This dissertation has been microfilmed exactly as received 67-16,326
PAUL, Balbir Singh, 1925- A STUDY ON THE PHARMACODYNAMICS OF DIHYDROQUINIDINE IN CANINE.
The Ohio State University, Ph.D,, 1967 Pharmacology
University Microfilms, Inc., Ann Arbor, Michigan A STUDY ON THE PHARMACODYNAMICS
OF DIHYDROQUINIDINE IN CANINE
DISSERTATION
Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University
By
Balbir Singh Paul, B.V.Sc., M.V.Sc.
*******
The Ohio State University 1967
Approved by
Adviser Department of Veterinary Physiology and Pharmacology ACKNOWLEDGMENTS
I am highly indebted to Dr. Thomas E. Powers,
Professor in the department of Veterinary Physiology and
Pharmacology for his excellent guidance, encouragement and the sincere interest shown by him throughout the entire course of my graduate program-and research.
My sincere thanks are due to Drs. S. Dutta and
B. Marks for cooperation and affording facilities and guid ance during the course of my research. Special thanks are also due to Drs. C. R. Smith, R. W. Redding and R. S. Ray for giving their valid criticism and guidance during the course of my dissertation writing.
The numerous illustrations were drawn by Mr. B. Kramer and Miss Diana Walker, medical artists. The statis tical computations in part, for this study, were done in the statistical laboratory at The Ohio State University.
Technical assistance was given by Mrs. Dassie
Sprecher, Messrs. Thomas Heading and Robert Slobody.
I am pleased to have been able to fulfill the long
desire of my late father. Dr. Ram Singh, in acquiring my
graduate education. I also extend my sincere appreciation
to my devoted wife, Inder, for her enthusiasm, encourage ment and many sacrifices in behalf of my educational pursuit. ii VITA
August 5, 1925 Born - Amritsar, India
1946...... B.V.Sc., The University of Panjab, Lahore, India
1946-1948.... Veterinary Surgeon, Veterinary Hospital, Sulu, India
1948-1958.... Instructor, Department of Pharmacology, College of Veterinary Science and Animal Husbandry, Hissar, India
1958-195 9.... Graduate Student, Department of Veterinary Physiology and Pharma cology, The (ftiio State University, Columbus, Ohio
1959-196 4 .... Assistant Professor and Professor, of Pharmacology, College of Veterinary Science, Panjab Agricultural University, Hissar, India
1961...... M.V.Sc., Panjab University, Chandigarh, India
1964-1967.... Graduate Student, The Ohio State University, Columbus, Ohio
FIELDS OF STUDY
Major Field; Veterinary Pharmacology
iii CONTENTS
Page ACKNOWLEDGMENTS...... il
VITA...... iii
TABLES...... vii ILLUSTRATIONS...... x
INTRODUCTION;...... 1
CHAPTER
I. REVIEW OF LITERATURE...... 5
History of cinchona alkaloids
Chemical structure of quinidine and dihydroquinidine
Comparison of cardiovascular actions of qui-nidine and dihydroquinidine
Cardiovascular responses to quinidine with special emphasis on conduction
Distribution of cinchona alkaloids in blood and tissues
Concentrâtion-Activity relationship of quinidine
Metabolism and excretion of cinchona alkaloids
II, MATERIAL AND METHODS...... 32
Purity and identification of DHQ-H^
Methods of preparation of drug solutions dihydroquinidine and dihydroquinidine-H^
XV Page
Methods of determination of the specific activity of DHQ-IP
Studies on distribution and effects of DHQ on myocardial conduction velocity
Surgical technique
Electrode assembly
Recording system --
Renal clearance
Biliary excretion
Extraction of unchanged drug from various tissues and biological fluids
Radiometric analysis
III. RESULTS...... 54
Identification of DHQ-E?
Determination of specific activity of DHQ-H®
Effect of dihydroquinidine on myocardial conduction velocity
Distribution of dihydroquinidine in various body compartments
Disappearance of DHQ from plasma
Tissue distribution
Excretion of DHQ
Renal excretion
Renal clearance
Biliary excretion
Metabolite concentrations of DHQ in various tissues and biological fluids Page
Tissues
Biological fluids
IV, DISCUSSION...... 128
Experimental techniques
Effect of DHQ on myocardial conduction velocity
Distribution in various body compartments
Excretion and metabolism
SUMMARY...... 167
BIBLIOGRAPHY...... 170
Vi TABLES
Table Page
1. Determination of the specific activity of DHQ-Hr used in destribution studies...... 57
2« Determination of the specific activity of DHQ-oH^ used in excretion studies...... 59
3. Conduction time in milliseconds (msec) from the stimulus to each recording point (1-14) 1 mm apart during different time intervals after DHQ administration in experiment #1, ...... — 60
4. Conduction time in milliseconds (msec) from the stimulus to each recording point (1-14) 1 mm apart during different time intervals after DHQ administration in experiment #2...... 61
5. Conduction time in milliseconds (msec) from the stimulus to each recording point (1-14) 1 mm apart during different time intervals after DHQ administration in experiment #3...... 62
6. Conduction time in milliseconds (msec) from the stimulus to each recording point (1-14) 1 mm apart during different time intervals after DHQ administration in experiment #4, ..... 63
7. Conduction time in milliseconds (msec) from the stimulus to each recording point (1-14) 1 mm apart during different time intervals after DHQ administration in experiment #5...... 64
8. Effect of DHQ on myocardial conduction velo city ...... 72
9. Dosage of DHQ and DHQ-H^ in each experiment of 64 minutes distribution studies...... 73
vii Table Page 10. Arterial, venous and coronary sinus plasma concentrations of DHQ in experiments 1 to 4 during different time intervals, ••••••••••••• ...... 75
11. Arterial, venous and coronary sinus plasma concentrations of DHQ in experiment number 5 during different time intervals...... 76 12. Arterial plasma concentrations of DHQ..... 77
13• Venous plasma concentrations of D H Q , 78
14. Coronary sinus plasma concentrations of DHQ...... 79
15. Tissue distribution of DHQ at 64 minutes post injection in experiments 1 to 5...... 94
16. Tissue distribution of DHQ at 64 minutes post injection...... 95
17. Tissue distribution of DHQ at 6 hours post injection in experiments 6 to 8.,.....,,.., 97
18. Tissue distribution of DHQ at 6 hours post injection...... 98
19. Cumulative renal excretion of DHQ during 64 minutes...... 106
20. Dosage of DHQ and DHQ-H^ for experiments on excretion (renal and biliary) . , 108
21. Renal clearance studies on DHQ, Dog #1,.... 110
22. Renal clearance studies on DHQ, Dog #2.... « 111
23. Renal clearance studies on DHQ, Dog #3.... « 112
24. Renal clearance studies on DHQ, Dog #4..... 113
25. Renal clearance studies on DHQ, Dog #5 ..... 114
26. Renal clearance studies on DHQ, Dog #6..... 115
viii Table Page 27. Renal clearance studies on DHQ, Dog #7 ..... 116
28. Cumulative renal excretion of DHQ during 117
29. Cumulative biliary excretion of DHQ diirlnc 6 hours ...... 119
30. Concentration of metabolites of DHQ in various tissues at the end of 64 minutes in the distribution experiments...... 123
31. Concentration of metabolites of DHQ in plasma 124
32. Concentration of metabolites of DHQ in urine...... 125
33. Concentration of metabolites of DHQ in bile...... 127
ix ILLUSTRATIONS
Figure Page
1. Nomenclature used for identification of various quinidine and dihydroquinidine structures...... 10
2. Scheme for the route of metabolism of various cinchona alkaloids (proposed -by Brodie et al. 1951) A, shows the channel adopted by quinidine or quinine. B, shows the channel adopted by cinchonine and cinchonidine ...... 29
3. Illustrates the arrangement of the experiment pertaining to the study on the effect of dihydroquinidine on myocardial conduction velocity and its distribution and excretion. The various appliances employed are labeled.., 38
4. Illustrates the schematic diagram on the arrangement of the multipolar electrode assembly and its application on the right myocardium...... 41
5. Radio-chromatographic scan of a thin layer chromatoplate of DHQ-H^ and standard DHQ, using a solvent system consisting of chloroform and diethylamine (8:2), Note the radio peak adjacent to the positive area of the standard unlabeled DHQ, marked as U.V (standard DHQ visible under ultra violet light)...... 56
6. Electrograros recorded from 14 points on the epicardium during control period and at 30 seconds, 1 minute and 2 minutes (after intravenous injection of DHQ (10 mg/kg)... 66
7. Continuation of Figure 6. Electrograms recorded during 4, 16, 32 and 64 minutes post injection period of DHQ admini stration are shown...... 68
X Figure 3E^ge
8. Graphie representation of distance between electrodes and stimulus artifact (2-15 mm) against the conduction time (msec) between the stimulus artifact and nadir or peak of electrograms from each bipolar electrode. The records are shown both before and during various post injectional periods of DHQ...... 71
9. Disappearance rate of dihydroquinidine from the arterial blood plasma...... 82
10. Disappearance rate of dihydroquinidine from venous blood plasma...... 84
11. Disappearance rate of dihydroquinidine from coronary sinus blood plasma...... 86
12. Composite graph showing the comparative rates of disappearance of DHQ from the arterial, venous and coronary sinus blood plasma...... 90
13. Illustrates the comparison between arterial-venous and arterial-coronary sinus differences in plasma concentration at different time intervals...... 92
14. The distribution of DHQ in cardiac, smooth and skeletal muscular tissues at 64 minutes and 6 hours post injectional periods...... 100
15. The distribution of DHQ in various excretory organs and spleen at 64 minutes and 6 hours post injectional periods...... 103
16. The distribution of DHQ in various endocrine organs and brain at 64 minutes and 6 hours post injectional periods ...... 105 17. Excretion of DHQ in urine and bile for 6 hours. Cumulative urinary excretion is taken from the mean of 6 experiments and cumulative biliary excretion is taken from an average of 3 experiments ...... 121
xi INTRODUCTION
This is a study on the pharmacodynamics of dihydroquinidine, one of the antifibrillatory agents present in cinchona alkaloids. The antifibrillatory use of quinidine, another member of the cinchona alkaloids, resulted from the clinical studies made by Frey in 1918
(21). The actual impetus for Frey * s studies was obtained from the experience of one of Wenckebach's patients, who found that he could avert his attack of atrial fibrillation by self administration of quinine.
The action of commercial quinidine has been attributed by some investigators to be related to its dihydroquinidine contents (73). It is also reported that dihydroquinidine is three and one-half times more effective as an antifibrillatory agent than quinidine
(64). The ultimate mechanism of the antifibrillatory activity of dihydroquinidine or quinidine is still obscure.
The use of radio-labeled drugs enables the detection of submicrogram concentrations of the drug in tissues. DHQ-H^ was produced for this study by the catalytical reduction of quinidine in the presence of tritium gas. In the past several workers have studied the distribution of quinidine in blood and tissue by using colorimetric and fluorometric analytical techniques (34,
40, 63, 77). No relationship of the biological effects with the plasma or tissue concentrations were found« These studies were limited by the lack of sensitivity of the analytical tests employed.
In spite of the fact that dihydroquinidine is much more potent than quinidine, very little work has been done on its pharmacodynamic properties. Recently some work on its distribution and excretion has been reported in the cat (86).
Due to the lack of information about DHQ, the study of the pharmacodynamics of dihydroquinidine with the use of radio-labeled technique in the dog was selected for this investigation.
The predominant pharmacological action of the antifibrillatory agents is upon conduction velocity of the
cardiac tissue. For this reason, the recording of the
conduction time of an artificially induced excitatory
impulse through various points of the myocardium was selected as the pharmacological parameter to study dihydroquinidine. A specially designed fourteen paired
bipolar electrode assembly was used to study this effect.
The dose of unlabeled dihydroquinidine that was
capable of producing an appreciable effect on conduction 3 velocity was experimentally determined and mixed with tritium labeled dihydroquinidine for investigations in this study• After injection of this predetermined dose, a study on the pharmacological action, distribution, excretion and metabolism of dihydroquinidine was made.
The plasma concentrations in arterial, venous and coronary sinus blood were determined during different post injection periods. From this information, the decay rate for each system was determined. The decay rate of the drug in each of these vascular systems was derived from this information and a correlation of the three resultant curves were made.
The distribution of dihydroquinidine in various chambers of the heart and other tissues was studied at the end of sixty-four minutes and six hours in order to establish the uptake of the drug. Tissue to plasma ratio were derived from the previous results.
The mechanism of excretion of quinidine and especially dihydroquinidine is still obscure. In an
attempt to describe the mechanism of excretion, detailed
studies on renal and biliary clearances of metabolites
and non-metabolites were made.
