A new method for determination of in vivo pA2 using infusions of naloxone to steady-state blood concentrations

A New Method for Determination of In Vivo pAz Using Infusions of Naloxone to Steady-State Blood Concentrations

MICHAEL YEADON AND IAN KITCHEN

A method onist

is described

naloxone,

for the measurement

infused

to predicted

The method has been applied of opioid plasma order

agonists.

clearance infusion

oxone

were

The

rate constants

pA2 determinations tained

by bolus

mates

of in vivo

biological bolus

by Schild injection. pA,

antagonist

whence

plasma analysis

and

could

dosing,

rests upon

appropriate

be applied

which

many

In vivo pA2; Nsaxolone;

widely

Infusion;

of the

doses

different

from

more

antagonists,

of the problems

is currently

effects and zero

concentrations

provides

to other

depressant

of nal-

the infusion

significantly

described

in rats.

determination

loading

The predicted

were

antag-

concentrations

measurement.Using

The approach

overcoming

blood

of the respiratory

are calculated.

by direct

responses,

Key Words:

to antagonism

basis of the technique

of the antagonist

verified

of pAz in vivo for the opioid

steady-state

inherent

protocol those

accurate species, in the

obestiand

use of

employed.

Opioid

receptors

INTRODUCTION The pA, of a competitive antagonist logarithm of the molar concentration dose of agonist

to that of half the dose,

of the competitive agonist. Measures

was defined by Schild (1947) as the negative of an antagonist that reduces the effect of a

antagonist

for

of pA, have central

and is an indirect

the receptor(s)

importance

measure

that mediate

in pharmacology,

of the affinity

the effect

in particular

of the for dis-

cerning the heterogenity or otherwise of receptors, and in the opioid field measures of pA2 with naloxone in isolated tissues in vitro (Hughes et al., 1975) have provided evidence isolated

of the multiplicity tissue

is known,

and the assumption

receptor is achieved. However, extension 1969) has a number oxone concentration of antagonist

Address

Surrey, reprint

and Toxicology, Received

The

determination

is made that equilibrium

of the pAz concept

Thus

the result

of Biochemistry,

United

Kingdom

requests

to: Dr.

University

October

receptors.

of pA, in an

because the molar concentration

to the whole

of naloxone

of the antagonist

at the

animal

et al.,

(Takemori

of drawbacks, the most important being that estimates of nalat the opioid receptor sites cannot be made, since bolus doses

are given.

From the Department Guildford,

of opioid

bath is straightforward

1987;

Ian Kitchen,

of Surrey, revised

Division

Guildford,

is not a true pAz but an apparent value of of Pharmacology Department Surrey

of Biochemistry,

GU2 5XH,

and accepted December

and Toxicology,

United

University

Division

of Surrey,

of Pharmacology

Kingdom.

1987.

29 lournal

ol Pharmacological

cl1988

Elievier

Mrthodc

Science Puhlishlng

LO, 29-G CII

(19881

Inc.,51Vanderhllt

Avenur,, New York. NY IOllli

30

M. Yeadon and I. Kitchen pA2, and this thermore,

renders

direct comparisons

with

isolated

the time to peak effect of both agonist

termined

to ensure

one response

comparability

tissue

data impossible.

and antagonist

of the effects of two doses,

per animal in the presence

of antagonist

must

first

Furbe de-

and, in addition,

may be obtained

only

using such

a protocol. We

describe

antagonist

dose-response The

here

a new method

and permits curves

method

that provides

considerable

savings

may be constructed

has been developed

for

steady-state

both

from

results

obtained

pAZ measurements

AND

since

in a single

of naloxone

opioid induced respiratory depression but could equally petitive agonists and alternative pharmacological effects. MATERIALS

concentrations

in cost and time,

of

entire animal.

reversal

be applied to other

of

com-

METHODS

A protocol for infusion of naloxone to steady-state was developed based on estimates of naloxone clearance determined from measures of biological half-life of naloxone trations

and literature

Alfentanil Belgium,

hydrochloride

and naloxone

chemicals

were obtained

ethane-anesthetized strain

values

achieved were verified

(300-450

Biological

of distribution.

measurement

hydrate was a gift from Janssen hydrochloride from

was a gift from

and tidal volume

vein in volumes

with 0.2 ml saline. (Janssen, related

concen-

Pharmaceuticals, NY, USA.

albino

rats,

purity

Beerse, All other

available.

