edoc

Development and application of a LC-MS/MS method for the analysis of plasma bioavailabilities of different cannabinoids after the administration of "Cannabis sativa L." extracts and MarinolTM

Grauwiler, Sandra Béatrice. Development and application of a LC-MS/MS method for the analysis of plasma bioavailabilities of different cannabinoids after the administration of "Cannabis sativa L." extracts and MarinolTM. 2008, PhD Thesis, University of Basel, Faculty of Science.

[img]
Preview
PDF
760Kb

Official URL: http://edoc.unibas.ch/diss/DissB_8333

Downloads: Statistics Overview

Abstract

A lively interest in the cannabis plant can be verified for a long time. As a drug in the
traditional medicine, different pieces of the cannabis plant were used against a
palette of diseases such as pain (head- and stomach-ache), menstrual problems and
diarrhoea. Further, it was used as a sedative and to induce sleep [1].
At the beginning of the 20th century, a scientific interest for cannabis has emerged.
Research was done to detect the pharmacokinetic and pharmacodynamic effects of
cannabis. The discovery of the endogenous cannabinoid system opened a broad
field for research. This system gradually helped to better understand the molecular
mechanisms of the cannabis effects. Links to other modulating or regulatory systems
in our body are now possible [2].
The special applications of cannabis in traditional medicine, have to be clinically
investigated with the scientific knowledge of today. Some applications are already
established. The United States Food and Drug Administration (FDA) approved
MarinolTM (a soft gelatine capsule containing THC dissolved in sesame oil) to treat
nausea and vomiting associated with cancer chemotherapy in patients who have
failed to respond adequately to conventional therapies. The antiemetic effects are
comparable to conventional therapy such as domperidone [3]. Furthermore, the FDA
approved MarinolTM to treat appetite loss associated with weight reduction in people
with acquired immunodeficiency syndrome (AIDS). It was shown in recent studies
that THC or cannabis preparations have a promising potential as a releasing factor,
in moving disorders and as a pain reducer in patients suffering from multiple
sclerosis. Therefore further research is required.
Worldwide, the use of natural cannabis products for medical purposes is practically
not allowed. In contrast, drugs containing synthetic cannabinoids like dronabinol, a
synthetic THC are often exempt from these restrictions. Synthetic products, however,
have a disadvantage: they do not contain a well-balanced combination of active
substances which can be found in the natural cannabis plant. Patients having
consumed natural cannabinoids for their medical therapy report more adverse effects
after the administration of synthetic cannabis preparations [4, 5]. The principle of
phytotherapy is the treatment with a mixture of bioactive compounds. The idea is that
a complex pathophysiological process can be influenced more effectively and with
fewer adverse effects by a combination of several low-dosage extract compounds
than by a single isolated compound [6]. Therefore, it is important to develop and
clinically investigate oral cannabis extract formulations to prove pharmacodynamic
and pharmacokinetic properties and to compare them with the existing synthetic THC
products and against placebo is important.
The aim in project one of the present work (publication 3, chapter 3.3) was to
approve the performance of an open, randomised, single-center, three-periods crossover
study with different, standardised Cannabis sativa L. extract capsule
formulations and MarinolTM, to analyse the pharmacokinetics and pharmacodynamics
of the cannabinoids and to evaluate the best Cannabis sativa L. extract capsule
formulation in this clinical phase I study for a possible future implementation as a
new, concomitant medication in cancer, HIV and AIDS therapies.
In the first study part, the heating-effect on the relative content of cannabinoids in the
Cannabis sativa L. extract capsule formulation was assessed. Data were compared
to the commercial formulation MarinolTM. The reason for this is that in naturally grown
Cannabis sativa L., up to 95% of the occurring total cannabinoids (THCtot) are in the
form of D9-tetrahydrocannabinolic acid A (THCA-A). By heating, THCA-A is
quantitatively decarboxylised to phenolic THC [7]. Although THCA-A is described as
pharmacologically inactive and devoid of psychotropic effects [7], reports of popular
medicinal use of unheated cannabis or cannabis preparations show pharmacological
effects often accompanied with a lower rate of adverse effects (anecdotal reports). It
also possesses some anti-inflammatory and analgesic effects [8]. Recently, it was
shown that unheated cannabis extracts were able to inhibit tumor necrosis factor
alpha in macrophage culture and peripheral marcrophages after LPS stimulation [9].
In the second study part, the effect of different Cannabis sativa L. extract capsule
formulations, containing different concentrations of TPGS, on the bioavailabilities of
different active cannabinoids was assessed. The reason for this is that the enteral
absorption of cannabinoids under optimal conditions would be up to 95%, but due to
the extensive liver first-pass metabolism and the poor solubility the effective,
measured bioavailability is very low (10-20%) [10]. Further the activity of Pglycoprotein
(P-gp), a membrane efflux transporter also expressed in the intestine,
may reduce the oral bioavailability of cannabinoids. Therefore, it is important to
increase the bioavailabilities of oral drugs with substances possessing absorption
enhancement, drug solubilising and inhibiting effects on P-gp. Previous work has
shown that D-a-tocopheryl polyethylene glycol 1000 succinate (TPGS) has an
accelerating effect on gastrointestinal transit and a modulating influence on drug
absorption in humans [11]. A P-gp inhibition could be demonstrated [12-14].