In view of the fact that the antifibrillatory
drugs owe their activity mainly to the unchanged drug
(10, 52), it was considered necessary to obtain some
estimate of the rate of metabolism of dihydroquinidine. 4
Accordingly, an attempt vas made to extract the unchanged , drug from various tissues, plasma, urine and bile with dichlorethane * From these data an estimate of the per cent of the drug metabolized at certain time intervals was made. CHAPTER I
REVIEW OF LITERATURE
History of cinchona alkaloids
Quinidine is a stereoisomer of quinine and is one of the natural alkaloids found in cinchona bark. It is present to the extent of 0.25 to 1,25 percent (12).
Cinchona (fever bark) is the dried bark of the stem or the root of cinchona species. Cinchona trees are indigenous to Andes of Ecuador and Peru, being found at an altitude of 3000 to 9000 feet. In addition, they are cultivated in the East Indies, India and in Central and
South America. There are 36 known species and hybrids of cinchona. Trees, six to nine years old, yield the maximum amount of alkaloids from the bark.
The use of cinchona bark in Peru remained dormant for one hundred and twenty-five years after its discovery in 1513. During this time, the natives of Peru remained ignorant about the medicinal value of the drug. A variety of colorful and fanciful versions regarding its discovery for medicinal use exist.
One of the popular stories about the medicinal use of cinchona bark is that an Indian, overcome with fever. 6 was cured after being forced to drink stagnant water in which fallen cinchona trees had macerated for some time.
Following this discovery of its value, an Indian medicine
man near Loxa taught a Jesuit missionary the use of the
drug. Gradually its use spread among the other Canizares
of Loxa. One of the Canizares sent the bark to Juan de
Vega, who at the time was treating the Countess of Cinchon,
wife of the Count of Cinchon, the Viceroy of Peru, for
tertian fever. The Countess recovered and her miraculous
cure resulted in the naming of the bark, "cinchona bark"
and also in the introduction of cinchona into» Europe in
1639 (26).
The use of cinchona was further propagated by
Jesuit fathers, who were the main importers and
distributors of cinchona in Europe. Some of the early
names given to the drug were Countess bark, Jesuit*s bark.
Cardinales bark (named after an eminent philosopher of
Rome) and Peruvian bark.
For nearly a century, the drug was dispensed
predominantly by charlantans in the form of secret
remedies (6). For this reason, its use was controversial
for a long time.
The cinchona was first officially recognized in
1677, when it was included in the edition of the London
Pharmacopoeia as as "Cortex Peruanus," For nearly two centuries, the bark was employed in medicine as a powder, extract, or infusion. After Pelletier and Caventon
isolated quinine and cinchonine from cinchona in 1820, the
use of these alkaloids in medicine became increasingly popular (24),
These alkaloids have been used in medicine as
analgesics, antipyretics, sclerosing agent in the treatment
of vericose veins, and as local anesthetics. They were
also employed to initiate labor and parturition. Cardiac muscle depression is the most qualitatively different
property possessed by the cinchona alkaloids, Quinidine
is specifically known for this cardiac depressant effect
(36),
Van Heynin-Gen first described quinidine in 1848
and Pasteur gave it its present name in 1853 (24).
The use of preparations of cinchona in the
treatment of cardiac irregularities is generally considered
as one outstanding contribution to modern medicine. The earliest recorded medical reference to the use of cinchona
in cardiac disease is by a French physician, Jean Baptiste,
who used quinine successfully in the treatment of a
disorder which he referred to as ’’rebellious palpitation,"
This condition is recognized today as atrial fibrillation
(87).
Another important reference concerning the use of
quinine in cardiac irregularities is given by a Viennese 8 clinician, Oppolzer, in 1872. In his discourse on the treatment of internal diseases, he begins his chapter on the treatment of heart disease with the statement: "The best and most powerful factors in dealing with heart patients are three, rest, digitalis and quinine" (83).
Wenckebach, a Viennese cardiologist learned of the use of quinine in atrial fibrillation quite by accident.
One of his patients, a merchant from the East Indies, told him that he could frequently avert his attacks of atrial fibrillation by taking the common remedy for malaria, quinine (cited by Beckman) (2). Frey (21) an associate of Wenckebach, greatly Impressed by this observation, performed investigations on the effect of various cinchona derivatives on atrial fibrillation and found quinidine to be the most effective. His observations were quickly confirmed by others.
Levy (43) used quinidine sulphate orally to treat atrial fibrillation with the restoration to normal rhythm in eight out of nineteen patients. Following this, the use of quinidine in atrial fibrillation became established
(42, 48, 89).
Chemical structure of quinidine and dihydroquinidine
The bark of cinchona trees (Cinchona suecirubra,
C. ledgeriana, C. calisaya; and hybrids of these) contain 9 numerous alkaloids, of which quinine, quinidine, cinchonine and cinchonidine are the most abundant and important* An average commercial yield of the cinchona alkaloid is about
4.9 percent quinine, 0.1 percent quinidine, 0.4 percent cinchonidine and 0.3 percent cinchonine. These alkaloids are all closely related in structure and medicinal action.
Quinidine is a d-stereoisomer of quinine. The chemical structure and its various components are shown in Figure 1. Its basic nucleus, 9-rubanol is derived from the parent compound, ruban. This nomenclature was proposed by Rabe (57) to simplify the naming of this type of compound and to indicate its origin from the rubiaceae family. Nine-rubanol is composed of a quinoline ring structure linked by a secondary carbinol (alcohol) to a quinuclidine ring. There is a methoxyl group (-OCB^) in
6* position on the quinoline ring and a vinyl group
(-CIP=CH2) in the 3-position on the quinuclidine ring.
However, demethoxy alkaloids (cinchonidine and cincho nine) differ from quinine and quinidine only by the absence of methoxyl group in the former (88).
Cinchonidine and cinchonine are much weaker myocardial depressants. This emphasized that the methoxyl group plays an important quantitative role, although it is not essential qualitatively (15).
Because of quinine's physical properties it has been used in many medicinally related areas. Quinine y\ 10 OH I H -C -
V ^ / O V if RUBAN 9-R U B A N O L /V,N. V QUINOLINE NUCLEUS QUINUCLIDINE NUCLEUS OH H I CH3 0 CgH 6 CHo I 5CH-
N+ CHzCHg I H QUINIDINE
N. \ \ H H H H ^C— C— H ■C —CH H I H H^ H^
DIHYDROQUINIDINE TRITIATED DIHYDROQUINIDINE
FIG 1: NOMENCLATURE USED FOR IDENTIFICA TION OF VARIOUS QUINIDINE AND DI HYDROQUINIDINE STRUCTURE 11 possesses the physical property of high fluorescence. This_ property has enabled the development of special techniques for determining its concentration in blood * tissue, and urine. In addition, its property of polarizing light has led to its use in the manufacture of polaroid glasses and to its incorporation in skin preparations used to protect the epidermis from the rays of the sun (16).
Dihydroquinidine or hydroquinidine is an alkaloid of cinchona, steroisomeric with hydroquinine (33). It is usually prepared by hydrogenation of quinidine, and in this way the vinyl group in the 3-position on the quinu clidine ring is saturated (Figure 1).
Comparison of cardiovascular actions of quinidine and dihydroquinidine
Dihydroquinidine was first reported by Lewis et al. in 1922 (46) to be the chief contaminant of commercial quinidine and present in it usually to the extent of about 20 percent.
The action of commercial quinidine sulphate was attributed to dihydroquinidine by Van Dongen and Sanchs
(73). This was the earliest record and it was reported that pure quinidine appeared to be without effect in increasing resistance to fibrillation in cat heart by electrical stimuli. However, Welsman (80), who studied the depressor action of these drugs, mentioned in a footnote that dihydroquinidine failed to restore normal cardiac 12 rhythm to any of eight patients with chronic auricular fibrillation.
Following World War II, Scot et al. (64) made detailed comparative pharmacological studies on pure quinidine, dihydroquinidine and commercial quinidine sulphate. Their studies indicated that dihydroquinidine was decidedly more potent. It was approximately 3.5 times more effective than pure quinidine in raising the threshold voltage needed to produce permanent ventricular fibrillation in cats. A dose of 1 mg per kg administered intravenously was determined to be the threshold dose for dihydroquinidine, while with pure quinidine, the same effect was produced with 3.5 mg per kg. The results on the effect of these drugs on blood pressure showed that there was very little difference in the degree of depressor action between pure quinidine and dihydroquini dine. However, the doses required for marked depressor effect were much higher than those required to raise the threshold voltage in ventricular fibrillation. Further more, toxicity studies (median lethal dose) made in mice showed that dihydroquinidine was approximately 18 percent more toxic than quinidine.
Although the acute toxicity of dihydroquinidine in mice is about 18 percent greater than that of pure quinidine, the therapeutic index of DHQ is greater than 13 quinidine since dihydroquinidine requires a much smaller dose to be effective.
Cardiovascular responses to quinidine with special emphasis on conduction
Quinidine is generally regarded as a myocardial depressant drug, since it depresses excitability, conduction velocity and contractility.
As a result of the development of micro-electrode techniques, the effects of quinidine on the intracellular cardiac fiber action potentials could be studied. Vaughan Williams (74), studied simultaneously the effect of quinidine in various concentrations on contraction, conduction velocity and intracellular action potential from single fibers in isolated rabbit atria.
The analysis of his data shows that at concentrations of
10 mg per liter, which was regarded as equivalent to therapeutic serum levels in man, there was a great reduction in conduction velocity and also in the rate of rise of the action potential. There was a significant prolongation in effective refractory period (terminal phase of repolarization), without any such effect on absolute refractory period (measured as the time from the peak of the action potential to half repolarization, i.e., first half of repolarization phase). At this concentration, the number of contractions were reduced by not more than one 14 fourth. However, the contractions were greatly reduced at concentrations of 16 mg per liter or above.
At high concentrations of quinidine the atria became totally inexcitable, although there was no significant fall in resting potential. This shows that the effective refractory period bad been infinite, even though the repolarization was complete.
Another observation made in this study was that acetylcholine accelerated the rate of rise of the action potential, even in the presence of high concentrations of quinidine. This may relate to the direct action of acetylcholine on the phase of depolarization.
The effect of quinidine on the action potential of
the cells of 8-A node of isolated rabbit atria showed that quinidine in therapeutic concentrations, 6 mg per liter
in the perfusion solution, caused an increase in the duration of the action potential and also a decrease in the
slope of slow diastolic depolarization (84). The
suppression of normal pacemaker activity may be disadvan
tageous in some cases as it may force the heart into escape
mechanism such as idioventricular rhythms and ventricular tachycardias (16),
In similarly isolated preparations of ventricular
tissue, these effects of quinidine on depolarization and
repolarization phases have been observed by different
workers. 15
Johnson (37) reported that the predominant effect of quinidine was to reduce the maximum rate of rise of membrane action potential of ventricular muscle, however, this effect was occasionally accompanied by a decrease in the membrane resting potential. Further, quinidine also lengthened the duration of action potential, which was in some instances largely the result of an increase in the duration of the repolarization phase, but in others was also the result of an increase in the length of depolari zation phase.
Qualitatively similar suppressant effect on spontaneous pacemaker activity of the Purkinje fibers by this drug have also been reported by Hoffman (29), and
Weidmann (78),
Recently the effect of quinidine and other drugs on the conduction velocity through the myocardium in intact dogs was studied by Hamlin et al. (31). Their results showed that within 30 seconds following intravenous administration of total of 0.3 gm of quinidine, there was a marked suppression of myocardial conduction velocity from a mean of 230 mm per second to a mean of 166 mm per second.
One of the mechanisms by which quinidine may depress or abolish ectopic impulse generation is by increasing the threshold to electrical stimuli. The threshold at a fully recovered state of excitability was 16 increased by 10 to 25 percent in isolated rabbit atria by concentrations of 6 mg per litter (84).
Further it has been reported that there was no change in the threshold in the intact dog heart until doses as high as 10 mg/kg were used. Doses higher than 10 mg/kg produced major depression of excitability of all cardiac tissues (24).
The depressant effect of quinidine upon the electro cardiogram is by the prolongation of P-R interval and the
QRS duration. These electrocardiographic signs are attributed to the increased duration of electrical systole and to the diminished intraventricular conduction velocity
(24). An increase in the temporal depression of ventri cular refractory period and idioventricular impulse generation may result from the use of large doses of quinidine (27).
The vasodepressor effect of quinidine is manifested only by large doses. Hypotensive effects may be attributed to peripheral vasodilatation and/or reduction in cardiac output (20, 50).
The mode of action of quinidine at the cellular level has long been a paradox. Recently much attention has been paid to the transport of potassium. Holland (30) and
Armitag (1) have found that by lowering the concentration of external potassium, there was a reduction in the depressant effect of quinidine on isolated rabbit atria. 17 Similar results have been found in the isolated, perfused rabbit heart (76)• It has been observed that perfusion of quinidine (0.01 mg/ml)in a normal concentration of potassium (5.6 m £q/L) caused a significant decrease in the maximal rate of depolarization, but by lowering the potassium to 0.8 m Eq/L, in the presence of the same concentration of quinidine, there was a significant increase in the maximal rate of depolarization. From these observations, it may be concluded that quinidine decreases the efflux (outflow) of potassium by the cardiac fiber and thereby prolongs the duration of action potential.
