidural anesthesia.
Postpartum bleeding may be increased by administration of oxytocin; this effect may be related to reports of oxytocin-induced thrombocytopenia, afibrinogenemia, and hypoprothrombinemia. By carefully controlling delivery, the incidence of postpartum bleeding may be minimized.
For more Drug Warnings (Complete) data for Oxytocin (22 total), please visit the HSDB record page.

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Pharmacodynamics
Oxytocin is a nonapeptide, pleiotropic hormone that exerts important physiological effects. It is most well known to stimulate parturition and lactation, but also has important physiological influences on metabolic and cardiovascular functions, sexual and maternal behaviour, pair bonding, social cognition, and fear conditioning. It is worth noting that oxytocin receptors are not limited to the reproductive system but can be found in many peripheral tissues and in central nervous system structures including the brain stem and amygdala.

C43H66N12O12S2

1007.1873

1006.436

50-56-6

6233-83-6 (acetate)

439302

L-Cysteinyl-L-tyrosyl-L-isoleucyl-L-glutaminyl-L-asparaginyl-L-cysteinyl-L-prolyl-L-leucylglycinamide cyclic (1®6)-disulfide; or Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH2 (Disulfide bridge:Cys1-Cys6)

CYIQNCPLG-NH2 (Disulfide bridge:Cys1-Cys6)

White to off-white solid powder

1.3±0.1 g/cm3

1533.3±65.0 °C at 760 mmHg

192-194°C

881.1±34.3 °C

0.0±0.3 mmHg at 25°C

1.554

Endogenous Metabolite

-4.26

12

15

17

69

1870

9

S1C([H])([H])[C@@]([H])(C(N2C([H])([H])C([H])([H])C([H])([H])[C@@]2([H])C(N([H])[C@]([H])(C(N([H])C([H])([H])C(N([H])[H])=O)=O)C([H])([H])C([H])(C([H])([H])[H])C([H])([H])[H])=O)=O)N([H])C([C@]([H])(C([H])([H])C(N([H])[H])=O)N([H])C([C@]([H])(C([H])([H])C([H])([H])C(N([H])[H])=O)N([H])C([C@]([H])([C@@]([H])(C([H])([H])[H])C([H])([H])C([H])([H])[H])N([H])C([C@]([H])(C([H])([H])C2C([H])=C([H])C(=C([H])C=2[H])O[H])N([H])C([C@]([H])(C([H])([H])S1)N([H])[H])=O)=O)=O)=O)=O

XNOPRXBHLZRZKH-DSZYJQQASA-N

InChI=1S/C43H66N12O12S2/c1-5-22(4)35-42(66)49-26(12-13-32(45)57)38(62)51-29(17-33(46)58)39(63)53-30(20-69-68-19-25(44)36(60)50-28(40(64)54-35)16-23-8-10-24(56)11-9-23)43(67)55-14-6-7-31(55)41(65)52-27(15-21(2)3)37(61)48-18-34(47)59/h8-11,21-22,25-31,35,56H,5-7,12-20,44H2,1-4H3,(H2,45,57)(H2,46,58)(H2,47,59)(H,48,61)(H,49,66)(H,50,60)(H,51,62)(H,52,65)(H,53,63)(H,54,64)/t22-,25-,26-,27-,28-,29-,30-,31-,35-/m0/s1

(2S)-1-({(4R,7S,10S,13S,16S,19R)-19-amino-7-(2-amino-2-oxoethyl)-10-(3-amino-3-oxopropyl)-16-(4-hydroxybenzyl)-13-[(1S)-1-methylpropyl]-6,9,12,15,18-pentaoxo-1,2-dithia-5,8,11,14,17-pentaazacycloicosan-4-yl}carbonyl)-N-{(1S)-1-[(2-amino-2-oxoethyl)carbamoyl]-3-methylbutyl}pyrrolidine-2-carboxamide

α-Hypophamine; Oxytocic hormone; Uteracon; Syntocinone; Pitocin; Synpitan

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: ~100 mg/mL (99.3 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)