Metabolite Of Aspirin Research Articles

Salicylate, an aspirin metabolite, specifically inhibits the current mediated by glycine receptors containing α1‐subunits

Aspirin or its metabolite sodium salicylate is widely prescribed and has many side effects. Previous studies suggest that targeting neuronal receptors/ion channels is one of the pathways by which salicylate causes side effects in the nervous system. The present study aimed to investigate the functional action of salicylate on glycine receptors at a molecular level. Whole-cell patch-clamp and site-directed mutagenesis were deployed to examine the effects of salicylate on the currents mediated by native glycine receptors in cultured neurones of rat inferior colliculus and by glycine receptors expressed in HEK293T cells. Salicylate effectively inhibited the maximal current mediated by native glycine receptors without altering the EC(50) and the Hill coefficient, demonstrating a non-competitive action of salicylate. Only when applied simultaneously with glycine and extracellularly, could salicylate produce this antagonism. In HEK293T cells transfected with either alpha1-, alpha2-, alpha3-, alpha1beta-, alpha2beta- or alpha3beta-glycine receptors, salicylate only inhibited the current mediated by those receptors that contained the alpha1-subunit. A single site mutation of I240V in the alpha1-subunit abolished inhibition by salicylate. Salicylate is a non-competitive antagonist specifically on glycine receptors containing alpha1-subunits. This action critically involves the isoleucine-240 in the first transmembrane segment of the alpha1-subunit. Our findings may increase our understanding of the receptors involved in the side effects of salicylate on the central nervous system, such as seizures and tinnitus.

Effect of salicylic acid and calcium on mitochondrial functions.

The rapid mitochondrial uptake of calcium followed by slow release in certain pathophysiological states associated with an increase in intracellular calcium, to normalize the cytoplasmic levels of free calcium, provides an important protective mechanism against calcium cellular toxicity. Salicylic acid, an in vivo metabolite of aspirin, inhibits the uptake and enhances the release of calcium by mitochondria, thereby increasing the levels of cytoplasmic free calcium. The Ca2+ induced mitochondrial swelling is enhanced in the presence of salicylic acid and in which turn leads to loss of biosynthesis of ATP. These results suggest that salicylic acid may promote cellular damage in pathophysiological states associated with increase in intracellular free calcium.

Emergent Aspirin Use in Cardiovascular Disease in the Emergency Department: Oral Dosing versus Rectal Suppositories

Emergent Aspirin Use in Cardiovascular Disease in the Emergency Department: Oral Dosing versus Rectal Suppositories

NS398 protects cells from sodium nitroprusside-mediated cytotoxicity through enhancing HO-1 induction independent of COX-2 inhibition

Heme oxygenase-1 (HO-1) plays a preventive role in oxidative stress. In contrast, COX-2 is involved in the pathogenesis of many inflammatory diseases, thus COX-2 inhibitor is believed to exert anti-inflammatory properties by blocking COX-2. Recently, however, salicylate the active metabolite of aspirin showed COX-independent anti-inflammatory effects through induction of HO-1. Thus, we hypothesized that HO-1 are induced as an adaptive response to sodium nitroprusside (SNP) and play a protective role against cytotoxicity. Moreover, we investigated the protective effect of NS398 known as a selective COX-2 inhibitor on SNP-mediated cytotoxicity, and whether the protective effect of NS398 is due to COX-2 inhibition or not. SNP induced cytotoxicity in a dose-dependent manner, which was enhanced by inhibition of HO-1, suggesting that HO-1 acts in an adaptive response to SNP. Interestingly, NS398 decreased SNP-mediated cytotoxicity whereas COX-2 siRNA did not. Furthermore, NS398 enhanced SNP-induced HO-1 induction even though NS398 alone failed to induce HO-1 protein expression. In addition, NS398 enhanced SNP-induced COX-2, even though NS398 effectively inhibited SNP-mediated PGE(2) production. These results demonstrate that NS398 exerts cytoprotective effects by inducing HO-1 independent of COX-2 inhibition.

