Clin. Cardio!' 2, 146-150 (1979) © G. Witzstrock Publishing House Inc.
Studies on Digitalis. 23. Blood-Brain Barrier of Digitoxin in Humans L.
STORSTEIN, M.D.,
A. K.
NORE, M.S.,
O.
SJAASTAD, M.D.
Medical Department B and Department of Neurology, University Clinics, Rikshospitalet, Oslo, Norway
Summary: Data on the cerebrospinal fluid concentrations of digitoxin in man have been lacking. Aim of the present study was to investigate serum and cerebrospinal fluid concentrations of digitoxin. Eleven patients with normal cerebrospinal fluid composition, normal serum albumin values, normal renal and hepatic function and normal hematologic tests were given a single dose of 0.6 mg digitoxin. Serum and cerebrospinal fluid were collected 16 h after the dose, and assayed by radio-immunoassay, serum digitoxin protein binding was measured with equilibrium dialysis. Mean serum digitoxin concentration was 12.4 ng/ml (SO 2.6) and mean CSF concentration 0.84 ngiml (SO 0.22) giving a CSF/serum ratio of 0.07 (SO 0.03). Serum digitoxin protein binding was 97.4% (SO 0.4) and the calculated CSF/free serum digitoxin ratio was thus 2.94 (SO 1.43). Free serum digitoxin concentrations are more relevant for pharmacologic effect than total serum digitoxin concentrations and also for passage into the central nervous system. Using free drug concentrations the ratio between digitoxin in serum and CSF is lower than reported values
Supported by grants from the Norwegian Council on Cardiovascular Diseases and Nitedals Taendstikfabrik, Hjelpestikkenes Medisinske Fond. Address for reprints: Liv Storstein, M.D. Medical Department B Rikshospitalet University Clinic Oslo, Norway 'eceived: December 6, 1978 'cepled: January 18, 1979
for digoxin indicating a higher penetrance of digitoxin into the central nervous system. This corresponds well with the higher lipid solubility of digitoxin. Post mortem studies have shown that concentrations are higher in cerebral tissue than in CSF for digoxin. Determination of CSF concentrations thus probably underestimates the penetrance of digitalis glycosides into the central nervous system, but may serve as a useful indicator of variance among digitalis glycosides. Keywords: digitalis, blood-brain barrier, digitoxin
Introduction The blood-brain barrier acts as a regulatory interface between blood and the central nervous system (13, 14,22, 33, 40), the brain and the cerebrospinal fluid (CSF) being the main compartments. Drugs pass into the central nervous system via the brain capillaries. Permeability to drugs is regulated by transendothelial diffusion in proportion to their lipid-solubility. Accessible for diffusion is the free, unbound drug fraction. Protein binding and lipid-solubility are thus factors which influence the extent of drug passage into the central nervous system (CNS). Binding of drugs to albumin as well as to plasma membranes depends on interactions between lipoid regions of the drug and the membrane in question and is also proportional to lipidsolubility. The cerebrospinal fluid is produced by secretion at the choroid plexus to 70% (6, 17, 30) and to 30% (32, 36, 37) from the capillary bed of the brain or by metabolic water production. Drug excretion into the CSF thus reflects penetrance through the blood-brain barrier, but has no direct bearing on the drug concentration at the site of central action.
L. Storstein et al: Blood-Brain Barrier of Digitoxin in Humans
The maJonty of cardiovascular drugs also exert therapeutic or toxic effects in the CNS. Data on the pharmacokinetic properties of the CNS are scarce in humans. Aim of the present investigation was to study the passage of the lipid-soluble and highly protein-bound glycoside digitoxin across the blood-brain barrier in man and correlate CSF to serum concentrations of total and free drug.
Patient Material Informed consent was obtained from 11 patients, 3 females and 8 males, with a mean age of 52 years undergoing diagnostic lumbal puncture. A single dose of 0.6 mg digitoxin was given as tablets (Digitoksin "NAF") 16 h before lumbal puncture. The following laboratory parameters were determined: Serum albumin, blood urea, serum creatinine, bilirubin, serum transaminases, hemoglobin, blood sedimentation rate, leukocyte counts. These parameters were within normal limits in all patients. The cerebrospinal fluid composition was also found to be normal in all patients.
Methods Laboratory Method A radioimmunoassay (RIA, Clinical Assays) was used for serum and CSF digitoxin determinations. In a previous paper (27) we described the validity of the method for serum, urine and CSF determinations and the interference of active digitoxin metabolites with the assay. CSF determinations have to be performed with a CSF standard
147
curve as serum standard curves would lead to underestimation of true CSF values. The digitoxosides and genin of digitoxin have almost the same affinity for the assay as digitoxin while digoxin, its digitoxosides and genin show low cross-reactivity. Serum digitoxin protein binding was studied in vitro with radioactive labeled digitoxin and equilibrium dialysis (24, 41). We have previously reported a mean value of 97.3% (SD 0.4) in an adult control group (41), a value in good accordance with the 97 % reported by Lukas and deMartino (24).
Statistical Methods Mean and standard deviations were calculated according to standard formulas. Linear regression analysis was used to correlate serum and CSF concentrations.
Results (Table I) Serum digitoxin protein binding was 97.4% (SD 0.4) in the 11 patients giving a mean free digitoxin fraction of 2.6%. The values were quite similar to those obtained in the control group. Serum digitoxin concentration had a mean value of 12.1 nglml (SD 2.6). The calculated free digitoxin concentration ranged from 0.18 to 0.43 nglml (mean 0.31). Cerebrospinal fluid digitoxin concentration was 0.84 nglml (SD 0.22), ranging from 0.58 to 1.24 nglml. CSFlserum ratios were 0.07 (SD 0.03) when using total serum digitoxin concentrations and 2.9 (SD 1.4) when using free digitoxin concentrations for the calculations. Correlation between CSF and total and free serum digitoxin concentration. No significant correlation was found (Fig. 1).
