Inhibition of natural killer cell function by marijuana components

The extent of modulation of host resistance mechanisms by marijuana components is not fully understood. Natural killer (NK) cells are a subpopulation of lymphoid cells and are important in host resistance mechanisms against malignant cells, virus-infected cells, and possibly pathogenic bacteria and fungi. We report that the marijuana component delta-9-tetrahydrocannabinol (THC) injected into mice results in a suppression of splenic NK activity. Furthermore, THC and the hydroxylated metabolite 11-hydroxy-delta-9-tetrahydrocannabinol (11-hydroxy-THC) suppress the NK activity of cultured murine splenocytes in a dose-dependent manner (range 1 X 10(-5) to 3.2 X 10(-5) M) without diminishing NK cell viability. The hydroxylated derivative appears to possess a more potent suppressive effect, in that it suppresses at lower concentrations than THC does and requires a shorter incubation time with the effector cells for its suppressive action. Purification of NK cells by Percoll density-gradient centrifugation suggests that both cannabinoids act directly on the natural killer cell population, resulting in suppression. Studies involving target binding analysis and calcium ionophore experiments suggest that cannabinoids do not suppress NK cell killing by the inhibition of effector/target binding or by disruption of calcium ion flux. These results suggest that two principal psychoactive cannabinoids can suppress natural killer cell function by interacting directly with the killer cells and disrupting cellular events postbinding and during the programming for lysis. Furthermore, the data suggest different modes of action for THC and the hydroxylated metabolite.     

Alcohol consumption decreases IL-2-induced NF-kappaB activity in enriched NK cells from C57BL/6 mice.

Previously we showed that ethanol (EtOH) consumption suppressed IL-2-induced cytolytic activity of murine splenic natural killer (NK) cells. Although IL-2 receptor signaling is involved in activation of NK cells, neither the mechanism for this activation nor the role of EtOH consumption in modulating activation is completely understood. In this study we show by electrophoretic mobility-shift assay (EMSA) that enriched splenic NK cells from EtOH-consuming C57BL/6 mice exhibit reduced NF-kappaB and AP-1 binding activity in response to IL-2 stimulation as compared to the water-drinking mice. Semiquantitative RT-PCR and real-time PCR analyses indicated that EtOH consumption inhibits the induction of perforin, granzyme A, and granzyme B in response to IL-2. Pyrrolidine dithiocarbamate (PDTC) and N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) blocked NFkappaB and AP-1 binding activity in nuclear extracts of IL-2-stimulated NK cells in an EMSA and also inhibited the IL-2-induced expression of perforin, granzyme A, and granzyme B gene expression in enriched NK cells. These inhibitors dramatically suppressed IL-2-stimulated NK cytolytic activity against YAC-1 lymphoma target cells. Taken together, these results suggest that NFkappaB and AP-1 are important regulators of NK cell cytolytic function through regulation of perforin, granzyme A, and granzyme B gene expression. The findings further suggest that the decreased cytolytic activity of IL-2-stimulated NK cytolytic activity in EtOH-consuming mice is due at least in part to impaired transactivation of these and possibly other genes involved in control of NK-cell target lysis.     

Plasma membrane inhibition of macromolecular precursor transport by THC.

10^?6to 10^?4 M of delta-9-tetrahydrocannabinol (THC) or of other cannabinoids, which all have in common the C ring of olivetol, inhibit in cultured lymphocytes incorporation of [³H]thymidine. The inhibitory effect of olivetol derivatives is related to their octanol-water partition coefficient (liposolubility). Within 15 min of incubation, THC inhibits precursor pool formation and macromolecular incorporation of thymidine, uridine and leucine. THC inhibits also [¹⁴C] aminoisobutyric acid uptake into the cell, but does not alter the cellular “leakage” of this amino acid analogue. Using the isotopic dilution technique with L 1210 murine lymphoma cells and human lymphocytes, it was observed that THC decreases [³H]thymidine uptake within fifteen seconds after addition of the drug to the culture. Experiments performed at 0° indicate that THC has no action on thymidine binding to the carrier. All of these observations suggest that THC in micromolar concentration inhibits DNA synthesis through a “non-specific” alteration of membrane configuration. This effect, due to the liposolubility of the drug, could induce eonfonnational changes of membrane-bound transport systems which would inhibit their function.     