Other possibilities of its action are that quinidine effects the sodium carrying mechanism (37, 78) and that it blocks the action of intracellularly released acetylcholine (30),
Distribution of cinchona alkaloids in blood and tissues
The earlier studies on the distribution of quinidine was made by Weiss and Hatcher in 1927 (82) in cats. Dosage ranging from total of 60 to 150 mg of quinidine sulphate were administered intravenously to cats.
The extraction of quinidine base from blood and tissues was done with chloroform and titrated with bromine water.
These studies indicated that about 95 percent of the administered dose of quinidine left the blood within five 18 minutes and thereafter the remaining concentration in the blood fell gradually. Only traces of quinidine remained in the blood after an interval of 3-4 hours. There was much individual variation in the disappearance rate from the blood. The lungs, liver.and kidney showed the highest concentration among the tissues and they attributed this to the binding of the drug to the capillaries of these organs. Ihey found that the fatal dose of quinidine in the cat was approximately 80 mg/kg, when injected at the rate of 5 mg/kg/min. The toxic dose was much smaller with a more rapid rate of injection.
There are a few interesting observations available from the comparative data on the concentration of quinine in the tissues of chicken, dog and rabbit, one hour after the intravenous injection of 10 mg/kg of quinine. The liver of rabbit showed 1/23 to 1/22 of the concentration found in the chicken and dog livers. However, the concentration of the drug in the bile of chicken was approximately three times that of the dog. Furthermore, the concentration of the drug in rabbit's lung was the highest as compared to chicken and dog lungs (40).
The distribution studies of various cinchona alkaloids in various fluids and body tissues of dogs after infusing them with the sulphates of quinine, quinidine, cinchonine and cinchonidine and attaining a concentration 19 plateau in the plasma for at least thirty minutes was done by Hiatt and Quin (34). The estimation of concentration of these alkaloids was done by colorimetric method. They found that the cerebrospinal fluid, erythrocytes, skeletal muscles and brain had very low tissue to plasma ratio (0.1 to 5.0). However, a very high ratio (10 to 40) for glandular tissues and lungs was seen. No information was made available for cardiac muscle to plasma concentration for quinidine in this study. These ratios (tissue: plasma concentrations) remained approximately the same, in spite of changes in the plasma concentration. These observations suggest that equilibration between tissues and plasma takes place rapidly. All of the cinchona alkaloid give similar results.
The oral administration of 50 mgm/kgm of quinidine sulphate to dogs also produced similar results in that concentration of quinidine in the tissues were much higher than in the plasma. The peak concentration in tissues and plasma was reached at the same time. However, the frequency of plasma determinations was not adequate in this study and as such it was not possible to determine exactly as to when the maximal tissue and plasma concen trations occurred and how long they remained maximal (77).
Weisman (79) studied the rate of elimination of quinidine in heart and other tissues in dogs after 20 administering a single dose of 100 mg. He found that the maximum concentration of the drug reached in thirty minutes, and no trace of the drug was seen after four hours.
With a higher oral dose of 585 mg, the maximum concen tration reached in one hour and there was no detection of the drug after seven hours. However, when the same dosage was given in repeated dosage, the time for attaining maximum concentration in the tissues and also for their elimination was increased.
The plasma concentration of quinidine observed after the administration of total of 0.6 gm of the drug by oral, intramuscular and rectal routes were 3.2, 2.68 and 0.89 mg per liter in an average time of two and one quarter, two and one-third and three and one-quarter hours respectively (38). It is suggested by these results that
the drug could be given by the intramuscular route when
circumstances preclude the use of the oral route.
Recently Scherlis et al, (63) made an extensive study in dogs on arterfo-coronary sinus concentration
differences of quinidine with the effect of varying
concentrations in the blood upon the myocardial concen
trations. It was found that, after rapid intravenous
administration of quinidine gluconate in varying doses, a
peak arterial level was attained in one minute, with a
rapid decline ensuing within 3-5 minutes and thereafter 21 a plateau occurred within 15-30 minutes. The arterial, venous and coronary sinus levels were similar in 30 minutes in all dogs. An interesting observation in this study was that the quinidine level in the coronary sinus usually exceeded the arterial level after the first minute and this persisted for 15-30 minutes. % i s finding is, however, debatable. It has been explained by Scherlis et al. that quinidine was pooled transiently or bound in the myocardium and was subsequently washed into the coronary sinus. The myocardial concentrations of quinidine were four to tenfold higher than the corresponding blood specimens and the absolute amount varied directly with the amount of quinidine injected. However, Conn and Luchi (9) reported that the tissue to plasma ratio is increased to ten when only the unbound drug is considered.
The information available on distribution of dihydroquinidine in blood and tissue is very limited.
Whitney (86) studied the distribution of tritium labeled dihydroquinidine in various tissue of the cat• He found that the ventricle of the heart had comparatively higher radioactivity than the atria and that both of these tissues had a greater concentration of the drug than skeletal or uterine muscle. The liver showed much higher radio activity than any other organ. The tissue plasma ratios achieved in the liver were also much higher than those 22 achieved in any other organ and this ratio increased with the lapse of time. The kidney had the next highest drug concentration in comparison to other organs, being particularly high during earlier time intervals. The thyroid retained relatively high concentration for a much longer time than any other organ.
The disappearance of the drug from venous plasma was in two phases, the first with a half life of approximately 2.5 minutes and the second with a half life of approximately 54 minutes.
Concentration-activity relationship of quinidine
Most of the information on the relationship between blood levels of quinidine and therapeutic or toxic effects were obtained by determining peripheral venous levels following oral or intravenous administration. However, no definite correlation between therapeutic effects and blood levels could be established from the available, though limited data.
Wegria and Boyle (77) attempted to establish a
relationship between plasma levels of quinidine and the
cardiac effects after administration of quinidine sulphate
to patients with chronic atrial fibrillation. "Miey found
that there were discrepancies between the intensity of
cardiac effect and quinidine plasma levels. The plasma 23 levels of the drug decreased faster than the intensity of its cardiac effects. Similarly no correlation between the plasma concentrations of quinidine and toxic symptoms manifested could he made (39).
Sokolow and Edgar (67) used quinidine in thirty patients with atrial fibrillation in an attempt to convert them to a sinus rhythm. Quinidine blood levels were determined by a photofluorometric method. They reported that successful conversion occur^d in 82 percent of their patients with a mean quinidine level of 5.9 mg per liter. Seventy-five percent of the patients were converted to the normal rhythm at plasma levels between 4 to 9 mg per liter, obtained by oral administration of 0.4 to 0.6 gm per person of quinidine sulphate every two hours for five days. However, in an earlier clinical study made on two patients by Delevett and Poindexter (14), it was reported that at a plasma quinidine concentration above
1.0 mg per liter, the attack of auricular fibrillation was alleviated and dosage which produced plasma levels below
1.0 mg per liter resulted in a recurrence of frequent attacks.
Further, some attempts have been made to correlate the pharmacological activity of quinidine with varying concentrations of quinidine in in vitro studies. At a concentration of 10 mg per liter, equivalent to therapeutic 24 serum levels of quinidine in man, there was a great reduction in conduction velocity and also in the rate of rise of the action potential (74).
Recently James and Nadeau (35) perfused the sinus node of the dog with quinidine in concentrations varying from 10"3 to 10^ mcg/ml in order to study its effect on the node. They proposed that quinidine has three actions:
(1) direct, (2) anticholinergic, and (3) antiadrenergic.
The direct action of quinidine (sinus arrest or any disorganization of sinus rhythm) was shown to be insigni ficant at,concentrations below 10.0 meg per ml. However, at 100 mcg/ml, which is considered to be above the therapeutic range, the rate of the sinus node was usually depressed and its activity often arrested. Further it was postulated that the concentration which suppresses the activity of sinus node, also causes similar suppression to the Â-Y node. At all concentrations of quinidine, there was a detectable anticholinergic effect, which was especially pronounced, often with a complete cholinergic block at 10 and 100 mcg/ml. However, in all circumstances the anticholinergic effect gradually subsided with return to normal cholinergic response in from 30 to 50 minutes.
Furthermore, their observations also showed that anti adrenergic effect of quinidine occurs at the same time and
for approximately the same duration as the anticholinergic 25 effect, which means that the two opposite effects are of comparable degree. There was a linear relationship between these effects and the concentration of quinidine. They postulated the action of quinidine to be at the receptor site and not due to inhibition of local release of effector substances•
Metabolism and excretion of cinchona alkaloids
Metabolism.— Although quinine and quinidine have been widely employed in the treatment of malaria and cardiac arrhythmias respectively, very little information exists concerning the metabolic fate of cinchona alkaloids in general and quinidine in particular.
Early studies on the metabolism of cinchona alkaloids have shown that quinine, quinidine, cinchonine and cinchonidine are extensively metabolized in man, and only a small fraction of the ingested drug is excreted unchanged in the urine (25, 70).
Kelsey and Geilling (40) studied the metabolism of quinine and quinidine in birds and mammals, both in vitro and in vivo. Under the conditions of the in vitro studies, quinine was more readily metabolized than quinidine by tissues of the rabbit and rat and also in in vivo studies in rabbits. In contrast to this, quinidine in in vitro studies in birds (chicken, turkey, pigeon, duck 26
and goose) appeared to be more readily metabolized of the
alkaloids. The rabbit metabolized quinine much more rapidly
than quinidine. Comparatively, the rate of metabolism in
the case of duck is slower than in the chicken. Furthermore,
the concentration of both unchanged quinine and quinidine
remaining in the tissues of the duck at the end of four hours were several times higher than those in the tissues of the
chicken.
Brodie et al. (4) have isolated the metabolic products
of each of the four principle cinchona alkaloids from human
urine. The separation of the products from each other and
from the parent drug was based on differences in their partition ratios in different extraction systems.
The various cinchona alkaloids and their metabolites
were extracted from the alkaline urine (pH 10) with ether.
This mixture was reextracted into an aqueous acid solution.
The components of this mixture were separated, after
alkalinization of the aqueous extract, into solvents of
varying polarity. The quinidine was found in the heptane
extract. Its presence was proven by counter-current
distribution, melting point and ultraviolet spectrophotometry.
The route of metabolism proposed by Brodie et al. (4)
for quinine and quinidine has been postulated to involve two
parallel pathways. The first pathway is the formation of a
carbostyril by the addition of oxygen to the quinoline ring;
the second pathway is the addition of oxygen to the 27 quinuclldine ring to form a monohydroxy non-phenolic derivative (Fig. 2A), This compound may further be oxidized in quinuclidine nucleus to form a dihydroxy derivative. The main metabolite of quinidine vas the non-phenolic monohydroxy derivative. It was present in smaller quantities than the unchanged quinidine.
The principal quinine metabolic product was also the non-phenolic monohydroxy derivative. However, the amount of this metabolite was comparable in amount to the unchanged quinine and three times the amount of carbostyril. The more soluble metabolites of both quinine and quinidine were also present in significant amounts. Similar results were reported by Conn and Luchi (9).
The metabolism of the demethoxy alkaloids (cinchonine and cinchonidine) differs from the methoxy alkaloids
(quinine and quinidine (Fig. 2B). With the demethoxy alkaloids there is no evidence of a metabolite in which only the quinuclidine ring is oxidized. However, the carbostyrils of the demethoxy alkaloids are oxidized by the second oxygen
(OH) in the quinuclidine nucleus.
Recently Conn and Luchi (9) studied metabolites of quinidine in the tissues of the dog and rabbit. The various
quinidine products were obtained from tissue extracts by
separation on thin layer chromatography. They were
identified by comparison of Rf values of quinidine and Figure 2.--Scheme for the route of metabolism of various cinchona alkaloids (proposed by Brodie et al. 1951). A shows the channel adopted by quinidine or quinine. B shows the channel adopted by cinchonine and cinchonidine.
28 29 CHr CH=CH, CH CH=CHr CH_ CH, CHOH- CHOH- OH \J N
C H ^ O - 2nd ^^3 [MHYDROXY- > DERIVATIVE PATHWAY (NON- \ / ^ N ^ PHENOLIC) Q U IN ID IN E I MONOHYDROXY TT m NON-PHENOLIC
PATHWAY CH=CH,
CHOH
CH.,0
OH
CARBOSTYRIL 131
B
-CHCHr Æ v -CH=CH2 -CH=CH,
CHOH CHOH- CH CHOH OH l y N
-OH
CINCHONINE ± CARBOSTYRIL IL CARBOSTYRIL IE OR CINCHONIDINE 30 authentic samples of metabolites isolated from human urine.
The relative concentration of each of the substances vas determined from ultraviolet spectroscopy. The studies on liver extraction showed that as much as 90 percent of quinidine was metabolized. However, in other tissues like heart, kidney, skeletal muscle and serum, the metabolized quinidine constituted only 30 to 35 percent of the total material. Among various metabolites, non-phenolic metabolite was 15 to 20 percent and 2-hydroxy quinidine was 10 to 15 percent. These results tend to show that the biotrans formation of the drug occurs almost exclusively in the liver.