Table I Individual values in 11 patients with mean, standard deviations (SD) standard error of mean (SEM) and coefficient of variation (CV%). SerumT - total serum digitoxin concentration, SerumF - free serum digitoxin concentration. protein binding free fraction (%) 1 2 3 4 5 6 7 8 9 10 11 mean SD SE CVO/C
serumT (ng/ml)
(ng/m!)
CSF (nglml)
serumF
CSF/serumT
CSF/serumF
2.8 3.0 3.1 2.8 2.6 2.6 2.4 1.7 2.3 2.7 2.4
9.4 13.0 12.9 9.8 13.3 10.0 10.3 10.4 18.5 12.0 13.8
0.26 0.39 0.40 0.27 0.35 0.26 0.25 0.18 0.43 0.32 Q.32
1.24 0.66 0.65 0.68 0.72 1.00 0.98 1.05 1.00 0.58 0.63
0.13 0.05 0.05 0.07 0.05 0.10 0.10 0.10 0.05 0.05 0.05
4.8 1.7 1.6 2.5 2.1 3.8 3.8 5.9 2.3 1.8 2.0
2.6 0.4 0.11 14.6
12.1 2.6 0.89 21.7
0.31 0.08 0.02 24.0
0.84 0.22 0.067 26.6
0.07 0.03 0.01 40.3
2.9 1.4 0.43 48.S
Clin. Cardiol. Vol. 2, April 1979
L. Storstein et al: Blood-Brain Barrier of Digitoxin in Humans
148 ng/ml
ng/ml
1.5
1.5
•
u c: (J
•
c:
'x B
1.0
'6>
'6 u..
en'
()
0.5 0.10
0.20
.•
0
-•
(J
c:
•
x
B
1.0
'6>
'6 u..
en
•• •
--
0.30
I 0.40
I
0
•
u c:
0
()
• 0.5
.••
I
0.50 ng/ml
8
Fig. I Left: Total serum digitoxin concentrations are plotted against CSF concentrations. plotted against CSF concentrations.
Discussion
10 12 14 16 18 20 ng/ml Right: Free serum digitoxin concentrations are
use a CSF standard curve or make no statement regarding this question, the importance of which was mentioned before. All ratios are calculated with total serum concentration. The use of free digoxin for the calculation would increase CSF serum ratios further by approximately 25 %. Children have the same ratios as adults (1). After a single dose of digoxin the drug was not traceable in the CSF in most of the patients. When calculating CSF/serum ratio in patients who got the drug 12 h before sampling (I), the ratio was lower than in patients on maintenance dosage. Beta-methyl-digoxin, a more lipid-soluble drug than di-
Blood-Brain Barrier for Digitalis Glycosides in Humans
Table II summarizes the findings reported in the literature. The data are partly calculated from basic information in the various studies. The CSF/serum ratios in patients on maintenance dosage with digoxin differ between 0.03 and 0.33 (1, 5, 10, 19, 38, 39). Methodologic aspects are not fully discussed, but as CSF concentrations are very low the sensitivity of the various radioimmunoassays is obviously a crucial point. Some authors use a serum curve while others
Table II
•
Data from the literature on the CSF/serum ratios for cardiac glycosides
glycoside
type of study
n
serum-conc. (ng/ml)
digoxin
steady state children (I) adults (I) adults (5) adults (38) adults (10)
8 11 6 10 14
single dose adults (1)
6
0.7 (0.3-1.0)
toxil;ty adults (39)
3
21. 9 (12.5-30.0)
f3 -methyl- digoxin
steady state (5)
6
(J.9 (0.5-1.2)
digitoxin
single dose adults (prescnt study) toxicity (39)
II 2
12.1 (9.4-lll.S)
1.5 U 2.4 1.1 1.1
(0.7-2.3) (0.5-2.2) (0.6-4.8) (0.6-1.9) (0.4-2.2)
Clin. CardioL Vol. 2, April 1979
CSF-conc. (nglml)
CSF/serum ratio
0.5 (O.3-U) 0.3 (~.6) 0.3 (0-1.0) 0.04 (0-0.2) 0.16 «l.O7-O.77)
0.33 0.31 0.10 0.04 0.14
0.12 (H-0.3)
0.17 (0-0.38)
3.7
0.17 (0.13-0.22)
(2.0-5.0)
(0. 13-{).56 ) (0-0.86) (0-0.27) (0-0.14) (0.05-0.41)
0.55 (0.26-0.11)
0.60 (O.30-0.113)
0.X4 (O.511-1.24) 0-8.0
(WJ.64
0.07 (0.05-0.13)
L. Storstein et al: Blood-Brain Barrier of Digitoxin in Humans goxin, has a 6 times higher penetrance into the eSF (5), the ratio being 0.60 compared to 0.10 for digoxin. These human data are in accordance with animal experiments showing a high penetrance across the barrier for betamethyl-digoxin (3, 8, 19). Digitoxin has a low eSF/serum ratio when total drug concentration is used for the calculations. Entry into the central nervous system is dependent on the concentration of free diffusible drug and it is therefore relevant to use free drug concentrations in estimating the blood-brain barrier (33). A high mean ratio of 2.94 was found when free drug concentration was used for the calculations. As discussed in a previous paper free digitoxin concentrations are also more relevant for uptake in the myocardium (42). Myocardial/free digitoxin serum concentration ratio was around 200 compared to 100 for digoxin. Penetrance across the blood-brain barrier is thus low for both digoxin and digitoxin although digitoxin has 20 times higher penetrance in accordance with its higher lipid solubility. Drug concentrations in the eSF do not necessarily reflect concentrations in various parts of the cerebrum. Bertier et al (4) reported twice as high digoxin concentrations in the choroid plexus (205 nglg tissue) as in ventricular myocardium (92 nglg), but the concentrations in the brain were lower (mean 25 ng/g). The same relationship between choroid plexus and brain was also found in infants.