Interactions of gallic acid, resveratrol, quercetin and aspirin at the platelet cyclooxygenase-1 level Functional and modelling studies

While resveratrol and quercetin possess antiplatelet activity, little is known on the effect of gallic acid on platelets. We studied the interactions of these three different polyphenols among themselves and with aspirin, at the level of platelet cyclooxygenase-1 (COX-1). Both functional (in vitro and in vivo) and molecular modelling approaches were used. All three polyphenols showed comparable antioxidant activity (arachidonic acid [AA]-induced intraplatelet ROS production); however, resveratrol and quercetin, but not gallic acid, inhibited AA-induced platelet aggregation. Gallic acid, similarly to salicylic acid, the major aspirin metabolite, prevented inhibition of AA-induced platelet function by aspirin but, at variance with salicylic acid, also prevented inhibition by the other two polyphenols. Molecular modelling studies, performed by in silico docking the polyphenols into the crystal structure of COX-1, suggested that all compounds form stable complexes into the COX-1 channel, with slightly different but functionally relevant interaction geometries. Experiments in mice showed that gallic acid administered before aspirin, resveratrol or quercetin fully prevented their inhibitory effect on serum TxB(2). Finally, a mixture of resveratrol, quercetin and gallic acid, at relative concentrations similar to those contained in most red wines, did not inhibit platelet aggregation, but potentiated sub-inhibitory concentrations of aspirin. Gallic acid interactions with other polyphenols or aspirin at the level of platelet COX-1 might partly explain the complex, and possibly contrasting, effects of wine and other components of the Mediterranean diet on platelets and on the pharmacologic effect of low-dose aspirin.

Damage to cellular and isolated DNA induced by a metabolite of aspirin

Aspirin has been proposed as a possible chemopreventive agent. On the other hand, a recent cohort study showed that aspirin may increase the risk for pancreatic cancer. To clarify whether aspirin is potentially carcinogenic, we investigated the formation of 8-oxo-7,8-dihydro-2’-deoxyguanosine (8-oxodG), which is correlated with the incidence of cancer, in cultured cells treated with 2,3-dihydroxybenzoic acid (2,3-DHBA), a metabolite of aspirin. 2,3-DHBA induced 8-oxodG formation in the PANC-1 human pancreatic cancer cell line. 2,3-DHBA-induced DNA single-strand breaks were also revealed by comet assay using PANC-1 cells. Flow cytometric analyses showed that 2,3-DHBA increased the levels of intracellular reactive oxygen species (ROS) in PANC-1 cells. The 8-oxodG formation and ROS generation were also observed in the HL-60 leukemia cell line, but not in the hydrogen peroxide (H 2O 2)-resistant clone HP100 cells, suggesting the involvement of H 2O 2. In addition, an hprt mutation assay supported the mutagenicity of 2,3-DHBA. We investigated the mechanism underlying the 2,3-DHBA-induced DNA damage using 32P-labeled DNA fragments of human tumor suppressor genes. 2,3-DHBA induced DNA damage in the presence of Cu(II) and NADH. DNA damage induced by 2,3-DHBA was enhanced by the addition of histone peptide-6 [AKRHRK]. Interestingly, 2,3-DHBA and histone peptide-6 caused base damage in the 5′-ACG-3′ and 5′-CCG-3′ sequences, hotspots of the p53 gene. Bathocuproine, a Cu(I) chelator, and catalase inhibited the DNA damage. Typical hydroxyl radical scavengers did not inhibit the DNA damage. These results suggest that ROS derived from the reaction of H 2O 2 with Cu(I) participate in the DNA damage. In conclusion, 2,3-DHBA induces oxidative DNA damage and mutations, which may result in carcinogenesis.

Salicylic Acid sans Aspirin in Animals and Man: Persistence in Fasting and Biosynthesis from Benzoic Acid

Salicylic acid (SA), which is central to defense mechanisms in plants and the principal metabolite of aspirin, occurs naturally in man with higher levels of SA and its urinary metabolite salicyluric acid (SU) in vegetarians overlapping with levels in patients on low-dose aspirin regimens. SA is widely distributed in animal blood. Fasting for major colorectal surgery did not cause disappearance of SA from plasma, even in patients following total proctocolectomy. A 13C6 benzoic acid load ingested by six volunteers led, between 8 and 16 h, to a median 33.9% labeling of urinary salicyluric acid. The overall contribution of benzoic acid (and its salts) to the turnover of circulating SA thus requires further assessment. However, that SA appears to be, at least partially, an endogenous compound should lead to reassessment of its role in human (and animal) pathophysiology.

