1. No category

The Distribution of Diazepam Urinary Metabolites in a Sample of Patients with Chronic Pain
Samantha O. Luk, B.S.1; Rabia S. Atayee, Pharm.D.,BCPS1,2; Joseph D. Ma, Pharm.D. 1,2; Brookie M. Best, Pharm.D., MAS1,3; Amadeo J. Pesce, Ph.D., DABCC4,5
1University
of California, San Diego (UCSD), Skaggs School of Pharmacy & Pharmaceutical Sciences, La Jolla, CA, 2Doris A. Howell Palliative Care Service, San Diego, CA, 3UCSD Department of Pediatrics, Rady Children’s
Hospital, San Diego, CA; 4Millennium Research Institute, San Diego, CA; 5Department of Pathology and Laboratory Medicine, UCSD School of Medicine, CA
Figure 1. Histogram and descriptive statistics of the fraction of diazepam metabolites
2000
1000
0.0
• 
• 
Diazepam and its metabolites bind to the allosteric benzodiazepine site on the GABA receptor3
•  Enhances GABA transmission and affect downstream processes3.
Extensive studies on diazepam and its metabolites in the blood4,6,7, but limited and conflicting data on
urinary values
•  Some argue that temazepam is the primary metabolite9,10
•  Some argue that oxazepam is the primary metabolite8,11
•  No known standard level of diazepam metabolites in the urine8,9,10,11
•  Nordiazepam has been established as the primary metabolite in the blood4,6,7
Difficult to interpret urine test results for diazepam and relate it to plasma concentrations in the body
0.2
0.4
0.6
0.8
Descriptiv
e statistic
Nordiazepa
m
Temazepa
m
Oxazepam
Mean
0.17
0.33
0.50
95%
Confidenc
e Intervals
(0.164,
0.168)
(0.328,
0.333)
(0.500,
0.506)
%CV
69.70%
48.42%
41.20%
c.
7000
[Creatinine] ≥ 20 mg/dL
927,772 specimens
3000
2000
1000
1
2
3
c.
7000
Taking diazepam only
34,549 specimens
Removed those without nordiazepam,
temazepam, and oxazepam readings
45,274 specimens
Graphical and statistical analysis
•  Creatinine corrected values were used
•  Creatinine corrects for the account for body mass, water intake, and hydration status12
•  Used these as a measure of metabolic activity:
•  The creatinine corrected fraction of diazepam metabolite to other diazepam metabolites
•  The fraction of diazepam metabolites to the total moles of excreted metabolites
•  Two-sample t-tests and graphical analyses were done with OriginPro 8.5.1 and Microsoft Excel 2010
•  Percent coefficient of variation (%CV) was used as a measure of variability
•  %CV =
​"#$%&$'& &()*$#*+%/-($% 6000
oxazepam > temaz epam
oxaz epam < temazepam
Number of subjects
1st or single visit
N=21,789
5000
4000
3000
2000
1000
0
-2
-1
0
1
2
log(oxazepam/temazepam)
-4
2000
-1
0
1
2
3
Oxazepam
-3
-9
-8
-7
-6
-5
-4
0.4
0.2
3
Figure 2. Histograms of the log of the fraction
of metabolite.
•  The lines on figures 2a–2c indicate the point
at which the metabolites would be equal.
•  Left of the line = metabolite in
denominator is greater
•  Right of the line = metabolite in
numerator is greater
•  Breakdown:
•  Nordiazepam < temazepam in 92% of
population
•  Nordiazepam < oxazepam in 82% of
population
•  Temazepam < oxazepam in 71% of
population
Blood
48
Schwartz, et al.,
19658
Urine
2
•  Oxazepam = major metabolite
•  ~30% oxazepam
•  ~10% temazepam
Arnold, 19759
Urine
4
•  Temazepam = major
metabolite
Chiba, et al.,
199510
Urine
4
•  Temazepam = major
metabolite
•  50% temazepam
•  29% nordiazepam
•  21% oxazepam
Smith-Kielland,
et al., 200111
Urine
27
•  Oxazepam = major metabolite
Current study
-7
-6
-5
-4
Urine
22, 509
-3
Log(Total moles)
b.
