Pharmacodynamic Model for Determining Last Use of Inhaled and Oral Cannabis Products

Abstract

The present invention provides a method for determining recent use of cannabis in human subjects, the method comprising collecting samples of whole blood separated in time utilizing a device that collects and stores capillary blood for LC-MS/MS analysis. In particular embodiments, the method is used by law enforcement personnel to collect evidence in driving under the influence investigations. In some embodiments, the method is utilized by employers in routine monitoring of workplace drug policy compliance among employees and in workplace accident investigations.

Claims

1. A method for determining recent use of cannabis in a subject, the method comprising collecting two or more whole blood samples separated in time by approximately 20 to 30 minutes, analyzing them for Δ.sup.9-THC, Δ.sup.9-THC metabolites, and other cannabinoids, computing specific pharmacokinetic parameters, and then evaluating the results based on the criteria of at least six parameters associated with the recent use of cannabis.

2. The method of claim 1, whereby a subject is evaluated by law enforcement personnel for driving under the influence (DUI) of cannabis by collecting two or more whole blood samples.

3. The method of claim 2, whereby the blood samples are collected using a device that automatically collects and stores capillary blood for later laboratory analysis.

4. The method of claim 3, whereby blood sample collection is performed in combination with other cannabis recent use detection technologies such as a Δ.sup.9-THC breathalyzer or oral fluid analysis device.

5. The method of claim 1, whereby an employee or prospective employee is tested for recent use of cannabis by an employer or prospective employer.

6. The method of claim 5, whereby the subject is tested for recent cannabis use by collecting two or more whole blood samples using a device that automatically collects and stores capillary blood for later laboratory analysis.

7. The method of claim 5, whereby the subject is tested for recent cannabis use by collecting two or more whole blood samples using a lancet or other means of blood collection.

8. The method of claim 5, whereby the subject is tested for recent cannabis use by collecting two or more samples of oral fluid or urine in addition to whole blood.

9. The method of any one of claims 2 through 8, whereby the samples collected are also analyzed for prescription and non-prescription drugs, including alcohol, benzodiazepines, barbiturates, opiates, tricyclic antidepressants, selective serotonin reuptake inhibitors, and antifungals.

10. A kit comprising blood collection devices, sample tubes, gloves, sample tube labels, and sample shipping container.

11. The kit of claim 10, wherein the blood collection devices automatically collect and store capillary blood for later laboratory analysis.

12. The kit of claim 10, wherein the blood collection devices are sterile, disposable lancets.

13. The method of claim 1, whereby the parameters used for determining recent use of cannabis are specifically adapted for cannabis edibles.

14. The method of any one of claims 2 through 8, whereby the samples are analyzed and assessed using recent use parameters specific for cannabis edibles.

15. A method for determining recent use of other drugs of abuse, for example, illegal synthetic cannabinoids, methamphetamine, opiates, benzodiazepines, barbiturates, and cocaine, the method comprising collecting two or more whole blood samples separated in time by approximately 20 to 30 minutes, analyzing them for key drug molecules and metabolites, computing specific pharmacokinetic parameters, and then evaluating the results based on the criteria of at least six parameters associated with the recent use of each respective drug compound.

16. The method of claim 15, whereby a subject is evaluated by law enforcement personnel for driving under the influence (DUI) of drugs by collecting two or more whole blood samples.

17. The method of claim 16, whereby the blood samples are collected using a device that automatically collects and stores capillary blood for later laboratory analysis.

18. The method of claim 15, whereby an employee or prospective employee is tested for recent use of drugs by an employer or prospective employer.

19. The method of claim 18, whereby the subject is tested for recent drug use by collecting two or more whole blood samples using a device that automatically collects and stores capillary blood for later laboratory analysis.

20. The method of claim 18, whereby the subject is tested for recent drug use by collecting two or more whole blood samples using a lancet or other means of blood collection.

21. The method of claim 18, whereby the subject is tested for recent drug use by collecting two or more samples of oral fluid or urine in addition to whole blood.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0022] FIG. 1. Metabolism of Δ.sup.9-THC. The major metabolites of Δ.sup.9-THC and the metabolic enzymes primarily responsible for their formation are shown. CYP=cytochrome P450; UGT=uridine 5′-diphospho-glucuronosyltransferase; gluc=glucuronic acid.