Differential roles of constitutively activated ERK1/2 and NF-kappa B in cytotoxicity and proliferation by human NK cell lines

Compared with freshly isolated peripheral blood natural killer (NK) cells, the YT and NK-92 cell lines are characterized by elevated cytolytic activity. The molecular mechanisms underlying the rapid proliferation and enhanced lytic activity of NK cell lines are poorly understood. Investigation of these cell lines revealed that ERK1/2 and NF-kappa B are constitutively activated, providing evidence that these two signaling pathways are differentially involved in cytolysis and proliferation. Furthermore, blocking ERK1/2 activation with the specific inhibitor, PD098059, inhibited cytolytic activity in both cell lines and reduced mRNA expression of cytolysis-related effector molecules such as Fas-L and IFN gamma, as measured by semi-quantitative RT-PCR. However, MTT colormetric analysis showed that treatment with the PD098059 inhibitor did not affect cell proliferation. Meanwhile, blockade of the NF-kappa B signaling pathway using MG132 inhibited cellular growth without impacting cytolytic capability. No synergistic interactions were observed between ERK1/2 and NF-kappa B after combination treatment with PD098059 and MG132, suggesting that these two signaling pathways likely affect cellular proliferation and cytotoxicity by NK cells differentially.     

Enhancement of the behavioral effects of endogenous and exogenous cannabinoid agonists by phenylmethyl sulfonyl fluoride

Marijuana's effects in humans are most often reported as intoxicating or therapeutic; yet, some humans report dysphoria or other negative affect. To evaluate whether differences in endocannabinoid levels might account for this variability, the present study examined whether sensitivity to cannabinoids changed when anandamide (AEA) metabolism was inhibited through administration of phenylmethyl sulfonyl fluoride (PMSF) a non-specific irreversible amidase inhibitor. Male Long Evans rats were trained to discriminate 3 mg/kg Δ(9)-tetrahydrocannabinol (THC) versus vehicle in 2-lever drug discrimination procedure. ED(50)s for THC and CP 55,940 were lower when administered with PMSF than alone. PMSF administration also potentiated characteristic cannabimimetic effects of THC in ICR mice. Potentiation of AEA's in vivo effects by PMSF were also observed, primarily as a consequence of PMSF inhibition of the enzyme fatty acid amide hydrolase. Enhancement of the effects of THC and CP 55,940 through this mechanism is unlikely, as these cannabinoids are predominantly metabolized through the P450 system. Mass spectrometry revealed that, in the presence of THC, endogenous AEA levels in the brain decreased and that this decrease was prevented by PMSF, suggesting that increased AEA levels may have acted additively with exogenously administered cannabinoids to increase cannabimimetic effects. These findings may account for the varying affect in response to marijuana in humans or cannabinoids in animals while also suggesting that metabolic inhibitors of AEA may potentiate marijuana's intoxicating effects in humans. This article is part of a Special Issue entitled 'Post-Traumatic Stress Disorder'.     

Immunoregulation of natural and lymphokine-activated killer cells by selenium

The effect of selenium (Se) on natural killer (NK) and lymphokine-activated killer (LAK) cell activities and proliferative responses of human lymphocytes was studied in vitro. Direct addition of Se at 1.0 microgram/ml final concentration to the mixture of target and effector cells during a 4 h cytotoxicity assay significantly suppressed the NK activity of normal lymphocytes. When lymphocytes were preincubated with Se at concentrations as low as 0.2 microgram/ml for a period of 48 h, a significant inhibitory effect on NK activity was observed. In the LAK cell assay, direct addition of Se at concentrations of 0.2-1.0 microgram/ml to a mixture of target and effector cells did not show any effects on LAK cell activity, whereas LAK cells generated in the presence of Se at 0.8 microgram/ml showed significant inhibition of their functions. Lymphocyte proliferative responses to T cell mitogens such as phytohemagglutinin (PHA) and concanavalin A (Con A) were also significantly suppressed by direct addition of Se at 0.5-1.0 microgram/ml. The inhibitory effect of Se was not due to nonspecific toxicity of effector cells as demonstrated by viability nor was the effect directed against target cells. These studies suggest that although Se is an essential micronutrient for various immune mechanisms, an excess of Se may have a deleterious effect on certain immunological functions. As these activities are considered to be important defense mechanisms against tumors and virus infections, a nutritional imbalance of Se could result in an increased risk of these disorders.     

