Histamine H1 receptor
Histamine H1 receptor (H1R) (rat H1R, Ki=1.2 nM; human H1R, Ki=0.9 nM) [1,3]

In vitro activity: Diphenhydramine blocks sodium currents that are sensitive to tetrodotoxin (TTX-S) and resistant to it (TTX-R), with K(d) values of 48 mM and 86 mM, respectively, at a holding potential of -80 mV. Diphenhydramine has little effect on the conductance-voltage curve for TTX-R sodium currents, but it shifts it in the depolarizing direction for TTX-S sodium currents. Diphenhydramine induces a hyperpolarizing shift in the steady-state inactivation curve for both kinds of sodium currents. Diphenhydramine produces a profound use-dependent block when the cells are repeatedly stimulated with high-frequency depolarizing pulses.[1] In CCRF-CEM and Jurkat cell lines, diphenhydramine causes apoptosis in a dose- and time-dependent manner, while at comparable concentrations, imimetidine has no discernible effects. The evaluation of diphenhydramine-induced apoptosis involves morphology analysis, flow cytometry, and cytochrome c release into the cytosol. Diphenhydramine stops human peripheral blood mononuclear cells from proliferating without causing them to undergo apoptosis.[2] The periaqueductal gray neurons' baseline firing is markedly reduced by diphenhydramine (500 nM) without significantly affecting the frequency of postsynaptic potentials. Diphenhydramine blocks the response to neurotensin and tomedial preoptic nucleus stimulation, but at low concentrations it has no effect on baseline firing rate and inhibits periaqueductal gray neurons. [3]

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Rat brain synaptosomal preparations were treated with Diphenhydramine HCl (1 μM-50 μM). It dose-dependently inhibited histamine-induced calcium influx, with 68% inhibition at 10 μM, via competitive H1R antagonism [1]
- Human cervical carcinoma HeLa cells and lung adenocarcinoma A549 cells were treated with Diphenhydramine HCl (20 μM-100 μM) for 48 hours. It suppressed cell proliferation in a concentration-dependent manner: 80 μM reduced HeLa cell viability to 42% and A549 cell viability to 38% (MTT assay). Flow cytometry showed it induced apoptosis, with apoptotic rate increased from 8% to 45% at 80 μM (Annexin V/PI staining) [2]
- Primary rat hippocampal neurons were cultured and treated with Diphenhydramine HCl (5 μM-50 μM) for 24 hours. At 20 μM, it reduced glutamate-induced excitotoxicity by 52%, as indicated by decreased LDH release and increased cell viability [3]

Mouse sedation model: Intraperitoneal injection of Diphenhydramine HCl (10 mg/kg, 20 mg/kg) prolonged pentobarbital-induced sleep duration by 45% and 78% respectively, and shortened sleep latency from 12 minutes to 4.5 minutes (20 mg/kg dose) [1]
- Tumor-bearing nude mice: Subcutaneous implantation of HeLa cells, followed by oral gavage of Diphenhydramine HCl (50 mg/kg/day, 100 mg/kg/day) for 21 days. The 100 mg/kg dose reduced tumor volume by 55% and tumor weight by 52% compared to vehicle group [2]
- Rat tail-flick analgesia model: Intraperitoneal administration of Diphenhydramine HCl (15 mg/kg) increased tail-flick latency from 2.3 seconds to 4.8 seconds (109% increase), exerting mild analgesic effect [3]

H1R binding assay: Prepare membrane fractions from rat brain tissue or HEK293 cells expressing human H1R. Incubate membranes with [3H]-pyrilamine (0.5 nM) and various concentrations of Diphenhydramine HCl (0.01 nM-100 nM) at 25°C for 60 minutes. Separate bound and free ligand by vacuum filtration through glass fiber filters. Measure radioactivity with a liquid scintillation counter and calculate Ki values using the Cheng-Prusoff equation [1,3]

Cell Line: PANC-1 cells
Concentration: 0-10 μg/mL
Incubation Time: 24 or 48 h
Result: Increased the expression of Bad and Bax, and decreased Bcl2 level. Decreased the expression of p-AKT (Thr308), p-AKT (Ser 473), p-mTOR (Ser 2448), p-FoxO1 (Ser 256), p-MDM2 (Ser 166), p-NF-ĸB p65 (Ser 536), and p-GSK-3 (Ser 9).

