Therefore, the increased cytotoxicity observed by the encapsulated terpenes in the MTT assay may be explained by the potentially promoted calcium-mediated cell death resulted from the increased intracellular Ca2+ concentrations. In disagreement with our findings, a recent report that tested different terpenoids, including MC , found that these terpenoids had no activation effect on human TRPV1 . Nevertheless, the discrepant findings may be explained by many variations in the assay conditions, which may include differences in TRPV1expression levels, and variations in the assay method, buffer composition, and experimental temperature used. Our findings suggest that the newly developed PLGA-based nanosystems can remarkably increase the ability of the tested cannabis-based terpenes to enhance the fluorescence intensity driven by the intercellular calcium ion influx. This effect is correlated with the activation of TPRV1 channels which indicates that the terpene NPs may have great potential for application in pain management. Since 2006 an US/European international study about the onsite oral fluid drug testing devices has shown that no device is reliable enough in order to be recommended for roadside screening of drivers. Standard neurological tests for gross motor coordination such as the observation of gait or one-leg stand, the ability to walk in a straight line or finger-to-nose or the examination of the pupillary light reflex using a halogen penlight are not reliable to determine if a driver is under the influence of alcohol and/or drugs because too much subjective.
In fact, driving under the influence of alcohol and drugs is worldwide considered an elevated risk for traffic safety and a crime. In Germany for example, like in other European countries, the legal requirements for assessing a driver’s fitness to drive are so far set out in national Road traffic Regulations. Therefore, trimming trays for weed during a routine traffic control as well as a medical examination assessing fitness to drive it is necessary to establish a method that helps to clarify the question whether an individual is under the influence of substances acting on the central nervous system . Several studies have already demonstrated the applicability of the non-invasive infrared pupillography as an objective measurement method to indicate the possible influence of drugs on CNS. Various pupil function parameters can change significantly between healthy individuals and subjects under the influence of drugs or medications. Based on previous results , the pupil examination by infrared pupillography can assess the neurological status of such individuals who warrant further clinical examination, including blood testing. The effects on pupil function by several illicit and prescription drugs have been already studied among which opiates , benzodiazepines ,cocaine and cannabinoids. However, the correct interpretation of parameters is not an easy task due to a partially large range of variation of corresponding parameters from individual to individual also according to an un-satisfactory definition of a “normal” pupil. The discriminatory power of pupil parameters also needs to be better investigated in order to improve the reliability of the method. The present study focuses on cannabis consumers. In fact, after alcohol, tetrahydrocannabinol is the most used substance among injured drivers. Cannabis can produce psychoactive effects and impair psychomotor performance in a wide of operative tasks, such as motor coordination, individual’s equilibrium, perception, and attention as long as 24 h after smoking .
Chronic health effects of cannabis are well-known including dependence syndrome, which could affect daily life functions and impairment of cognitive functions . Acute subjective effects can reduce the speed of visual or auditory stimuli increasing the risk of motor vehicle accidents. However, previous studies on cannabis-related effects on simulated driving performances did regularly not show relevant impairments of motor behavior, and it is only an assumption that documented deficits are related to attention or perception. Concentrations of 7–10 ng/ml THC in serum are thought to evoke comparable impairments to 0.5 g/kg blood alcohol concentration , which is the legal limit provided by most of the European Road traffic Regulations. Therefore, THC serum concentrations below 10 ng/mL should not increase the risk of a traffic accident, while THC consumption in doses up to 300mg per kilogram body weight can be considered to cause relevant cognitive and psychomotor impairments comparable to 0.5 g/l BAC. In our opinion these considerations have to be interpreted critically. In a recent study a defined THC concentration that leads to an inability to ride a bicycle could not be determined. In fact, estimates of risk are hard to obtain due to the rapid decline of THC concentrations in blood. Also for this reason, in several European countries, every case needs to be treated individually because of cutoff values for the absolute impairment to drive are not available for most of the common illicit drugs other than alcohol . Acute influence of cannabis on operative tasks of the individual can be assessed by the cannabis influence factor and a CIF of 10 was proposed as a threshold for a driving impairment. However, when using the CIF, it must be kept in mind that the formula to calculate the CIF might significantly disadvantage onetime consumers and treat regular consumers preferentially. The aims of this study were to assess: a) the differences in pupillography response between cannabis consumers after a washout period and no cannabis consumers and b) the dose related effects on pupillography parameters of cannabis in cannabis consumers. To evaluate the cannabis effects on the ability in driving, infrared pupillography was performed in individuals before and after inhalative consumption of cannabis.