[0023] FIG. 2. Typical Δ.sup.9-THC blood concentration profile after smoking in a recent cannabis user compared to a non-recent user.

[0024] FIG. 3. Clinical data summary: immediately after smoking to 20 minutes post-smoking. Following the analysis of whole blood samples for Δ.sup.9-THC and metabolites from 14 subjects given a 500-mg cannabis cigarette to smoke, pharmacokinetic data (0-20 minutes post-smoking) was subjected to a six-parameter recent use model and compared to the results from samples collected from 11 non-recent users over the same time interval. *p<0.05, **p<0.01.

[0025] FIG. 4. Clinical data summary: 120 minutes to 140 minutes post-smoking. Following the analysis of whole blood samples for Δ.sup.9-THC and metabolites from six subjects given a 500-mg cannabis cigarette to smoke, pharmacokinetic data (120-140 minutes post-smoking) was subjected to a six-parameter recent use model and compared to the results from samples collected from 11 non-recent users over the same time interval. *p<0.01

[0026] FIG. 5. Δ.sup.9-THC metabolite conversion. The conversion of Δ.sup.9-THC metabolites 11-OH-Δ.sup.9-THC to 11-nor-9-carboxy-Δ.sup.9-THC and 8β-hydroxy-Δ.sup.9-THC to 8β,11-dihydroxy-Δ.sup.9-THC in a recent cannabis user, after smoking a 500-mg cannabis cigarette (black bars), compared to a non-recent smoker (gray bars) are shown.

[0027] FIG. 6. Clinical data summary: 180 minutes to 200 minutes post-smoking. Following the analysis of whole blood samples for Δ.sup.9-THC and metabolites from six subjects given a 500-mg cannabis cigarette to smoke, pharmacokinetic data (180-200 minutes post-smoking) was subjected to a six-parameter recent use model and compared to the results from samples collected from 11 non-recent users over the same time interval. *p<0.05, **p<0.01.

[0028] FIG. 7. Cannabinoid and metabolite detection in non-recent users. The profile of detection of five different cannabinoids and Δ.sup.9-THC metabolites (Δ.sup.9-THC, CBN, 11-OH-Δ.sup.9-THC, Δ.sup.9-THC-COOH, 8β,11-dihydroxy-Δ.sup.9-THC) in 11 non-recent cannabis users is shown. ND=no detection.

[0029] FIG. 8. Confidence of predicting recent use. The percentages of subjects at each of three different time intervals after smoking a 500-mg cannabis cigarette for whom the described model had a recent use prediction confidence level of 95% (grey bars) and 99% (checkered bars) are shown.

[0030] FIG. 9. Cannabis smoking versus edibles: a case study. Positive recent use parameters following different time intervals after consumption through smoking (500-mg cannabis cigarette) or ingestion of a cannabis edible (30 mg Δ.sup.9-THC) in the same subject are shown.

DETAILED DESCRIPTION OF THE INVENTION

[0031] I. Introduction

[0032] This invention involves the development of a model and test for determining the time of last cannabis use based on the presence of key recent use indicators, including but not limited to CBN, CBG, Δ.sup.9-THCV, Δ.sup.9-THC-epoxides and Δ.sup.9-THC-glucuronide, pharmacokinetic information for Δ.sup.9-THC and its principal metabolites (11-OH-Δ.sup.9-THC, Δ.sup.9-THC-COOH, and 8β,11-dihydroxy-Δ.sup.9-THC), and concentration ratios of Δ.sup.9-THC to these metabolites, including the ratios between different metabolites. For the evaluation of recent cannabis use in a DUI investigation, blood specimens are collected on-site from a suspect who, in the judgment of law enforcement, after performing standard roadside sobriety testing, is impaired. The blood samples are collected using a device designed to collect capillary blood automatically into a receptacle containing appropriate anticoagulant additives so as to provide sufficient material to provide both a primary sample and a duplicate. At least two (2) samples are collected to provide the necessary pharmacokinetic information. The second blood sample is ideally collected approximately 20 to 30 minutes after collection of the first sample. The blood collection device operates in a fashion similar to lancing devices used by diabetics to collect blood for glucose testing; that is, the device will lance a suitable body part (e.g., the upper arm) so that a small quantity of blood, <250 may by collected by the device. The blood specimens are then shipped to the laboratory for testing.

