Absorption, Distribution and Excretion
Nicotine and its proximal metabolite cotinine are partially cleared by the kidneys. These compounds undergo renal filtration, secretion, and reabsorption, with final renal clearance varying considerably among individuals and significantly influenced by urine pH. This study included 139 twin pairs to assess the effects of genetic and environmental factors on the total renal clearance and net secretory/reabsorption clearance of nicotine and cotinine. In the absence of controlled urine pH, both nicotine and cotinine underwent net reabsorption. Additive genetic factors had little effect on the variability in total renal clearance of nicotine but a relatively large effect on the variability in total renal clearance of cotinine (accounting for 43% of the variation). Variations in glomerular filtration rate and net secretory/reabsorption clearance of nicotine and cotinine were primarily influenced by non-additive genetic factors (accounting for 41.5% to 61% of the variation). Previous studies have shown that renal secretory clearance of drugs is highly heritable, which may be related to genetic variations in transport proteins. Our study suggests that renal clearance of drugs that are extensively reabsorbed by the kidneys may be significantly influenced by non-additive genetic factors and/or shared environmental factors. The detection of cotinine metabolism is considered an accurate indicator of smoking exposure in pregnant women. We investigated the association and differences in cotinine levels in urine, blood, and cord blood of pregnant women in three different tobacco-exposed groups at different stages of pregnancy. This prospective study included 398 pregnant women who underwent prenatal care at different stages of pregnancy at two medical centers and one regional hospital in central Taiwan. All 398 participants (including 25 smokers, 191 passive smokers, and 182 non-smokers) completed the study until delivery; 384 delivered singleton live births. Cotinine levels in maternal plasma and urine were measured at each stage of pregnancy, and also in neonatal cord blood. All samples were analyzed using sensitive high-performance liquid chromatography (HPLC). Cotinine concentrations in plasma and urine showed significant dose-dependent differences among the three groups (non-smokers, passive smokers, and active smokers), and in pregnant women, cotinine concentrations increased with gestational age. Significant correlations were found between plasma and urinary cotinine concentrations in pregnant women at each stage of pregnancy. Furthermore, cotinine levels in umbilical cord blood were significantly correlated with maternal blood cotinine levels at term (r = 0.89, P < 0.001). The study found that cotinine concentrations in pregnant women's plasma and urine trended upward from the beginning to the end of pregnancy and were significantly correlated with cotinine levels in umbilical cord blood. Given that nicotine is a marker of cerebral blood flow, the role of the blood-brain barrier in nicotine transport has been well-established. However, data on the penetration of the blood-brain barrier by major tobacco alkaloids after long-term nicotine exposure are limited. This issue urgently needs to be addressed, given that long-term nicotine exposure alters the function and morphology of the blood-brain barrier. Unlike nicotine, cotinine (the major metabolite of nicotine) has been reported to be unable to cross the blood-brain barrier, but the distribution of cotinine in the brain after nicotine exposure has been well-established. Therefore, it is surprising that existing literature indirectly suggests that the distribution of cotinine in the central nervous system is secondary to the metabolism of nicotine in the brain. This report aims to clarify the blood-brain barrier transport of nicotine and cotinine in nicotine-naïve and nicotine-exposed animals. We used an in situ brain perfusion model to assess the blood-brain barrier uptake of [3H]nicotine and [3H]cotinine in nicotine-naïve animals and chronically exposed animals receiving continuous osmotic infusion of S-(-)nicotine (4.5 mg/kg/day) via an osmotic pump. Our data indicate that: 1) chronic nicotine exposure did not alter blood-brain barrier uptake of [3H]nicotine in an in situ perfusion model; 2) [3H]cotinine can cross the blood-brain barrier; and 3) similar to [3H]nicotine, chronic nicotine exposure did not alter blood-brain barrier transport of [3H]cotinine. To our knowledge, this is the first report to detail the uptake of nicotine and cotinine after chronic nicotine exposure and to quantify the rate at which cotinine crosses the blood-brain barrier.

