JapaneseJournal
Journal of Community
No.1,
1-8（2019）
Japanese
CommunityPharmacy
Pharmacy Vol.7,
Vol.7,
No.1,
1-8 (2019)《 Original Article 》

Hypnotic doses of benzodiazepine-GABAA receptor agonists may not only be
associated with the intrinsic receptor binding affinity, but also withpharmacokinetic parameters of drug exposure in the brain
Kazuharu Nakazawa1,2*, Hirotoshi Echizen2
Although the difference between the lowest and highest approved oral doses ofbenzodiazepine (BZD) hypnotics is almost 30-fold, it remains unclear whether the intrinsic
potency of the BZD-gamma-aminobutyric acid type A (GABAA) receptor agonist is solely
responsible for this variability. We conducted the present study to examine this issue. In
previous studies, we determined the half-maximum inhibitory concentration of the GABAA
receptor-chloride ion channel complex in human brain (hIC50) for seven BZDs and two nonBZD drugs with agonistic action on GABAA receptors. Hypnotic doses of these drugs and thebiomarkers of brain exposure of these drugs [plasma concentration-time curve (AUC) andmaximum drug concentration (Cmax)] were retrieved from the official prescribing information
provided by pharmaceutical companies. We also calculated the AUC and Cmax of the unbounddrug (AUCu and Cmax,u, respectively) as biomarkers of drug brain exposure, by multiplying
AUC and Cmax by the plasma unbound drug fraction (fu). In addition, we estimated theliposolubility (e.g., logD7.4) of the drugs by using a computer software (Marvin Sketch®). There
was a significant correlation between log hIC50 and log nitrazepam-equivalent hypnotic dosesof BZD-GABAA receptor agonists (r = 0.91, p < 0.01). A significant correlation was observed
between hIC50 and AUCu (r = 0.84, p < 0.01), between hIC50 and Cmax,u (r = 0.94, p < 0.01), andbetween logD7.4 and log hIC50 (r = -0.95, p < 0.001). We concluded that the differences in the
hypnotic doses of BZDs may be largely accounted for by the variability of hIC50, and to someextent by the differences in volume of distribution (Vd) and fu, as Cmax,u = fu・D/Vd. In
addition, hIC50 may be affect the lipophilicity of drugs (logD7.4), because drug transfer to thelipid-rich brain tissue is another factor associated with the potency of BZDs in the brain neurons.
Key words; benzodiazepine-GABAA receptor agonist, hypnotics, IC50,
lipophilicity, plasma unbound drug
Received February 17, 2019 ; Accepted May 10, 2019
1 Clinical Trial Office, National Hospital Organization Higashisaitama National Hospital, 4147 Kurohama, Hasuda,

Saitama 349-0196, Japan ,2 Department of Pharmacotherapy, Meiji Pharmaceutical University, 2-522-1 Noshio, Kiyose, Tokyo 204-8588, Japan ,*Corresponding author: Kazuharu Nakazawa, PhD, Department of Pharmacotherapy, 2-522-1 Noshio, Kiyose,Tokyo 204-8588, Japan Phone +81 42-495-8611
E-mail: k.n.ivmjup@jcom.zaq.ne.jp11★Vol7No1_日本地域薬局薬学会誌_本文.indd 1

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Nakazawa
BZD-GABA receptor
receptorbinding
binding
Nakazawaetetal.；Hypnotical.；Hypnoticdoses
doses and BZD-GABA

1． Introduction

elimination half-lives and plasma protein binding of
BZDs.

Owing

to

the

differences

in

the

Since the prototype benzodiazepine (BZD) drugs,

pharmacokinetics, the cumulative exposure ofchlordiazepoxide and diazepam, were introduced

BZDs in brain tissue [i.e., the area under the plasma

into clinical practice in the 1960s, more than 30

drug concentration-time curve (AUC)] or the

BZDs have been introduced into clinical use.