Pbarmacokinetic Data from Animal Studies Differences among cardiac glycosides in the penetration and action of cardiac glycosides on the central nervous system have been reported in a number of studies. Lipid-soluble glycosides like oleandrin, beta-methyl-digoxin and digitoxin in that order have a higher penetrance into the CNS than digoxin and ouabain, in various animal species (3,8, 19), Lage and Spratt (20) compared intravenous and intracerebral doses necessary to produce LD 50 for the digitoxosides, genins, epi- and keto-derivatives of digitoxin and digoxin in mice. Toxicity decreased with decreasing number of sugar molecules for both digitoxin and digoxin. The exception to this was digitoxigenin which had the lowest LD 50 of the compounds studied. Digitoxigenin always and digitoxin often produced convulsions before death whereas this reaction was not seen with the other glycosides studied. After intracerebral application all deaths were preceded by convulsions. Digoxin compounds were more toxic than digitoxin derivatives. Epi-and keto-derivatives had a much lower potency. The observed differences between intravenous and intracerebral LD 50 can be explained by the higher penetrance of digitoxin compounds into the CNS. Repke (34) has also shown that the passage into the CNS in rats is ten times higher after digitoxigenin than digitoxin.
149
Therapeutic and Toxic Digitalis Actions on the Central Nervous System Since Withering (46) it has been recognized that digitalis has toxic effects on the eNS. Vomiting, nausea, blurred and colour vision, weakness and mental disturbances all originate from various centers in the brain. The chemoreceptor trigging zone is responsible for the emetic action and ablation of this zone leads to emetic refractoriness (9). In man extracardiac symptoms of digitalis intoxication are as common as rhythm disturbances. In a largescale epidemiologic digitalis intoxication (22), fatigue, muscular weakness, psychic and visual disturbances were very common. We found that extracardiac symptoms were more common than rhythm disturbances (43), but that most patients with digitalis intoxication exhibited both extracardiac symptoms and rhythm disturbances. The central nervous system plays a role in the development of cardiac arrhythmias as pointed out with increasing frequency in recent years (12,16,29,31,35,44) and depression of the autonomic nervous system will lead to a lower incidence of ventricular Ii brillation (7, 11). The entire effect of digitalis glycosides on atrioventricular conduction is mediated through the autonomic nervous system and is abolished in humans with transplanted hearts (15, 21). The kinetics of the major digitalis effects, inotropic and electrophysiologic actions, are dissociated as shown in recent years (2, 18, 24, 28). We have correlated the pharmacokinetics of digitoxin to changes in contractility (paced dp/dt) and electrophysiology (heart rate, AV nodal funct~on, atrial refractoriness and ventricular monophasic actIOn potentials) in the dog and have found a highly significant linear correlation between contractility and serum concentrations. No such correlation could be found for electro physiologic effects indicating both rapid senzitation through baroreceptors and long-lasting effects which probably are mediated through the autonomic nervous system. Cardiac glycosides decrease the CSF secretion rate (45) and have been used therapeutically in infants (26). The important new understanding of the role of the central nervous system calls for further investigations into the pharmacokinetic properties of the brain and the exact mode and site of action of digitalis. Acknowledgment Expert technical assistance was given bv Anne Thune-Larsen . and Oystein Larsen.
References I. AHonen H, Andersson KE. lisalo E. Kanto J. Striimblad LG.
Wetrell G: Passage of digoxin into the cerebrospinal fluid in man. Acta Pharmacol 41, 193 (1977) 2. Amlie JP. Storstein L. Heldaas 0: Correlation between p~ar~acokinetics and electrophysiologic response to digitOXIn In the intact dog. Submitted to J Cardiovasc Pharmacal
Clin. Cardio!. Vo!' 2. April 1979
150
L. Storstein et al: Blood-Brain Barrier of Digitoxin in Humans
3. Benthe HF: Organverteilung verschiedener Herzglykoside. In: Digitalistherapie, Beitrage zur Pharmakologie und Klinik (Ed Jahrmarker H), Springer, Berlin (1975), p 25 4. Bertler A, Adersson KE, Wetrell G: Concentration of digoxin in choroid plexus. Lancet 2, 1453 (1973) 5. Bodem G, Boldt U, Ochs H: Konzentrationen von Digoxin und Beta-Methyldigoxin im Liquor und Plasma. Klin Wochenschr 55, 355 (1977) 6. Cutler RWP, Page L, Galicide J, Watters GV: Formation and absorption of cerebrospinal fliud in man. Brain 91, 707 (1968) 7. Erlij D, Mendez R: The modification of digitalis intoxication by excluding adrenergic influences on the heart. J Pharmacol Exp Ther 144, 97 (1964) 8. Flasch H, Heinz N: Konzentration von Herzglykosiden im Myokard and Gehirn. Arzneimittel-Forsch 26, 1213 (1976) 9. Gaitonde BB, McCarthy LE, Borison HL: Central emetic effects of digitalis in cats. J Pharmacol Exp Ther 147, 409 (1965) . 