[0033] The blood samples will be analyzed by the laboratory for Δ.sup.9-THC, 11-OH-Δ.sup.9-THC, Δ.sup.9-THC-COOH, 8β-hydroxy-Δ.sup.9-THC, 8β,11-dihydroxy-Δ.sup.9-THC, CBG, CBN, Δ.sup.9-THCV, Δ.sup.9-THC-glucuronide, and Δ.sup.9-THC epoxides using validated analytical methods; for example, high-performance liquid chromatography (HPLC), gas chromatography tandem mass spectrometry (GC-MS/MS) and liquid chromatography tandem mass spectrometry (LC-MS/MS). Once the concentrations of these compounds have been determined, the various Δ.sup.9-THC and metabolite ratios described above will be calculated so that a determination of the time of last cannabis use can be made.

[0034] The described model can be used by itself to assess recent use of cannabis, or it can be used in combination with other technologies, for example, a Δ.sup.9-THC breathalyzer or urine analysis. The application of this model is not restricted to just a two-point analysis. Three, four or more samples can be collected, for example, in a workplace setting to further strengthen the predictive accuracy of the model. Another advantage of this invention is that it can be used to assess recent use of cannabis edibles, unlike other technologies, for example, oral fluid analysis and Δ.sup.9-THC breathalyzers, which can be unreliable in this setting.

[0035] Yet a further advantage of this invention is in the setting of workplace drug testing in states or countries where recreational and/or medicinal use of cannabis has been legalized. For employers, the described model will better differentiate those employees who have used cannabis recently, thereby posing a potential safety risk, from employees who have used cannabis in the past, but not recently enough to be impaired. This would protect both the employer, who can identify those employees who pose a genuine threat to the business, and employees, who can avoid unjustified termination as a result of legal and responsible cannabis use. In addition to non-DUI investigations related to cannabis use, this invention can also be applied to DUI and non-DUI investigations related to the use of drugs other than cannabis; for example, determining recent use of illegal synthetic cannabinoids, methamphetamines, opiates, benzodiazepines, cocaine, and barbiturates.

[0036] Further refinement of the described model can take into account interactions between cannabis and prescription and non-prescription drugs that can lead to a prolongation of the psychoactive effects, and thus potential for impairment, of cannabis. Based on observations, for example, a prolongation of the half-lives of key cannabinoids, a secondary proprietary test for drug interactions is necessary.

[0037] A model based on multiple recent use parameters will be used to determine the probability that a suspect recently used cannabis and was driving within the established “impairment window” (within 4-5 hours after consumption). The greater the number of recent use parameters for which the subject is positive, the higher the statistical probability of recent cannabis use. The model is based on the following parameters that have been associated with recent use of cannabis, whether through smoking, vaping or ingestion of cannabis edibles, with one point being given for each parameter in which the suspect's samples are positive. This list is for illustration purposes only. As additional recent use parameters are identified, they may be added to the model.

[0038] The presence of Δ.sup.9-THC-glucuronide, CBG, CBN, Δ.sup.9-THCV, or Δ.sup.9-THC epoxides in plasma or whole blood.

[0039] A short Δ.sup.9-THC half-life (<1 hour), indicating distribution phase kinetics.

[0040] A short CBN half-life (<1 hour), indicating distribution phase kinetics.

[0041] A short 11-OH-Δ.sup.9-THC half-life (<1 hour), indicating distribution phase kinetics.

[0042] A ratio of 11-OH-Δ.sup.9-THC to Δ.sup.9-THC that is increasing by at least 25%.

[0043] A ratio of Δ.sup.9-THC-COOH to Δ.sup.9-THC that is increasing by at least 25%.

[0044] A ratio of Δ.sup.9-THC-COOH to CBN that is increasing by at least 25%.

[0045] A ratio of 8β,11-dihydroxy-Δ.sup.9-THC to Δ.sup.9-THC that is increasing by at least 25%.

[0046] An increasing plasma or whole blood Δ.sup.9-THC concentration, indicating recent consumption of cannabis edibles.