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Metabolism/Metabolites
Nicotinine and its major oxidative metabolites are partially metabolized via glucuronidation. Genetic variation in the UGT isoenzymes that catalyze glucuronidation activity suggests that differences in glucuronidation rates are partly determined by genetic factors. A twin study of nicotine pharmacokinetics assessed the relative contributions of genetic and environmental factors to individual differences in the glucuronidation rates of nicotine, cotinine, and trans-3'-hydroxycotinine. Two approaches were used to define the glucuronidation rate: one considered the variability in renal clearance, and the other assumed that the relative renal clearance of the parent drug and its glucuronide conjugate was the same across individuals. Previous definitions led to a high correlation between nicotine and cotinine glucuronidation rates, significantly influenced by both additive (heritable) and non-additive (dominant and epistatic) genetic effects. These findings suggest that genetic variation in UGT isoenzymes (acting in an additive and interactive manner) is a crucial factor determining individual differences in nicotine and cotinine metabolism via the glucuronidation pathway. Cotinine formation is the primary pathway for nicotine metabolism in smokers, and the primary pathway for cotinine metabolism is trans-3'-hydroxylation. Trans-3'-hydroxycotinine and its glucuronide conjugates account for 50% of nicotine metabolites excreted by smokers. Minor cotinine metabolites excreted by smokers include norcotinine and cotinine N-oxide, each accounting for less than 5% of the nicotine dose. P450 2A6 has been reported as a catalyst for cotinine metabolism. However, we report here that the major product of P450 2A6-catalyzed cotinine metabolism is N-(hydroxymethyl)norcotinine, a previously unknown human cotinine metabolite. We chemically synthesized N-(hydroxymethyl)norcotinine and demonstrated its stability under enzymatic reaction conditions. The [5-3H]cotinine metabolites catalyzed by P450 2A6 were quantitatively analyzed using radioactive flow high-performance liquid chromatography (HPLC). Based on HPLC analysis using three different chromatographic systems and co-elution with N-(hydroxymethyl)norcotinine standards, we identified N-(hydroxymethyl)norcotinine as the major metabolite. 5'-Hydroxycotinine and trans-3'-hydroxycotinine were minor products of P450 2A6-catalyzed cotinine metabolism, accounting for 14% and 8% of the total cotinine metabolites, respectively. N-(hydroxymethyl)norcotinine is a product of cotinine metabolism catalyzed by P450 2A13 in extrahepatic cells, but its content is relatively low. The major product of cotinine metabolism catalyzed by P450 2A13 is 5'-hydroxycotinine, which is produced at twice the rate of trans-3'-hydroxycotinine. All cotinine metabolites produced by these two enzymes were identified by LC/MS/MS analysis. The kinetic parameters of cotinine metabolism in P450 2A6 and P450 2A13 were determined. This study confirms that trans-3'-hydroxycotinine, the major metabolite of cotinine in smokers, is only a minor metabolite of cotinine metabolism catalyzed by P450 2A6. Nicotine is the main component of tobacco and plays a key role in smoking addiction. In the human body, nicotine is mainly metabolized to cotinine, and cotinine is further metabolized to trans-3'-hydroxycotinine. Recently, we demonstrated that heterologously expressed human CYP2A13 exhibits high activity in the metabolism of 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK), a nicotine-derived carcinogen. In this study, we found that CYP2A13-catalyzed NNK metabolism is competitively inhibited by nicotine and N'-nitrosonornicotinamide (NNN), indicating that nicotine and NNN are also substrates of CYP2A13. We further confirmed that human CYP2A13 is indeed a highly efficient enzyme capable of catalyzing the C-oxidation of nicotine to cotinine, with apparent K_m and V_max values of 20.2 μM and 8.7 pmol/min/pmol, respectively. CYP2A13 can also catalyze the 3'-hydroxylation of cotinine to produce trans-3'-hydroxycotinine, with apparent K_m and V_max values of 45.2 μM and 0.7 pmol/min/pmol, respectively. The importance of CYP2A13-catalyzed nicotine and cotinine metabolism in vivo remains to be determined. Nicotine plays an important role in smoking addiction, smoking cessation replacement therapy, and as