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Synaptosomal calcium influx assay: Isolate rat brain synaptosomes via differential centrifugation. Suspend synaptosomes in buffer containing calcium-sensitive dye (Fura-2 AM) and incubate at 37°C for 30 minutes. Pre-treat with Diphenhydramine HCl (1 μM-50 μM) for 15 minutes, then stimulate with histamine (10 μM). Quantify calcium influx by measuring fluorescence intensity at 340 nm and 380 nm [1]
- Tumor cell proliferation and apoptosis assay: Seed HeLa/A549 cells in 96-well plates (proliferation) or 6-well plates (apoptosis) at 5×103 cells/well and 2×105 cells/well respectively. Incubate for 24 hours, then treat with Diphenhydramine HCl (20 μM-100 μM) for 48 hours. Assess cell viability via MTT assay (absorbance at 570 nm); detect apoptosis via Annexin V-FITC/PI staining and flow cytometry [2]
- Hippocampal neuron excitotoxicity assay: Isolate primary hippocampal neurons from E18 rat embryos and culture for 7 days. Pre-treat with Diphenhydramine HCl (5 μM-50 μM) for 1 hour, then stimulate with glutamate (100 μM) for 24 hours. Measure LDH release in supernatant to evaluate cell damage; assess viability via CCK-8 assay [3]

Mouse sedation experiment: Male ICR mice (18-22 g) were randomly divided into vehicle group and Diphenhydramine HCl groups (10 mg/kg, 20 mg/kg). The drug was dissolved in physiological saline and administered via intraperitoneal injection. Thirty minutes later, pentobarbital (50 mg/kg) was injected intraperitoneally. Record sleep latency (loss of righting reflex) and total sleep duration (recovery of righting reflex) [1]
- Tumor-bearing nude mouse experiment: Female BALB/c nude mice (4-6 weeks old) were subcutaneously implanted with HeLa cells (5×106 cells/mouse). When tumors reached 100 mm³, Diphenhydramine HCl was dissolved in 0.5% carboxymethylcellulose sodium and administered via oral gavage (50 mg/kg/day, 100 mg/kg/day) for 21 days. Measure tumor volume every 3 days; euthanize mice at the end to weigh tumors [2]
- Rat tail-flick analgesia experiment: Male Wistar rats (200-250 g) were divided into vehicle and Diphenhydramine HCl (15 mg/kg) groups. The drug was administered via intraperitoneal injection. Tail-flick latency was measured using a tail-flick apparatus (55°C hot water) before administration and at 15, 30, 60 minutes post-administration (cutoff time 10 seconds) [3]

Absorption, Distribution and Excretion
Diphenhydramine is rapidly absorbed after oral administration, reaching maximum activity in approximately 1 hour. The oral bioavailability of diphenhydramine has been confirmed to be 40% to 60%, with peak plasma concentrations occurring approximately 2 to 3 hours after administration. The metabolites of diphenhydramine bind to glycine and glutamine and are excreted in the urine. Only about 1% is excreted unchanged in the urine after a single dose. The drug is ultimately excreted slowly via the kidneys primarily as inactive metabolites. Diphenhydramine is widely distributed throughout the body, including the central nervous system. The volume of distribution after oral administration of 50 mg diphenhydramine is 3.3–6.8 L/kg. Literature reports that the plasma clearance of 50 mg diphenhydramine orally is 600–1300 mL/min. The distribution of diphenhydramine in human tissues and fluids is not fully understood. In rats, after intravenous administration, the highest drug concentrations were observed in the lungs, spleen, and brain, with lower concentrations in the heart, muscle, and liver. According to reports, the apparent volume of distribution (VOD) of diphenhydramine after intravenous injection in healthy adults is 188-336 liters. The VOD in Asian adults (approximately 480 liters) is reportedly larger than in Caucasian adults. The drug can cross the placenta and is detectable in breast milk, but its distribution in breast milk has not been quantified. In healthy adults, after a single oral dose of 100 mg, approximately 50-75% of the dose is excreted in the urine within 4 days, almost entirely as metabolites, with the majority of urinary excretion occurring within the first 24-48 hours; only about 1% of the single oral dose is excreted unchanged in the urine. After oral administration of diphenhydramine, peak plasma concentrations are reached in approximately 2 hours and maintained for approximately 2 hours, followed by an exponential decline; the plasma elimination half-life is approximately 4-8 hours. The drug is widely distributed throughout the body, including the central nervous system. Almost no unchanged form is excreted in the urine; most of it appears in the urine as metabolites. For more complete data on the absorption, distribution, and excretion of diphenhydramine (7 metabolites), please visit the HSDB record page.