Comparison of pharmacokinetic and pharmacodynamic profiles of aspirin following oral gavage and diet dosing in rats

Aspirin is one of the oldest drugs and has been purported to have multiple beneficial effects, including prevention of cardiovascular disease and cancer, in addition to its original indication for treatment of inflammation, fever and pain. In cancer chemoprevention studies using animal models, two methods of aspirin administration have been employed: oral gavage and diet. The untested assumption was that exposure and the resultant pharmacological effects are similar for these two administration methods when dosing is normalized on the basis of mg/kg body weight/day. This study examined and compared time-dependent plasma and colon mucosal concentrations of aspirin metabolite salicylate (aspirin concentrations were below level of quantification), plasma thromboxane B 2 concentrations, and colon mucosal prostaglandin E 2 concentration following these two different dosing paradigms in rats. Diet dosing yielded relatively constant plasma and colon salicylate concentration vs. time profiles. On the other hand, oral gavage dosing led to a rapid peak followed by a fast decline in salicylate concentration in both plasma and colon. Nevertheless, the exposure as measured by the area under plasma or colon concentration–time curve of salicylate was linearly related to dose irrespective of the dosing method. Linear relationships were also observed between colon and plasma salicylate areas under the curve and between colon prostaglandin E 2 and plasma thromboxane B 2 areas under the curve. Therefore, more easily accessible plasma salicylate and thromboxane B 2 concentrations were representative of the salicylate exposure and prostaglandin E 2 pharmacodynamic biomarker in the target colon, respectively.

Histone deacetylase inhibitors and aspirin interact synergistically to induce cell death in ovarian cancer cells

Histone deacetylase inhibitors (HDIs) as well as non-steroidal anti-inflammatory drugs including aspirin show promise as antineoplastic agents. The treatment with both HDIs and aspirin can result in hyperacetylation of proteins. In this study, we investigated whether HDIs and aspirin interacted in inducing anticancer activity and histone acetylation. We found that the HDIs, suberoylanilide hydroxamic acid and sodium butyrate, and aspirin cooperated to induce cell death in the ovarian cancer cell line, A2780. The effect was synergistic, as evidenced by CI-isobologram analysis. However, aspirin had no effect on histone acetylation, neither in the absence nor presence of HDIs. To gain insight into the mechanism underlying the synergistic action of HDIs and aspirin, we employed the deacetylated metabolite of aspirin, salicylic acid, and the cyclooxygenase-1- and -2-selective inhibitors, SC-560 and NS-398, respectively. We found that HDIs and salicylic acid interacted synergistically, albeit less efficiently than HDIs and aspirin, to induce cancer cell death, suggesting that the acetyl and the salicyl moiety contributed to the cooperative interaction of aspirin with HDIs. SC-560 and NS-398 had little effect both when applied alone or in conjunction with HDIs, indicating that the combinatorial effect of HDIs and aspirin was not the result of cyclo-oxygenase inhibition. In conclusion, our study demonstrates that HDIs and aspirin synergize to induce cancer cell death and, thus, provides a rationale for a more in-depth exploration into the potential of combining HDIs and aspirin as a strategy for anticancer therapy.

Sodium salicylate switches glucose depletion-induced necrosis to autophagy and inhibits high mobility group box protein 1 release in A549 lung adenocarcinoma cells

Sodium salicylate, the active metabolite of aspirin, has been shown to exert anti-inflammatory activities by inhibiting the expression of various pro-inflammatory factors, and has potent anti-cancer effects against a number of human cancers including colon, lung, breast and leukemia. Necrotic cell death is emerging as one of the crucial factors that trigger an inflammatory response since during necrotic death the cell membrane is ruptured and the intracellular constituents including high mobility group box 1 (HMGB1) are released into the extracellular space, thereby activating an inflammatory response. In contrast, autophagic death is regarded as a form of tumour suppressive cell death, as indicated in tumour suppressors such as beclin 1 in autophagic pathways. To better understand the anti-inflammatory properties of sodium salicylate and its effect on necrotic cell death in A549 cells induced by glucose depletion (GD), a common characteristic of the tumour micro-environment, was examined. While GD induced mostly necrotic death in A549 cells, salicylate suppresssed GD-induced necrosis and HMGB1 release. In addition, salicylate shifted the cell death pattern to autophagy by inhibiting GD-induced Cu/Zn superoxide dismutase release and ROS production. These results indicate that the activity of salicylate to prevent necrotic death may contribute to its anti-inflammatory action and suppress tumour development possibly through switching the cell death mode from tumour-promoting necrotic cell death to tumour-suppressive autophagic cell death.