maximum exposure of drugs in the brain tissue [i.e.,Previous studies performed on animals (e.g.,

maximum drug concentrations (Cmax)] after therodents) revealed that the hypnotic effects of BZDs

administration of the same dose may differ

may be associated with their agonistic effects on theconsiderably between BZDs. In this report, we

gamma-aminobutyric acid type A (GABAA)

attempt to explain the apparent differences in thereceptor-chloride ion channel complex in cerebral

hypnotic doses of BZDs from the perspective ofneurons. The binding of BZDs to GABAA receptors

intrinsic GABAA receptor agonistic potency (i.e.,

promotes the opening of the chloride ion channels

hIC50), as well as the pharmacokinetic biomarkers

and the influx of chloride ions into neurons, therebyof BZDs.

leading to hyperpolarization of the neuronalmembranes

andreducing

theirliability2.

toMethods

depolarization by external stimuli. These areconsidered the molecular mechanisms associatedAt present, 12 BZDs are available as hypnotics inwith the sedative, hypnotic, and anxiolytic effects ofJapan. Excluding BZDs with active metabolites or

BZDs1,2).

prodrugs,

weanalyzed

seven

BZD

drugs

There is an approximately 30-fold difference in(brotizolam, estazolam, etizolam, flunitrazepam,

the oral doses of BZDs approved as hypnotics inlormetazepam, nitrazepam, and triazolam) and two

Japan. A previous in vitro study of post-mortem

non-BZD hypnotic (zopiclone and zolpidem) with ahuman brain tissues demonstrated that there was ansimilar mechanism of action to BZD drugs.

approximately 1,000-fold difference in the half

Nitrazepam-equivalent hypnotic doses of each drug

maximal inhibitory concentration (hIC50) of thewere obtained from a previous report2). We

GABAA receptor binding for BZDs3). In addition, it

retrieved approved hypnotic doses, AUC of plasma

was also shown that there was a significant

total (unbound plus bound) drug concentrations

correlation between hIC50 values and their clinical(AUC)

doses3,4). These findings appear to support theconcentration after a single oral dose (Cmax) fromhypothesis that clinical doses of BZDs are largely

the corresponding prescribing information provided

associatedtheby pharmaceutical companies5-14). In addition, data

pharmacological effects at the sites of action (e.g.,on plasma unbound fraction (fu) of the drugs were

hIC50).

retrieved from the prescribing information. AUC

withHowever,thethedifferences
variability

in
in

the

and

Cmax

maximum

plasma

total

drug

pharmacokinetics of BZDs may also be associatedandwith differences in the therapeutic doses of BZDs.

concentrations were calculated by multiplying AUC

For example, there are large differences in theand Cmax by fu of the respective drugs. As plasma

expressed

asplasmaunbound2
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JapaneseJournal
Journal of Community
No.1,
1-8（2019）
Japanese
CommunityPharmacy
Pharmacy Vol.7,
Vol.7,
No.1,
1-8 (2019)drug concentrations and related pharmacokinetic

analyzed with the exponential model. All statisticalparameters are expressed as non-SI unit (e.g.,

analyses were performed with JMP® Pro 11.0.0

μg/mL) in most of the prescribing informations used

(SAS Institute Japan) and values of p<0.01 werein the present study, they were converted to SI units

considered statistically significant.

(e.g., mole/L).
3.

We retrieved hIC50 values of the eight BZDs from

Resultsa previous report where they were measured in postmortem brain tissues using 3H-flunitrazepam as a

Table 1 summarizes the GABAA receptor binding

3)

ligand of the GABAA receptors . As the hIC50 forparameterszolpidem was not available from this report, it wasparameters such as AUC and Cmax, and approved

estimated from the relationship between half-

hypnotic doses, and logD7.4 for the nine BZDs thatmaximum inhibitory concentrations of BZDs

were currently approved in Japan for clinical use.

obtained from Sprague Dawley rats (rIC50) using

There was an approximately two-order difference in3

the receptor binding affinity assessed by rIC50 and

H-flumazenil as a ligand of the GABAA

receptors

15)

such

as

hIC50,

pharmacokinetic

hIC50 among the BZDs studied. In addition, there

and hIC50 for the corresponding BZDs,

as in the study of Kobayashi et al.3). Eight drugs

was(alprazolam, brotizolam, clonazepam, diazepam,

nitrazepam-equivalent dose of BZDs. Nevertheless,flunitrazepam,

there was an excellent correlation between the twolorazepam,

triazolam,andapproximately

40-fold

differencesinbinding parameters: hIC50 = 1.34・rIC501.16 (r= 0.97,

zopiclone) were used for the correlation analysis.
The physicochemical properties (e.g., molecularp<0.01, n=8). When the rIC50 value of 14.0

weight, lipophilicity, and solubility) of the BZDs

nmoles/L for zolpidem15) as substituted into thewere estimated by using the MarvinSketch®

equation, we were able to estimate that hIC50 was besoftware (version 17.13). Specifically, the V-g