[0047] Approximately equal concentrations of Δ.sup.9-THC and 11-OH-Δ.sup.9-THC in plasma or whole blood, indicating recent consumption of cannabis edibles.

[0048] Approximately equal concentrations of Δ.sup.9-THC and Δ.sup.9-THC-COOH in plasma or whole blood.

[0049] A Δ.sup.9-THC plasma or whole blood concentration>5 ng/mL combined with a Δ.sup.9-THC-COOH/11-OH-Δ.sup.9-THC concentration ratio<20.

[0050] Consistently higher concentrations of Δ.sup.9-THC-COOH compared to Δ.sup.9-THC and 11-OH-Δ.sup.9-THC, indicating recent consumption of cannabis edibles.

[0051] In summary, a pharmacologic model will be developed for the assessment of recent cannabis use. As a theoretical example, a subject who is positive for at least four (4) recent use parameters out of a total of six (6) has an approximately 99% probability of having used cannabis recently, a test result that can be used as further evidence to support a law enforcement officer's determination of driver impairment.

[0052] II. Definitions

[0053] Unless specifically indicated otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this invention belongs. In addition, any method or material similar or equivalent to a method or material described herein can be used in the practice of the present invention. For purposes of the present invention, the following terms are defined.

[0054] The terms “a,” “an,” or “the” as used herein not only include aspects with one member, but also include aspects with more than one member. For instance, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a parameter” includes a plurality of such parameters and reference to “the metabolite” includes reference to one or more metabolites known to those skilled in the art, and so forth.

[0055] The terms “subject,” “patient,” or “individual” are used herein interchangeably. The term “sample” includes, whole blood, plasma, serum, oral fluid and urine.

[0056] As used herein, the term “consumption” includes smoking, vaping, sublingual administration, oral administration, topical contact, and administration as a suppository. One skilled in the art will know of additional methods of administering cannabis and cannabis-derived compounds.

[0057] The term “cannabinoid” refers any member of the broad class of phytocannabinoid compounds derived from the cannabis plant (Cannabis spp.), or their synthetic equivalents, including, but not limited to Δ.sup.8-THC, Δ.sup.9-THC, CBD, CBG, CBC, Δ.sup.9-THCV, CBN and any of their associated carboxylic acid forms.

[0058] The term “metabolite” includes any of the products resulting from the metabolism of cannabinoids within the body, including but not limited to 11-OH-Δ.sup.9-THC, 11-nor-9-carboxy-Δ.sup.9-THC, 8β-hydroxy-Δ.sup.9-THC, 8β,11-dihydroxy-Δ.sup.9-THC, Δ.sup.9-THC-glucuronide, and Δ.sup.9-THC epoxides.

[0059] Δ.sup.8-THC and Δ.sup.9-THC refer to, respectively, Δ.sup.8-tetrahydrocannabinol and Δ.sup.9-tetrahydrocannabinol.

[0060] 11-OH-Δ.sup.9-THC refers to the metabolite 11-hydroxy-Δ.sup.9-tetrahydrocannabinol.

[0061] Δ.sup.9-THC-COOH refers to the metabolite 11-nor-9-carboxy-Δ.sup.9-tetrahydrocannabinol.

[0062] Δ.sup.9-THCV refers to the cannabinoid Δ.sup.9-tetrahydrocannabivarin.

[0063] CBN refers to the cannabinoid cannabinol.

[0064] CBD refers to the cannabinoid cannabidiol.

[0065] CBG refers to the cannabinoid cannabigerol.

[0066] CBC refers to the cannabinoid cannabichromene.

[0067] The term “illegal synthetic cannabinoid” refers to any of the illegal cannabinoid-like designer drugs that have been identified, including but not limited to, Spice, K2, synthetic marijuana, AK-47, Mr. Happy, Scooby Snax, Kush, and Kronic.

[0068] III. Description of the Method

[0069] The method relies on the use a statistical model to derive the probability that a subject recently used cannabis or some other substance of abuse for which testing is desired. Comprising the model are parameters that have been associated with the recent use of cannabis or other compounds of interest. At least six parameters are used, but more may be added as they are identified to strengthen the statistical power of the model. The subjects' pharmacokinetic data are used to determine whether the particular subject is positive, for which a value of “1” is assigned or negative, for which a value of “0” is assigned, in each of the parameters. An appropriate statistical test is then applied to determine whether the subjects' average scores are significantly different from the hypothetical average of “0,” which would represent someone who has not used the substance recently.