10. Gayes 1M, Greenblatt DJ, Lloyd BL, Hermatz JS, Smith lW: Cerebrospinal fluid digoxin concentrations in humans. J Clin Pharmacol 18, 16 (1978) II. Gillis RA, Corr PB, Pace DG, Evans DE, DiMicco J, Pearle DL: Role of the nervous system in experimentally induced arrhythmias. Cardiology 61, 37 (1976) 12. Gillis RA, Quest JA: Neural actions of digitalis. Ann Rev Med 29, 73 (1978) 13. Goldman EE: Die auBere und innere Sekretion des gesunden und kranken Organismus im Lichte der "vitalen Flirbung". Beitr Klin Chir 64, 192 (1909) 14. Goldman EE: Vitalflirbung am Zentralnervensystem. Beitrag zur Physiologie des Plexus choroideus und der Hirnhaute, Berlin 1913 15. Goodman DJ, Rossen RM, Cannon DS, Rider AK, Harrison DC: Effect of digoxin on atrioventricular conduction: Studies in patients with and without cardiac autonomic innervation. Circulation 51, 251 (1975) 16. Hashimoto K, Kimura T, Kubota K: Study of the therapeutic and toxic effects of ouabain by simultaneous observations on the excised and blood-perfused sinoatrial node and papillary muscle preparations and the in situ heart of dogs. J Pharmacol Exp Ther 186, 463 (1973) 17. Heisey SR, Held D, Pappenheimer JR: Bulk flow and diffusion in the cerebrospinal fluid system of the goat. Am J Physiol 203, 763 (1962) 18. Kim YI, Noble RJ, Zipes DP: Dissociation of the inotropic effect of digitalis from its effect on atrioventricular conduction. Am J Cardiol 36, 459 (1975) 19. Kuhlmann J, Rietbrock N, Schnieders B: Tissue distribution of cardiac glycosides. In: Cardiac glycosides (Eds Bodem G, Dengler HJ), Springer, Berlin-Heidelberg-New York (1978), p 109 20. Lage GL, Spratt JL: Structure-activity correlation of the lethality and central effects of selected cardiac glycosides. J Pharmacol Exp Ther 152, 501 (1966) 21. Leachman RD, L'okkinos DVP, Cabrera R, Leatherman LL, Rochelle DG: Response of the transplanted, denervated human heart to cardiovascular drugs. Am J Cardiol 27, 272 (1971 ) 22. Lely AH, van Enter CHJ: Non-cardiac symptoms of digitalis intoxication. Am Heart J 3. 149 (1972) 23. Lewandowsky M: Zur Lehre von der Cerebrospinalflussigkeit. Z Klin Med 40. 480 ( 19(0) 24. Lukas DS, DeMartino AG: Binding of digitoxin and some related cardenolides to human plasma proteins. J Clin Invest 48. 1041 (1969)
25. LiiHmann H, Peters T: Cardiac glycosides and contractility. The myocardium. Adv Cardiol 12, 174 (1974) 26. Neblett CR, McNeel DP, Waltz TA, Harrison GM: Effect of cardiac glycosides on human cerebrospinal-fluid production. Lancet 2, 1008 (1972) 27. Nore AK, Storstein L, Amlie JP, Larsen A: Studies on digitalis. 19. Determination of digitalis glycosides in serum, urine and cerebro-spinal fluid by a micromethod radioimmunoassay. Submitted for publication to Clin Chern 28. Okita GT, Richardson F, Roth-Schecter B: Dissociation of the positive inotropic action of digitalis from inhibition of sodium- and potassium-activated adenosine triphosphatase. J Pharmacol Exp Ther 185, 1 (1973) 29. Pace DG, Gillis RA: Neuroexcitatory actions of digoxin in the cat. J Pharmacol Exp Ther 199, 583 (1976) 30. Pappenheimer JR, Heisey SR, Jordan EF, Downer J: Perfusion of the cerebral ventricular system in unanesthetized goats. Am J Physiol 203, 763 (1962) 31. Pearle DL, Gillis RA: Effect of digitalis on the response of ventricular pacemakers to sympathetic nerve stimulation. Am . J Cardiol 34, 704 (1974) 32. Pollay M, Curl F: Secretion of cerebrospinal fluid by the ventricular ependyma of the rabbit. Am J Physiol 213, 1031 (1967) 33. Rapoport S: Blood-brain barrier in physiology and medicine. Raven Press, New York (1976) 34. Repke K: Metabolism of cardiac glycosides. In: Proceedings of the First International Pharmacological Meeting (Ed WiIbrandt W) Vol. 3. Pergamon Press, New York-Stockholm (1963), p 47 35. Roberts J, Kelliher GJ, Lathers CM: Role of adrenergic influences in digitalis-induced ventricular arrhythmia. Life Sci 18,665, (1976) 36. Sahar A: Choroidal origin of cerebrospinal fluid. Isr J Med Sci 8, 594 (1967) 37. Sato 0, Bering EA: Extra-ventricular formation of cerebrospinal fluid. Brain Nerv 19, 883 (1967) 38. Schott GD, Holt DW, Hayler AM: Penetration of digoxin into the cerebrospinal fluid. Postgrad Med J 52, 700 (1976) 39. Somogyi G, Kaldor A, Jankovich A: Studies of the digitalis level of the cerebrospinal fluid. The effect of digitalis glycosides on the nervous system. Int Z Klin Pharmakol Ther Toxikol 4, 421 (1971) 40. Stem L, Gautier R: Recherches sur Ie liquide cephalo-rachidien. I. Les rapports entre Ie liquide cephalo-rachidien et la cirulation sanguine. Arch Int Physiol 17, 138 (1921) 41. Storstein L: Studies on digitaliS. V. The influence of impaired renal function, hemodialysis and drug interaction on serum protein binding of digitoxin and digoxin. Clin Pharmacal Ther 20, 6 (1976) 42. Storstein L: Studies on digitalis. X. Digitoxin metabolites in human myocardium and relationship between myocardial and serum concentrations in patients on maintenance treatment. Clin Pharmacol Ther 21, 395 (1977) 43. Storstein 0, Hansteen V, Hatle L, Hillestad L. StoTStein L: Studies on digitalis. XIII. A prospective study of 649 patients on maintenance treatment with digitoxin. Am Heart J 93. 434 (1977) 44. Weaver LC, Akera T. Brody TM: Digoxin toxicity: primary sites of drug action on the sympathetic nervous system. J Pharmacol Exp Ther 197, I (1976) 45. Weick K: Secretion of cerebrospinal fluid by choroid plexus of the mbbit. Am J Physiol 205. 617 (1963) 46. Withering W: An account of the foxglove. Swinney. Birmingham (1785)
C1in. Cardiol. Vol. 2, April 1979
Studies on Digitalis. 23. Blood-Brain Barrier of Digitoxin in Humans L.
