The lessons learned from past drug development efforts in DR tell us that a more rational strategy is required to discover and test novel drug candidates as well as the need for better transition from preclinical to clinical first-in-human studies. To these ends, we present a hypothetical pathway (summarizing the discussions above) illustrating how CBD exerts its pharmacodynamic action to reduce retinal inflammation in diabetes (). The elements in this figure can serve as a useful foundation to map the future biomarker development strategies in clinical proof of concept studies of CBD treatment of DR.
The postulated anti-inflammation mechanism of action by CBD in treatment of DR
Insofar as the putative genetic markers are concerned, a hereditary component has been reported both for the prevalence and severity of DR [73
]. A number of candidate genes encoding proteins involved in aldose reductase pathway, major histocompatibility complex and immunity, glucose transporters, cell communication and the extracellular matrix, endothelial function and nitric oxide synthases have been studied [76
] (). Thus far, these findings do not always corroborate each other but this is also a commonly observed scenario in candidate gene studies that will still demand future replication efforts, to account for variations in characteristics of study populations, substructures created by molecularly heterogenous phenotypic representations and classification of DR severity, among other confounding factors. Moreover, candidate drug targeted pathway-related genetic variants add up to this challenge considering the multi-factorial nature of the studied phenotype (i.e., phenotypes on drug efficacy and toxicity). Genome-wide association studies in well characterized patient populations evaluated with standardized DR severity grading system will be essential to evaluate the findings from candidate gene studies published to date. These broader genome wide inquiries can also inform to establish the link between postulated molecular mechanisms and clinical pharmacodynamics of CBD after administration in patients with DR.
Candidate genes studied in relation to susceptibility for diabetic retinopathy.
In regards to drug metabolism and distribution, it is also important to bear in mind the pertinent biomarker pathways that can potentially impact the CBD concentrations in the systemic circulation or locally in the retina [78
]. Importantly, the phase I metabolism of the classical cannabinoids (including THC, cannabinol and CBD) has been shown to depend primarily on the cytochrome P450 mixed-function oxidases, CYP2C9 and CYP3A4 in human hepatic microsomes [79
]. The impact of the CYP2C9
polymorphisms on the clinical pharmacokinetics of orally administered THC was studied in 43 healthy volunteers [80
]. The results revealed that subjects carrying a coding variant (Ile359Leu) (CYP2C9
) had decreased total clearance for THC and may thereby express enhanced therapeutic and adverse effects of orally administered THC. CYP2C9
encodes an enzyme with 3–30 fold lower activity in comparison to the wildtype (CYP2C9
) depending on the specific substrate [81
]. Although the quantitative contributions of CYP2C9 and CYP3A4 enzymes to CBD metabolism have not been elucidated, based on the structural similarity of THC, cannabinol and CBD, the impact of the CYP2C9
polymorphism on the potential pharmacokinetics of orally administered CBD can potentially be anticipated.
In contrast to the phase I metabolism, very little is known about the phase II metabolism of classical cannabinoids [82
]. Phase II metabolites appear to be mainly conjugates of the phase I metabolites with glucuronic acid, catalyzed by the activity of glucuronosyltransferases. The conjugates are the main metabolites of THC or CBD found in urine [83
In addition to the potential impact of genetic variation of phase I and phase II enzymes on CBD metabolism, the inhibitory/inductive effect of CBD on phase I and phase II drug metabolism activity and the role of genetic variation in baseline (constitutive) drug metabolizing enzyme activity should be considered with respect to the (prediction) of the risk for drug-drug interactions with CBD during its future development. To this end, CBD has been shown to inhibit CYP2C and CYP3A enzyme activities in rat and mouse hepatic microsomes [84
Ocular pharmacokinetics of a drug is another potential challenge to consider in personalized/targeted drug development as both systemic and local administration routes might involve a genetically determined interindividual variability. Corneal epithelium and blood-retina barrier constitutes the primary regulating sites of xenobiotic access into ocular tissues. Moreover, the presence of both phase I and phase II drug metabolizing enzymes and transporter proteins, including organic anion and cation transporters as well as efflux proteins, have been shown in several ocular tissues in different ranges, retinal pigment epithelium and ciliary body being the primary detoxification cites for xenobiotics [85
]. The impact of genetic variation of these components on ocular drug pharmacokinetics has not been studied yet and this aspect certainly requires substantial thoughtful attention in future ocular drug development in relation to efficacy, dose finding and safety.
Insofar as the primary molecular targets for CBD (for applications in DR) and biomarker development are concerned, inhibition of adenosine reuptake via ENT1 in rat microglia is postulated as the mechanism of action for the anti-inflammatory effects of CBD [35
] (). The gene encoding the human ENT1, SLC29A1
, is mapped to chromosome 6p21.1-21.2 and several polymorphisms have been identified in the promoter and coding regions of the gene [87
]. Notably, a 1.37-fold increased in vivo
(white blood cells) expression of the transporter has been shown for individuals heterozygous for the variant -1345C/ -1050G/ -706C haplotype compared to individuals homozygous for the wild-type -1345C/ -1050G/ -706G haplotype [88
]. However, these results obtained from white blood cells may not reflect the activity of the ubiquitously expressed ENT1.
A potential interindividual difference in ubiquitous or ocular ETN1
expression in relation to these haplotypes deserves further investigation.