28.9 nmoles/L. As no values for hIC50 are reported

method 16) was used for estimating liposolubility ofin literature, we used this value and undertook

drugs with log D values at pH 7.4 (log D7.4), and thefurther analysis as described below.

formula proposed by Yalkowsky et al. was used to

As for pharmacokinetic parameters of BZDs

estimate aqueous solubility of drugs with log S(AUC and Cmax) of the BZDs at the respective

values at pH 7.4 (log S7.4)17). An index of theapproved doses, there were also large differenceshydrophilic-lipophilic(120- and 54-fold) among the drugs.

balance

(HLB)

was

estimated with the Davies’ formula18).

For LogD7.4, there was an eight-fold difference

A correlation between hIC50 and rIC50 wasamong the drugs. Despite these large between-drug

analyzed with an exponential model (y=a・xb). A

differences in BZD receptor binding affinity,

relationship between the log nitrazepam-equivalentapproved doses, pharmacokinetic parameters that

doses of the BZDs and the log hIC50 was also

are associated with drug exposure to the brain, andanalyzed with the exponential model. Relationships

physicochemical properties, there were significant

between log AUC or AUCu and log hIC50 and thosecorrelations between the parameters. For example,

between log Cmax or Cmax,u and log hIC50 were

there was a significant correlation between the log
33★Vol7No1_日本地域薬局薬学会誌_本文.indd 3

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Nakazawa
BZD-GABA receptor
receptorbinding
binding
Nakazawaetetal.；Hypnotical.；Hypnoticdoses
doses and BZD-GABA

Table 1

In vitro affinity of BZDs to BZD-GABAA receptor binding in human brain (hIC50)
and their pharmacokinetic parameters of drug exposure in the brain.

CompoundhIC50 3)(nmoles/L)

Nitrazepamequivalentdose 2)(mg)

Nitrazepamequivalentdose 2)(moles)

Brotizolam

1.2

0.25

Estazolam

28.9

Etizolam*

AUC 5-14)

Cmax 5-14)

total
conc.
(nmoles/L*h)unboundconc.
(nmoles/L
*h)

total
conc.
(nmoles/L)

unboundconc.
(nmoles/L)

LogD7.4 16)

0.6

76.3

7.6

9.5

0.9

3.1

2.0

6.8

11813.3

2362.7

381.4

76.3

2.51

4.6

1.5

4.4

560.0

39.2

62.6

4.4

3.4

Flunitrazepam

5.9

1.0

3.2

512.0

109.6

51.1

10.9

2.58

Lormetazepam

2.0

1.0

3.0

182.0

15.7

16.7

1.4

3.26

Nitrazepam

24.8

5.0

17.8

4874.3

731.1

141.9

21.3

2.67

Triazolam

1.7

0.25

0.7

39.2

4.3

7.2

0.8

3.31

Zopiclone

103.6

7.5

19.3

1365.6

423.3

183.3

56.8

0.41

Zolpidem**

28.9

10

13.1***

1597.3

63.9

390.4

15.6

3.08

hIC50 = half-maximum inhibitory concentrations of BZDs to the BZD-GABAA receptor complex binding in the human brain
tissues, AUC= area under the plasma concentration-time curves, Cmax = maximum plasma concentration after a single oral
administration*For AUC and Cmax of etizolam, those obtained after an oral administration of 1.0 mg were multiplied by a factor of 1.5, as nodata were available for those after the administration of 1.5 mg.
**The hIC50 value for zolpidem was estimated from the equation hIC50 = 1.34・rIC501.16 and the rIC50 value of 14 nmoles/L
obtained from the study15). rIC50= half-maximum inhibitory concentrations of BZDs to the BZD-GABAA receptor complex
binding in the rat brain tissues.
***The molecular weight of zolpidem tartrate that is contained in the commercially available formula is 764.87. The
pharmacokinetic parameters were calculated using the weight of the zolpidem bas

Half-maximum inhibitory concentrations of BZDs to the BZD-GABAA
receptor complex binding in human brain tissue (nmoles/L)

Figure 1 Correlation between the log nitrazepam-equivalent doses of BZD hypnotics and the log half-

maximum inhibitory concentrations of BZDs to the BZD-GABAA receptor complex binding in the humanbrain tissues. Micromoles equivalent to 5 mg of nitrazepam (17.8 μmole) were plotted on the y axis. There
was a significant correlation between the two parameters: y = 0.90・x0.73, r=0.91, p<0.01.