STORSTEIN, M.D.,
A. K.
NORE, M.S.,
O.
SJAASTAD, M.D.
Medical Department B and Department of Neurology, University Clinics, Rikshospitalet, Oslo, Norway
Summary: Data on the cerebrospinal fluid concentrations of digitoxin in man have been lacking. Aim of the present study was to investigate serum and cerebrospinal fluid concentrations of digitoxin. Eleven patients with normal cerebrospinal fluid composition, normal serum albumin values, normal renal and hepatic function and normal hematologic tests were given a single dose of 0.6 mg digitoxin. Serum and cerebrospinal fluid were collected 16 h after the dose, and assayed by radio-immunoassay, serum digitoxin protein binding was measured with equilibrium dialysis. Mean serum digitoxin concentration was 12.4 ng/ml (SO 2.6) and mean CSF concentration 0.84 ngiml (SO 0.22) giving a CSF/serum ratio of 0.07 (SO 0.03). Serum digitoxin protein binding was 97.4% (SO 0.4) and the calculated CSF/free serum digitoxin ratio was thus 2.94 (SO 1.43). Free serum digitoxin concentrations are more relevant for pharmacologic effect than total serum digitoxin concentrations and also for passage into the central nervous system. Using free drug concentrations the ratio between digitoxin in serum and CSF is lower than reported values
Supported by grants from the Norwegian Council on Cardiovascular Diseases and Nitedals Taendstikfabrik, Hjelpestikkenes Medisinske Fond. Address for reprints: Liv Storstein, M.D. Medical Department B Rikshospitalet University Clinic Oslo, Norway 'eceived: December 6, 1978 'cepled: January 18, 1979
for digoxin indicating a higher penetrance of digitoxin into the central nervous system. This corresponds well with the higher lipid solubility of digitoxin. Post mortem studies have shown that concentrations are higher in cerebral tissue than in CSF for digoxin. Determination of CSF concentrations thus probably underestimates the penetrance of digitalis glycosides into the central nervous system, but may serve as a useful indicator of variance among digitalis glycosides. Keywords: digitalis, blood-brain barrier, digitoxin
Introduction The blood-brain barrier acts as a regulatory interface between blood and the central nervous system (13, 14,22, 33, 40), the brain and the cerebrospinal fluid (CSF) being the main compartments. Drugs pass into the central nervous system via the brain capillaries. Permeability to drugs is regulated by transendothelial diffusion in proportion to their lipid-solubility. Accessible for diffusion is the free, unbound drug fraction. Protein binding and lipid-solubility are thus factors which influence the extent of drug passage into the central nervous system (CNS). Binding of drugs to albumin as well as to plasma membranes depends on interactions between lipoid regions of the drug and the membrane in question and is also proportional to lipidsolubility. The cerebrospinal fluid is produced by secretion at the choroid plexus to 70% (6, 17, 30) and to 30% (32, 36, 37) from the capillary bed of the brain or by metabolic water production. Drug excretion into the CSF thus reflects penetrance through the blood-brain barrier, but has no direct bearing on the drug concentration at the site of central action.
L. Storstein et al: Blood-Brain Barrier of Digitoxin in Humans
The maJonty of cardiovascular drugs also exert therapeutic or toxic effects in the CNS. Data on the pharmacokinetic properties of the CNS are scarce in humans. Aim of the present investigation was to study the passage of the lipid-soluble and highly protein-bound glycoside digitoxin across the blood-brain barrier in man and correlate CSF to serum concentrations of total and free drug.
Patient Material Informed consent was obtained from 11 patients, 3 females and 8 males, with a mean age of 52 years undergoing diagnostic lumbal puncture. A single dose of 0.6 mg digitoxin was given as tablets (Digitoksin "NAF") 16 h before lumbal puncture. The following laboratory parameters were determined: Serum albumin, blood urea, serum creatinine, bilirubin, serum transaminases, hemoglobin, blood sedimentation rate, leukocyte counts. These parameters were within normal limits in all patients. The cerebrospinal fluid composition was also found to be normal in all patients.
Methods Laboratory Method A radioimmunoassay (RIA, Clinical Assays) was used for serum and CSF digitoxin determinations. In a previous paper (27) we described the validity of the method for serum, urine and CSF determinations and the interference of active digitoxin metabolites with the assay. CSF determinations have to be performed with a CSF standard
147
curve as serum standard curves would lead to underestimation of true CSF values. The digitoxosides and genin of digitoxin have almost the same affinity for the assay as digitoxin while digoxin, its digitoxosides and genin show low cross-reactivity. Serum digitoxin protein binding was studied in vitro with radioactive labeled digitoxin and equilibrium dialysis (24, 41). We have previously reported a mean value of 97.3% (SD 0.4) in an adult control group (41), a value in good accordance with the 97 % reported by Lukas and deMartino (24).
Statistical Methods Mean and standard deviations were calculated according to standard formulas. Linear regression analysis was used to correlate serum and CSF concentrations.
Results (Table I) Serum digitoxin protein binding was 97.4% (SD 0.4) in the 11 patients giving a mean free digitoxin fraction of 2.6%. The values were quite similar to those obtained in the control group. Serum digitoxin concentration had a mean value of 12.1 nglml (SD 2.6). The calculated free digitoxin concentration ranged from 0.18 to 0.43 nglml (mean 0.31). Cerebrospinal fluid digitoxin concentration was 0.84 nglml (SD 0.22), ranging from 0.58 to 1.24 nglml. CSFlserum ratios were 0.07 (SD 0.03) when using total serum digitoxin concentrations and 2.9 (SD 1.4) when using free digitoxin concentrations for the calculations. Correlation between CSF and total and free serum digitoxin concentration. No significant correlation was found (Fig. 1).