The effects of cannabinoids on P-glycoprotein transport and expression in multidrug resistant cells

Cannabis is the most widely used illicit drug in the world. Cannabinoids are used therapeutically by some patients as they have analgesic, anti-emetic and appetite stimulant properties which palliate adverse symptoms. Use of these agents in an oncology setting raises the question of whether they act to modulate the effectiveness of concurrently administered anti-cancer drugs. The transporter, P-glycoprotein (P-gp) confers multiple drug resistance (MDR) by effluxing a diverse array of anti-cancer agents. This study was undertaken to examine the effect of cannabinoids on P-gp. Unlike the known P-gp inhibitor, PSC833, short 1h exposure to three plant-derived cannabinoids, cannabinol (CBN), cannabidiol (CBD) and Delta(9)-tetrahydrocannabinol (THC) and the synthetic cannabinoid receptor agonist, WIN55, 212-2 (WIN) did not inhibit the efflux of the P-gp substrate Rhodamine 123 (Rh123) in either a drug-selected human T lymphoblastoid leukaemia cell line (CEM/VLB(100)) or in a mouse fibroblast MDR1 transfected cell line (77.1). However, in CEM/VLB(100) cells, prolonged 72 h exposure to the cannabinoids, THC and CBD, decreased P-gp expression to a similar extent as the flavonoid, curcumin (turmeric). This correlated with an increase in intracellular accumulation of Rh123 and enhanced sensitivity of the cells to the cytotoxic actions of the P-gp substrate, vinblastine. Taken together, these results provide preliminary evidence that cannabinoids do not exacerbate P-gp mediated MDR. Further, plant-derived cannabinoids are moderately effective in reversing MDR in CEM/VLB(100) cells by decreasing P-gp expression.     

Inhibition of a phosphodiesterase III in the lysis-sensitive target-induced elevation of cyclic AMP (cAMP) in human natural killer cells

Natural killer (NK) cells are lymphocytes that are capable of destroying tumor cells and virally infected cells (cytolysis) without prior sensitization. When cyclic AMP (cAMP) is elevated artificially in NK cells, it is a potent inhibitor of their cytolytic function. Recently, we have shown that when NK cells are exposed to a range of lysis-sensitive (LS) tumor target cells, there is an increase in intracellular cAMP levels in the NK cells over a 60-min period. There is no increase in NK-cell cAMP in response to lysis-resistant (LR) tumor target cells. We determined that this cAMP elevation is due, in part, to an LS target-induced activation of adenylyl cyclase (AC), and that the AC-activation component appears to require a protein tyrosine kinase (PTK) activity. In the present study, we demonstrated that an LS target-induced inhibition of phosphodiesterase (PDE) is also contributing to the overall elevation of cAMP. Direct measurement of PDE activity showed an inhibition in lymphocytes that were exposed to LS targets but not in those exposed to LR targets. The inhibition of PDE activity was maximal by 30 min. Lymphocytes were exposed to targets and then lysed, so that PDE activity could be measured. Addition of class-selective inhibitors of PDE (at levels sufficient to completely block that class of PDE) to the lysate focused the measurement of PDE activity on those classes of PDE that were unaffected by the selective inhibitor. Using the PDE IV selective inhibitor rolipram and the PDE III selective inhibitors trequinsin and milrinone, we showed that a PDE III is being inhibited in lymphocytes by exposure to LS targets. As PDE III is known to be inhibited by elevated cyclic GMP (cGMP) levels, increased cGMP in NK cells following exposure to LS targets was a possible mechanism by which a PDE III in NK cells might be inhibited. However, when we measured cGMP levels in control and LS target-stimulated lymphocytes, we saw no change.     