High total moles
0.8
Oxazepam = 0
•  In a large population of pain patients, the mean percentage of nordiazepam to temazepam to
oxazepam in the urine was 17% to 33% to 50%, which is seen in most subjects
•  Different from previous studies
•  86.80% of the population made all three metabolites
0.33%
0.6
0.71%
0.4
0.45%
•  13.20% had one or two of the three possible metabolites
•  Usually had low total moles
•  Maybe were at the beginning or end of the dosing interval
86.80%
0.2
0.29%
0.0
Temazepam
0.2
CONCLUSIONS
•  The amount of each metabolite made is not dependent on the total excreted metabolite
•  Suggests the diazepam metabolism pathways are not saturated at therapeutic doses
Nordiazepam
Low total moles
0.1
•  Oxazepam = major
metabolite
•  50% oxazepam
•  33% temazepam
•  17% nordiazepam
Medium high total moles
Medium low total moles
0.0
Major findings
•  Nordiazepam = major
metabolite
Log(Total moles)
0.6
-8
# of subjects
-3
Figure 3. Semi-logarithmic scatterplots of the log of total
moles versus the fraction of metabolite.
•  No relationship between the fraction of metabolite and the total
excreted metabolite.
•  Fraction of nordiazepam is less than the fraction of temazepam
and oxazepam (fig. 3a, 3b, 3c)
•  There is more overlap between the temazepam and oxazepam
fraction (fig 3b, 3c)
-9
Analyte
Greenblatt, et al.,
19894
0.2
Log(Total moles)
1.0
3000
-2
0.4
0.0
ox azepam < nordiazepam
log(oxazepam/nordiazepam)
log(temazepam/nordiazepam)
oxaz epam = temazepam
N=subjects
-5
4000
0
0
0
-6
a.
1000
-1
-7
1.0
Fraction of temazepam
Number of subjects
Number of subjects
temazepam > nordiazepam
4000
-2
-8
Nordiazepam = 0
oxazepam < nordiazepam
temazepam < nordiazepam
5000
Temazepam
0.6
RESULTS: METABOLITE DISTRIBUTION ANALYSIS
oxazepam = nordiazepam
6000
Specimen collection
•  Urine specimens from1st or single visit patients with chronic pain from 2008-2011
•  Urine concentrations analyzed by liquid chromatograph tandem mass spectrometry (LC-MS/MS) at Millennium
Laboratories
•  Retrospective study on de-identified urine specimens
Starting population
1,025,138 specimens
b.
temaz epam = nordiazepam
Nordiazepam
0.8
0.0
-9
5000
METHODS
Reported taking diazepam,
nordiazepam, temazepam, or oxazepam
55,402 specimens
0.2
0.8
Figure 1. Histogram and descriptive statistics of the fraction of diazepam metabolites
•  Mean urinary percentage: nordiazepam (17%) < temazepam (33%) < oxazepam (50%)
•  All three means were significantly different from each other (p<0.00001)
•  Subjects with fraction of oxazepam = 1 (N = 1580, 7% of the total population) were not found to be
significant outliers and analyses were still significant when these values were excluded
a.
0.4
1.0
Fraction of metabolite (metabolite/total)
The aim of this study was to examine the urinary distribution of diazepam metabolites in a
population of pain patients reported to be on diazepam and not any of its metabolites.
0.6
0.0
RESULTS: FRACTION OF METABOLITE
OBJECTIVES
0.8
DISCUSSION: COMPARISION WITH OTHER STUDIES
Study
1.0
Fraction of temazepam
3000
b.b.
1.0
Table 1. Summary of descriptive statistics
(N =21,789)
4000
0
• 
a.
Nordiazepam
Temazepam
Oxazepam
*
5000
RESULTS: METABOLITE TO TOTAL MOLES
Fraction of nordiazepam
• 
Diazepam is used as an adjuvant
to opioid therapy to reduce painrelated anxiety and serves as a
muscle relaxant in pain patients1,2,3
Metabolized to either nordiazepam
or temazepam by various
cytochrome P450 enzymes
•  N o r d i a z e p a m a n d
temazepam are further
metabolized to
oxazepam4,5
•  All three metabolites are
pharmacologically active
Number of subjects
• 
RESULTS: FREQUENCY DISTRIBUTION
Fraction of oxazepam
BACKGROUND
0.3
0.4
0.5
0.6
0.7
0.8
0.9
4.18%
7.25%
Oxazepam
•  Diazepam urinary population data allows us to better understand how to interpret urine
values
1.0
Fraction of nordiazepam
Temazepam = 0
Figure 4. Scatterplots (non-stratified and stratified for total moles) and Venn diagram of metabolite
distribution.