University

Ur-

of Surrey

of Naloxone

frequency

acting potent,

naloxone

Du Pont,

Sigma and were of the highest

(1.6 g/kg, i.p.1 male Wistar

were measured

connected via a Fleisch (00) pneumotachometer pressure transducer. Agonists were administered right jugular

The

of plasma concentrations.

g) were used in all experiments.

Half-life

Respiratory

for volume by direct

Naloxone

synthetic

1982) produced depression

a tracheal cannula

of 0.1 ml saline (0.9% w/v) and washed in immediately was given via the left jugular

opioid

vein. Alfentanil,

analgesic of the 4-anilinopiperidine

an immediate

of both

using

to a Grass Instruments volumetric via an indwelling cannula into the

respiratory

apnea of dose-related frequency

(fentanyl)

duration

and tidal volume,

a rapidseries

and a doseand thus

of

minute volume. A dose of alfentanil was chosen that reduced minute volume by approximately 30% at the measurement point of 15 s post-dosing. This dose was given three times with a 5-min interval between doses, and the mean value of minute volume

depression

was defined as 100% response

to the standard dose of alfentanil.

The animal was then dosed with the opioid antagonist naloxone i.v. bolus and after 5 min, and subsequently at IO-min intervals

(200 pg/kg) as an for 110 min, the

standard dose of alfentanil was administered and the respiratory response noted. This procedure was repeated four times, and the mean responses at each time point were used to construct a plot from which an estimate of naloxone half-life was drawn (Figure Protocol

1).

for Infusion

of Naloxone

to Steady-state

An estimate of the apparent volume of distribution of naloxone in the rat was taken from the work of Tepperman et al. (1983). If a drug is infused at a constant

In Vivo pA2 Determinations

IB

r= -0.991

\

S1ope=-921~10-~= -k&303 I+=242~10-~ min-1

\

t.1/2=0.693/ke=33minutes.

\

IA

1

‘50

I

I

I

I

I

I

60

70

00

90

100

110

Naloxone

Time after

(mind

FIGURE 1. Kinetics of naloxone elimination from the rat after i.v. bolus administration. Rats were prepared for respiratory studies as described in the text. The mean of three responses to a standard dose of alfentanil producing approximately 30% depression of minute volume was defined as 1.0. After naloxone (200 &kg), repeated dosing with alfentanil at lo-min intervals produced responses whose magnitude gradually increased towards the prenaloxone control (log 1.0 = 0). The half-life of naloxone was calculated from the slope of the regression line fitted to the terminal phase as illustrated. The plot is representative of the four determinations made. rate (C,) (Cl,)

k. (the zero order rate constant), then a steady-state blood concentration will be reached in a time independent of the k,and the rate of plasma clearance of naloxone, but equal to approximately five times the elimination half-life,

t%. The value of the plateau

concentration

reached

C,

= k&I

Cl,

= k,/VD

is given by

P

where

Vd is the apparent volume of distribution and k, (the first order elimination rate constant) is 0.693/t%. Thus, any value of CS; may be approached by a simple calculation

of ko. However,

it is impractical

to infuse

the antagonist

for five elimi-

nation half-lives in order to obtain a steady-state concentration. The desired C5; is best obtained by administration of a loading bolus dose of naloxone followed by a continuous infusion at the rate that alone would have produced the same CS& given over five half-lives. In this way, the required C& is theoretically reached immediately, although it is prudent to allow at least one half-life in order that a distributional equilibrium be reached.

31

32

M. Yeadon and I. Kitchen Preliminary experiments produced the following estimates of the required pharmacokinetic parameters for naloxone in the rat: t% = 32.7 t 2.5 min, k, = 2.12 x IO-* i 0.16 x lop2 mini’. With the estimate of VD taken to be 8000 ml/kg, Cl, = 170 mliminikg. Thus if a CS; of 0.125 kg/ml is required, then a bolus loading dose of naloxone

of 1 mg/kg is given, followed

170 = 21.25 t.r,glmin/kg. Other in bolus chosen.

loading

by a continuous

CS; values were obtained

dose and infusion

rates.