a potential drug for treating various diseases such as Parkinson's disease, Alzheimer's disease, and ulcerative colitis. Absorbed nicotine is rapidly and extensively metabolized and excreted in urine. One of the main pathways of nicotine metabolism is C-oxidation to cotinine, a process catalyzed by CYP2A6 in the human liver. Cotinine is subsequently metabolized to trans-3'-hydroxycotinine by CYP2A6. Nicotine and cotinine undergo glucuronidation primarily via UGT1A4, and partially via UGT1A9, to produce N-glucuronide. Trans-3'-hydroxycotinine undergoes glucuronidation primarily via UGT2B7, and partially via UGT1A9, to produce O-glucuronide. Approximately 90% of ingested nicotine is excreted as these metabolites and nicotine itself. Nicotine metabolism is a crucial factor determining nicotine clearance. In recent years, our understanding of individual differences in nicotine metabolism has deepened. Extensive data indicate that inter-individual differences in cotinine production are closely related to polymorphisms in the CYP2A6 gene. Furthermore, differences exist in cotinine production levels and the frequency of the CYP2A6 allele among different ethnic groups. Because CYP2A6 gene polymorphisms significantly affect nicotine clearance, it is speculated that they may be associated with smoking behavior or lung cancer risk. The glucuronidation metabolic pathway of nicotine, cotinine, and trans-3'-hydroxycotinine in the human body may be one of the reasons for inter-individual differences in nicotine metabolism. Known metabolites of cotinine in the human body include norcotinine, trans-3'-hydroxycotinine, cotinine N-glucuronide, and 5'-hydroxycotinine.
Biological half-life
Cotinine levels in infants are higher than in older children or adults exposed to the same dose of environmental tobacco smoke. One hypothesis to explain this difference is that the elimination half-life of cotinine in the urine of infants and older children differs. Urine was collected at admission, 12 hours, 24 hours, and 48 hours, and cotinine levels were subsequently measured and normalized by adjusting for creatinine excretion. This study calculated the elimination half-life of cotinine in the urine of 31 infants and 23 older children. The median half-life of cotinine in infants was 28.3 hours (range 6.3–258.5 hours), and the median half-life in older children was 27.14 hours (range 9.7–99.42 hours). The Mann-Whitney U test showed no significant difference in the median urinary cotinine half-life between the two age groups (P = 0.18). Multiple linear regression analysis showed no significant correlation between the urinary cotinine half-life and the adjusted cotinine level (P = 0.24). Our results support the hypothesis that higher urinary cotinine levels in infants are due to greater exposure rather than slower metabolism. This study also investigated the effects of race, menthol cigarette preference, body composition, and alcohol consumption history on the urinary cotinine half-life 6 days after smoking cessation in African American and white women. A 7-day inpatient study protocol was conducted at the Comprehensive Clinical Research Center, allowing free smoking on day 1 and smoking cessation on days 2–7 (n = 32). Plasma cotinine levels were measured every 8 hours during the study. The mean half-life of cotinine was 21.3 hours, similar to the previously reported 18–20 hours. Three women still had plasma cotinine levels above 14 ng/mL 136 hours after quitting smoking. Host factors associated with prolonged cotinine half-life included African American menthol cigarette smokers, shorter duration of alcohol consumption, and larger lean body mass; these factors explained 52.0% of the variation in cotinine half-life. Among menthol smokers, there were no significant differences in baseline cotinine levels and cotinine half-life between white and African American women. Cotinine is the major metabolite of nicotine, and its half-life in vivo (approximately 15–40 hours in humans) is longer than that of nicotine (2–3 hours), thus serving as a stable biomarker of nicotine exposure. [1]
- After nicotine absorption, cotinine is distributed in various bodily fluids (urine, saliva, blood): in adolescents, detectable levels of cotinine in these fluids are associated with self-reported smoking behavior, and its concentration varies with smoking frequency (e.g., occasional smoking versus daily smoking). [1]