Table I Individual values in 11 patients with mean, standard deviations (SD) standard error of mean (SEM) and coefficient of variation (CV%). SerumT - total serum digitoxin concentration, SerumF - free serum digitoxin concentration. protein binding free fraction (%) 1 2 3 4 5 6 7 8 9 10 11 mean SD SE CVO/C
serumT (ng/ml)
(ng/m!)
CSF (nglml)
serumF
CSF/serumT
CSF/serumF
2.8 3.0 3.1 2.8 2.6 2.6 2.4 1.7 2.3 2.7 2.4
9.4 13.0 12.9 9.8 13.3 10.0 10.3 10.4 18.5 12.0 13.8
0.26 0.39 0.40 0.27 0.35 0.26 0.25 0.18 0.43 0.32 Q.32
1.24 0.66 0.65 0.68 0.72 1.00 0.98 1.05 1.00 0.58 0.63
0.13 0.05 0.05 0.07 0.05 0.10 0.10 0.10 0.05 0.05 0.05
4.8 1.7 1.6 2.5 2.1 3.8 3.8 5.9 2.3 1.8 2.0
2.6 0.4 0.11 14.6
12.1 2.6 0.89 21.7
0.31 0.08 0.02 24.0
0.84 0.22 0.067 26.6
0.07 0.03 0.01 40.3
2.9 1.4 0.43 48.S
Clin. Cardiol. Vol. 2, April 1979
L. Storstein et al: Blood-Brain Barrier of Digitoxin in Humans
148 ng/ml
ng/ml
1.5
1.5
•
u c: (J
•
c:
'x B
1.0
'6>
'6 u..
en'
()
0.5 0.10
0.20
.•
0
-•
(J
c:
•
x
B
1.0
'6>
'6 u..
en
•• •
--
0.30
I 0.40
I
0
•
u c:
0
()
• 0.5
.••
I
0.50 ng/ml
8
Fig. I Left: Total serum digitoxin concentrations are plotted against CSF concentrations. plotted against CSF concentrations.
Discussion
10 12 14 16 18 20 ng/ml Right: Free serum digitoxin concentrations are
use a CSF standard curve or make no statement regarding this question, the importance of which was mentioned before. All ratios are calculated with total serum concentration. The use of free digoxin for the calculation would increase CSF serum ratios further by approximately 25 %. Children have the same ratios as adults (1). After a single dose of digoxin the drug was not traceable in the CSF in most of the patients. When calculating CSF/serum ratio in patients who got the drug 12 h before sampling (I), the ratio was lower than in patients on maintenance dosage. Beta-methyl-digoxin, a more lipid-soluble drug than di-
Blood-Brain Barrier for Digitalis Glycosides in Humans
Table II summarizes the findings reported in the literature. The data are partly calculated from basic information in the various studies. The CSF/serum ratios in patients on maintenance dosage with digoxin differ between 0.03 and 0.33 (1, 5, 10, 19, 38, 39). Methodologic aspects are not fully discussed, but as CSF concentrations are very low the sensitivity of the various radioimmunoassays is obviously a crucial point. Some authors use a serum curve while others
Table II
•
Data from the literature on the CSF/serum ratios for cardiac glycosides
glycoside
type of study
n
serum-conc. (ng/ml)
digoxin
steady state children (I) adults (I) adults (5) adults (38) adults (10)
8 11 6 10 14
single dose adults (1)
6
0.7 (0.3-1.0)
toxil;ty adults (39)
3
21. 9 (12.5-30.0)
f3 -methyl- digoxin
steady state (5)
6
(J.9 (0.5-1.2)
digitoxin
single dose adults (prescnt study) toxicity (39)
II 2
12.1 (9.4-lll.S)
1.5 U 2.4 1.1 1.1
(0.7-2.3) (0.5-2.2) (0.6-4.8) (0.6-1.9) (0.4-2.2)
Clin. CardioL Vol. 2, April 1979
CSF-conc. (nglml)
CSF/serum ratio
0.5 (O.3-U) 0.3 (~.6) 0.3 (0-1.0) 0.04 (0-0.2) 0.16 «l.O7-O.77)
0.33 0.31 0.10 0.04 0.14
0.12 (H-0.3)
0.17 (0-0.38)
3.7
0.17 (0.13-0.22)
(2.0-5.0)
(0. 13-{).56 ) (0-0.86) (0-0.27) (0-0.14) (0.05-0.41)
0.55 (0.26-0.11)
0.60 (O.30-0.113)
0.X4 (O.511-1.24) 0-8.0
(WJ.64
0.07 (0.05-0.13)
L. Storstein et al: Blood-Brain Barrier of Digitoxin in Humans goxin, has a 6 times higher penetrance into the eSF (5), the ratio being 0.60 compared to 0.10 for digoxin. These human data are in accordance with animal experiments showing a high penetrance across the barrier for betamethyl-digoxin (3, 8, 19). Digitoxin has a low eSF/serum ratio when total drug concentration is used for the calculations. Entry into the central nervous system is dependent on the concentration of free diffusible drug and it is therefore relevant to use free drug concentrations in estimating the blood-brain barrier (33). A high mean ratio of 2.94 was found when free drug concentration was used for the calculations. As discussed in a previous paper free digitoxin concentrations are also more relevant for uptake in the myocardium (42). Myocardial/free digitoxin serum concentration ratio was around 200 compared to 100 for digoxin. Penetrance across the blood-brain barrier is thus low for both digoxin and digitoxin although digitoxin has 20 times higher penetrance in accordance with its higher lipid solubility. Drug concentrations in the eSF do not necessarily reflect concentrations in various parts of the cerebrum. Bertier et al (4) reported twice as high digoxin concentrations in the choroid plexus (205 nglg tissue) as in ventricular myocardium (92 nglg), but the concentrations in the brain were lower (mean 25 ng/g). The same relationship between choroid plexus and brain was also found in infants.