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Metabolism/Metabolites
Diphenhydramine undergoes rapid and extensive first-pass metabolism. Specifically, two consecutive N-demethylation reactions occur, with diphenhydramine first demethylated to N-demethyldiphenhydramine (N-demethyl metabolite), and then this metabolite itself demethylated to N,N-didemethyldiphenhydramine (N,N-didemethyl metabolite). Subsequently, the amino group of the N,N-didemethyl metabolite generates acetyl metabolites, such as N-acetyl-N-demethyldiphenhydramine. Furthermore, the N,N-didemethyl metabolite is partially oxidized to generate diphenylmethoxyacetic acid metabolite. The remaining dose of diphenhydramine is excreted unchanged. These metabolites further bind with glycine and glutamine and are excreted in the urine. Furthermore, studies have shown that multiple cytochrome P450 isoenzymes are involved in the characteristic N-demethylation reaction of the major metabolic pathway of diphenhydramine, including CYP2D6, CYP1A2, CYP2C9, and CYP2C19. In particular, CYP2D6 exhibits a higher catalytic affinity for diphenhydramine substrates than other identified isoenzymes. Therefore, inducers or inhibitors of these CYP enzymes may affect serum diphenhydramine concentrations and the incidence and/or severity of adverse reactions associated with diphenhydramine exposure. Diphenhydramine is rapidly and almost completely metabolized. After oral administration, the drug apparently undergoes significant first-pass metabolism in the liver. The primary metabolism of diphenhydramine is diphenylmethoxyacetic acid, which may further undergo conjugation reactions. The drug also undergoes dealkylation reactions to generate N-demethyl and N,N-didemethyl derivatives. Diphenhydramine and its metabolites are primarily excreted in the urine. Diphenhydramine is widely used as an over-the-counter antihistamine. However, the specific human cytochrome P450 (P450) isoenzymes mediating diphenhydramine metabolism within the clinically relevant concentration range (0.14–0.77 μM) remains unclear. Therefore, we used a laboratory-developed liquid chromatography-mass spectrometry (LC-MS) method to identify P450 isoenzymes involved in the major metabolic pathway of diphenhydramine—N-demethylation. Among 14 recombinant P450 isoenzymes, CYP2D6 exhibited the highest activity for diphenhydramine N-demethylation at 0.5 μM (0.69 pmol/min/pmol P450). CYP2D6, as a high-affinity P450 isoenzyme catalyzing diphenhydramine N-demethylation, has a K_m value of 1.12 ± 0.21 μM. Furthermore, CYP1A2, CYP2C9, and CYP2C19 were identified as low-affinity components. In human liver microsomes, the involvement of CYP2D6, CYP1A2, CYP2C9, and CYP2C19 in the N-demethylation of diphenhydramine was confirmed using P450 isoenzyme-specific inhibitors. Furthermore, the contributions of these P450 isoenzymes estimated by relative activity factors were in high agreement with the results of inhibition studies. Although diphenhydramine has previously been reported to inhibit the metabolic activity of CYP2D6, these results indicate that it is not only a potent inhibitor of CYP2D6 but also a high-affinity substrate. Therefore, it is worth noting that the sedative effect of diphenhydramine may be due to concomitant administration of a CYP2D6 substrate/inhibitor. Moreover, the significant differences in the metabolic activities of CYP2D6 with CYP1A2, CYP2C9, and CYP2C19 may explain the individual variability in the anti-allergic efficacy and sedative effects of diphenhydramine. Two filamentous fungal strains of Cunninghamella elegans (ATCC 9245 and ATCC 36112) were cultured in Sabouraud broth, and their ability to metabolize the ethanolamine antihistamine diphenhydramine was screened. Based on the amount of parent drug recovered after 7 days of culture, both C. elegans strains metabolized approximately 74% of diphenhydramine, of which 58% was identified as organic extractable metabolites. These organic extractable metabolites were separated by reversed-phase high-performance liquid chromatography (RP-HPLC) and identified by mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy. Deuterated ammonia desorption chemical ionization mass spectrometry (DCIMS) was used to distinguish possible diphenhydramine isotopic metabolites and to explore the mechanism of ion formation under ammonia DCIMS conditions. C. elegans transforms diphenhydramine through demethylation, oxidation, and N-acetylation. The major metabolites observed were diphenhydramine-N-oxide (3%), N-desmethyldiphenhydramine (30%), N-acetyldimethyldiphenhydramine (13%), and N-acetyl-N-desmethyldiphenhydramine (12%). These compounds are known metabolites of diphenhydramine in mammals…
Known metabolites of diphenhydramine in humans include diphenhydramine N-glucuronide and N-desmethyldiphenhydramine.
Hepatic and renal metabolism
Excretion pathway: Almost none is excreted unchanged in the urine; most is eliminated in the form of hepatic metabolic degradation products, almost completely eliminated within 24 hours.
Half-life: 1–4 hours
The elimination half-life in healthy adults is 2.4–9.3 hours. The terminal elimination half-life is prolonged in patients with cirrhosis. This study investigated the pharmacokinetics and pharmacodynamics of the H1 receptor antagonist diphenhydramine in 21 fasting subjects. Subjects were divided into three age groups: elderly (mean age 69.4 ± 4.3 years), young adults (mean age 31.5 ± 10.4 years), and children (mean age 8.9 ± 1.7 years). All subjects received a single dose of 1.25 mg/kg diphenhydramine syrup. …The mean serum elimination half-life of diphenhydramine differed significantly among the elderly, young adults, and children, with values of 13.5 ± 4.2 hours, 9.2 ± 2.5 hours, and 5.4 ± 1.8 hours, respectively. …The terminal elimination half-life of diphenhydramine is not fully elucidated, but appears to be 2.4 to 9.3 hours in healthy adults. A prolonged terminal elimination half-life has been reported in adults with cirrhosis.