Further study endpoints were a) to assess the relative bioavailabilities of THC and its
metabolites assessed as area under the plasma concentration/time curve from time T
= 0 h extrapolated to infinity (AUC(0-¥)), b) to assess the relative tolerability and
safety of six different oral formulations of 20 mg THCtot (THC and THCA-A), c) to
assess the effect of six different oral formulations of 20 mg THCtot on psychomotor
function assessed as simulator assisted evaluation of driving ability, d) to assess
repetitive heart rate, blood pressure and a visual analogue scale (VAS) for
psychotropic effects.
The pharmacokinetics of the cannabinoids was highly variable between the subjects.
Due to this variability, no statistically significant differences between the AUC of the
different forms could be detected, neither in part I nor in part II of the study. Addition
of different amounts of TPGS resulted in an increase in relative bioavailability of the
sum of cannabinoid metabolites (THC + 11-OH-THC + THC-COOH + CBN) to
122.5% (7.5% TPGS), 134.9% (0.5% TPGS) and 135.9% (5% TPGS) compared with
the AUC of the unheated extract (=100%) in study part I. The administration of
cannabis extracts as well as the addition of TPGS leads to a qualitatively different
pattern of cannabinoid metabolites. After administration of the unheated extract, a
significantly higher proportion of THC AUC and a significantly lower THC-COOH AUC
of all cannabinoids were observed compared to the heated extract or MarinolTM. After
administration of the synthetic MarinolTM, no plasma concentrations of CBD could be
detected. This was expected, since THC is not converted to CBD in vivo and is found
only in cannabis plants. Heating of extracts decreased the proportion of CBD
significantly. The future approach will address further research. Further, clinical
studies with the 0.5% or 5% TPGS Cannabis sativa L. extract capsule formulations
may be helpful. The study should be placebo controlled and later tested in the future
patient group.
In the present work, only the pharmacokinetics of the study are described, evaluated
and discussed. The pharmacodynamic results are reported in two separate
publications.
The aim in project two (publication 1, chapter 3.1) was the development of a sensitive
high-performance liquid chromatographic separation method with tandem-mass
spectrometry detection for the simultaneous detection of THC and its major
metabolites 11-OH-THC and THC-COOH as well as the components CBD and CBN
in human EDTA-plasma and urine. Optimal conditions for the analysis method, such
as extraction procedure, matrices, column, quality controls, wavelength, mobile
phases, run time, optimal separation (gradient, retention times), temperature,
voltages, vacuum and internal standards, resulting in the best sensitivity and
selectivity, were developed in preliminary experiments. The validation of the method
was performed according to the FDA Good Laboratory Practice guidelines,
containing linear measuring range, quantification, lower limit of quantification (LLOQ),
lower limit of detection (LLOD), quality controls, precision, accuracy, recovery,
stability and matrix effects. In conclusion, the described high-performance liquid
chromatographic separation method with tandem-mass spectrometry detection
showed a satisfactory overall analytical performance well suited for applications in
medical science. The combination of SPE/LLE, LC and APCI-MS/MS represents an
attractive alternative to the well-established technique of GC-MS.
In project three (publication 2, chapter 3.2), the sensitivity and specificity of two
immunoassays (CEDIA, FPIA) were established in urinary samples from volunteers
receiving oral synthetic THC or Cannabis sativa L. extracts. Urinary THC-COOH
excretion was evaluated by the immunoassays with a cut-off value of 50 ng/ml as
well as the described LC-MS/MS method (gold standard) with a cut-off value of 15
ng/ml. It was demonstrated that LC-MS/MS is an excellent confirmation method for
immunoassays allowing the qualitative and quantitative detection of many
cannabinoids. The ROC analysis indicated that the FPIA test discriminates better
between users and non-users than the CEDIA test. The results of both
immunoassays show that the National Institute on Drug Abuse (NIDA) set general
immunoassay cut-off of 50 ng/ml is possibly not applicable for analysis of samples
from persons consuming the Cannabis sativa L. extracts orally instead of smoking. It
has to be discussed, whether a lower cut-off value would be advantageous. It is
supposed that metabolite concentrations differ strongly depending on the route of
application. The amount and appearance of different metabolites may disturb the
immunoassay methods. The hydrolysation procedure showed a total transformation
of the THC-COOH-glucuronides to THC-COOH confirmed by the nearly 100%
agreement of the concentrations in the different samples analysed with the two
immunoassays and the LC-MS/MS comparisons. The glucuronide is automatically
detected together with THC-COOH and it is direct de-glucuronated in the APCI unit
of the detector.
The present work is structured into a theoretical and a publication section. The
theoretical section gives an overview about cannabis, mass spectrometry, assay
validation and GLP-guidelines related to the aspects used in the work of this thesis.
The publication section describes the results of the investigations, submitted for
publication to different scientific journals.
Advisors:Drewe, Jürgen
Committee Members:Scholer, André and Huwyler, Jörg
Faculties and Departments:05 Faculty of Science > Departement Pharmazeutische Wissenschaften > Ehemalige Einheiten Pharmazie > Klinische Pharmazie (Drewe)
Item Type:Thesis
Thesis no:8333
Bibsysno:Link to catalogue
Number of Pages:134
Language:English
Identification Number:
Last Modified:30 Jun 2016 10:41
Deposited On:13 Feb 2009 16:32

Repository Staff Only: item control page