Inhibition of monoamine oxidase activity by cannabinoids

Brain monoamines are involved in many of the same processes affected by neuropsychiatric disorders and psychotropic drugs, including cannabinoids. This study investigated in vitro effects of cannabinoids on the activity of monoamine oxidase (MAO), the enzyme responsible for metabolism of monoamine neurotransmitters and affecting brain development and function. The effects of the phytocannabinoid Δ⁹-tetrahydrocannabinol (THC), the endocannabinoid anandamide (N-arachidonoylethanolamide [AEA]), and the synthetic cannabinoid receptor agonist WIN 55,212-2 (WIN) on the activity of MAO were measured in a crude mitochondrial fraction isolated from pig brain cortex. Monoamine oxidase activity was inhibited by the cannabinoids; however, higher half maximal inhibitory concentrations (IC₅₀) of cannabinoids were required compared to the known MAO inhibitor iproniazid. The IC₅₀ was 24.7 μmol/l for THC, 751 μmol/l for AEA, and 17.9 μmol/l for WIN when serotonin was used as substrate (MAO-A), and 22.6 μmol/l for THC, 1,668 μmol/l for AEA, and 21.2 μmol/l for WIN when phenylethylamine was used as substrate (MAO-B). The inhibition of MAOs by THC was noncompetitive. N-Arachidonoylethanolamide was a competitive inhibitor of MAO-A and a noncompetitive inhibitor of MAO-B. WIN was a noncompetitive inhibitor of MAO-A and an uncompetitive inhibitor of MAO-B. Monoamine oxidase activity is affected by cannabinoids at relatively high drug concentrations, and this effect is inhibitory. Decrease of MAO activity may play a role in some effects of cannabinoids on serotonergic, noradrenergic, and dopaminergic neurotransmission.     

Granulysin: a novel antimicrobial

Granulysin is a novel lytic molecule produced by human cytolytic T-lymphocytes (CTLs) and natural killer (NK) cells. It is active against a broad range of microbes, including Gram-positive and -negative bacteria, parasites and Mycobacterium tuberculosis. It is functionally related to other antibacterial peptides, like defensins and magainins, but is structurally distinct. It has structural similarity to porcine NK-lysin and to amoebapores made by Entamoeba histolytica. Synthetic peptides derived from granulysin have differential activity against eukaryotic cells and bacteria. Selective bactericidal peptides may have therapeutic roles as novel antibiotics.     

Drug discrimination properties and cytotoxicity of the cannabinoid receptor ligands

The worldwide distribution of smokable herbal mixtures called "Spice" that contain synthetic cannabinoids with a pharmacological activity similar to delta 9-tetrahydrocannabinol (delta 9-THC) has been reported. The synthetic cannabinoids induce behavior and have biochemical properties similar to naturally occurring cannabinoids such as delta 9-THC. In drug discrimination procedures, animal behavior is differentially reinforced depending on the presence or absence of specific drug stimuli. This review seeks to establish an animal model to serve as a discriminative stimulus of the synthetic cannabinoids, to determine whether this discriminative stimulus is identical to that of delta 9-THC. Much data have been obtained in drug discrimination experiments with various synthetic cannabinoids. In the discriminative study, synthetic cannabinoids such as CP-55,940 and WIN-55,212-2 were substituted for delta 9-THC in rats trained to discriminate delta 9-THC from the vehicle. These discriminative effects of synthetic cannabinoids were antagonized by CB1 antagonist SR-141,716A. The discriminative effects of synthetic cannabinoids may overlap with the delta 9-THC cue mediated by CB1 receptors. In in vitro study using NG 108-15 cell lines, synthetic cannabinoids have produced strong cytotoxicities that were suppressed by pretreatment with the CB1 receptor antagonist. Furthermore, pretreatment with caspase inhibitors suppressed these synthetic-cannabinoid-induced cytotoxicities in NG 108-15 cells. These findings indicate that the cytotoxicity of synthetic cannabinoids towards NG 108-15 cells is mediated by the CB1 receptors and further suggest that caspase cascades may play an important role in the cytotoxicities induced by these synthetic cannabinoids. In conclusion, synthetic cannabinoid abuse could be a health hazard for humans.     

Granulysin: a novel antimicrobial

Granulysin is a novel lytic molecule produced by human cytolytic T-lymphocytes (CTLs) and natural killer (NK) cells. It is active against a broad range of microbes, including Gram-positive and -negative bacteria, parasites and Mycobacterium tuberculosis. It is functionally related to other antibacterial peptides, like defensins and magainins, but is structurally distinct. It has structural similarity to porcine NK-lysin and to amoebapores made by Entamoeba histolytica. Synthetic peptides derived from granulysin have differential activity against eukaryotic cells and bacteria. Selective bactericidal peptides may have therapeutic roles as novel antibiotics.     