Excretion.— Earlier reports showed that a relatively small percentage of quinidine was excreted in urine in relation to dose given (67).
Ditlefsen (17) made a comparative study in normal individuals and patients with congestive heart failure for blood concentration and renal excretion after intramuscular administration of a total of 0.84 gm of quinidine chloride.
The excretion of the drug in the urine of the patients was comparatively less and also delayed as compared to the control group. This resulted in the concentration of the drug in the blood of patients being considerably higher than in normal individuals. 31 The excretion studies of tritiated dihydroquinidine made in three cats have shown that less than one-third of the total excretion was renal and more than two-thirds was fecal, "Die mean total recovery in excretion was 67,3 percent of administered dose (86). CHAPTER II
MATERIALS AMD METHODS
Purity and identification of DHQ-B^
Thin layer chromatography.--Eastman prepared silica gel chromatogram sheets,^ which were used in this study. The 20 x 20 cm sheets were cut into strips of
20 X 5 cm size. Two points located two centimeters apart on the bottom of each strip were marked, one was employed for applying DHQ standard and the other DHQ-B^. The TLC sheets were not activated before use.
The developing agent consisted of chloroform- diethylamine (8:2) was mixed at room temperature. The chromatographic tank was lined with filter paper, then saturated with the solvent system one-half an hour before chromatogram development in order to assure equilibration.
Procedure.— In chromatograms, an ascending technique of the mobile phase solvent was used. A very small quantity of the samples of labeled DHQ and standard
DHQ were applied by means of micropipettes on the respective spots marked on the chromatogram sheet. The
^Eastman chromatogram sheets, Type E301R.
32 33 chromatogram was attached to a glass plate and Inserted into the chromatographic tank. The chromatographic development was performed at room temperature for a period of 45 minutes. The chromatograms were removed and air dried.
Methods of visualization and scanning of radio activity material.— In order to locate the material, the 2 Chromatograms were visualized under short-wave ultra violet light. The ultraviolet positive area with Rf value equivalent to pure dihydroquinidine was marked with a pencil.
The chromatogram was next scanned in a Packard
Radio Chromatogram Scanner^ to show that the radioactivity
coincided with the chromatographed material.
Methods of preparation of drug solutions
Dihydroquinidine^ solution (unlabeled).— Dihydro- 5 quinidine was weighed on a semi-micro analytical balance
and then a solution was prepared by adding 0.1 N sulphuric
acid drop by drop, with constant stirring. Usually it
^Mineral light ultraviolet lamp, Model, Short Wave OVS-12.
^Packard Model 7200.
^Supplied by Merck, Sharp and Dohme Research Laboratory, Rahaway, New Jersey,
^Mottle, type H-16. 34 required approximately 6.0 ml of 0.1 N sulphuric acid to dissolve 200 mg of dihydroquinidine. The pH of this acidic solution (pH 3,0 to 3,2) was neutralized to pH 6.5 by the gradual addition of 0.1 N sodium-hydroxide. The final concentration of the solution employed for distribution and excretion studies ranged from 9 to 11 mg per milliliter.
Dihydroquinidine-H^ (labeled).-— DHQ-H? was obtained g from a commercial laboratory. One milliliter of the stock solution having 10 mg of DHQt.1^ in an ethanol was pipetted into a serum bottle. Methanol was removed over low beat and with nitrogen gas passing through the bottle. After the methanol was removed, 0.65 ml of 0.1 N sulphuric acid was added to each 10 mg of the free DHQ-H^. The total volume of the resultant solution was made to 10 ml by the addition of distilled water. The specific activity of each solution used for distribution and excretion studies was determined.
Methods of determination of the specific activity
DHQ~H? in distribution studies.— The 0.1 ml of
DHQ-H^ solution containing 100 meg of DHQ-H^ was placed in
100 ml volumetric flask. Methanol, the solvent of this
aliquot, was evaporated by slow head and nitrogen and then
^New England Nuclear Corporation, Boston, Mass. 35 its volume was adjusted to 100 ml by the addition of a sufficient amount of distilled water. The radioactivity of the solution was determined by placing 0.1 and 0.2 ml of this solution in separate vials containing Bray's counting solution (3). These volumes represented 0.1 and 0.2 micrograms of total D H Q - ^ respectively.
DHQ-]^ in excretion studies.— The same procedure as mentioned above was adopted for determination of the specific activity of employed in excretion studies.
Two vials were counted for each of the following concen trations: 0.1 meg, 0.2 meg, and 0.5 meg of total DHQ-I^.
Calculations for the amount of DHQ recovered from various body fluids and tissues in relation to radio activity.— In these investigations, a mixture of DHQ unlabeled (10 mg/kg) and DHQ-I^ (15 uc/kg « 31,2 to 38.5 mcg/kgm) was administered to each dog. Accordingly, the specific activity of the mixture can be represented as follows:
10 mg (10,000 micrograms) « 15 microcuries
1 microcurie - 222 x 10^ DPM of radio activity 4 Therefore, 15 microcuries » 15 x 222 x 10 DFH
or 15 X 222 x 10^ DPM is represented by 10,000 meg
of DHQ or 3330 DPM represents 1 microgram (meg). 36 Studies on distribution and effects of dihydroquinidine on myocardial conduction velocity
The arrangement of the experiment pertaining to the study of the effect of DHQ on myocardial conduction, distribution in various body compartments and excretion is shown in Figure 3,
Surgical technique,— Five female mongrel dogs, weighing 9 to 13 kg were anaesthetized with sodium pentobarbital (approximately 30 mg/kg of body weight) administered intravenously. The animals were intubated and ventilations were maintained with a respirator.?
Thoracotomy was performed through the fifth inter costal space. To expose the thoracic cavity, several ribs were severed at the vertebral border and costochondral junction and removed from the side of the thorax exposing the lungs and heart. "Rie pericardium was removed, and a specially-designed multipolar electrode assembly was sutured on the right ventricle so that its length was parallel and approximately 3 cm anterior to the right anterior descending coronary artery.
Electrode assembly.— A multipolar electrode assembly 2.5 cm long and 0.5 cm wide was constructed of flexible plastic,® The arrangement of the assembly is
?Model No. 607, Harvard Apparatus Co., Inc. Mass.
®Duro Plastic, Woodhill Chemical Corp., Cleveland, Ohio. Figure 3.— Illustrates the arrangement of the experiment pertaining to the study on the effect of dihydroquinidine on myocardial conduction velocity and its distribution and excretion. The various appliances employed are labeled.
37 KIM06RAPH CAMERA INFUSION QBIP
FOR INJECTION OF DfWG
URINARY CATH FOR VENOUS . SAM
RESPIRATOR PUMP
ULATOR
w OD 3 9 shown in Figure 4. It consisted of 14 pairs of differ ential bipolar electrodes and a stimulating electrode# The stimulating electrode consisted of tips of two wires 2 mm apart and was located at one end of the assembly. The bipolar electrodes (14 pairs) consisted of 28 (0.04 mm diameter) insulated copper wires which protruded in a linear fashion from the plastic, beginning at 2 mm from the stimulating electrode. The copper wires except for their tips were imbedded in the plastic. The tips being bare thus contacted the myocardial when the assembly was attached to the heart. The interelectrodal distance from center to center between each lead point member of a bipolar pair is approximately 0.5 mm* The distance between two adjacent electrodes is approximately 1 mm.
Leads taken from each adjacent recording pair, thus formed 14 close bipolar recording leads.
Cannulation technique.— The femoral veins and arteries of both rear legs were cannulated by means of polyethylene tubing (0.095 inch diameter) which were fitted with a three-way disposable plastic stopcock. The cannulas were kept patent by flushing them with heparinized normal saline solution. One of the cannulated femoral veins was employed for infusing the 5 percent mannitol solution in distilled water (approximately 4 drops/min/kg body wt.) and also for injecting DHQ-DHQ-H® mixture. The femoral Figure 4.--Illustrates the schematic diagram on the arrangement of the multipolar electrode assembly and its application on the right myocardium.
40 41
EPICARDIUM
SUTURE
\STIMULATING ELECTRODE
MYO RECORDING CARDIUM ELECTRODES
RIGHT {VENTRICLE
SUTURE EPICARDIUM
MYO- I CARDIUM 42 arterial cannula on the same side was also employed for recording blood pressure with the Statham water manometer.
The cannulas in the vessels of the opposite rear leg were
inserted for a distance of about 5 cm and were employed
for collecting arterial and venous samples.
The coronary sinus was cannulated by inserting an
18-gauge Rochester needle into the right atrial appendage
and guiding it into the coronary sinus. The Rochester
needle in turn was attached to a polyethylene tube by means
of an adapter. Small holes were made at the end of plastic
tubing of the Rochester needle to facilitate the withdrawal
of coronary sinus blood samples. The tube was sutured to
the right atrial wall in order to keep the coronary cannula
in position. The position of the catheter in the coronary
sinus was always ascertained at the end of each experiment.
The bladder was catheterized with a Foley catheter
(size 12FR) and maintained in position by inflating the
balloon of the catheter by the injection of 5 ml of
distilled water. Catheterization was aided by a vaginal
speculum and the catheter was guided into urethra with a
silver flexible probe.
Timings of various samples.— With the aid of three
assistants, arterial, venous and coronary blood samples
were withdrawn simultaneously at intervals of one-half,
one, two, four, eight, sixteen, thirty-two and sixty-four 43 minutes* The collection of samples was completed within ten seconds. About 2 ml of each blood sample was collected with a two and a half milliliter disposable syringe and transferred slowly into small heparinized specimen tubes.
The samples were spinned and plasma was separated* Four urine samples were also collected in continuation during this period and the time interval of these samples were
0-5, 6-13, 16-30 and 31-64 minutes* The total volume of each sample was noted and a small quantity from each sample was set aside for analysis*
The heart was artificially driven by a stimulator® through the stimulating wires attached to the electrode assembly*
Square wave pulses of 3 millisecond duration and of a voltage and frequency great enough to dominate the sinus rhythm were employed* Lead II, Af and V-10 electrocardio grams were recorded continuously and were made visible on the beam of the Sanborn^® recording apparatus. The electrocardiograms gave a guidance that the heart was being paced artificially throughout the experiment*
Recording system*— The basic recording system consists of four Tectronix 502,^^ dual beam oscilloscopes,
®Model 54A, Grass Instrument Co* Quincy, Hass.
l®8anborn 550H Polybeam recorder.
l^Tektronix 502, Tektronix, Beaverton, Oregon. 44 which share a common trigger source from a Tektronix 162 wave form generator and Tektronix 161^^ pulse generator.
The oscilloscopes were triggered and swept synchronously.
Time marks at 5 M. Sec. interval were fed into the upper trace of all oscilloscopes. The cluster of four dual beam oscilloscope screens was photographed with a Grass kymograph camera.
Bipolar electrograms were recorded with sharp negative or positive going deflections. Attempts were made to record them at the same time intervals as when the blood samples were taken. One technician was assigned to take the photographs of the electrograms recorded on the screen of the oscilloscope.
*nie interpretation of the bipolar electrograms was accomplished by measuring the temporal relationship of each bipolar electrogram with the stimulating signal, which was recorded on one of the simultaneously triggered channels. The time in milliseconds between the peak of each electrogram and the stimulus signal was measured.
Tissue sampling.— At the end of 64 minutes, the experiment was terminated by sacrificing the animal with the injection of pentobarbital. , ...... — rw------— — — " . — ...... — Tektronix 161 and 162, Tektronix, Beaverton, Oregon,
l^Model 64, Grass Instrument Co., Quincy, Mass, arG zn o'V'e
- x z d o t > “t a. iL xaecS.
6£fe.x»3E>3Le
^ tk z m e ^
zag%aa,aara^y s V a. ar dL e- d
x >jei:q — i x ^
- %_ "fcy
' ■ ted. oaa. st
^ “ ]P b y
^ - 3 -fc. c> f > j p ^ d
w X % 3L d %
-Ta i :K t z j & r e *
^ '- na j
- Is 44 which share a common trigger source from a Tektronix 162 wave form generator and Tektronix 161^^ pulse generator.
The oscilloscopes were triggered and swept synchronously.
Time marks at 5 H, Sec. interval were fed into the upper trace of all oscilloscopes. The cluster of four dual beam oscilloscope screens was photographed with a Grass 1 o kymograph camera,**
Bipolar electrograms were recorded with sharp negative or positive going deflections. Attempts were made to record them at the same time intervals as when the blood samples were taken. One technician was assigned to take the photographs of the electrograms recorded on the screen of the oscilloscope.
The interpretation of the bipolar electrograms was accomplished by measuring the temporal relationship of each bipolar electrogram with the stimulating signal, which was recorded on one of the simultaneously triggered channels. The time in milliseconds between the peak of each electrogram and the stimulus signal was measured.