An infusion

Validation of Estimate of C, by Measurement Naloxone Concentration by HPLC Rats were

prepared

as described,

given

infusion

rate of 0.125

by proportionate flow

x

reduction

rate of 10 t_d/min was

of Plasma

a bolus

loading

dose

of 1 mg/kg and

infused with naloxone at 21.25 pg/min/kg body weight for a period equal to three elimination half-lives. The concentration of naloxone in rat serum was determined by HPLC after extraction and lyophilization. The extraction recoveries of naloxone and the closely ments

related

to be identical;

the assay-to-assay Blood

morphine

morphine

recovery

was obtained

ticoagulant.

compound thus

After 1 hr clotting

and collected

ethyl

acetate (I :I0

by preliminary standard

into tubes

time at room temperature,

to give a concentration

added 2.5 ml saturated

shown

experi-

to calculate

of naloxone.

by decapitation

at 2000 g, 4”C, for 20 min. A 5-ml aliquot morphine

were

was used as an internal

sodium

of the serum

so obtained

of 62.5 ng/ml. To the spiked

bicarbonate

v/v). The mixture

buffer

was shaken

containing

no an-

the tubes were centrifuged was spiked

serum

with

sample was

(pH 8.9) and 15 ml propan-2-011

vigorously

for 15 min,

after which

the phases were separated by low-speed centrifugation. The aqueous layer was discarded. To the organic layer was added 5 ml hydrochloric acid (0.01 M), and this was shaken the aqueous oliethyl chloric

centrifugation,

layer was re-extracted

with

the organic

sodium

layer was discarded,

bicarbonate

buffer

and

and propan-2-

acetate as above. To the resulting organic phase was added 1 ml hydroacid (0.01 M), shaken for 5 min, then centrifuged. The final aqueous layer

was retained solution

for 10 min. After

and neutralized

was freeze-dried

HPLC solvent. Morphine and naloxone

by addition

to zero

volume

were separated

of 50 j.~l sodium and the residue by reverse

hydroxide

(0.2 M). This

resuspended

phase HPLC

in 100 ~1

using

a C18 t.~-

Bondapak column (3.9 x 150 mm) eluted isocratically with an aqueous solution of acetic acid, ammonia, and acetonitrile (6:0.25: 5%; v/v), at a flow rate of 1 mlimin and detected by UV absorption injected prior to the extracted

at 280 nm. Morphine and naloxone standards were materials, and standard curves were constructed.

The amount of naloxone in the extract was found by multiplying the value calculated by interpolation of the naloxone peak height onto the naloxone standard curve by the reciprocal of the recovery of morphine, which was in turn calculated by interpolation of the morphine peak height onto the morphine standard curve.

Determination Estimates

of pAz In Vivo Using Bolus or Infusion

of pA, were made by measuring

Protocols

the shift in the position

response line (or dose-ratio), at three or more antagonist plasma concentrations (infusion studies) and constructing

of the log dose-

doses (bolus Schild plots.

studies)

or

In Vivo pA2 Determinations Rats were dosed with depression

of minute

10 min were allowed response relationship

alfentanil

volume.

to establish

After a bolus

a dose-response

relationship

for the

dose of 50, 200, or 800 t_@kg naloxone

for the activity of the antagonist was then obtained at increased

to maximize. A further dosedoses of alfentanil and dose-

ratio calculated. Only one dose-ratio was obtained per animal using this naloxone bolus protocol. In the infusion protocol, four dose-response curves were obtained from

each animal,

determinations by infusion for

incrementing

rates of 1.06,4.25,

naloxone

was allowed

an equilibrium, tablishment

the naloxone

C;

by means of loading doses of 50,150, or 17 t_@min/kg. were

of the dose-response

dose-response

curve were

A period

minute

at 15 s post-dosing

maintained

relationship.

successive

equivalent

The

agonist

followed

to one half-life

to permit

during

attainment

the subsequent

comparison

taken to be the dose producing with

dose-ratio

or 600 pgikg naloxone

after the start of each infusion

and the infusions

volume

between

points

a 50%

on each

depression

and the dose producing

of

re-esof

an apnea

of 6 s duration. Five to nine estimates of the dose-ratio at three different naloxone doses or steady-state concentrations were obtained. Schiid plots were constructed and slope

and pAz determined.