Pbarmacokinetic Data from Animal Studies Differences among cardiac glycosides in the penetration and action of cardiac glycosides on the central nervous system have been reported in a number of studies. Lipid-soluble glycosides like oleandrin, beta-methyl-digoxin and digitoxin in that order have a higher penetrance into the CNS than digoxin and ouabain, in various animal species (3,8, 19), Lage and Spratt (20) compared intravenous and intracerebral doses necessary to produce LD 50 for the digitoxosides, genins, epi- and keto-derivatives of digitoxin and digoxin in mice. Toxicity decreased with decreasing number of sugar molecules for both digitoxin and digoxin. The exception to this was digitoxigenin which had the lowest LD 50 of the compounds studied. Digitoxigenin always and digitoxin often produced convulsions before death whereas this reaction was not seen with the other glycosides studied. After intracerebral application all deaths were preceded by convulsions. Digoxin compounds were more toxic than digitoxin derivatives. Epi-and keto-derivatives had a much lower potency. The observed differences between intravenous and intracerebral LD 50 can be explained by the higher penetrance of digitoxin compounds into the CNS. Repke (34) has also shown that the passage into the CNS in rats is ten times higher after digitoxigenin than digitoxin.
149
Therapeutic and Toxic Digitalis Actions on the Central Nervous System Since Withering (46) it has been recognized that digitalis has toxic effects on the eNS. Vomiting, nausea, blurred and colour vision, weakness and mental disturbances all originate from various centers in the brain. The chemoreceptor trigging zone is responsible for the emetic action and ablation of this zone leads to emetic refractoriness (9). In man extracardiac symptoms of digitalis intoxication are as common as rhythm disturbances. In a largescale epidemiologic digitalis intoxication (22), fatigue, muscular weakness, psychic and visual disturbances were very common. We found that extracardiac symptoms were more common than rhythm disturbances (43), but that most patients with digitalis intoxication exhibited both extracardiac symptoms and rhythm disturbances. The central nervous system plays a role in the development of cardiac arrhythmias as pointed out with increasing frequency in recent years (12,16,29,31,35,44) and depression of the autonomic nervous system will lead to a lower incidence of ventricular Ii brillation (7, 11). The entire effect of digitalis glycosides on atrioventricular conduction is mediated through the autonomic nervous system and is abolished in humans with transplanted hearts (15, 21). The kinetics of the major digitalis effects, inotropic and electrophysiologic actions, are dissociated as shown in recent years (2, 18, 24, 28). We have correlated the pharmacokinetics of digitoxin to changes in contractility (paced dp/dt) and electrophysiology (heart rate, AV nodal funct~on, atrial refractoriness and ventricular monophasic actIOn potentials) in the dog and have found a highly significant linear correlation between contractility and serum concentrations. No such correlation could be found for electro physiologic effects indicating both rapid senzitation through baroreceptors and long-lasting effects which probably are mediated through the autonomic nervous system. Cardiac glycosides decrease the CSF secretion rate (45) and have been used therapeutically in infants (26). The important new understanding of the role of the central nervous system calls for further investigations into the pharmacokinetic properties of the brain and the exact mode and site of action of digitalis. Acknowledgment Expert technical assistance was given bv Anne Thune-Larsen . and Oystein Larsen.
References I. AHonen H, Andersson KE. lisalo E. Kanto J. Striimblad LG.
Wetrell G: Passage of digoxin into the cerebrospinal fluid in man. Acta Pharmacol 41, 193 (1977) 2. Amlie JP. Storstein L. Heldaas 0: Correlation between p~ar~acokinetics and electrophysiologic response to digitOXIn In the intact dog. Submitted to J Cardiovasc Pharmacal
Clin. Cardio!. Vo!' 2. April 1979
150
L. Storstein et al: Blood-Brain Barrier of Digitoxin in Humans
3. Benthe HF: Organverteilung verschiedener Herzglykoside. In: Digitalistherapie, Beitrage zur Pharmakologie und Klinik (Ed Jahrmarker H), Springer, Berlin (1975), p 25 4. Bertler A, Adersson KE, Wetrell G: Concentration of digoxin in choroid plexus. Lancet 2, 1453 (1973) 5. Bodem G, Boldt U, Ochs H: Konzentrationen von Digoxin und Beta-Methyldigoxin im Liquor und Plasma. Klin Wochenschr 55, 355 (1977) 6. Cutler RWP, Page L, Galicide J, Watters GV: Formation and absorption of cerebrospinal fliud in man. Brain 91, 707 (1968) 7. Erlij D, Mendez R: The modification of digitalis intoxication by excluding adrenergic influences on the heart. J Pharmacol Exp Ther 144, 97 (1964) 8. Flasch H, Heinz N: Konzentration von Herzglykosiden im Myokard and Gehirn. Arzneimittel-Forsch 26, 1213 (1976) 9. Gaitonde BB, McCarthy LE, Borison HL: Central emetic effects of digitalis in cats. J Pharmacol Exp Ther 147, 409 (1965) . 