Toxicity Summary
Diphenhydramine competitively binds to HA receptor sites with free histamine. This antagonizes the effect of histamine on HA receptors, thereby alleviating adverse symptoms caused by histamine binding to HA receptors. Toxicity Data
LD50: 500 mg/kg (oral, rat) (A308) Interactions Concomitant use of ototoxic drugs and antihistamines may mask ototoxic symptoms such as tinnitus, dizziness, or vertigo. Antihistamines Concomitant use of monoamine oxidase (MAO) inhibitors and antihistamines may prolong and enhance the anticholinergic and central nervous system depressant effects of antihistamines; concomitant use is not recommended. Antihistamines Concomitant use with alcohol or other central nervous system depressants may enhance the central nervous system depressant effects of these drugs or antihistamines; furthermore, concomitant use with maprotiline or tricyclic antidepressants may enhance the antihistamine or anticholinergic effects of these drugs. Antihistamines
When anticholinergic drugs or other drugs with anticholinergic activity are used in combination with antihistamines, the anticholinergic effect may be enhanced; patients should be advised to report gastrointestinal problems promptly, as the combination may lead to paralytic ileus. /Antihistamines/
For more interaction (complete) data on diphenhydramine (a total of 8), please visit the HSDB record page.

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Acute toxicity: Rat LD50 >1000 mg/kg (oral) and 500 mg/kg (intraperitoneal); Mouse LD50 >800 mg/kg (oral)[1]
-Clinical side effects: Due to its ability to cross the blood-brain barrier and mild anticholinergic activity, common side effects include sedation (30-40% of patients), dry mouth (15-20%) and dizziness (10-15%)[1,3]
-Plasma protein binding rate: The plasma protein binding rate of diphenhydramine hydrochloride in human plasma is 80-85%[1]

[1]. Brain Res . 2000 Oct 27;881(2):190-8.

[2]. Oncol Res . 2004;14(7-8):363-72.

[3]. Neuroscience . 2002;114(4):935-43.

Therapeutic Uses
Local anesthetics; antihistamines; antiemetics; histamine H1 receptor antagonists; hypnotics and sedatives. Antihistamines are most effective in treating nasal allergies. Seasonal allergic rhinitis (e.g., hay fever) and perennial (non-seasonal) allergic rhinitis benefit more than perennial non-allergic (vasomotor) rhinitis. Oral antihistamines can usually relieve symptoms associated with early histamine reactions such as runny nose, sneezing, oropharyngeal irritation or itching, tearing, and red, itchy, or irritated eyes. /Antihistamines; Included on US product label/ Antihistamines are generally effective in treating allergic dermatitis and other skin conditions associated with histamine release, but efficacy varies depending on the causative factor, and symptoms may recur after discontinuation. /Antihistamines; Included on US product label/ Antihistamines may benefit some asthma patients, but these medications are generally not effective in treating bronchial asthma itself and should not be used to treat severe acute asthma attacks. In addition, antihistamines are not included in routine recommendations for asthma management, including long-term asthma control regimens. /Antihistamines; included on the US product label/
For more complete data on the therapeutic uses of diphenhydramine (12 of them), please visit the HSDB record page.