Tissue sampling.— At the end of 64 minutes, the experiment was terminated by sacrificing the animal with the injection of pentobarbital.
■ ■ ■ yn~ ’ ...... ■' "" ' " ' " **^Tektronix 161 and 162, Tektronix, Beaverton, Oregon.
13jiodel 64, Grass Instrument Co., Quincy, Mass. 45
The following tissue samples were rapidly removed and weighed on a torsion^^ balance: left auricle, left ventricle, right auricle, right ventricle, liver, lung, spleen, adrenal, kidney, ovary, uterus, thyroid and skeletal muscle. The skeletal muscle sample was obtained from lateral head of the quadriceps. Each tissue sample was removed with a pair of scissors and forceps and blotted between two layers of gauze prior to weighing.
The samples were then placed on a preweighed triangularly cut-glazed paper for weighing. The sample weights varied from 30 to 80 milligrams.
Drug administration.— DHQ (unlabeled) and DHQ-H®
(labeled) at the rates of 10 mg/kg and 15 uc/kg respec tively were mixed in the barrel of a sterile 12 ml capacity syringe for each experiment. The syringe was fitted on a stand and attached to the intravenous infusion drip by means of a three-way stopcock. The infusion was stopped during the time of injection of the drug mixture, which required 10-20 seconds. After injecting the drug mixture, the electrically run time-clock was set to note timings for various samples and recordings.
^^odel No* 5702, Roller-Sroith, Inc., Newark, New Jersey. 46 Excretion Studies
Renal clearance Experimental technique,— -Six female and one male mongrel dogs weighing from 9 to 12 kg were anaesthetized with pentobarbital solution and placed in lateral recumbency. A Cournand needle was placed in the jugular vein and attached to 5 percent dextrose intravenous drip with a three-way stopcock. Blood samples were also taken from the jugular vein.
Cannulation of one of the femoral veins with a
Cournand needle was employed for infusing inulin solution.
The femoral vein of the opposite hind leg was used for injecting DHQ-DHQ-B^ mixture. The bladder was catheterized with a Foley retention catheter for collecting urine samples and the same procedure for catheterization was adopted as mentioned earlier.
Infusion of Dextrose 5 percent and Inulin 5 percent solution.— Dextrose intravenous infusion was started just prior to inulin infustion. The dextrose infusion was regulated at the rate of 2 drops/min/kg body weight.
Before starting the inulin infusion, control urine and blood samples were taken. 47
Using a 50-milliliter glass syringe, the inulin solution was infused with an infusion withdrawal pump^^ at a slow speed of 0.191 ml per minute (speed 8). An equilibration period of 45 minutes of infusion was performed prior to the collection of the first samples of urine and
blood.
The mixture of dihydroquinidine (10 mg/kg) and
dihydroquinidine-H® (15 uc/kg) was injected into the
cannulated femoral vein, thirty minutes after the start
of inulin infusion.
Collection of blood and urine samples.— The first
sample of urine was continuously collected from the time
of injection of DHQ-DHQ-H^ mixture up to 30 minutes.
Emptying and washing of the bladder was started approxi
mately two minutes prior to the end of the collection
period. By means of a 30 ml syringe attached to a urinary
catheter, urine was withdrawn from the urinary bladder.
Twenty ml of distilled water was infused as a wash and
immediately withdrawn. This procedure was repeated twice
and then followed by the injection of 20 ml of air and
then removal of any urine-water mixture remaining. Fifteen
ml blood sample was obtained with a new syringe and emptied
into a heparinized centrifuge tube.
l^Harvard Apparatus Company, Dover, Mass. 48
Subsequent blood and urine samples were taken after every thirty minutes or sixty minutes for a period of three and one half hours from the time of the injection of drug mixture. In one experiment urine and blood samples were taken in a similar fashion up to 6 hours with a time interval of one hour sampling used after the end of three hours, BloOd samples were spun down on a centrifuge for separating the plasma.
Inulin concentration in urine and blood samples was determined by chemical methods (65).
Glomerular filteration rate was calculated by the formula : ”inV ■ Pin
Cfn » clearance on inulin
Uin “ concentration of inulin in urine (mcg/ml)
V = volume of urine produced in a minute (ml/min)
P " concentration of inulin in plasma (mcg/ml)
The concentration of DHQ in various urine and blood samples was calculated after determining the radioactivity of these samples.
Biliary excretion
Bile duct cannulation.— Three female dogs, weighing
9.0 to 10.0 kg were anaesthetized with pentobarbital and laparotomy was performed. The common bile duct was 49 exposed. In one of the earlier experiments, the position of common bile duct was ascertained by passing a catheter through the ampulla of Vater (duodenum) into the common bile duct (54).
A polyethylene tube about 20 cm long and 0.095 inch diameter was passed into the common bile duct and it was guided into gall-bladder. The distal end of the polyethylene tube was perforated to facilitate withdrawal of bile samples. The position of the catheter in the bile duct and gall-bladder was secured by fastening a ligature around the entrance in the bile duct.
The femoral veins of both rear legs were cannula ted.
One of them was used for dripping dextrose 5 percent
(2 drops/min/kg) and also for injecting the drug mixture and the other for drawing blood samples.
Collection of bile and urine samples.— Bile samples
from gall-bladder were taken by washing and emptying the
gall-bladder, which was done in almost the same way as bladder washing in renal clearance studies. The first sample was taken continuously up to 30 minutes after
injecting the drug. By means of 10 ml syringe, attached to
the biliary catheter, as much bile as possible was with
drawn. Then 10 ml of distilled water was infused and
immediately withdrawn. This washing and emptying was
repeated three more times. Last of all 10 ml of air was 50 injected and withdrawn along with any hile-water mixture remaining. All the washings were saved in the collecting jar. Subsequent continuous bile samples were taken after every 30 minutes until three hours after injection time and thereafter, every hour until six hours after the Injection time. The bile samples were diluted to 50 and 100 ml with distilled water according to the time interval of 30 and
60 minutes respectively.
In all the experiments, bladder was also catheterized and urine samples were collected continuously at 60-minute intervals for six hours.
Extraction of the unchanged drug (non-metabolite) from various tissues and biological fluids
Heart, kidney and liver.— Two to four gram samples of heart, kidney and liver were weighed and sufficient
10 percent sucrose solution was mixed to make the final concentration to 10 percent. The mixture was homogenized in a tube with the electrically driven pestle. The homogenates were centrifuged for one hour at a speed of
40,000 rpm. The supernatant from each homogenate was separated and subjected to extraction procedures. The radioactivity in an aliquot from the supernatant was con sidered to represent the total activity in the sample.
Extraction.— Earlier, various techniques to extract unchanged DHQ were employed e.g., the whole supernatant 51 or urine and plasma were lypholyzed and shaken with benzene and later benzene was evaporated, following this radio activity of the whole material was counted. Chloroform extraction was also attempted. The results of these procedures were not entirely satisfactory.
Extraction with dichlorethane gave comparatively better results and the following procedure was adopted.
The various supernatants were filtered through
glass wool and their pH was adjusted to 10.5 to 11.0 with ammonium hydroxide. Ammonium hydroxide is considered
better alkalinizing agent for releasing the base from
cinchona alkaloids (Takern et al., 1961). Five ml of each
of the alkaline supernatant was put in a separatory
funnel and 5 ml of dichlorethane was mixed in each. The mixture in each separatory funnel was shaken for five minutes with an electric shaker. The dichlorethane phase was separated from the aqueous phase and marked as first
extract. The remaining aqueous phase was shaken two more
times with an equal volume of dichlorethane and the
dichlorethane layer separated each time being marked as
second and third extracts respectively. In some of the experiments extraction for the fourth time was also made.
An aliquot of 0.2 ml in duplicate from each of
the extract was counted for radioactivity. The radio
activity in the second extraction was approximately 20 52
percent of the first extract and subsequent extractions
gave radioactivity less than five percent of the first
extract•
Biological fluids,— -Similar procedure for the
extraction of the unchanged drug from urine, plasma and
bile samples was adopted as for the tissues. Five ml of
urine, plasma and bile samples were alkalinized to pH 10,5 with ammonium hydroxide and extracted with dichlorethane.
In the excretion studies, urine and bile samples were already well diluted and there was no problem of
emulsion formation during shaking with dichlorethane.
However, in some of the samples, particularly of bile,
there was some emulsion formation during shaking. This
problem was solved by centrifugation of the mixture and
later separating the extracted (dichlorethane) layer.
Radiometric analysis,— Bray's solution (3) was
employed for counting tritium activity as it has excellent
solvent properties and permits a rather higher counting
efficiency. It contains a mixture of naphthane, 60 grams,
1,4-bis (5 phenyl-e-oxazolyl) benzene (POPOP), 200
milligrams, 2,5-diphenyl oxazole (PPG), 4 grams, methanol,
100 milliliters, ethylene glycol, 20 milliliters and P-
dioxane to make 1 liter.
Liquid Scintillation Spectrometer^® was employed for
the determination of the radioactivity of various samples. ■ Yg ' " ' — Model No. 4312, Packard Instruments Co., Inc. 53 Various plasma, urine, bile and other extracted material samples were prepared by pipetting 0.2 ml of these fluids into the liquid scintillation counting vials containing Bray’s solution, whose background activity was predetermined. Samples of various tissues earlier weighed were also put into the counting vials. All the samples were held for 12 to 24 hours in the freezer counting chamber of the liquid scintillation spectrometer.
Sufficient counts were taken to assure an error of less than 5 percent in the ratio between background counts and total counts (49). The pre-set counting was always two thousand or ten minutes, whichever was earlier. Each sample was counted for a minimum of two times. Some of the samples showed low efficiency, while counted. This was due to quenching from color and immisdibility of biological samples in the organic solvents used for liquid scintillation counting. In such samples, after initial counting, one milliliter of toluene (internal standard) with a known specific activity was added and the samples were again counted to determine the counting efficiency. CHAPTER III
RESULTS
Identification of DHQ-H^
The purity of the tritinted dihydroquinidine was identified by running its chromatogram simultaneously with the standard unlabeled dihydroquinidine (thin layer chroma tography) and later scanning the chromatogram. DHQ-H^ was found to be identical to its standard, "Riis is illustrated in Figure 5, It can be seen that the radioactive peak of DHQ-H^ has the same R^ as the flourescent spot of the unlabeled DHQ. Less than 1 percent of the labeled material remains at the origin.
Determination of the specific abtivity of DHQ-H^
Distribution studies.— Table 1 shows the individual data and method of calculation of the specific activity of
DHQ-H^. Duplicate samples of 0.1 and 0.2 micrograms were
counted. The average specific activity of all samples was found to be 0,482 microcuries per microgram of DHQ-B^,
Excretion studies,— Similar calculations for the
determination of the specific activity of DHQ-K? employed
54 55
Figure 5.~«.Radio-chromatographic scan of a thin layer chromatoplate of DHQ-H^ and standard DHQ, using a solvent system consisting of chloroform and diethyl- amine (8:2). Note the radio peak adjacent to the positive area of the standard unlabeled DHQ, marked as U.V (standard DHQ visible under ultraviolet light). 56 ORIGI N
/—\
SOLVENT FRONT TABLE 1
DETERMINATION OF THE SPECIFIC ACTIVITY OF DIHYDROQUINIDINE-H^ (DHQ-H^) USED IN DISTRIBUTION STUDIES
Amount of DHQ-h 3 in Counts Effic Disintegrations Disintegrations Microcuries the Vial per iency per Minute per Minute per . -(.meg) .... Vial M n u t e Factor in San5)le per meg Microgram
0.1 1 21247.9 5.1 108364.2 1083642.0 0.488 0.1 2 20000.0 5.2 104000.0 1040000.0 0.468
0.2 1 43456.7 5.1 221629.1 1108145.5 0.499
0.2 2 41234.5 5 .1 210295.9 1051479.5 0.474
Efficiency factor determined from the external standardization curve of the machine. Specific activity of D i h y d r o q u i n i d i n e factpr x ^ 3.7 X 10^ X 60 DPM Average = 0.482.
VJI 58 for excretion studies is shown in Table 2» Duplicate samples of 0.1 meg, 0.2 meg and 0.5 meg were counted and the average specific activity of all samples calculated was
0.389 microcuries per microgram of DHQ-H?.
Effect of dihydroquinidine on myocardial conduction velocity
Conduction velocity was determined in five dogs.
Tables 3 to 7 show the measurement of the conduction time in milliseconds from the stimulus to each recording point during various post-injectional periods in the individual experiments. The first recording point is located 2 mm from the stimulating electrode and the subsequent points are 1 mm apart. The conduction time was measured from the end of the stimulus to the peak or nadir on each electrogram.
Figures 6 and 7 illustrate the effect of DHQ on the conduction time in one of the experiments during different time intervals after the administration of the drug. It
is evident from these curves that a marked effect on the prolongation of conduction time occurred within the first two minutes after which the effect diminished but did not return to the control level even after sixty-four minutes.