The cannabis consumption was standardized according to methods for clinical research involving cannabis administration. Participants in the CC group were invited to smoke a non tobacco cigarette. Each joint contained 300mg of THC per kilogram of body weight. The test persons were instructed to consume the joints in the following way: 4-s inhalation,10-s holding breath, and 15-s exhalation. A maximum of three joints could be consumed. Cannabis was imported for the trial with the allowance from the German Federal Opium Agency, an authorized import agency of the Dutch medical cannabis flos from Bedrocan, Netherlands . Acute adverse effects of cannabis administration such as the tachycardia, orthostatic hypotension, pulmonary irritation, motor in-coordination, cognitive impairment, anxiety, paranoia, psychosis, have been evaluated.The infrared pupillography was applied in the study as described in previous articles. The measurements were taken using a developed version of the previous described puillograph, the F2D2 – a portable device, which is more suitable to be used during routine police check-points. The device measures the horizontal pupil diameter using a two dimensional camera and determines the diameter of the pupil with a resolution of 0.02 mm at a measuring frequency of 25 Hz over a period of 2 s on the basis of the different reflexivity of the iris and pupil. The eye is continuously and diffusely illuminated with two infrared diodes in order to not induce pupillary light reflex and to magnify the pupil, The duration of the stimulus can be freely selected between 0.1 and 0.2 s and it was set up at 200 ms. The brightness of the stimulus can also be freely selected between 254 and 1. The luminous intensity values have no unit of measurement and range between 0.22 and 56 lx measured at the vertex of the cornea. For our investigation we chose 60 and 254 PLR values on the basis of the previous research studies. It can be seen that the major differences between the study groups are more apparent when the parameters are detected at these luminance values.
Once the measurement has been taken, pupil movement is shown in graphic form on the connected laptop. At the same time,trimming tray with screen special software breaks down the pupil reaction into various parameters according to predetermined measurement points and intervals. On the monitor, the tester gains a first impression of the pupil and its ability to react and can also read off the parameters calculated in the respective window. The research study for the CC group included a baseline recording consisting of pupillographic analysis and drug blood test with evaluation of the main active THC metabolites along with 11-OH-THC and the main secondary metabolite THC-COOH, repeated four times at 2 , 4 , 6 and 8 hours from baseline. The CC group assumed cannabis at T2, T3, and T4. The drug blood test and the pupillographic analysis were performed 20 min after the assumption. At T5 no cannabis was administrated but only drug blood test and pupillographic analysis were performed. The Ctr group underwent to drug blood test and pupillographic analysis only at baseline.Infrared pupillography can represent a standardized and objective method for measuring the pupil function and assessing the influence of substances acting on the CNS. In fact, the pupillary light reflex can be significantly altered by psychoactive substances, like cannabis. In the present study, all four pupillary parameters seemed to be changed according to a weakened pupil function due to an acute administration of cannabis. Although pupil size as well as the latency depend on age, no relevant age-related bias in pupil diameter and latency were found in age and gender distribution between the CC and Ctr groups. In accordance with previous studies, acute cannabis administration may cause pupil constriction, and conjunctival injection , self-limiting effects, which do no require any particular treatment. In particular, velocity of contraction and latency time were altered in proportion to the daily use of cannabis. Although the simultaneous influence of amphetamine/MDMA and cannabis has been found to hinder the opthalmological effects caused by cannabis, such results cannot be confirmed by the present two test individuals under the influence of amphetamine/MDMA. According to other studies investigating neurological impairment by drugs long-term effects can be appreciated especially in THC “heavy users”. Probably cannabis causes long term effects and addiction because the active substance stimulates the same reward pathways of other drugs. The effect of cannabis on the pupil parameters investigated at T4 was probably related to the different metabolization of THC when it accumulates in the body. In fact, high concentrations of cannabinoids are metabolized through ways other than the microsomal system and cytochromes and the resulting products could have different targets with different biological roles. Furthermore, the effects detected during the study could be due to some phenomena of molecular mimicry, or to receptor desensitization phenomena or, again, to the possible effect of cannabinoids active on receptors other than CB1 and CB2.
The acute effect recorded over two hours after the last dose, when this should be terminated, can be explained by the fact that in the usual consumer the kinetics of elimination of the active substance is different . Our findings demonstrate that the velocity of contraction was correlated with BMI while the latency time and the reaction time were significantly and inversely correlated with the BMI. Although there is still no evidence, it seems that this phenomenon can be related to the lipophilic nature of THC and, consequently, to the possible accumulation at high concentrations in adipose tissue which increases with increasing BMI and lower blood concentration of active molecule and reduced toxicological influence on the pupil. The results of the study show that almost all pupil parameters are reliable indicators for the detection of subjects under the acute effect of cannabis, at the comparison with a control group after light stimulation of the same intensity. However, to understand the effects of cannabis on the ability to drive, it would be necessary to establish ranges of measured metabolites related to the deficit grade in driving a vehicle, as is the case for alcohol. The demonstration of a correlation between the quantity of the blood metabolites and the pupillary effect should be performed on a larger sample due to the high intra- and inter-individual variability.Although the integrity of the pupil function is not always correlated with the ability to drive and the examination of the pupil size and function are not sufficient to evaluate the fitness for driving, we can affirm that the pupil function is a useful objective indicator of neurological conditions. Considering the inter individual differences in pupil physiology and the changes during the study, further research studies should be performed possibly by using more precise devices with higher measuring frequency in order to enhance the sensitivity in recording the differences of the pupillographic response.