DDVP markedly decreases the expression of granzyme B and granzyme 3/K in human NK cells

Natural killer (NK), lymphokine-activated killer (LAK), and cytotoxic T lymphocyte (CTL) cells kill target cells by the directed release of cytolytic granules that contain perforin, granzymes, and granulysin. We have found previously that dimethyl 2,2-dichlorovinyl phosphate (DDVP, dichlorvos), an organophosphorus pesticide, significantly decreased the expression of perforin, granzyme A (GrA), and granulysin and inhibited NK, LAK, and CTL activities. To further explore the mechanism of organophosphorus pesticide-induced inhibition of cell-mediated cytolysis, we examined whether organophosphorus pesticides affect the expression of GrB and Gr3/K in NK cells. We used an interleukin-2 (IL-2) independent human NK cell line, NK-92CI. We confirmed that NK-92CI cells express intracellular GrB and Gr3/K by flow cytometry, that NK-92CI cells are highly cytotoxic to K562 cells with a chromium release assay, and that DDVP significantly inhibited cytolytic activity of NK-92CI cells in a dose- and time-dependent manner. We found that DDVP significantly decreased the expression of GrB and Gr3/K in NK-92CI cells in a dose- and time-dependent manner by flow cytometry. Immunocytochemical results showed that DDVP significantly decreases the level of GrB positive granules in NK-92CI cells, which may be due to degranulation. Taken together, DDVP significantly inhibits NK activity and reduces the intracellular GrB and Gr3/K levels in NK cells.     

The quasi-morphine withdrawal syndrome: effect of cannabinol, cannabidiol and tetrahydrocannabinol

Delta-9-tetrahydrocannabinol (THC), the main psychoactive principle of cannabis, has been shown to attenuate the exhibition of signs of the quasi-morphine withdrawal syndrome in rats. Cannabinol (CBN) showed the same activity but required a dosage of approximately eight times that of THC to produce an equivalent effect. Cannabidiol was without effect at the dosage levels used. The efficacy of these cannabinoids and the potency differences recorded in this study are in accord with their effects on other behaviours, both in experimental animals and in man. The activity of THC and CBN was not affected by the narcotic antagonist, naloxone.     

Effects of delta 9-tetrahydrocannabinol on prostaglandin formation in brain

In order to investigate the effects of cannabinoids on prostaglandin (PG) formation in the mammalian central nervous system, slices were prepared from different regions of guinea pig brain and incubated with (-)-trans-delta 9-tetrahydrocannabinol (THC) with or without acetylcholine (ACh). The amounts of prostaglandins E (PGE) and (F (PGF) released into the medium were measured by radioimmunoassay. Incubation of cerebral cortex slices for 1 hr with concentrations of THC over the range of 0.8-16 microM significantly inhibited the formation of both PGE and PGF by up to 50% of control levels. Incubation of cortical slices with delta 8-THC, a psychoactive THC congener, resulted in similar dose-dependent inhibitions in both PGE and PGF levels. Congeners of THC devoid of psychoactivity, namely, cannabidiol and the inactive stereoisomer of THC, were less potent inhibitors of PGE synthesis. In marked contrast to the inhibitory effect of the psychoactive cannabinoids, PGF levels were elevated in the presence of the nonpsychoactive congeners. The effects of THC on PG formation were compared in different brain regions. In striatal slices, 0.8 microM THC significantly stimulated PG formation but did not affect PG levels in the cerebellum. In order to gain insight into the interaction between THC and endogenous neurotransmitters, we compared the effect of THC on the ACh-induced increase in PG formation in the cerebral cortex and the cerebellar cortex. In the cerebral cortex, 0.8 microM THC abolished the ACh-induced increase in PGE levels and inhibited the rise in PGF by 70%. In contrast, THC did not significantly affect the ACh-induced rise in PG levels in the cerebellum. The results indicate that cannabinoids alter both the basal and ACh-enhanced formation of PGE and PGF in the brain and that these effects are structurally and regionally specific.     

Effects of delta-9-tetrahydrocannabinol, 11-hydroxy-delta-9-tetrahydrocannabinol and cannabidiol on neuromuscular transmission in the frog

Intracellular recording techniques were used on neuromuscular junctions of the sartorius muscle of the frog, in vitro, to define the synaptic pharmacology of delta-9-tetrahydrocannabinol (THC), 11-hydroxy-THC and cannabidiol (CBD). The frequency of miniature endplate potentials was increased by THC, decreased by CBD and was unaffected by 11-hydroxy-THC, whereas the amplitude of the miniature endplate potentials was depressed by all three cannabinoids. In addition, the mean quantum content of the endplate potential (m) was first increased and then decreased by THC and 11-hydroxy-THC, but CBD produced only depression. Changes in m and the frequency of the miniature endplate potential indicated presynaptic sites of drug action and reduction of the amplitude of the miniature endplate potential suggested a postsynaptic site. The findings suggest possible mechanisms of action for the central excitatory and depressant properties of the cannabinoids.     