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Japanese
Journalof Community
Pharmacy
Vol.7, receptor
No.1, 1-8（2019）
Nakazawa
et al.；Hypnotic
doses
and BZD-GABA
binding

Half-maximum inhibitory concentrations of BZDs to the BZD-GABAA receptor complex
binding in human brain tissue (nmoles/L)

Figure 2 Correlation between the log AUC of total plasma concentrations of BZDs and the log

half-maximum inhibitory concentrations of BZDs to the BZD-GABAA receptor complex binding inhuman brain tissue. (A): y = 76.00・x1.00, r = 0.83, p< 0.01. Correlation between the log AUC of
plasma unbound BZDs and the log half-maximum inhibitory concentrations of BZDs to the BZDGABAA receptor complex binding in human brain tissue. (B): y = 6.56・x1.15, r=0.84, p< 0.01.

Half-maximum inhibitory concentrations of BZDs to the BZD-GABAA receptor complex
binding in human brain tissue (nmoles/L)

Figure 3 Correlation between the log Cmax of plasma total BZDs and the log half-maximum

inhibitory concentrations of BZDs to the BZD-GABAA receptor complex binding in human brain tissue.
(A): y = 9.37・x0.88, r = 0.91, p<0.01. Correlation between the log Cmax of plasma unbound BZDs andthe log half-maximum inhibitory concentrations of BZDs to the BZD-GABAA receptor complex
binding in human brain tissue. (B): y = 0.79・x1.04, r = 0.94, p<0.01.

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Nakazawa
et al.；Hypnotic
doses Pharmacy
and BZD-GABA
receptorbinding
Japanese
Journalof Community
Vol.7,
No.1, 1-8
(2019)

nitrazepam-equivalent doses of BZDs (y) and log

and Cmax. Overall, the relationship between Cmax,uhIC50 (x): y = 0.90・x0.73, r=0.91, p<0.01 (Figure 1).

and hIC50 showed the highest r value (0.94) of the

Furthermore, there were significant correlations

pharmacokinetic biomarkers of drug exposure to thebetween log AUC (y) and log hIC50 (x) and between

brain. As there was 3–5-fold difference in plasma

the log AUCu (y) and the log hIC50 (x): y = 76.00・

protein binding of BZDs (Table 1), not only hIC50,

x1.00, r=0.83, p<0.01 and y =6.56・x1.15, r=0.84,

but also fu is associated with the variability of thep<0.01, respectively (Figures 2A and 2B). Similarly,

hypnotic doses of BZDs. Collectively, for a newly

there were significant correlations between the log

developed BZD drug, its hypnotic Cmax,u may be

Cmax (y) and the log hIC50 (x) and between the log

predicted by hIC50, and the corresponding hypnotic

Cmax,u (y) and log hIC50 (x): y =9.37・x0.88, r=0.91,

dose after a single dose may be approximated in

p<0.01and y =0.79 ・ x

1.04

accordance, r= 0.94, p<0.01,

with

the

basic

pharmacokinetic

respectively (Figures 3A and 3B).

equations, as D = Cmax,u・Vd,u or Cmax・Vd.

There were significant (p<0.01) correlations

There were significant correlations between the

between the log hIC50 and the respective

hIC50 values and the physicochemical parameters ofphysicochemical parameters: for log D7.4, log S7.4,

BZDs (i.e., log D7.4, log S7.4, and HLB). Theseand HLB; correlation coefficients were r=-0.95,

physicochemical properties were associated with

0.97，and 0.90, respectively (data are not shown).

lipophilicity and liposolubility of BZDs. As thebrain tissue is lipid-rich, BZDs with higher

4.

