European Journal of Pharmacology, 33 (1975) 205--209 © North-Holland Publishing Company, Amsterdam -- Printed in The Netherlands
Short communication EFFECT OF CHRONIC PENTOBARBITAL TREATMENT ON BLOOD--CEREBROSPINAL FLUID KINETICS* MICHIKO OKAMOTO and NORMAN R. BOISSE
Department of Pharmacology, Cornell University Medical College, New York, N. Y., U.S.A. Received 21 May 1975, accepted 5 June 1975
M. OKAMOTO and N.R. BOISSE, Effect of chronic pentobarbital treatment on blood--cerebrospinal fluid kinetics, European J. Pharmacol. 33 (1975) 205--209. Cats were made tolerant and physically dependent by twice daily equieffective anesthetic dosing with sodium pentobarbital intragastrically for 5 weeks. Serial blood and CSF samples were simultaneously collected on day 1 and day 35 and analyzed for pentobarbital by a gas chromatographic method. Pentobarbital penetration into CSF was rapid and unchanged by chronic treatment. The concentration in blood and CSF increased significantly with chronic treatment; however, the relative CSF/blood concentration ratios were unchanged. Pentobarbital
Blood--CSF pharmacokinetics
1. Introduction A reproducible method of barbiturate physical dependency production has been established based upon an equieffective anesthetic dosing regimen in cats with sodium pentobarbital (Rosenberg and Okamoto, 1974). Characteristics of the tolerance developed by such chronic pentobarbital dosing have been reported (Okamoto et al., 1975). By pharmacokinetic analysis of blood pentobarbital concentrations, it was possible to identify and quantitatively characterize two types of tolerance, namely dispositional and functional tolerances (cf. Kalant et al., 1971). The present study determines the effect of chronic pentobarbital treatment upon its penetrability into the CNS. If there were any significant alteration in the penetrability of pentobarbital during chronic treatment this should be quantitatively disclosed and considered in assessing most accurately the extent of disposi* This investigation was supported by Research Grant No. R01-DA-00591 from the National Institute on Drug Abuse.
Barbiturate tolerance
tional and functional tolerances. Therefore, experiments were designed to compare the pattern of movement of pentobarbital between blood and cerebrospinal fluid (CSF) on the first day (termed 'acute') and last day (termed 'chronic') of a 5 week treatment regimen in self-controlled animals. 2. Materials and methods 2.1. Chronic cannulation o f cisterna cerebellomedullaris
Adult cats (2.0--3.0 kg) of either sex were used. Under sodium pentobarbital anesthesia, cisternal cannulae were implanted for chronic use based on the methods of Yaksh et al. {1973) and Feldberg et al. (1970), except the approach for cannulation was modified. A hole slightly larger than the 18 gauge needle diameter was drilled through the center line of the interparietal bone, 2--3 mm anteriorally from the lambdoidal ridge and parallel to the supraoccipital bone process. A Butterfly-19 infusion set (Abbott) was used as a cisternal cannula. The cannula was inserted in the hole parallel to
206 the supra-occipital bone process along the internal wall of dulla into the cisterna cerebellomedullaris. The cannula was secured to the skull by dental acrylic cement. No motor disturbances or other observable after-effects were seen following cisternal cannulation.
2. 2. Chronic sodium pentobarbital dosing The method used for treating cats chronically with sodium pentobarbital has been described in detail previously (Rosenberg and Okamoto, 1974; O k a m o t o et al., 1975). Sodium pentobarbital was administered twice daily, AM and PM, through a polyethylene t u b e that had been surgically implanted into the stomach through the abdominal wall. A series of neurological tests (Okamoto et al., 1975) were performed at preset times each day to test the degree of CNS depression. These were used to determine the peak CNS depression achieved each day and the degree of recovery before dosing. Based on these results, each dose was individually adjusted to produce a preset anesthetic level of peak CNS depression throughout the chronic treatment period. The initial dose of sodium pentobarbital was 40 mg/kg. The total duration of treatment was 5 weeks.
2.3. Blood and cerebrospinal fluid pentobarbital pharmacohinetic study Blood and CSF samples were collected simultaneously from animals on their first and last days of chronic treatment. Duplicate blood samples (100 pl each) were collected from the saphenous vein at 1/4, 1 / 2 , 1 , 2, 4, 6, 8 and 10 hr after the morning dose of sodium pentobarbital. One hundred pl of CSF were collected simultaneously into a 500 pl Hamilton Syringe by needle insertion through the chronically implanted cisternal cannula into cisterna cerebellomedullaris. The depth of this needle insertion was determined at the time of the first sample collection for each animal and its level was marked for subsequent samplings. The analytical method for pentobarbital involves a single step extraction into chloroform
M. OKAMOTO, N.R. BOISSE followed by evaporation of the solvent. Dried extracts are reconstituted with chloroform containing hexobarbital as an internal standard (Okamoto et al., 1975). Gas chromatographic analysis was performed on a Perkin--Elmer Model 990 equipped with a hydrogen flame ionization detector, an automatic solvent-free injection system (AS-41) and a data reduction system (PEP-l). Separation of the barbiturates was achieved with 3% OV-17 on Gas Chrom Q 100/120 mesh packed in a 6 foot glass column. GC operating conditions described by Adams (1972) were slightly modified; temperature programming began at the time of injection with an initial temperature of 180°C, a 24 ° C/ min rate, and a final temperature of 280°C held for 8 min. Recovery of pentobarbital from blood and CSF is 98--100% complete. The sensitivity of this analytical system is approximately I pg/ml using 100 pl of biofluid sample.