[0070] For example, the following six parameters have been associated with the recent use cannabis: (1) a short Δ.sup.9-THC half-life (<1 hour), indicating distribution phase kinetics; (2) a short CBN half-life (<1 hour), indicating distribution phase kinetics; (3) a ratio of 11-OH-Δ.sup.9-THC to Δ.sup.9-THC that is increasing by at least 25%; (4) a ratio of Δ.sup.9-THC-COOH to Δ.sup.9-THC that is increasing by at least 25%; (5) a ratio of Δ.sup.9-THC-COOH to CBN that is increasing by at least 25%; and (6) a ratio of 8β,11-dihydroxy-Δ.sup.9-THC to Δ.sup.9-THC that is increasing by at least 25%. After determining positivity or negativity for each of these six parameters, a Student's t-test with two-sample equal variance, two-tailed distribution, and significance level of 0.05 is then applied. The null hypothesis for this test is that there is no difference between the subject's average for the six parameters and a hypothetical non-recent user. The result of this test will return the probability (p) that the test subject could have come from a population of non-recent users. Thus, a p-value of 0.05 indicates a 5% probability of non-recent use, meaning a 95% probability of recent use of cannabis. A p-value of 0.01 indicates a 1% probability of non-recent use, which translates to a 99% probability of recent use of cannabis. When using a model comprised of six parameters, a subject who is positive for three parameters has a 95% probability of recent use. A subject who is positive for four or more parameters has a 99% probability of recent use. A p-value result of <0.05 is considered statistically significant. Other statistical tests may be suitable and may be employed in the evaluation. This model may be adapted to test for the recent use of other drugs of interest, and additional parameters may be added to this model to strengthen its statistical power.

IV. EXAMPLES

[0071] The following examples are offered to illustrate, but not to limit, the claimed invention.

[0072] A total of 20 human subjects were included in a clinical trial designed to evaluate the feasibility of the described method for determining recent use of cannabis. Also evaluated in this trial was the safety and feasibility of an experimental blood draw device, compared to a standard lancet device, that automatically collects and stores whole blood in a tube containing anticoagulant. Subjects were asked to describe their experience with both types of blood draw devices. Blood samples were collected prior to smoking and then at various time points up to 200 minutes post-smoking, focusing on three major time intervals: (1) 0 to 20 minutes post-smoking; (2) 120-140 minutes post-smoking; and (3) 180-200 minutes post-smoking. For cannabis edibles, blood samples were collected up to eight hours after ingestion.

Example 1

Determining Recent Use of Cannabis—Immediately After Smoking to 20 Minutes Post-Smoking

[0073] The goal of examining the 20-minute period immediately after smoking was to assess the ability of the described model to detect recent use of cannabis when the evidence of recent use should be at or near maximal levels.

[0074] A total of 14 subjects had blood samples collected immediately after smoking and 20 minutes post-smoking. Following written informed consent, subjects were given a cannabis cigarette to smoke that contained approximately 500 mg of a high-potency cannabis strain. Subjects were instructed to smoke the entire cigarette, or as much as they could, within a 10-minute period. Blood samples were collected using a either a standard lancet device (13 subjects) or the experimental blood draw device described above (1 subject). Blood samples were then extracted and analyzed for Δ.sup.9-THC, Δ.sup.9-THC metabolites, and other cannabinoids by liquid chromatography tandem mass spectrometry (LC-MS/MS). Using the results, the following six parameters were then calculated: Δ.sup.9-THC half-life; CBN half-life; 11-OH-Δ.sup.9-THC/Δ.sup.9-THC ratio; Δ.sup.9-THC-COOH/Δ.sup.9-THC ratio; Δ.sup.9-THC-COOH/CBN ratio; 8β,11-dihydroxy-Δ.sup.9-THC/Δ.sup.9-THC ratio. These six parameters were also computed for the 11 subjects from whom baseline blood samples were collected 20 minutes apart prior to smoking.