[1]. An assessment of the validity of adolescent self-reported smoking using three biological indicators. Nicotine Tob Res. 2003 Aug;5(4):473-83.

Therapeutic Uses

Therapeutic Category: Antidepressant. /Experimental Therapy/
/Experimental Therapy/ Cotinine is the main metabolite of nicotine in the human body, and its duration of action in the body is much longer than that of nicotine. Recent studies have shown that cotinine has unique pharmacological properties, including potential cognitive enhancement, antipsychotic activity, and cytoprotective effects. Since this metabolite is generally less potent than nicotine in the body, we considered whether some of the therapeutic effects of cotinine might be related to its ability to reduce nicotine receptor desensitization compared to nicotine. We conducted experiments on freely moving rats in cages equipped with instruments to continuously measure mean arterial blood pressure. The ganglion stimulant dimethylphenylpiperazine could increase mean arterial blood pressure by a maximum of 25 mmHg. Slow (20 minutes) intravenous infusion of nicotine (0.25–1 μL) did not cause a change in resting mean arterial blood pressure, but the pressor response induced by subsequent injection of dimethylphenylpiperazine was significantly attenuated in a dose-dependent manner, up to 51%. Pre-infusion of an equal dose of cotinine produced the same degree of maximal inhibition as dimethylphenylpiperazine. A single intravenous injection of nicotine also caused a dose-dependent increase in mean arterial blood pressure, reaching a maximum of 43 mmHg after the highest tolerated dose. In contrast, injections of cotinine up to 13 times the highest dose of nicotine did not cause a significant change in mean arterial blood pressure. These results suggest a disconnect between nicotine receptor activation and receptor desensitization, and indicate that the pharmacological effects of cotinine may be mediated through partial desensitization or through non-ganglionic subtypes of nicotine receptors.

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- Biomarker Background: Cotinine is the major inactive metabolite of nicotine (produced by the metabolism of nicotine in the liver by cytochrome P450 enzymes). Its presence in biological samples (urine, saliva, blood) is widely used to verify self-reported smoking status, especially in adolescents, as adolescent self-reporting may be inaccurate. [1] - Detection threshold for smoking verification: This study used different concentrations of cotinine in bodily fluids to distinguish between smokers and non-smokers: 1. Urine cotinine: ≥100 ng/mL was the threshold for confirming active smoking (sensitivity: ~92%, specificity: ~95%); 2. Saliva cotinine: ≥10 ng/mL was the threshold (sensitivity: ~88%, specificity: ~93%); 3. Blood cotinine: ≥10 ng/mL was the threshold (sensitivity: ~90%, specificity: ~94%). [1]
- Validation results: In adolescents, the levels of cotinine in urine, saliva, and blood of self-reported "current smokers" were significantly higher than those of self-reported "non-smokers" (medians: 450 ng/mL, 35 ng/mL, and 28 ng/mL, respectively, compared to <10 ng/mL, <1 ng/mL, and <1 ng/mL for non-smokers). The concordance between self-reported smoking and cotinine detection results was highest in urine cotinine detection (Kappa coefficient: 0.82). [1]

176.21

176.21

176.094

486-56-6

854019

Light brown to brown <40°C powder,>42°C liquid

1.1±0.1 g/cm3

316.0±0.0 °C at 760 mmHg

40-42ºC

166.7±25.9 °C

0.0±0.6 mmHg at 25°C

1.556

-0.23

0

2

1

13

205

1

O=C1N(C)[C@H](C2=CC=CN=C2)CC1

UIKROCXWUNQSPJ-VIFPVBQESA-N

InChI=1S/C10H12N2O/c1-12-9(4-5-10(12)13)8-3-2-6-11-7-8/h2-3,6-7,9H,4-5H2,1H3/t9-/m0/s1

(5S)-1-methyl-5-pyridin-3-ylpyrrolidin-2-one

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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

Ethanol : ~120 mg/mL (~680.97 mM)
DMSO : ~65 mg/mL (~368.86 mM)

Solubility in Formulation 1: ≥ 2.17 mg/mL (12.31 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 21.7 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: ≥ 2.17 mg/mL (12.31 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 21.7 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: ≥ 2.17 mg/mL (12.31 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 21.7 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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