ABSTRACT
Δ9-tetrahydrocannabinol (THC) is a psychoactive phytocannabinoid found in the Cannabis sativa plant. THC is primarily metabolized into 11-hydroxy-Δ9-tetrahydrocannabinol (11-OH-THC) and 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (COOH-THC), that may themselves be psychoactive. There is very little research-based evidence concerning the pharmacokinetics and pharmacodynamics of 11-OH-THC as an individual compound. Male C57BL/6 mice were treated with THC or 11-OH-THC via i.p injection, tail vein i.v injection, or oral gavage (p.o), and whole blood compound levels were measured to determine pharmacokinetic parameters (Cmax, Tmax, t1/2, AUC, Vd, ClS, k and F) while also monitoring changes in catalepsy, body temperature, and nociception. 11-OH-THC achieved Tmax at 30 min for all routes of administration. The maximum concentration at 30 min was not different between i.v. and i.p. routes, but the p.o. Cmax was significantly lower. THC had a 10 min time to the maximum concentration - which was the first blood collection time point - for i.v. and i.p., and 60 min for p.o. with a lower Cmax for i.p. and p o. compared to i.v When accounting for circulating compound levels and ED50 responses, these data suggest that 11-OH-THC was 153% as active as THC in the tail-flick test of nociception, and 78% as active as THC for catalepsy. Therefore, 11-OH-THC displayed equal or greater activity than the parent compound THC, even when accounting for PK differences. Thus, the THC metabolite 11-OH-THC likely plays a critical role in the bioactivity of cannabis; understanding its activity when administered directly will aid in the interpretation of future animal and human studies. Significance Statement In this study we establish that the primary metabolite of THC - 11-OH-THC - displays equal or greater activity than THC in a mouse model of cannabinoid activity when directly administered and even when accounting for route of administration, sex, pharmacokinetic, and pharmacodynamic differences. These data provide critical insight into the bioactivity of THC metabolites that will inform the interpretation of future cannabinoid research and represent a model for how THC consumption and metabolism may affect cannabis use in humans.
ABSTRACT
Gram-negative bacteremia is a major cause of global morbidity involving three phases of pathogenesis: initial site infection, dissemination, and survival in the blood and filtering organs. Klebsiella pneumoniae is a leading cause of bacteremia and pneumonia is often the initial infection. In the lung, K. pneumoniae relies on many factors like capsular polysaccharide and branched chain amino acid biosynthesis for virulence and fitness. However, mechanisms directly enabling bloodstream fitness are unclear. Here, we performed transposon insertion sequencing (TnSeq) in a tail-vein injection model of bacteremia and identified 58 K. pneumoniae bloodstream fitness genes. These factors are diverse and represent a variety of cellular processes. In vivo validation revealed tissue-specific mechanisms by which distinct factors support bacteremia. ArnD, involved in Lipid A modification, was required across blood filtering organs and supported resistance to soluble splenic factors. The purine biosynthesis enzyme PurD supported liver fitness in vivo and was required for replication in serum. PdxA, a member of the endogenous vitamin B6 biosynthesis pathway, optimized replication in serum and lung fitness. The stringent response regulator SspA was required for splenic fitness yet was dispensable in the liver. In a bacteremic pneumonia model that incorporates initial site infection and dissemination, splenic fitness defects were enhanced. ArnD, PurD, DsbA, SspA, and PdxA increased fitness across bacteremia phases and each demonstrated unique fitness dynamics within compartments in this model. SspA and PdxA enhanced K. pnuemoniae resistance to oxidative stress. SspA, but not PdxA, specifically resists oxidative stress produced by NADPH oxidase Nox2 in the lung, spleen, and liver, as it was a fitness factor in wild-type but not Nox2-deficient (Cybb-/-) mice. These results identify site-specific fitness factors that act during the progression of Gram-negative bacteremia. Defining K. pneumoniae fitness strategies across bacteremia phases could illuminate therapeutic targets that prevent infection and sepsis.