Various mathematical models were fitted by means
of a computer to the data of conduction time versus
distance at various time intervals following the injection TABLE 2
DETEBMIHATION OF THE SPECIFIC ACTIVITY OP DIHYDROQUINIDINE-H^ (DHQ-H^) USED IN EXCRETION STUDIES
Amount of DHQ-H^ in Counts Effic Disintegrations Disintegrations Microcuries the Vial per iency per Minute per Minute per (meg) Vial Minute Factor in Sample per meg Microgram
0.1 1 16465.7 5.3 87268.2 872682.0 0.393
0.1 2 16143.3 5.4 87173.8 871738.0 0.392
0.2 1 33692.8 5.2 175202.5 876012.5 0.394
0.2 2 32251.2 5.3 170931.3 854656.5 0.384
0.5 1 74434.2 5.7 424274.9 848549,8 0.382
0.5 2 75198.4 5 .7 428630.8 857261.6 0.386 Efficiency factor determined from the external standardization curve of the machine.
Specific activity of Dlhydroqulnldlne-H^ = .ÇM fagtor x.Welgit _ 3.7 X 10 X 60 DPM
Average = O.389.
53 TABLE 3
CONDUCTION TIME IN MILLISECONDS (msec) FROM THE STIMULUS TO EACH RECORDING POINT (l-l4) 1 mm APART DURING DIFFERENT TIME INTERVALS AFTER DHQ ADMINISTRATION, EXPERIMENT #1
Recording ' *“ Points______1 2 3 4 5 6 7 8 9 10 11 12 13 l4 Conduction time (msec) Control 8 l6 20 25 28 28 31 39 4l 42 45 45 4? 47
30 seconds
1 minute
2 minutes 11 18 24 34 4l 47 56 57 6l 67 68 68 68 68
4 minutes 8 12 17 24 32 38 46 53 55 57 64 65 66 66
8 minutes 13 18 23 28 35 39 43 46 48 54 55 57 58 58
16 minutes 13 19 21 27 34 37 44 49 54 58 58 59 59 59
32 minutes 12 18 25 29 34 37 43 46 49 49 53 55 56 56
64 minutes 12 17 22 27 33 37 43 44 53 53 53 53 53 53
o\ o TABLE 4
CONDUCTION TIME IN MILLISECONDS (msec) FROM THE STIMULUS TO EACH RECORDING POINT (l-l4) 1 mm APART DURING DIFFERENT TIME INTERVALS AFTER DHQ ADMINISTRATION, EXPERIMENT #2
Recording Points 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Conduction time (msec) Control 10 l4 20 22 30 33 35 4o 42 45 48 52 56 60
30 seconds 16 23 30 37 43 50 56 57 65 68 73 76 77 77 1 minute
2 minutes 69 74 78 79 80 80 81
4 minutes 14 15 19 22 30 38 44 52 59 61 66 68 68 68
8 minutes 8 15 20 23 28 37 44 52 59 61 66 66 68 70 l6 minutes 12 14 21 28 34 39 46 55 60 61 65 65 65 67
32 minutes — —
64 minutes
o\ H TABLE 5
CONDUCTION TIME IN MILLISECONDS (msec) PROM THE STIMULUS TO EACH RECORDING POINT (l-l4) 1 mm APART DURING DIFFERENT TIME INTERVALS AFTER DHQ ADMINISTRATION, EXPERIMENT #3
Recording Points 1 2 3 ^ 5 6 7 8 9 10 11 12 13 Conduction time (msec) Control 5 10 l6 20 23 30 37 42 4$ 51 53 56 56 56
30 seconds 10 l4 20 26 33 35 4l
1 minute 20 28 34 4o 47 58 68 78 80 85 88 95 96 97 2 minutes
4 minutes
8 minutes
16 minutes
32 minutes 18 25 34 36 4o 46 50 57 6l 65 67 67 69 70 64 minutes 9 l4 21 25 27 34 Ao 45 50 52 55 57 60 60 TABLE 6
CONDUCTION TIME IN MILLISECONDS (msec) FROM THE STIMULUS TO EACH RECORDING POINT (l-l4) 1 mm APART DURING DIFFERENT TIME INTERVALS AFTER DHQ ADMINISTRATION, EXPERIMENT #4
Recording Points 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Conduction time (msec) Control 10 13 20 25 29 34 4l 50 50, 50 50 50 50 50
30 seconds 10 12 19 25 31 37 43 64 69 74 78 78 78 78
1 minute 15 20 25 34 4o 50 6o 70 72 79 81 81 81 81
2 minutes 15 20 25 34 43 52 65 70 75 82 82 82 82 82
4 minutes 13 19 24 28 42 51 6o 68 73 76 76 76 76 76
8 minutes 10 l6 22 31 4o 48 58 65 70 70 70 70 70 70 l6 minutes 10 15 22 27 36 45 55 60 62 62 62 62 62 62
32 minutes 9 13 15 21 33 42 47 53 55 55 55 55 55 55
64 minutes 9 13 20 26 34 38 49 52 52 52 52 52 52 52
cy\ U) TABLE 7
CONDUCTION TIME IN MILLISECONDS (msec) PROM THE STIMULUS TO EACH RECORDING POINT (l-l4) 1 imn APART DURING DIFFERENT TIME INTERVALS AFTER DHQ ADMINISTRATION, EXPERIMENT #5
Recording Points 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Conduction time (msec) Control 9 13 16 20 23 27 31 33 38 4o 42 44 45 45
30 seconds 10 13 16 20 25 27 31 44 49 54 54 60 65 69
1 minute 18 21 25 29 33 38 42 68 72 78 80 86 89 92
2 minutes 18 21 27 25 35 39 48 49 53 58 63 66 76 76
4 minutes 16 21 25 28 32 4o 45 51 56 62 67 69 73 76
8 minutes 14 16 21 25 32 39 44 49 54 60 64 70 70 72 l6 minutes 15 20 26 31 34 45 46 52 57 63 68 72 75 75
32 minutes 11 l6 24 26 31 36 43 48 52 56 60 64 65 66
64 minutes 14 20 26 32 35 4l 48 53 55 59 62 65 65 65
© 65
Figure 6.— Slectrograns recorded front 14 points on the epicardium during control pefiod and at 30 seconds, 1 minute and two minutes after intravenous injection of DHQ (10 mg/kg body weight).
Electrograms from 1 to 14 points of the recording electrode during control and post injection periods are shown from top to bottom in their respective columns. Vertical time pips occur every 5 msec. Stimulus artifact is seen as a square wave at the beginning of some of the traces. The nadir of the rapid negative-going deflection signals the time of arrival of the induced activation process at the area subjacent to the electrode.
Potential S.F in horizontal columns shows the time of stimulus application for the electrograms recorded at points 1 through 7 on the recording electrode for each vertical column.
Potential S.B in horizontal columns similarly shows the time of stimulus application for the electrograms recorded at point 8 through 14 on the recording electrode for each vertical column. Time pips in electrograms 1-7 are synchronous with those on S.F and time pips of 8-14 are synchronous with those on S.B within each vertical column.
Notice the increase in the duration between stimulus artifact and the nadir of the deflection (conduction time) at all points within 30 seconds and marked effect at 1 and 2 minutes. 66
CONTROL 30 SECONDS 1 MINUTE 2 MINUTES 67
Figure 7,— Continuation of the Figure 6, Electrograms recorded during 4, 8, 32, and 64 minutes post injection period of DHQ administration are shown. Notice the increase in the conduction time persisting till 64 minutes. 6$
I Z S = MINUTES 32 MINUTES 64 MINUTES 69 of the drug. The best fits were sets of two linear models for the control and for each post injection time interval. Figure 8 is the example for this representation.
The fiist linear model of each set represents the data derived from two to nine millimeters distance from the stimulating electrode. The second linear model conforms to the data derived from ten to fifteen millimeters away from the stimulus.
The formula assumed to be the model for this system was Y - a + bx, in which Y is equal to the conduction time in milliseconds, x is equal to the electro- dal distance from the stimulus, a is the Y intercept and b is the slope of the line or the conduction time for
the excitatory wave to travel one millimeter.
The conduction velocity (expressed as mm/sec) was calculated by the following formula; conduction velocity •» b The estimates of a and b are, shown in Table 8
along with the calculated conduction velocities and
their percent reduction from the control during various
post injection periods.
Distribution of DHQ in Various Body Compartments
Table 9 shows the details of dosage of unlabeled
dihydroquinidine and DHQ-H^ used in various distribution 70
Figure 8,--Graphic representation of distance between electrodes and stimulus artifact (2-13 mm) against the conduction time (msec) between the stimulus artifact and the nadir or peak of the electrograms from each bipolar electrode* The records are shown both before and during various post injectional periods of DEQ. 90-1 71
80 H
70 H
32 M. 64 M. to 604 3 3 3 2 CONTROL I— to 50 H Q: LÜ h- Li. < iim U 404 ÜJ to
304
204
T T T T T- —I 2 4 6 8 10 16 Mm. FROM STIMULUS TABLE 8
ESTIMATES OF THE CONDUCTION TIME AND CONDUCTION VELOCITY AT VARIOUS TIME INTERVALS IN RELATION TO ELECTRODAL DISTANCE FOLLOWING INTRAVENOUS ADMINISTRATION OF DHQ
2 to 9 mm 10 bo 1 5 ram j/er uent or Per Cent or Time Reduction in Reduction in Post Estimate Conduction Conduction Estimate Estimte Conduction Conduction Injection Of . Velocity Velocity of Velocity Velocity fminutes) (b) S.D. mm/sec from Control (a) (b) S.D. ram/sec from Control(a)* Control -0.22 4.48 0.22 223.2 26.81 1.7 0 0.5 588.2 1/2 -1.99 5.89 0.53 169.7 23 .9 34.95 2.7 2 1,15 367,6 3 5 .9 1 -0,31 7.34 0.63 136.2 38.9 46.55 3.0 0.76 333,3 43 .2 2 -0.39 6.69 0.47 149,4 33.6 47,25 2 .0 5 0.95 487,8 17.0 4 -2.79 6.34 0.4o 157.7 29 .3 40.58 2 .1 5 0.69 467,9 20.6 8 -2.21 6.09 0.34 164.2 2 5 .5 40.00 1 .9 0 0,75 526.3 10,6 16 -0.98 6.04 0.23 165,5 2 5 .4 44.60 1 ,4 7 0.57 680.3 -15,5 32 0.31 5.45 0.24 183.4 17 .9 39.46 1 .5 4 0.70 649.7 -10.4
64 1.2 6 5.50 0.36 181.8 18.0 - - — — — —
*a is the y intercept, and b is the slope of the line for the mathematical formula y = a + bx, in which y is equal to the conduction time in milliseconds, and x is the electrodal distance from the stimulus.
13 TABLE 9
SHOWING THE DOSAGE OP DHQ AND DHQ-H^ IN EACH EXPERIMENT OF 64 MINUTES DISTRIBUTION STUDIES
Total Dose of DHQ Total Dose of Weight of Tritiated Disintegrations Rate of Infusion of the Animal Unlabeled Labeled per Minute 5 Per Cent Mannitol Experiment (kg) (mg) Cu. c ) of Tritium (drops per minute)
1 9.5 95.0 142.5 3 .1 6 X 108 40
2 11.3 113.0 169.5 3 .7 6 X 108 45
3 9.5 95.0 142.5 3 .1 6 X 10® 40
4 1 2 . 0 1 2 0 .0 180.0 3 .9 9 X 10® 45
5 12.7 127.0 190.5 4.2 X 10® 46
O Specific activity of DHQ-H = 0.482 microcuries/micrograin.
1 microcurie = 3.7 x 10 x 60 DPM. 74 experiments for 64 minutes. The total dose of DHQ has been shown in milligrams and that of DHQ-E^ in microcuries, along with its disintegrations per minute value. Specific activity of DHQ-H^ employed in these experiments was
0.482 microcuries per microgram.
Disappearance of DHQ from plasma.— The rate of disappearance of DHQ from arteirial, venous and coronary sinus in blood samples was determined in five dogs at one half, one, two, four, eight, sixteen, thirty-two and sixty-four minutes following the intravenous administra tion of DHQ and DHQ-H^ mixture. Table 10 gives the individual data on the arterial, venous and coronary sinus concentrations in experiments 1 to 4. In experiment
No. 5 (Table II) the concentrations for arterial. Venous and coronary sinus were exceptionally high and will be discussed separately.