RESULTS Morphine sorbance

and naloxone

with

3A). Routinely, in the extracts.

retention

standards

times

both compounds Extraction

at 86.4 and 83.2%,

sharp,

symmetrical

4 and 7 min,

peaks

respectively

were well separated from other absorbing

recoveries

respectively,

produced

of approximately of morphine

validating

Amount

and naloxone

the use of the recovery

of ab(Figure

materials

were very similar of the morphine

of drug on column (pg).

FIGURE 2. Standard curves for the measurement of morphine and naloxone by HPLC. Morphine and naloxone standards were separated by reverse-phase HPLC on a p-Bondapak Cls column. Isocratic efution with 6% acetic acid, 0.25% ammonia, and 5% acetonitrite in water gave retention times of approximately 4 min and 7 min respectively. The drugs were detected by their absorbance at 280 nm, and standard curves were always linear.

33

34

M. Yeadon and I. Kitchen

internal standard to calculate naloxone recovery. Standard curves were drawn of peak height against amount of material on column, as shown in Figure 2. The HPLC chromatogram of a typical rat serum extract after administration of a loading dose of 1 mglkg and infusion of 21.25 fq/min/kg naloxone is shown in Figure 3B, and the calculated value of naloxone C, actually obtained was 0.136 P,g/ml. The mean value of the naloxone C, from four experiments was 0.123 + 0.006 &g/ml, which compares very favorably with the value predicted to be reached using the pharmacokinetic parameters above of 0.125 pgiml. The mean body weight and starting respiratory values of the rats used in the naloxone bolus and infusion protocols were very similar. The slopes of the alfentanil dose-response lines before and after naloxone were not significantly different, and such plots were routinely linear within the range of responses observed. The slopes of the Schild plots were never significantly different from unity, and the PAZ was determined from Kg as described by Tallarida and Jacob (1979) in order to minimize the effects of small, chance variations in the Schild plot slope on pA2 derived from the intercept on the ordinate (Table 1). Determinations of pAz by either method (Table 1, Figure 4A) were in close agreement. The Schild plots for naloxone antagonism of alfentanil-induced apnea and depression of minute volume obtained by bolus or infusion of antagonist are shown in Figure 4.

B

A

Retention

time

(minutesl.

FIGURE 3. Typical HPLC elution profiles for morphine and naloxone. Morphine (0.5 ug) and naloxone (0.5 Pg) standards (A) and profile of a typical extract of rat serum after infusion to steady state with naloxone (B). The extract contained approximately 0.68 Pg naloxone compared with the predicted value of 0.625 pg, and also contained 0.3125 f.~gmorphine as internal standard.

In Vivo pAZ Determinations 2,5-

2.0Y2 L

IS-

% 5

IO-

E 0.5-

”

50

60

70

8.0

-Log Naloxone dose (moles/kg)

2-S-

F

TO-

A 5 ;

IS-

E z

l-O-

0.5-

-log[Naloxonel (predictedsteady state in blood) respiratory effects. Dose FIGURE 4. Schild plots for the antagonism of alfentanil-induced ratios were obtained as described in Methods. The points are the mean f s.e. mean of 59 determinations of dose ratio for antagonism of apnea (closed circles) and of minute volume depression (open circles) for bolus doses of naloxone (A) and for naloxone infused to steady state concentrations (B). The lines were fitted to the weighted means of each point by linear regression.

35

36

M. Yeadon

and I. Kitchen

TABLE 1

Antagonism

by Naloxone

of Alfentanil-Induced

NALOXONE

SCHILD

Minute

volume

Effects

NALOXONE

S,LOPE

BOLUS

Apnea

PLOT

Respiratory

SCHILD

INFUSIONS

7.9 2 0.07

0.90

? 0.16

9.1 t

7.5 i- 0.06

1.04

k 0.15

8.8

PLOT

SLOPE

0.05

1.12

-t 0.11

? 0.04

0.90

L 0.09

depression Values made

are mean

at three

2 s.e. mean of 5-9 determinations

different

naloxone

doses or plasma

of apparent

pAz (bolus) or pA> (infusion)

concentrations.