10. Gayes 1M, Greenblatt DJ, Lloyd BL, Hermatz JS, Smith lW: Cerebrospinal fluid digoxin concentrations in humans. J Clin Pharmacol 18, 16 (1978) II. Gillis RA, Corr PB, Pace DG, Evans DE, DiMicco J, Pearle DL: Role of the nervous system in experimentally induced arrhythmias. Cardiology 61, 37 (1976) 12. Gillis RA, Quest JA: Neural actions of digitalis. Ann Rev Med 29, 73 (1978) 13. Goldman EE: Die auBere und innere Sekretion des gesunden und kranken Organismus im Lichte der "vitalen Flirbung". Beitr Klin Chir 64, 192 (1909) 14. Goldman EE: Vitalflirbung am Zentralnervensystem. Beitrag zur Physiologie des Plexus choroideus und der Hirnhaute, Berlin 1913 15. Goodman DJ, Rossen RM, Cannon DS, Rider AK, Harrison DC: Effect of digoxin on atrioventricular conduction: Studies in patients with and without cardiac autonomic innervation. Circulation 51, 251 (1975) 16. Hashimoto K, Kimura T, Kubota K: Study of the therapeutic and toxic effects of ouabain by simultaneous observations on the excised and blood-perfused sinoatrial node and papillary muscle preparations and the in situ heart of dogs. J Pharmacol Exp Ther 186, 463 (1973) 17. Heisey SR, Held D, Pappenheimer JR: Bulk flow and diffusion in the cerebrospinal fluid system of the goat. Am J Physiol 203, 763 (1962) 18. Kim YI, Noble RJ, Zipes DP: Dissociation of the inotropic effect of digitalis from its effect on atrioventricular conduction. Am J Cardiol 36, 459 (1975) 19. Kuhlmann J, Rietbrock N, Schnieders B: Tissue distribution of cardiac glycosides. In: Cardiac glycosides (Eds Bodem G, Dengler HJ), Springer, Berlin-Heidelberg-New York (1978), p 109 20. Lage GL, Spratt JL: Structure-activity correlation of the lethality and central effects of selected cardiac glycosides. J Pharmacol Exp Ther 152, 501 (1966) 21. Leachman RD, L'okkinos DVP, Cabrera R, Leatherman LL, Rochelle DG: Response of the transplanted, denervated human heart to cardiovascular drugs. Am J Cardiol 27, 272 (1971 ) 22. Lely AH, van Enter CHJ: Non-cardiac symptoms of digitalis intoxication. Am Heart J 3. 149 (1972) 23. Lewandowsky M: Zur Lehre von der Cerebrospinalflussigkeit. Z Klin Med 40. 480 ( 19(0) 24. Lukas DS, DeMartino AG: Binding of digitoxin and some related cardenolides to human plasma proteins. J Clin Invest 48. 1041 (1969)
25. LiiHmann H, Peters T: Cardiac glycosides and contractility. The myocardium. Adv Cardiol 12, 174 (1974) 26. Neblett CR, McNeel DP, Waltz TA, Harrison GM: Effect of cardiac glycosides on human cerebrospinal-fluid production. Lancet 2, 1008 (1972) 27. Nore AK, Storstein L, Amlie JP, Larsen A: Studies on digitalis. 19. Determination of digitalis glycosides in serum, urine and cerebro-spinal fluid by a micromethod radioimmunoassay. Submitted for publication to Clin Chern 28. Okita GT, Richardson F, Roth-Schecter B: Dissociation of the positive inotropic action of digitalis from inhibition of sodium- and potassium-activated adenosine triphosphatase. J Pharmacol Exp Ther 185, 1 (1973) 29. Pace DG, Gillis RA: Neuroexcitatory actions of digoxin in the cat. J Pharmacol Exp Ther 199, 583 (1976) 30. Pappenheimer JR, Heisey SR, Jordan EF, Downer J: Perfusion of the cerebral ventricular system in unanesthetized goats. Am J Physiol 203, 763 (1962) 31. Pearle DL, Gillis RA: Effect of digitalis on the response of ventricular pacemakers to sympathetic nerve stimulation. Am . J Cardiol 34, 704 (1974) 32. Pollay M, Curl F: Secretion of cerebrospinal fluid by the ventricular ependyma of the rabbit. Am J Physiol 213, 1031 (1967) 33. Rapoport S: Blood-brain barrier in physiology and medicine. Raven Press, New York (1976) 34. Repke K: Metabolism of cardiac glycosides. In: Proceedings of the First International Pharmacological Meeting (Ed WiIbrandt W) Vol. 3. Pergamon Press, New York-Stockholm (1963), p 47 35. Roberts J, Kelliher GJ, Lathers CM: Role of adrenergic influences in digitalis-induced ventricular arrhythmia. Life Sci 18,665, (1976) 36. Sahar A: Choroidal origin of cerebrospinal fluid. Isr J Med Sci 8, 594 (1967) 37. Sato 0, Bering EA: Extra-ventricular formation of cerebrospinal fluid. Brain Nerv 19, 883 (1967) 38. Schott GD, Holt DW, Hayler AM: Penetration of digoxin into the cerebrospinal fluid. Postgrad Med J 52, 700 (1976) 39. Somogyi G, Kaldor A, Jankovich A: Studies of the digitalis level of the cerebrospinal fluid. The effect of digitalis glycosides on the nervous system. Int Z Klin Pharmakol Ther Toxikol 4, 421 (1971) 40. Stem L, Gautier R: Recherches sur Ie liquide cephalo-rachidien. I. Les rapports entre Ie liquide cephalo-rachidien et la cirulation sanguine. Arch Int Physiol 17, 138 (1921) 41. Storstein L: Studies on digitaliS. V. The influence of impaired renal function, hemodialysis and drug interaction on serum protein binding of digitoxin and digoxin. Clin Pharmacal Ther 20, 6 (1976) 42. Storstein L: Studies on digitalis. X. Digitoxin metabolites in human myocardium and relationship between myocardial and serum concentrations in patients on maintenance treatment. Clin Pharmacol Ther 21, 395 (1977) 43. Storstein 0, Hansteen V, Hatle L, Hillestad L. StoTStein L: Studies on digitalis. XIII. A prospective study of 649 patients on maintenance treatment with digitoxin. Am Heart J 93. 434 (1977) 44. Weaver LC, Akera T. Brody TM: Digoxin toxicity: primary sites of drug action on the sympathetic nervous system. J Pharmacol Exp Ther 197, I (1976) 45. Weick K: Secretion of cerebrospinal fluid by choroid plexus of the mbbit. Am J Physiol 205. 617 (1963) 46. Withering W: An account of the foxglove. Swinney. Birmingham (1785)
C1in. Cardiol. Vol. 2, April 1979