•  Arrows and the corresponding legends on figure 4a indicate where nordiazepam, temazepam, or
oxazepam equal zero
•  Those that are missing one of the three possible metabolites in the urine usually have low moles
•  Fig. 4b describes the percentage of the population with urine readings for the diazepam metabolites
•  As seen in fig. 4b, 86.80% have all three possible urinary diazepam metabolites, making up the majority
of the subjects in fig 4a.
•  From fig. 4b, 13.20% of the population makes only one or two of the three possible metabolites
•  Usually have low total excreted metabolite (fig 4a. and fig 4b.)
•  90% of these subjects with < 3 metabolites had oxazepam in their urine
ACKNOWLEDGEMENTS AND DISCLOSURES
Thank you to UCSD Skaggs School of Pharmacy and Pharmaceutical Sciences, Millennium Research Institute and
Elizabeth Gonzales for their generous help. Thank you to Neveen Barakat, Sophie Bordson, Natalie Elder, Alex Guo,
Michelle Hughes, Katie Moy, Stephanie Tse, and David Yee for all their support, feedback, and assistance. Disclosure:
Dr. Pesce is an employee of Millennium Laboratories, Inc.
References
1.  Baselt, RC. Disposition of Toxic Drugs and Chemicals in Man. Biomedical Publications, Foster City, CA, 2008.
2.  Davies JM, Rosen M. Intramuscular diazepam in labour. A double-blind trial in multiparae. Br J Anaesth. 1977 Jun;49(6):601-4.
3.  Di Piero V, Ferracuti S, Sabatini U, Tombari D, Di Legge S, Pantano P, Cruccu G, Lenzi GL. Diazepam effects on the cerebral responses to tonic pain: a SPET study. Psychopharmacology (Berl). 2001 Nov; 158(3):
252-8.
4.  Greenblatt DJ, Ochs HR, Lloyd BL. Entry of diazepam and its major metabolite into cerebrospinal fluid. Psychopharmacology (Berl). 1980;70(1):89-93
5.  Andersson T, Miners JO, Veronese ME, Birkett DJ. Diazepam metabolism by human liver microsomes is mediated by both S-mephenytoin hydroxylase and CYP3A isoforms. Br J Clin Pharmacol. 1994 Aug; 38(2):
131-7.
6.  Friedman H, Abernethy DR, Greenblatt DJ, Shader RI. The pharmacokinetics of diazepam and desmethyldiazepam in rat brain and plasma. Psychopharmacology (Berl). 1986;88(3):267-70.
7.  Greenblatt DJ, Harmatz JS, Friedman H, Locniskar A, Shader RI. A large-sample study of diazepam pharmacokinetics. Ther Drug Monit. 1989 Nov;11(6):652-7.
8.  Schwartz MA, Koechlin BA, Postma E, Palmer S, Krol G. Metabolism of diazepam in rat, dog, and man. J Pharmacol Exp Ther. 1965 Sep;149(3):423-35.
9.  Arnold E. A simple method for determining diazepam and its major metabolites in biological fluids: application in bioavailability studies. Acta Pharmacol Toxicol (Copenh). 1975 Apr;36(4):335-52.
10.  Chiba K, Horii H, Chiba T, Kato Y, Hirano T, Ishizaki T. Development and preliminary application of high-performance liquid chromatographic assay of urinary metabolites of diazepam in humans. Chromatogr B
Biomed Appl. 1995 Jun 9;668(1):77-84.
11.  Smith-Kielland A, Skuterud B, Olsen KM, Morland J. Urinary excretion of diazepam metabolites in healthy volunteers and drug users. Scand J Clin Lab Invest. 2001 May;61(3):237-46.
12.  Heit HA, Gourlay DL. Urine drug testing in pain medicine. J Pain Symptom Manage. 2004 Mar;27(3):260-7.
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