3. Results Ideally, biofluid samples of the 'acute' and 'chronic' phases of treatment would have been collected from the same animals to most effectively compare the rate and extent of pentobarbital penetration into the CNS. However, among 13 paired cases studied, 5 pairs were not self~controlled. In these 5 'chronics', the cisternal cannulae were implanted after the morning dose about 5 days before the termination of chronic treatment. All animals treated b y this method required progressively higher morning and total daily doses to produce a constant level of peak CNS depression as the chronic treatment progressed (Okamoto et al., 1975). In this group of animals, the average morning dose was 40 mg/kg (preset) for the 'acute' and 57.9 + 2.4 (S.E.) mg/kg for the 'chronic'. The average total daily dose was 52.9 + 2.9 mg/kg for the 'acute' and 109.2 + 6.4 mg/kg for the 'chronic'. Accordingly, this increase in dose averaged 1.46 + 0.05 fold for morning doses and 2.12 + 0.14 fold for the total daily doses.
CHRONIC PENTOBARBITAL BLOOD--CSF KINETICS 504030-
~20=
0u .~IO-
:.o: Z L
~ TIME
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~I
PENTOBARIIITAL
110
112
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Fig. 1. Pentobarbital c o n c e n t r a t i o n curves in blood and CSF after the m o r n i n g dose on the first day ('acute') and the last day ( ' c h r o n i c ' ) of a 5 week pentobarbital t r e a t m e n t . Abscissa: t i m e in hr after morning dose of s o d i u m pentobarbital. Ordinate: pentobarbital b l o o d ( ) and CSF ( . . . . . . ) concentrations in p g / m l on a log scale. Solid circles (e) represent c o n c e n t r a t i o n s in 'acute'. O p e n circles (o) represent c o n c e n t r a t i o n s in 'chronic'.
Results of the analysis of b l o o d and CSF pentobarbital concentrations in 'acute' and 'chronic' are shown in fig. 1. Sodium pentobarbital was well absorbed from the intragastric route as indicated b y the rapid increase in pentobarbital concentration in blood (upper two curves). Although quite variable among animals, the average rate of rise of blood pentobarbital concentrations between 15 and 30 min after dosing provides an estimate for the rate of absorption. These rates were 0.8571 + 0.1550 log (#g/ml)/hr for the 'acute' and 0.5270 + 0.1493 log (~zg/ml)/hr for the 'chronic'; these values did not differ significantly (p > 0.1). The peak blood concentrations were invariably achieved between 1 and 2 hr for b o t h groups. The average peak blood concentrations achieved b y the 'acute' and 'chronic' were 21.55 + 1.21 and 30.87 + 2.47 pg/ml respectively; these values were significantly different (p < 0.05). Following the peak, blood concentrations declined following first order elimina-
207
tion kinetics as evidenced by the linear correlation of time and log concentration of pentobarbital in this figure. The average half-life was significantly shorter (p < 0.05) in the 'chronic' as compared to the 'acute'; these half-lives were 5.68 + 0.56 and 12.34 + 1.60 hr, respectively. The lower t w o curves in fig. 1 describe CSF pentobarbital concentrations. The rate of penetration of pentobarbital into CSF was estimated from the rate of rise of CSF pentobarbital concentrations between 15 and 30 min after dosing for each animal. The average rates of penetration were 0.8905 + 0.2174 log (pg/ml)/hr for the 'acute' and 0.7955 +- 0.1635 log (/~g/ml)/hr for the 'chrSnic'; these values did. not differ significantly (p > 0.1). The peak CSF concentrations were reached at 1 hr post drug which is slightly faster than the time required for bloods to reach their peaks. The peak CSF pentobarbital concentration achieved b y the 'chronic' group was significantly higher (p < 0.05) than that achieved b y the 'acute'; these values were 13.76 + 0.88 and 9.82 + 1.14 pg/ml, respectively. After reaching their peak, CSF pentobarbital concentrations declined following first order elimination kinetics. The average half-life was significantly shorter (p < 0.05) in the 'chronic' as compared to the 'acute'; these half-lives were 6.28 + 0.46 and 11.60 +- 1.36 hr, respectively. To determine h o w well changes in CSF pentobarbital concentration.s are reflected in blood-concentrations, the kinetic characteristics of t h e s e t w o compartments were compared for both the 'acute' and 'chronic'. No significant difference (p > 0.1 ) was found in the rate of rise of pentobarbital concentrations between 15 and 30 min for b o t h 'acute' and 'chronic'. Similarly, rates of elimination from blood did not differ significantly (p > 0.1) from CSF values for both 'acute' and 'chronic'. In contrast, peak blood concentrations of pentobarbital were approximately twice as high as CSF concentrations for both groups. Further, to explore a possible change in the partitioning of pentobarbital between blood and CSF following chronic treatment, the logarithmic concentration ratios of CSF to blood
208
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M. OKAMOTO, N.R. BOISSE
0.800-
m 0.700-
T
T
mO
~'J 0.60 Oam Oo Z L
0.500 TIME
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PENTOBARBITAL
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Fig, 2. Pentobarbital CSF/blood concentration ratio curves after the morning dose on the first day ('acute') and the last day ('chronic') of a 5 week pentobarbital treatment. Abscissa: time in hr after morning dose of sodium pentobarbital. Ordinate: pentobarbital CSF/blood log concentration ratios. Solid circle (o) represent the ratios on the 'acute'. Open circles (©) represent the ratios on the 'chronic'.