Evaluation of aspirin metabolites as inhibitors of hypoxia-inducible factor hydroxylases

Known and potential aspirin metabolites were evaluated as inhibitors of oxygen-sensing hypoxia-inducible transcription factor (HIF) hydroxylases; some of the metabolites were found to stabilise HIF-alpha in cells.

PALM KERNEL OIL BLENDS AS SUPPOSITORY BASES IN THE DELIVERY OF ASPIRIN

Rectal delivery of drugs has been proven to be effective in terms of drug absorption and distribution comparable with other routes such as oral, buccal, sublingual or even nasal. In this study, two new suppository bases were developed using combinations of locally sourced hydrogenated palm kernel oil, hydrogenated palm kernel stearin and hydrogenated palm kernel olein with mixtures of stearic acid and glyceryl monostearate. When formulated with aspirin, these bases produced suppositories with acceptable characteristics. These aspirin suppositories were tested on twelve healthy subjects after an approval from the Medical Ethics Committee, University of Malaya had been procured. We quantified aspirin from the urine samples of the subjects to determine the relative availability of the different suppository preparations relative to an oral dose. The excretion of salicylic acid, one of the metabolite of aspirin in human urine taking aspirin was quantified. The F value was found to range from 1.16 to 1.38. Hence, the excretion results showed that these palm kernel oil blends are suitable suppository bases.

The aspirin metabolite salicylate enhances neuronal excitation in rat hippocampal CA1 area through reducing GABAergic inhibition

Salicylate is the major metabolite and active component of aspirin (acetylsalicylic acid), which is widely used in clinical medicine for treating inflammation, pain syndromes and cardiovascular disorders. The well-known mechanism underlying salicylate's action mainly involves the inhibition of cyclooxygenase and subsequent decrease in prostaglandin production. Recent evidence suggests that salicylate also affects neuronal function through interaction with specific membrane channels/receptors. However, the effect of salicylate on synaptic and neural network function remains largely unknown. In this study, we investigated the effect of sodium salicylate on the synaptic transmission and neuronal excitation in the hippocampal CA1 area of rats, a key structure for many complex brain functions. With electrophysiological recordings in hippocampal slices, we found that sodium salicylate significantly enhanced neuronal excitation through reducing inhibitory GABAergic transmission without affecting the basal excitatory synaptic transmission. Salicylate significantly inhibited the amplitudes of both evoked and miniature inhibitory postsynaptic currents, and directly reduced γ-aminobutyric acid type A (GABA A) receptor-mediated responses in cultured rat hippocampal neurons. Together, our results suggest that the widely used aspirin might impair hippocampal synaptic and neural network functions through its actions on GABAergic neurotransmission. Given the capability of aspirin to penetrate the blood–brain barrier, the present data imply that aspirin intake may cause network hyperactivity and be potentially harmful in susceptible subpopulations.

Salicylates and their Spectrum of Activity

Salicylate is a signaling molecule in plants. It also exhibits signaling activities in mammalian cells. Experimental and clinical data indicate that salicylates have a spectrum of activities, including antithrombotic, anti-inflammatiory, anti-neoplastic, and anti-microbial actions. Aspirin, a synthetic derivative of salicylic acid, is widely used in treating human diseases. The antithrombotic action is unique to aspirin and not shared by other salicylates as only aspirin possesses the property to acetylate COX-1. Other actions of salicylates are attributed to salicylate, a major metabolite of aspirin in vivo. Salicylates are active in controlling inflammation and tumor growth by altering gene expressions. They suppress the expression of pro-inflammatory genes by inhibiting the DNA binding activities of transcription activators such as NF-κB, AP-1 and C/EBPβ. Their actions appear to be concentration related. Salicylates at high concentrations non-selectively inhibit the binding activities of diverse transactivators. At pharmacological concentrations, salicylates inhibit C/EBPβ binding and C-Rel. Transcriptional suppression by salicylates is mediated by kinase inhibition. Salicylates at high concentrations inhibit diverse classes of kinases and, paradoxically, activate some kinases. However, at micromolar concentrations, salicylates inhibit p90 ribosomal S6 kinase (RSK) and p70 S6 kinase (S6K). The mechanism by which salicylates inhibit kinases is unclear. We propose that salicylates at mM concentrations non-selectively inhibit ATP binding to kinases. At micromolar concentrations, they inhibit substrate binding to a selective family of kinases including RSK and S6K. Further structural analysis will yield valuable information which will be useful in designing new anti-inflammatory and antineoplastic drugs. Keywords: Aspirin, salicylate, cyclooxygenase, tumorigenesis, inflammation, NF-κB, C/EBP, p90 ribosomal S6 kinase