[0075] FIG. 3 shows the number of parameters associated with recent use of cannabis for which each subject was positive or negative. All 14 subjects showed statistically significant evidence (p<0.01 in 13 subjects; p<0.05 in 1 subject) of recent cannabis use based on the described model, while none of the blood samples collected from 11 subjects prior to smoking showed evidence of recent use.

Example 2

Determining Recent Use of Cannabis—120 to 140 Minutes Post-Smoking

[0076] The goal of examining the time period approximately two hours after smoking was to evaluate the ability of the described model to predict recent use of cannabis in a more realistic setting, where someone smokes cannabis, decides to drive about an hour later, and is then tested for suspicion of DUI about two hours after having smoked.

[0077] A total of six subjects had blood samples collected at 120 minutes and 140 minutes post-smoking. Following written informed consent, subjects were given a cannabis cigarette to smoke that contained approximately 500 mg of a high-potency cannabis strain. Subjects were instructed to smoke the entire cigarette, or as much as they could, within a 10-minute period. Blood samples were collected using a either a standard lancet device (5 subjects) or the experimental blood draw device described above (1 subject). Blood samples were then extracted and analyzed for Δ.sup.9-THC, Δ.sup.9-THC metabolites, and other cannabinoids by LC-MS/MS. Using the results, the following six parameters were then calculated: Δ.sup.9-THC half-life; CBN half-life; 11-OH-Δ.sup.9-THC/Δ.sup.9-THC ratio; Δ.sup.9-THC-COOH/Δ.sup.9-THC ratio; Δ.sup.9-THC-COOH/CBN ratio; 8β,11-dihydroxy-Δ.sup.9-THC/Δ.sup.9-THC ratio. These six parameters were also computed for the 11 subjects from whom baseline blood samples were collected 20 minutes apart prior to smoking.

[0078] FIG. 4 shows the number of parameters associated with recent use of cannabis for which each subject was positive or negative. All six subjects were positive for at least four recent use parameters, which was statistically significant evidence (p<0.01, all subjects) of recent cannabis use based on the described model, while none of the blood samples collected from 11 subjects during a 20-minute interval prior to smoking showed evidence of recent use.

[0079] FIG. 5 shows the conversion of 11-OH-Δ.sup.9-THC to 11-nor-9-carboxy-Δ.sup.9-THC and the conversion of 8β-hydroxy-Δ.sup.9-THC to 8β,11-dihydroxy-Δ.sup.9-THC in a recent cannabis smoker compared to an non-recent smoker up to 140 minutes post-smoking. The relative levels of 11-nor-9-carboxy-Δ.sup.9-THC and 8β,11-dihydroxy-Δ.sup.9-THC are clearly increasing in the smoker, while they remain relatively unchanged in the non-recent smoker.

Example 3

Determining Recent Use of Cannabis—180 to 200 Minutes Post-Smoking

[0080] The goal of evaluating the time period approximately three hours after smoking was to evaluate the ability of the model to predict recent use when pharmacologic evidence of such should be waning, but impairment is still possible.

[0081] A total of eight subjects had blood samples collected at 180 minutes and 200 minutes post-smoking. Following written informed consent, subjects were given a cannabis cigarette to smoke that contained approximately 500 mg of a high-potency cannabis strain. Subjects were instructed to smoke the entire cigarette, or as much as they could, within a 10-minute period. Blood samples were collected using a either a standard lancet device (7 subjects) or the experimental blood draw device described above (1 subject). Blood samples were then extracted and analyzed for Δ.sup.9-THC, Δ.sup.9-THC metabolites, and other cannabinoids by LC-MS/MS. Using the results, the following six parameters were then calculated: Δ.sup.9-THC half-life; CBN half-life; 11-OH-Δ.sup.9-THC/Δ.sup.9-THC ratio; Δ.sup.9-THC-COOH/Δ.sup.9-THC ratio; Δ.sup.9-THC-COOH/CBN ratio; 8β,11-dihydroxy-Δ.sup.9-THC/Δ.sup.9-THC ratio. These six parameters were also computed for the 11 subjects from whom baseline blood samples were collected 20 minutes apart prior to smoking.

[0082] FIG. 6 shows the number of parameters associated with recent use of cannabis for which each subject was positive or negative. At this time interval post-smoking, only four of the eight subjects showed statistically significant evidence of recent cannabis use (p<0.01 in three subjects; p<0.05 in one subject) based on the described model. None of the blood samples collected from 11 subjects collected over a 20-minute interval prior to smoking showed evidence of recent use.