DISCUSSION The determination of pA 2, a quantitative measure of antagonism, has long been used in opioid and other areas of pharmacology both to classify drugs and receptors and to investigate

receptor

sponses

to drugs.

Experiments

hitherto

have employed

al., 1969,

1972;

comparisons stantly

involvement of this

a protocol

Pazos and Florez,

in the mediation type in the opioid

of bolus 1983;

of pA2, since the blood antagonist

changing.

In an attempt to circumvent

the time to peak effect of both antagonist whereby

both drugs

centrations directly

dosing

Heyman

are maximally

of both agonist

proportional

antagonist

concentration

this problem,

and agonist

is unknown

(Takemori

sites

et

complicates

most workers At this

re-

animal

(Takemori

which

and condetermine

and devise a dosing

at the receptor

administered

biological

in the whole

et al., 1986),

active at the same time.

and antagonist

to the doses

with

of diverse field

time,

protocol the con-

are assumed

et al., 1972).

to be

However,

even at this point, the concentration of antagonist is unknown, and thus the value obtained from the Schild plot is not a true measure of the affinity of the antagonist for the receptor sites that mediate the effects of the agonist. With naloxone to known or estimated steady-state blood concentrations, PA;? values sured

in

thus ‘tro.

obtained

appear to more closely

For example,

KB (pAz =

reflect

antagonist

the infusion of however, the affinities

mea-

- loglOKB) for the pair naloxone/alfentanil

using the ! eripherally mediated apnea as the respiratory response is 1 .I & 0.13 nM, compares favorably with naloxone KB determinations on the electrically stim-

which

ulated guinea-pig

ileum,

(1.2 i

0.03 nM, Hughes

et al., 1975). This

tissue

contains

predominantly p-opioid receptors, and the apneic respiratory response to opioid agonists given as an i.v. bolus is believed to be a k opioid receptor mediated phenomenon (Pazos and Florez, 1983). Thus it appears that this new technique may permit direct interpretation of in vivo PA:! data in the light of previously determined in vitro data. It is believed to be a reasonable assumption that use of bolus doses and infusion rates to obtain C, values less than that at which the plateau blood naloxone concentration check was made will produce the calculated desired concentration. converse is not necessarily correct, although Tepperman et al. (1983) reported

The that

the pharmacokinetic parameters for naloxone elimination in the rat are unchanged for naloxone doses up to 5 mg/kg. It was not practical to perform direct checks of naloxone blood concentration at the lowest predicted values used because of the

In Vivo pAz Determinations relative

insensitivity

of the Schild values

of the analytical

plots

are strong

were achieved.

The

method,

evidence

slopes

although

the linearity

that proportional

of the plots

and unit slopes

changes

also confirm

in naloxone

that the nature

CG

of the

antagonism is competitive. The method of infusing an antagonist permits construction of full dose-response curves in a single animal in presence and absence of the antagonist, even when the agonist

has a relatively

tration

long duration

of action,

does not appear to alter significantly

by the parallelism

of serial

dose-response

upward or downward

curvature

with

it is unnecessary

this

technique,

agonist,

as long as responses

since the blood naloxone

over many half-lives. lines

concen-

is suggested

and by the fact that no progressive

of such dose-response

lines was seen. Additionally

to determine

are measured

This

time

at a consistent

to peak activity

of the

time after dosing.

When

pAz is determined

by the method of bolus dosing with antagonist,

only one response

may meaningfully

be obtained per animal after the single dose of antagonist

is given.

The comparison of pA, values for antagonism of alfentanil-induced apnea or depression of minute volume (Table I) shows that the two methods of antagonist dosing produce widely nique therefore viding

differing estimates of apparent antagonist affinity. This new techaffords considerable savings both in cost and time as well as pro-

a quantitative

antagonist

measure

binding

affinity

of naloxone

than do existing

antagonism, methods

which

more closely

of measurement

reflects

of apparent

pAz in vivo. It is clear that this technique may also be applied to the study of opioid receptor involvement in other physiological functions.

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JS, Koslo RJ, Mosberg

reca F (1986) Estimation at supraspinai Studies

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J, Kosterlitz

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JW

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37