Salicylates Trigger Protein Synthesis Inhibition in a Protein Kinase R-like Endoplasmic Reticulum Kinase-dependent Manner

The non-steroidal anti-inflammatory drug aspirin and its metabolite, sodium salicylate, have profound effects on cellular protein synthesis and cell physiology. However, the underlying mechanism by which they cause these responses remains unclear. We show here that salicylates induce phosphorylation of the alpha-subunit of eukaryotic translation initiation factor 2 (eIF2alpha), resulting in the inhibition of mRNA translation in cells. Exposure of cells to acetyl salicylic acid resulted in strong activation of eIF2alpha stress-activated protein kinase R-like endoplasmic reticulum kinase (PERK). Analysis of fibroblasts with a targeted deletion of the perk gene revealed that PERK is indispensable for triggering the phosphorylation of eIF2alpha as well as the inhibition of protein synthesis induced by salicylates. Although salicylate treatment did not trigger activation of inositol-requiring enzyme 1, there was an increased expression of the pro-apoptotic transcription factor CHOP-(gadd153), a downstream event to eIF2alpha phosphorylation known to mediate endoplasmic reticulum stress-mediated responses. Thus, salicylates selectively trigger an endoplasmic reticulum stress-responsive signaling pathway initiated through activation of PERK to induce their cellular effects.

Effect of salicylic acid on invasion of human vascular endothelial cells byStaphylococcus aureus

Invasion of vascular endothelial cells by Staphylococcus aureus is associated with diverse complications and recurrent infection. Little is known about the effect of salicylic acid, the major metabolite of aspirin, on the interaction between S. aureus and vascular endothelial cells. We examined the adhesion of S. aureus strain 8325-4 cultured with or without salicylic acid to human umbilical vein endothelial cells (HUVECs), and the ability of the strain to invade these cells. Strain 8325-4 cells grown in salicylic acid were significantly less adherent to and invasive in HUVECs. Production of cytokine interleukin (IL)-6 was lower from the HUVECs infected with clinical isolates of S. aureus cultured in salicylic acid compared with those unexposed to salicylic acid. This study raises the possibility of using salicylic acid as an adjuvant therapeutic agent in the treatment of S. aureus bacteremia to prevent its complications or recurrence.

Suppressive effect of post- or pre-treatment of aspirin metabolite on mitomycin C-induced genotoxicity using the somatic mutation and recombination test in Drosophila melanogaster

In our previous paper, we found that aspirin suppressed in a somatic mutation and recombination test (SMART) of mitomycin C (MMC) in Drosophila melanogaster. In order to reveal the mechanism of bio-antimutagenicity and/or preventive effect of aspirin, we evaluated the suppressive ability of each aspirin metabolite, such as salicylic acid (SA), salicyluric acid (SUA), gentisic acid (GA), gentisuric acid (GUA) and 2,3-dihydroxybenzoic acid (DHBA), in SMART in D. melanogaster using post- and pre-treatments. As for the post-treatment, SA reduced the numbers of large single and twin spots. GA reduced the small single and large single spots, and GUA reduced the single spots, large single and twin spots. The inhibition of GUA is slightly stronger than that of any other metabolites; the inhibition percentage is 49 at the dose of 5 mg/bottle. On the other hand, as for the pre-treatment, aspirin, SUA, GA and DHBA reduced the numbers of small single spots. SUA, GE and DHBA reduced the number of large single spots. Aspirin and its metabolites did not reduce the number of twin spots. The results of the present study suggest that SA, GA and GUA repair or replicate DNA-damage by MMC and SUA, GA, GUA and DHBA prevent DNA-damage by MMC. It is suggested that secondary cancer is prevented by aspirin post-treatment without losing the medicinal effectiveness (anti-tumor activity). Therefore, we consider there are effective doses and/or administration timing of aspirin and MMC to prevent secondary cancer.