In Tables 12 to 14 the results on four experiments in the form of the number of samples, average concentration of DHQ per milliliter of plasma and the average percent of the total dose per 100 ml of plasma at each time inter val for each of arterial, venous and coronary plasma samples have been presented, Ihe values of disintegration per minute were converted to micrograms and calculations for this conversion have already been shown. The mean value in micrograms for each time interval has been shown with a range of minimum to maximum and the standard deviation. TABLE 10
ARTERIAL, VENOUS AND CORONARY SINUS PLASMA CONCENTRATIONS OF DHQ IN EXPERIMENTS 1 TO 4 DURING DIFFERENT TIME INTERVALS
Experiment 1 Experiment 2 Experiment 3 Experiment 4 Time S ^ C T T 7 6 X 7 “ IS X 7 CT“ (min.) mcg/ml mcg/ml mcg/ml mcg/ml mcg/ml mcg/ml mcg/ml mcg/ml mcg/ml mcg/ml mcg/ml mcg/ml
1/2 16.7 8.0 6.8 25.9 13.9 12.9 — — 13.7 — — 39.5 2.5 17.4
1 8,7 6.1 6.6 l6.0 10.5 10.6 27.5 9.4 11.5 15.0 5.9 10.7
2 6.4 4.5 5.9 13.2 8.3 12.2 13.4 9.1 11.3 9.3 4.7 8.3
4 5.5 4.5 5.2 9.6 7.5 8.7 7.4 9.9 6.8 3.6 8.0
8 4.7 3.3 4.6 7.7 5.5 5.6 7.8 5.4 7.4 6.0 4.0 7.9
16 4,2 3.2 3.8 6.6 3.2 6.0 6.7 5.0 6.6 5.8 4.0 5.9
32 3.6 2.8 3.0 5.4 4.1 5.2 5.8 4.9 5.2 5.6 4.2 5.1
64 3.4 3.0 3.4 5.1 2.9 4.4 5.2 4.2 4.4 4.4 4.1 3.9
A = Arterial, V = Venous, C - Coronary Sinus.
ui 76
TABIE 11
ARTERIAL, VENOUS, AND CORONARY SINUS PLASMA CONCENTRATIONS OF DHQ IN EXPERIMENT 5 DURING DIFFERENT TIME INTERVALS
Time A V C (minutes ) mcg/ml mcg/ml mcg/ml
1/2 100.9 28.4 37.7
1 55.5 24.0 37.7
2 33.5 21.9 29.9
4 ' 25.9 19.3 2 6 .1 8 24.3 l4.i 2 6 .8 16 21.1 14.1 24.2
32 17.4 13.7 17.8
64 16.4 11.8 18.5
A = Arterial, V = Venous, C = Coronary sinus. TABLE 12
ARTERIAL PLASMA CONCENTRATIONS OF DIHYDROQUINIDINE
meg of DHQ/ml Number S.D. Average Per Cent Time of Sanç>les Minimum Maximum Average + of Total Dose/lOO ml
1/2 minute 3 16.7 39.5 27.3 +11.4 2.6
1 minute . 4 8.7 27.5 l6.8 ± 7.8 1.6
2 minutes 4 6.4 13.4 10.5 + 3.3 1.0
4 minutes 4 5.5 9.6 7.6 ± 1.8 0.7
8 minutes 4 4.7 7.8 6.4 ± 1.6 0.6 l6 minutes 4 4.2 6.7 5.8 ± 1.1 0.5 32 minutes 4 3.6 5.8 5.1 + 1.0 0.47
64 minutes 4 3.4 5.2 4.5 + 0.8 0.42
->3 TABLE 13
VENOUS PLASMA CONCENTRATIONS OF DIHYDROQUINIDINE
meg of DHQ/ml Number S.D. Average Per Cent Time of Sanç>les Minimum Maximum Average ± of Total Dose/100 ml
1/2 minute 4 2.5 13.9 9.5 +5.4 0.9
1 minute 4 5.9 10.5 7.9 +2.3 0.7
2 minutes 4 4.5 9.1 6.6 +2.4 0.6
4 minutes 3 3.6 7.4 5.1 +1.9 0.5
8 minutes 4 3.3 5.5 4.5 +1.1 0.4 l6 minutes 4 3.2 5.0 3.8 +0.8 0.38
32 minutes 4 2.8 4.9 4.0 +0.9 0.39
64 minutes 4 2.9 4.2 3.5 +0.7 0.33
00 TABLE 14
CORONARY SINUS PLASMA CONCENTRATIONS OP DIHYDROQUINIDINE
Number mcg of DHQ/ml S.D. Average Per Cent Time of Sangles Minimum Maximum Average + of Total Dose/100 ml
1/2 minute 3 6.8 17.4 12.4 +5.2 1.1
1 minute 4 6.6 11.5 9.8 +2.2 0.9
2 minutes 4 5.9 12.2 9.4 +2.9 0.8
4 minutes 4 5.2 9.9 7.6 +1.9 0.7
8 minutes 4 4.6 7.9 5.6 +1.6 0.5 l6 minutes 4 3.8 6.6 5.5 +1.6 0.5
32 minutes 4 3.0 5.2 4.5 +1.0 0.45
64 minutes 4 3.4 4.4 4.0 +0.5 0.4 80 The peak levels in arterial, venous and coronary sinus samples were reached in one half minute time. The range of peak levels in the arterial samples was 16.7 to
39.5 mcg/ml, in the venous samples was 2.5 to 13.9 mcg/ml and in the coronary sinus samples was 6.8 to 17.4 mcg/ml.
The levels in arterial samples fell abruptly within the first two minutes and thereafter, the fall was gradual with a plateau within 16 to 32 minutes. The levels in the venous and coronary sinus samples, however, fell gradually with a plateau approximately at the same time as that of arterial sample. At the end of 64 minutes, the average concentrations in arterial, venous and coronary sinus samples were 4.5, 3.5 and 4.0 mcg/ml respectively.
Various mathematical models for the disappearance of arterial, venous and coronary sinus concentrations against time were made. In these models Y = concentration of DHQ in arterial,venous andcoronary sinus plasma sample, a ■ Y-intercept, b ■ slope of the line and x = time. The models fitted were:
1. Y " a + bx
2. Y “ a + b
3. log of Y » a + bx The best fit was No. 3. Figures 9 to 11 are the graphic representations for this model as it applies to the disappearance of DHQ 81
Figure 9,— Disappearance rate of dihydro quinidine from the arterial blood plasma. 82
2.6 < I/) < 0. U_ o
£ ARTERIAL O O
_j < I- z
Ll. O u o z ÜJ u a: lij 1.4 M IN U TES CL
O 10 20 30 40 5 0 60 64 MINUTES 83
Figure 10.•— Disappearance rate of dihydro quinidine from venous blood plasma. PERCENTAGE OF INITIAL DOSE PER 100 ml. OF PLASMA
w 01 O) 00 CD O —4 . _l_ ^ ' n »»•*
0) I
< N) m m o t/i z O c z (/) c CO H O m c/i
O
01 o
0) o CO 2 85
Figure 11.— Disappearance rate of dihydro quinidine from coronary sinus blood plasma. 86
< 2 (/) <
ü_ O
CORONARY — O O
û: ÜJ CL ÜJ to O û
ü_ O ÜJ O g Z !Tk= 2.6 MIN UTES ÜJ U CL ÜJ CL 20 30 40 50 60 64 MINUTES 87 from the plasma of arterial, venous and coronary sinus samples respectively. The percent of the dose remaining in the plasma is plotted against time on a semi-logarithmic scale. An average figure of 4 experiments was taken.
These graphs reveal that the resulting curves can be resolved into two straight line components of different slopes.
For the determination of biological half-lives from the arterial, venous and coronary sinus plasma disappearance graphs, the line B in each case was extended to the ordinate and the extrapolated values for one half, two and four minutes were subtracted from the actual values (line A)• The dotted line C drawn through these points was used to determine the biological half life before equilibration with the tissues. According to the calculations made from the slope of these lines, the biological half life of the faster component is approxi mately 1.4 minutes for arterial, 1.6 minutes for venous and 2.6 minutes for coronary blood samples. The biological half life for the slow component was calculated by extending the line B toward abscissa. It was 187 minutes for arterial, 232 minutes for venous and 140 minutes for the coronary sinus blood samples.
A comparative rate of disappearance of DHQ from arterial, venous and coronary vascular system at different 88 time Intervals has been shown in Figure 12. Arterial and coronary sinus levels were essentially similar at 16 to
64 minutes, with levels ranging from 5 to 5.8 mcg/ml of plasma representing 0.4 to 0.5 percent of the total dose per 100 ml of plasma.
In experiment 5 (Table 11) which was not included in Tables 12 to 14, the plasma concentrations of DHQ in arterial, venous and coronary sinus samples were exception ally high. Ttie peak levels attained in one half minute in arterial, venous and coronary sinus samples were 100.9,
28,4 and 37.7 mcg/ml respectively. The pattern of disappearance of the drug from the plasma was quite comparable with other experiments. The arterial and coronary sinus levels were essentially similar at 4 minutes, but after that coronary sinus levels were slightly higher than arterial. At the end of 64 minutes, the arterial, venous and coronary sinus levels were 16,4,
11.8 and 18.5 mcg/ml respectively.
The average (4 experiments) differences of concentrations of DHQ in arterial and coronary sinus and arterial and venous blood samples at different time intervals are shown in Figure 13. The arterial-coronary sinus difference declined very sharply within two minutes and at 4 minutes, it was slightly below 0. After this, the difference, though insignificant, gradually increased with the time. The arterial-venous difference declined gradually. 89
Figure 12.— Composite graph showing the compara tive rates of disappearance of DHQ from the arterial, venous and coronary sinus blood plasma. 90
2.6
_J 0_
ARTERIAL CORONARY VENOUS
1.0 -
_J
10 20 30 40 50 GO 64 MINUTES 91
Figure 13.— Illustrates the comparison between arterial-venous and arterial-coronary sinus differences in plasma concentration at different time intervals. 10-1
ARTERIAL-CORONARY SINUS DIFFERENCE 0: ÜJ % % ARTERIAL-VENOUS DIFFERENCE
% % % (T y 5
I/) 2 < % q : I g O O ; q : U I I % I %
8 16 32 64 MINUTES 93
Tissue distribution
64 minutes,— Five dogs studied earlier for the effect of DHQ on conduction velocity and its disappearance
from the plasma were sacrificed at the end of 64 minutes and 14 different tissues were removed for distribution studies*
The concentration of DHQ in the tissues for all
the expe;riments expressed in microgram/gram of tissue, and
tissue to plasma ratio is shown in Table 15. In Table 16,
the number of samples, average concentration (mcg/gm) with a range (minimum to maximum), standard deviation and
average percent of the total dose per 100 grams of the
tissue is shown.
The tissue to plasma ratio was calculated for each
experiment as:
Cone, of DHQ/gm of tissue » mcg/gm (tissue) Cone, of DHQ/ml of plasma mcg/ml (plasma)
The average tissue to plasma ratio shown in the
Table 16 was taken from four experiments and experiment
5, where plasma concentration was exceptionally high is
not included. The percent of total dose per 100 grams
of each tissue was calculated as follows:
mcg/100 gm V Percent total dose/100 gm. = — " v '"-.'" ---- :--- r ^ total dose (meg) TABLE 15 TISSUE DISTRIBUTION OF DHQ AT 64 MINUTES POST INJECTION IN EXPERIMENTS 1 THROUGH 5
Experiment 1 Experiment 2 Experiment 3 Experiment 4 Experiment 5 ÿissue Tissue Tissue Tissue Tissue Name of DHQ Plasma DHQ Plasma DHQ Plasma DHQ Plasma DHQ Plasma No. the Tissue mcg/gm Ratio mcg/gm Ratio mcg/gm Ratio mcg/gm Ratio mcg/gm Ratio 1 Plasma 3.4 1.0 5.1 1.0 5.2 1.0 4.4 1.0 16.4 1 2 Left Auricle 9.0 2.6 17.7 3.5 13.2 2.5 12.5 2.8 15.8 0.9 6 3 Left Ventricle 17.0 5.0 17.0 3.3 17.0 3.3 12.9 2.9 17.0 1.0 4 Right Auricle 9.4 2.7 13.8 2.7 15.8 3.0 13.5 3.0 14.1 0.86 5 Right Ventricle 17.6, 5.2 21.8 4.3 14.8 2.8 11.8 2.7 15.5 0.9 4 6 Liver 23.5 6.9 24.9 4.9 42.2 8.1 24.0 5.4 3 0 ,8 1.9
7 Lung — — 36.2 7.1 — — — - 36.3 8.2 28.1 1.7 8 Spleen 27.9 8.2 30 .2 5.9 29 .0 5.6 19.2 4.4 23.5 1.4 9 Adrenal 26.4 7.8 26.7 5.2 25.9 5.0 13.4 3.0 21.4 1.3
10 Kidney 39.6 11.6 40.1 7.9 30.9 5.9 — — *“"• 25.4 1.5 11 Ovary 10.3 3.0 12.7 2.4 10.6 1.9 10,4 2.4 7.1 0.43 12 Uterus 8.7 2.5 7.0 1.4 6.4 1.2 7.3 1.6 7.8 0.57 13 Thyroid — — 5.0 0.9 7.5 1,4 3.7 0.84 7.3 0.44 14 Brain 3.4 1.0 4.4 0.86 4.6 0.88 5.2 1.1 5.5 0.33
15 Skeletal Muscle — — — - 3.9 0.76 4.3 0.83 3.7 0.84 3.2 0.2 TABLE 16
TISSUE DISTRIBUTION OF DHQ AT 64 MINUTES POST INJECTION
meg of DHQ per Number gram of Tissue Average Mean Tissue Name of of S.D. Per Cent of to No. the Tissue Animals Minimum Maximum l^ean + Dose/100 ml Plasma Ratio 1 Plasma 4 3.4 5.2 4.5 40.83 0.42 1.0
2 Left Auricle 5 9 .0 17.7 13.6 ±3 .3 1.5 2 .8
3 Left Ventricle 5 12.9 1 7 .0 16.1 +1.8 1.8 3.6
4 Right Auricle 5 9.4 1 5 .8 13.3 ±2.5 1.5 2.8 5 Right Ventricle 5 11.8 21.8 16.3 ±3.7 1.8 3.7 6 Liver 5 2 3 .5 42.4 2 9 .1 ±7.9 2.6 6.3 7 Lung 3 28.1 36.3 33.5 +6.6 2.8 7.6 8 Spleen 5 19.2 3 0 .2 2 5 ,9 ±4.5 2 .9 6 .0 9 Adrenal 5 1 3 .4 2 6 .4 22.7 +5.6 2.5 5.2 10 Kidney 4 2 5 .4 40.1 34.0 +7.1 3.1 8.4 11 Ovary 5 7.1 12.7 10.2 +2.0 1.1 2.4 12 Uterus 5 6.4 8.7 7.4 40.26 0.82 1,6 13 Thyroid 4 3.7 7.5 5.8 +1.8 0.5 1 1 .0 l4 Brain 5 3.4 5.5 4.6 40.26 0.51 0,95 15 Skeletal Muscle 4 3.2 4.3 3.7 +0.13 0.32 0.80 vo 96 6 hours,— The three dogs (6 to 8) employed for biliary excretion studies were sacrificed at the end of six hours. Ten tissues, namely: left auricle, left ventricle, right auricle, right ventricle, liver, lung, spleen, adrenal, kidney and thyroid were selected for distribution deter mination. The results are tabulated in Tables 17 and 18.