Discussion

liposolubility may have higher concentration at thebinding sites. However, it remains unclear whether

In the present study, we found that there was athis principle is applicable to other hypnotic drugs

significant correlation (r=0.91, p<0.01) between

with non-BZD structures. Further studies are

hIC50 values obtained from in vitro ligand binding

required to answer this question.

study and nitrazepam-equivalent hypnotic doses of

There present study has limitations. As only nine

BZD-GABAA receptor complex agonists for the

hypnotic drugs of BZD-GABAA receptor agonists

therapeutic doses were established via clinical trials.

are available in Japan, our analysis on theThis finding was consistent with our understanding

relationship between hIC50 and clinical doses andthat differences in the hypnotic doses of BZDs were

the pharmacokinetic biomarkers of brain drug

largely accounted for by their intrinsic potency at

exposure was performed on only a small number ofthe site of action. In addition, there were significant

drugs. As a result, further studies are required tocorrelations between hIC50 and pharmacokinetic

confirm the external validity with use of other BZDs

biomarkers of drug exposure to the brain tissue

approved as hypnotics in other countries.

(AUC and Cmax). Furthermore, the correlation

Recently, there has been a surge of interest

coefficients for the relationship between hIC50 andregarding a formulary system of drugs by all

the above drug exposure biomarkers expressed as

stakeholders (e.g., physicians and pharmacists) ofunbound plasma drug concentrations (AUCu andhealthcare in order to establishing policies for theCmax,u) were equal to or greater than those for AUC

economical and safe use of drugs19). The formulary7
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Japanese
Journalof Community
Pharmacy
Vol.7, receptor
No.1, 1-8（2019）
Nakazawa
et al.；Hypnotic
doses
and BZD-GABA
bindingsystem may be useful for recommending a small

3.

Kobayashi T, Kiuchi Y, Shimizu H, Takeuchi J,

number of drugs among those with similar

Ogata H, Tohru M, A study on theindications. As the implementation of a formulary

benzodiazepine receptors in post-mortem

system would require pharmacists to estimate thehuman brain., Ann Rep Pharmacopsychiatr

equivalent doses of drugs, the approach proposed in

Res Found., 1989: 20: 113-119 (in Japanese).

the present article based upon the information

4.

Möhler H, Okada T, The benzodiazepine

available in the prescribing information andreceptor in normal and pathological humaninterview forms may be useful for pharmacists

brain, Br J Psychiatr., 1978: 133: 261-268.

workingin

both

hospitals

andcommunity

5.

pharmacies.
In

Brotizolam® tablet “Sawai” Pharmaceuticalinterview form, 6th ed., Sawai Pharmaceuticalconclusion,

werevealed

that inCo., Ltd., 2017.

vitro

pharmacodynamic potency of BZDs (hIC50) and in6.

Estazolam® tablet “Amel” Pharmaceuticalvivo pharmacokinetic biomarkers representing druginterview

exposure to the brain (fu, AUCu, and Cmax,u) may

Pharmaceutical Industry Co., Ltd., 2017.
7.

collectively contribute to the variability of hypnotic

Etizolam®

form,

6th

ed.,

Kyowa

tablet “SW” Pharmaceutical

doses of the BZDs. Our preliminary findings on theinterview form, 6th ed., Sawai Pharmaceutical

relationship between physicochemical properties ofCo., Ltd., 2017.

BZDs and hIC50 should be studied in greater depth

8.

in the future.

Flunitrazepam® tablet “Amel” Pharmaceuticalinterview

form,

7th

ed.,

Kyowa

Pharmaceutical Industry Co.,Ltd., 2017.
Acknowledgement

9.

Evamyl® tablet Pharmaceutical interview form,
9th ed., Bayer Yakuhin, Ltd., 2017.

10. Nelbon®

We would like to thank Editage (www.editage.jp)
for English language editing.

tablets“DSC”

Pharmaceuticalinterview form, 11th ed., Daiichi Sankyo
company, Ltd., 2017.
11. Triazolam® tablet “Nichiiko” Pharmaceutical

Conflicts of Interest

interview
The authors have no conflict of interest to be

form,

12th

ed.,

Nichi-Iko

Pharmaceutical Co., Ltd., 2017
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doses Pharmacy
and BZD-GABA
receptorbinding
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No.1, 1-8
(2019)

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