[0083] FIG. 7 shows that Δ.sup.9-THC, CBN and the three Δ.sup.9-THC metabolites included in the parameters used to determine recent use in these examples were still detected in the majority of the 11 subjects from whom blood samples were collected during a 20-minute interval prior to smoking; however, when subjected to the recent use parameters defined in these examples, the data did not meet the established criteria in any subject.

[0084] FIG. 8 shows the degree of confidence in predicting recent use of cannabis in the three time intervals evaluated post-smoking. Within two hours post-smoking, the described model predicted recent use in all subjects with at least 95% confidence, and in most subjects with at least 99% confidence. At three hours post-smoking, the model was capable of predicting recent use in half of the subjects with at least 95% confidence.

[0085] The results from these three examples demonstrate that the described model accurately determines recent use of cannabis within approximately two hours post-smoking and is still about 50% effective in identifying recent use within three hours post-smoking. As shown in FIG. 7, the key cannabinoids and metabolites included in the parameters used to determine recent use in these examples were still detected in almost all of the 11 subjects prior to smoking. This is an important point because non-recent cannabis users will show detectable levels of several or all the compounds evaluated in these studies. An effective test for the recent use of cannabis must be able to discriminate between past use and recent use, which carries the risk of impairment. The results presented in these examples demonstrate that the described model can successfully determine recent use of cannabis within a background of non-recent use.

Example 4

Determining Recent Use of Cannabis—Smoking versus Edibles Case Study

[0086] The goal of this case study was to assess the value of the model in predicting recent use in the setting of cannabis edibles, the popularity of which is increasing and the impairment from which is delayed compared to smoking.

[0087] As a clinical case study, a subject was studied to determine recent use of cannabis following ingestion of a cannabis-extract infused chocolate cannabis edible containing 30 mg of Δ.sup.9-THC compared to smoking a cannabis cigarette containing approximately 500 mg of high-potency cannabis. Following written informed consent, two baseline blood samples were collected 30 minutes apart using a lancet device. The subject was then given three cannabis edibles to eat, each containing 10 mg of Δ.sup.9-THC. Blood samples were then collected every 30 minutes up to eight hours post-administration using a lancet device. After extraction and analysis, the following six parameters were then calculated: Δ.sup.9-THC half-life; CBN half-life; 11-OH-Δ.sup.9-THC/Δ.sup.9-THC ratio; Δ.sup.9-THC-COOH/Δ.sup.9-THC ratio; Δ.sup.9-THC-COOH/CBN ratio; 8β,11-dihydroxy-Δ.sup.9-THC/Δ.sup.9-THC ratio. The results were compared to those obtained after the subject smoked a cannabis cigarette in a previous study.

[0088] FIG. 9 shows the number of parameters associated with recent use of cannabis for which this subject was positive or negative at each time interval after smoking or consuming a cannabis edible. While convincing evidence of recent use appeared within the first 30 minutes after smoking, no such evidence was observed until three hours after the subject had consumed the cannabis edible. This is consistent with published data regarding the delayed absorption of cannabinoids following edible cannabis administration compared to smoking. Importantly, this subject displayed an identical pattern of positive recent use parameters between three and six hours after consuming the cannabis edible compared to the first three hours after smoking.

[0089] The results of this case study demonstrate that the described model can also be used to determine recent use of cannabis edibles, which may prove valuable given that use of edible cannabis products is increasing. Due to the comparatively very low levels of Δ.sup.9-THC following ingestion of edibles compared to smoking, Δ.sup.9-THC breathalyzers may prove unreliable, besides the fact that using Δ.sup.9-THC alone to assess recent use is problematic at best. While oral fluid analysis may be useful in determining recent use within the past 24 hours, it cannot be used to determine impairment. Furthermore, due to contamination of the oral cavity, saliva or oral fluid analysis is unreliable for determining recent use of cannabis following consumption of edibles.

[0090] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, one of skill in the art will appreciate that certain changes and modifications may be practiced within the scope of the appended claims. In addition, each reference provided herein is incorporated by reference in its entirety to the same extent as if each reference was individually incorporated by reference.