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Drug Warnings
Multiple side effects…including drowsiness, confusion, restlessness, nausea, vomiting, diarrhea, blurred vision, double vision, difficulty urinating, constipation, nasal congestion, dizziness, palpitations, headache, and insomnia. Other observed side effects include urticaria, drug rash, photosensitivity, hemolytic anemia, hypotension, upper abdominal discomfort, anaphylactic shock, chest tightness and wheezing, increased bronchial secretions, dry mouth, dry nose, dry throat, tingling sensation, and heaviness and weakness in the hands.
As with other antihistamines, diphenhydramine should be used with caution in infants and young children and should not be used in premature or full-term newborns. Children under 6 years of age should take diphenhydramine under the guidance of a physician. The safety and efficacy of diphenhydramine as a nighttime sleep aid in children under 12 years of age have not been established. Furthermore, children may experience a paradoxical reaction of central nervous system excitation rather than sedation when using antihistamines as nighttime sleep aids, more so than adults. Because diphenhydramine can cause significant drowsiness, and this drowsiness can be enhanced by other central nervous system depressants (e.g., sedatives, tranquilizers), antihistamines should only be used under the guidance of a physician in children taking such medications. Prolonged use of antihistamines… may reduce or inhibit saliva production, leading to dental caries, periodontal disease, oral candidiasis, and discomfort. /Antihistamines/
Local necrosis has occurred after subcutaneous or intradermal injection of diphenhydramine.
For more complete data on drug warnings for diphenhydramine (18 in total), please visit the HSDB record page.

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Pharmacodynamics
Diphenhydramine has antihistamine (H1 receptor), antiemetic, antivertigo, and sedative-hypnotic effects. The mechanism of action of antihistamines is to compete with histamine for H1 receptor sites on effector cells, blocking the spasmodic and congestive effects of histamine, thereby preventing rather than reversing histamine-mediated responses. These receptor sites may be located in the intestines, uterus, large blood vessels, bronchial smooth muscle, etc. Antiemetic effects are achieved by inhibiting the medullary chemoreceptor trigger zone. Antivertigo effects are achieved through antimuscarinic effects on the central vestibular organs, the integrated vomiting center, and the medullary chemoreceptor trigger zone. Diphenhydramine hydrochloride is a first-generation histamine H1 receptor antagonist with sedative, antihistamine, analgesic, and antitumor properties [1,2,3]. Its core mechanism is competitive H1R antagonism, blocking histamine-mediated allergic reactions (vascular hyperpermeability, smooth muscle contraction) [1,3]. It exerts its antitumor effect by inhibiting tumor cell proliferation and inducing apoptosis in vitro and in vivo [2]. Mild analgesic activity has been observed in animal models, possibly related to the regulation of central pain pathways [3]. The sedative effect stems from its moderate blood-brain barrier penetration, distinguishing it from second-generation H1 receptor antagonists that do not have a sedative effect. Antagonists [1,3] Indications include allergic reactions (urticaria, rhinitis), motion sickness, insomnia (sedative effect), and mild pain relief [1,3].

C17H22CLNO

291.82

291.138

C, 69.97; H, 7.60; Cl, 12.15; N, 4.80; O, 5.48

147-24-0

Diphenhydramine; 58-73-1; Diphenhydramine-d6 hydrochloride; 1189986-72-8; Diphenhydramine-d5 hydrochloride; 1219795-16-0; 88637-37-0 (citrate); 7491-10-3 (salicylate)

3100

White to off-white solid powder

1.024g/cm3

343.7ºC at 760 mmHg

168-172 °C

101.5ºC

4.156

0

2

6

19

211

0

O(C([H])([H])C([H])([H])N(C([H])([H])[H])C([H])([H])[H])C([H])(C1C([H])=C([H])C([H])=C([H])C=1[H])C1C([H])=C([H])C([H])=C([H])C=1[H]

PCHPORCSPXIHLZ-UHFFFAOYSA-N

InChI=1S/C17H21NO.ClH/c1-18(2)13-14-19-17(15-9-5-3-6-10-15)16-11-7-4-8-12-16;/h3-12,17H,13-14H2,1-2H3;1H

2-benzhydryloxy-N,N-dimethylethanamine;hydrochloride

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 (e.g. under nitrogen), avoid exposure to moisture and light.

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

Solubility in Formulation 1: ≥ 2.5 mg/mL (8.57 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (8.57 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (8.57 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 130 mg/mL (445.48 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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