Feeding Effects on Hypocalcemia Induced by Salicylic Acid Derivatives in the Rat

The present experiments were carried out to examine feeding effects on hypocalcemia induced by salicylic acid derivatives. When a large dose of aspirin was administered orally to rats of different ages (up to 45 weeks of age), hypocalcemia was observed at all ages. Because hypocalcemia was induced in both the fasting and non-fasting rats by intravenous injection of salicylic acid (SA, main metabolite of aspirin), hunger sensation was unlikely to be the cause of hypocalcemia induced by SA derivatives. Aspirin-induced hypocalcemia was maintained under fasting conditions and recovered to a normal calcium range by refeeding a normal diet. On the other hand, hypocalcemia was not resolved but strengthened by refeeding a calcium-deficient diet, indicating that hypocalcemia induced by SA derivatives is not due to the inhibition of calcium absorption from the intestine. Thus, local hormones from the gastrointestinal tract and/or calcitonin secreted by feeding stimulation appear to be involved in hypocalcemia, which can be masked by a sufficient supply of dietary calcium from the intestine. In conclusion, the present study suggests that taking calciumenriched meals before administrating a large dose of aspirin is necessary to prevent or minimize hypocalcemia.

Authors’ response: Relationships between the adverse effects of aspirin and polymorphisms in genes encoding aspirin metabolizing enzymes

We thank Miners & Day for their interest in our work and for their critical comments on our paper [1]. We agree with Miners & Day that the enzymes UGT1A6 and CYP2C9 may not be the main enzymes involved in the metabolism of aspirin [2]. However, in answer to the comments of Minors & Day, we want to address following: 1) Our main goal was to study possible associations between genetic polymorphisms in UGT1A6 and CYP2C9, both involved in the metabolism of aspirin, and gastrointestinal complaints as caused by cardiovascular dosed (80–100 mg) aspirin or one of its metabolites [1]. This does not necessarily need to be the main metabolite(s) of aspirin. It is not irrational to hypothesize, that the highly reactive product(s) of a minor oxidative metabolic pathway, often catalyzed by CYPs (here CYP2C9), may cause more damage or complaints as compared with a main metabolic product. 2) The data on metabolism of aspirin in humans, as given by Minors & Day, are based on studies by Hutt et al.[2], giving an oral dose of 900 mg aspirin, whereas our patients received 80–100 mg aspirin daily. This difference in dose may result in a different pattern of metabolites in urine from that given by Minors & Day, and since glucuronidation is often capacity limited, a low dose of aspirin may result in a larger contribution of glucuronide metabolites. (3) Minors & Day state that the UGTs involved in glucuronidation of salicylic acid are not characterized unambiguously, but by referring to the study of Keuhl et al.[3] they suggest that UGT1A6 may only be just one of several UGTs involved. However, apparent Kms for both phenolic- and acyl-glucuronidation of salicylic acid are lowest for UGT1A6, suggesting that this enzyme may be most relevant in the glucuronidation of low-dose aspirin.

Quantitative assessment of seminal vesicle and prostate drug concentrations by use of a noninvasive method

The male genital tract is a complex collection of anatomically and biochemically distinct compartments that contribute to the ejaculate. Understanding the pharmacokinetics in these compartments should inform rational therapeutics involving these glands. Nineteen men were administered a single dose of 600 mg chloroquine (base) and 975 mg aspirin before providing a semen sample by masturbation with fractionation into a 5-compartment collection device. Fractions were assayed for fructose (unique seminal vesicle marker), prostate-specific antigen (unique prostate marker), salicylate, and chloroquine. Seminal vesicle and prostate concentrations of salicylate and chloroquine were estimated via a novel analytic method involving a multilevel latent-variable model implemented by use of Bayesian methods. The geometric mean chloroquine semen/blood ratio was 4.02 (95% confidence interval [CI], 2.36-6.86); for salicylate, the primary metabolite of aspirin, the semen/blood ratio was 0.10 (95% CI, 0.08-0.14). The estimated mean prostate/seminal vesicle ratio for salicylate, 0.38 (95% CI by Bayesian methods, 0.12-0.73), was consistent with our hypothesis that salicylate would achieve higher concentrations in the seminal vesicle than in the prostate. Chloroquine, however, did not demonstrate a statistically significant seminal vesicle/prostate difference (4.41; 95% CI by Bayesian methods, 0.14-30.52). We successfully demonstrated the quantitative, noninvasive estimation of drug concentrations in the prostate gland fluid distinct from the seminal vesicle fluid using our optimized method of split-ejaculate collection and a novel mixed-effects model with Bayesian estimation. Our methods can be applied to gland-specific quantitation of drugs and other substances of interest, thus enabling pharmacokinetic, pharmacodynamic, and pathophysiologic studies to inform rational therapeutics within different glands of the male genital tract.