Decreased lymphocyte blastogenesis,IL2 production and NK activity following nifedipine administration to healthy humans

Summary The effects of a single oral dose of nifedipine on part of the immune response in healthy humans has been investigated in terms of two different immune functions: T lymphocyte proliferation and NK activity. Both functions are known to require calcium ions.Ten healthy subjects were bled before and 30 min, and 4 and 24 h after receiving 10 mg nifedipine. Lymphocyte proliferation, both in mitogen-activated lymphocyte cultures, and in autologous and allogeneic mixed lymphocyte reactions, was significantly reduced (up to 48%) 30 min after drug administration and reverted to normal 4 h later. The inhibition could be attributed to reduction in IL2 production by the T cells isolated 30 min following the administration of nifedipine, since they normally express IL2-receptors. The addition of recombinant IL2 of 200 U·ml^–1 to the cell cultures restored their responsiveness.NK activity was significantly reduced 30 min and 4 h after drug administration and returned to normal at the 24th h. This function was also restored by the addition of IL2.The data suggest that calcium channel blockers may inhibit, at least transiently, lymphocyte functions in vivo.List of abbreviations AMLC Autologous mixed lymphocyte cultures - CD Cluster determinants - ConA Concanavallin A - CTL Cytotoxic T lymphocytes - GAM-FITC Fluorescein isothiocyanate-conjugated goat anti-mouse immunoglobulins - HLA-class II DP-DQ-DR antigens - IL2 Interleukin 2 - mAb Monoclonal antibodies - MHC Major histocompatibility gene complex - MLC Mixed lymphocyte cultures - NK Natural killer activity - non- T/T type AMLC Autologous mixed lymphocyte culture in which non-T cells are used as stimulators - PBL Peripheral blood lymphocytes - T/T type AMLC Autologous mixed lymphocyte culture in which PHA-activated T lymphocytes are used as stimulators     

The intracellular localization of 9 -tetrahydrocannabinol in liver and its effects on drug metabolism in vitro

In the hepatic cell Δ⁹-tetrahydrocannabinol (THC) is localized in nuclei and microsomes. The intracellular binding of THC affects hepatic drug metabolism: nuclei markedly reduce the metabolism of THC by hepatic microsomes; THC inhibits the microsomal oxidation of aminopyrine and hexobarbital, the conjugation of estradiol and p-nitrophenol and enhances the reduction of p-nitrobenzoic acid. The metabolism of THC, in vitro, is strikingly inhibited by SKF-525A but not by desipramine, nortriptyline and iprindole which are potent inhibitors of the oxidation of other drugs.     

Cannabinoids in postmortem toxicology

Cannabinoids are often excluded from postmortem toxicology screens due to their ubiquitous nature, interpretative difficulties and unanswered questions regarding their postmortem redistribution. In this study, we review 30 postmortem cases where a drug screen gave a positive cannabinoids result and a confirmation identified Δ?-tetrahydrocannabinol (THC), 11-hydroxy-Δ?-tetrahydrocannabinol (11-OH-THC), and/or 11-nor-9-carboxy-Δ?-tetrahydrocannabinol (THC-COOH) in peripheral (BL-P) or cardiac/central blood (BL-C) and/or urine (UR). Had cannabinoids not been included in these toxicologic evaluations, incomplete or erroneous inferences would have been drawn in a substantial number of cases regarding cause/manner of death. THC was detected in 28 BL-C and in all 30 BL-P. THC and THC-COOH were confirmed present in 2 and 23 UR, respectively. 11-OH-THC was detected in 4 BL-C, 6 BL-P, and 0 UR. The mean THC concentrations in BL-C and BL-P were 8.0 and 15.8 ng/mL, respectively. The mean THC-COOH concentrations in BL-C and BL-P were 55.2 and 60.6 ng/mL, respectively. The mean 11-OH-THC concentrations in BL-C and BL-P were 17.0 and 12.5 ng/mL, respectively. Postmortem interval (PMI) for each case was determined and evaluated in relation to BL-C/BL-P concentration ratios with THC-COOH exhibiting a possible trend. This study is the first of its kind and demonstrates the usefulness of cannabinoid analyses as part of death investigations. Furthermore, it provides distribution data that will improve the ability of toxicologists and pathologists to evaluate cannabinoid concentrations in human postmortem specimens.