were calculated individually and then averaged; these averages were plotted in fig. 2. As shown b y this figure, the CSF/blood concentration ratios for both 'acute' and 'chronic' rose rapidly after dosing to reach a peak at I hr. Between 1 and 2 hr the ratios declined slightly to a level that remained relatively constant for the remainder of the observation period. Although statistically insignificant (p > 0.1), the C S F / blood ratios attained b y the 'chronic' were slightly higher than the values attained b y the 'acute' at every sampling time suggesting a slightly greater partitioning of pentobarbital into CSF following chronic treatment. The peak CSF/blood concentration ratios attained were 0.734 + 0.021 for the ' a c u t e ' a n d 0.765 + 0.019 for the 'chronic'. Plateau ratios at 4--10 hr post drug were 0.671 + 0.010 for 'acute' and 0.673 + 0.007 for 'chronic' animals.
4. Discussion
While an alteration in the CNS penetrability of barbiturates during tolerance and physical dependence development has not been sufficiently studied to permit a decisive conclusion (Ebert et al., 1964; Timer et al., 1966; Biich et al., 1969), this study was undertaken to exam-
ine the effect of chronic pentobarbital dosing upon its penetrability into the CNS and its distribution between blood and CNS compartments. Although brain tissues would have been ideal for estimating CNS concentrations of pentobarbital, CSF was chosen as a more practical alternative to meet the need for repeated sampling from the same animal. The development of a chronically implanted cisternal cannula has permitted the simultaneous serial sampling of blood and CSF in the same animal both at the start and at the end of a 5 week period of pentobarbital dosing. Following chronic intragastric administration of sodium pentobarbital, peak blood and CSF concentrations were higher than the acute; however, the distribution of pentobarbital between blood and CSF remained unchanged. In agreement with these findings, Ebert et al. (1964) were able to demonstrate that the distribution o f the more hydrophilic barbiturate barbital was unaltered by chronic barbital dosing. In the previous work of O k a m o t o et al. (1975), functional tolerance was identified and characterized on the basis of an increase in blood pentobarbital concentration required to maintain equi-effective peak effect. An implicit assumption in this study was that blood pentobarbital concentration changes are proportional to concentration changes in the CNS. However, if any alteration in the degree of pentobarbital penetration from blood into CNS occurred during chronic treatment this should be corrected in order to accurately assess functional tolerance. The present study confirms that blood pentobarbital concentrations are a precise indicator of drug concentrations in a CNS compartment because b o t h the distribution and the time relationship between these two compartments remained unchanged following chronic treatment.
Acknowledgements The authors acknowledge the technical instruction of Dr. Masaji Matsuzaki (N.Y. State D A C C Testing
CHRONIC
PENTOBARBITAL
BLOOD---CSF K I N E T I C S
and Research Lab.) for the initial development of the chronic cisternal cannulation method and wish to thank Mr. Robert Rosen and Mr. Robert Brunson for their technical assistance.
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209 the unanesthetized cat. Effect of calcium on body temperature, J. Physiol. 207,403. Kalant, H., A.E. LeBlanc and R.J. Gibbins, 1971, Tolerance to and dependence on, some non-opiate psychotropic drugs, Pharmacol. Rev. 23, 135. Okamoto, M., H.C. Rosenberg and N.R. Boisse, 1975, Tolerance characteristics produced during the maximally tolerable chronic pentobarbital dosing in the cat, J. Pharmacol. Exptl. Therap. 192, 555. Rosenberg, H.C. and M. Okamoto, 1974, A method for producing maximal pentobarbital dependence in cats: Dependency characteristics,in: Drug Addiction: Neurobiology and Influences on Behavior, Vol. 3, eds. J.M. Singh and H. Lal (Symposia Specialists,Miami) p. 89. Timer, M.: V. Licurici and M. Lazarescu, 1966, Incorporation du barbital C 14 chez les rats dans les conditions de l'accoutumance ~ certain barbituriques, Med. Pharmacol. Expts. 14, 24. Yaksh, T.L., L.A. Fedele and H.I. Yamamura, 1973, Effects of repeated withdrawal of cerebrospinal fluid by cisternal puncture on cisternal protein levels in the unanesthetized cat, Physiol. Behav. 10, 149.