The tissues were grouped into three general groups for inter-tissue comparison and also for time-interval comparison of concentration in them . Duncan * s Multiple
Range Test was also applied on the various tissue concen trations at 64 minutes and 6 hours.
Group 1 represents cardiac, smooth and skeletal muscles and their comparative distribution are shown in
Figure 14. The cardiac tissue contained a higher concentration than the smooth and skeletal muscle. Smooth muscle of the uterus contained a greater concentration than skeletal muscle. Among cardiac tissues, the ventricles had a higher concentration than the auricles.
No sample was taken from smooth and skeletal muscle at the end of 6 hours, therefore no comparison in these tissues can be made at these two time intervals. The tissue to plasma ratio dropped from a high value at 64 minutes in auricular and ventricular tissues to lower values at
6 hours. TABLE 17
TISSUE DISTRIBUTION OF DHQ AT 6 HOURS POST INJECTION IN EXPERIMENTS 6 THROUGH 8
Experiment 6 Experiment 7 Experiment 8 Tissue Tissue Tissue Name of DHQ Plasma DHQ Plasma DHQ Plasma No. the Tissue mcg/gm Ratio mcg/gm Ratio mcg/gm Ratio 1 Plasma 6,0 1.0 4.1 1.0 5.2 1.0 2 Left Auricle 7.2 1.2 6.4 1.7 10.8 2.0 3 Left Ventricle 8.2 1.3 9.2 2.2 13.1 2.5 4 Right Auricle 6.9 1.1 6.9 1.7 11.6 2.2 5 Right Ventricle 7.0 1.1 9.3 2.3 11.9 2.3 6 Liver 24.4 4.0 17.0 4,1 21.2 4.1 7 Lung 31.1 5.2 28.1 6.8 30.5 5.8 8 Spleen 10.0 1.7 10.2 2.5 11.1 2.1 9 Adrenal 12.0 2.0 10.2 2.5 13.5 2.5 10 Kidney 24.0 4.0 13.4 3.3 19.8 3.8 11 Thyroid 3.8 0.63 3.8 0.9 4.0 0.72
VO -4 TABLE 18
TISSUE DISTRIBUTION OF DHQ AT 6 HOURS POST INJECTION
Mean Number Micrograms per Gram of Tissue Average Per Cent Tissue Name of of of the Total Dose S D to Plasma the Tissue Animals Minimum Maximum Mean * * per 100 gm tissue No. + Ratio
1 Plasma 3 4.1 6,0 5.1 +0.93 0.5 1.0
2 Left Auricle 3 6.4 10.8 8.1 +2.3 0.76 1.6
3 Left Ventricle 3 8.2 13.1 10.1 +2.5 0.95 2.0
4 Right Auricle 3 6.9 11.6 8.4 +2.7 0.79 1.7
5 Right Ventricle 3 7.0 11.9 9.4 +2.4 0.88 1.9
6 Liver 3 17.0 24.4 20.8 ±3.7 1.96 4.0
7 Lung 3 28.1 31.1 29.9 +0.93 3.2 5.9
8 Spleen 3 10.0 11.1 10.4 ±0.55 0.98 2.3
9 Adrenal 3 10.2 13.5 11.9 +1.6 1.1 2.3
10 Kidney 3 13.4 24.0 19.0 ±5.3 1.8 3.7
11 Thyroid 3 3.8 4.0 3.8 40.0 0.35 0.75
% 99
Figure 14.— The distribution of DHQ in cardiac, smooth and skeletal muscular tissues at 64 minutes and 6 hours post injectional periods. loa
I I 64 MIN UTES
1 5 6 HOURS
TISSUE PLASMA RATIO LiJ Z> (/) I/) li. O 2 < 10- rIO tr O £T U CL < to CL 2 < 0: < O 2 O CL < U 5 - - 5 a. 2 LÜ Z 3 U) to I-
- 1
LEFT RIGHT LEFT RIGHT SMOOTH SKELETAL VENTRICLE VENTRICLE AURICLE AURICLE MUSCLE MUSCLE 101
In Figure 15, a comparison of the distribution in
Group 2, consisting of the excretory organs (liver, kidney and lung) and spleen is made• The kidney contained the highest concentration in this group, followed by the lung and liver and then the spleen. The rate of decrease in both tissue concentration and tissue to plasma ratio at the end of 6 hours is most marked in the spleen and kidney and the least in the lung. Comparatively, concentrations in the kidney had decreased more than liver concentrations at the end of 6 hours.
The various endocrine glands (adrenal, thyroid and ovary) and brain constitutes Group 3. Their comparative distribution is shown in Figure 16. The concentration in ovary and brain were determined for 64 minutes only. The concentration in brain was the least.
The concentration in the ovary was greater than in the thyroid but less than in the adrenal. The drop in tissue to plasma ratio from 64 minutes to 6 hours was more marked in the adrenal than in thyroid.
Excretion of DHQ
Renal excretion.— The amount of DHQ excreted in the urine was determined in the sixty-four minutes distribution experiments. Table 19 shows the individual data on the cumulated amount of the drug excreted in five, fifteen, thirty and sixty-four minutes. The cumulated 102
Figure 15.— The distribution of DHQ in various excretory organs and spleen at 64 minutes and 6 houre post injectional periods. 103
3 5-1 64 MINUTES
6 HOURS
3 0 - TISSUE PLASMA RATIO
Lü ZD U) CO 2 5 - h~ i Li_ % O 2
< 2 0 - ce % O
Q: UJ CL % 15- ri 5 co o < I- CL < O CL O CL < U 10- -1 0 2 co < _J I CL R 5 - -5 UJ z> co co
0 I •o LIVER KIDNEY LUNG SPLEEN 104
Figure 16.— The distribution of DHQ in various endocrine organs and brain at 64 minutes and 6 hours post injectional periods. 105
25-1
64 MINUTES
111 6 HOURS =) CO 20 - œ h- TISSUE PLASMA RATIO ü_ O 2 < 15- ce O ce ÜJ O CL !< co 1 0- r10 ce 2 < < ce 2 CD co O < ce U 5- - 5 2 W 3 CO
O 0 H ADRENAL THYROID OVARY BRAIN TABLE 19
CUMULATIVE RENAL EXCRETION OF DHQ DURING 64 MINUTES
0-5 Minutes 0-15 Minutes O-30 Minutes 0-64 Minutes Per Cent Per Cent Per Cent Per Cent Amount of Total Amount of Total Amount of Total Amount of Total Experiment (mgs) Dose (mgs) Dose (mgs) Dose (mgs) Dose
1 0.040 0.042 0.89 0.84 1.7 1.8 3.5 3,7
2 0.045 0.047 o.4i 0.43 1.15 1.2 2.6 2.7
3 0.12 0.1 1,5 1.25 4.6 3.8 6.3 5,2
4 0.01 0.007 0.55 0.43 1.1 0.9 2.1 1,7
Mean 0.052 0.049 0.81 0.73 2.13 1.92 3.62 3,3
H o o\ 107 amount of the drug excreted has been shown in milligrams and also as the percent of the total dose excreted• An average of 3.3 percent of the total dose was excreted at the end of 64 minutes.
Renal clearance.— To determine the mechanism of excretion by the kidney, renal clearance studies were performed on seven dogs. In these experiments clearance of the non-metabolite (N.M.) and the metabolite CM) of the drug at varying intervals was studied. The details of dosage of unlabeled and labeled DHQ for each experiment is given in Table 20. In experiment 7, a comparatively lower dose of the unlabeled drug was used in order to compare the pattern of clearance with the higher dose.
The expected total amount filtered was determined by assuming that 80 percent of the drug is bound with plasma proteins, and 20 percent is unbound (7, 8).
G.F.R, X plasma cone, Expected total amount filtered « 5
The denominator 5 is the resultant of multiplying 20 G.F.R. by 20 percent unbound drug ( ). Similarly, the amount reabsorbed was determined as expected amount which was calculated as;
Expected amount reabsorbed - Expected total amount filtered minus amount in urine. TABLE 20 DOSAGE OF DHQ AND DHQ-H^ FOR EXPERIMENTS ON EXCRETION (RENAL AND BILIARY)
Rate of Weight Total Dose of Duration Infusion of of Nature of Tritiated Disintegrations of 5 Per Cent Animal Excretion Unlabeled Labeled per Minute of Experiment Dextrose Experiment (kg) Studies ... (mg).... (-Me) Tritium (hours) (drops/min. Renal 1 10.9 Clearance 109.0 163.5 3.6 X 10® 3 22 It 2 9 .1 91.0 136.5 3 .0 X 10® 6 20 It 3 11 .8 118.0 177.0 3.9 X 10^ 3 24 It 4 10 .4 104.0 156.0 3.5 X 10^ 3 22 II 5 9 .5 95.0 142.5 3 .2 X 10^ 3 20 II 6 9 .1 91.0 136.5 3 .0 X 10^ 3 18 II 7 1 1 .4 — — 388.6 8 .6 X 10® 3 24 Biliary 91.0 3 .0 X 10® 6 20 8 9.1 and Renal 136.5 II 9 9.5 95.0 142.5 3 .2 X 10® 6 20 II 10 10.0 100.0 150.0 3 .3 X 10& 6 20 Specific activity of DHQ-H^ = O .389 microcuries/microgram. 1 microcurie = 3.7 x 10^ x 60 DPM.
ë CO 109 The clearance of the nonmetabolite and metabolite was calculated as:
Amount in urine (rocg/min/kg) Clearance = Plasma concentration (mcg/ml)
Table 21 to 27 give the individual data on renal clearance for all seven dogs. In all the experiments clearance were run after an equilibration period of sixty minutes. In experiments 1 to 3, the clearances were run at thirty minute intervals for a total period of three hours, except in experiment No, 2, where it was run for six hours. In experiments 4 to 6, the renal clearances were run at hourly intervals for a total period of three hours.
Cumulative renal excretion.--The data on individual
experiments on cumulative renal excretion for six hours
is shown in Table 28. These figures were taken from
experiments on renal clearance. It is apparent from this
table that at the end of six hours, the total amount of
the drug excreted was 23.6 percent of the total dose.
Biliary excretion.--The studies on biliary
clearance for six hours were made in three dogs. Urine
was also collected in these experiments for cumulative
renal excretion studies. Table 20 ( No. 8 to 10) gives
the details of dosage for unlabeled and labeled DHQ used TABLE 21
RENAL CLEARANCE STUDIES ON DHQ, DOG 1
Nature Plasma Expected Amount in Expected of the Cone. G.F.R. Total Amt. Urine Amount Clearance Time Drug mcg/ml ml/min/kg Filtered* mcg/mln/kg Reabsorbed* ml/min/kg
60 N.M. 15.6 5.8 18.1 5.5 12.6 0.35 Minutes M. 0.2 0.25 2.3 -2.05 11.5
90 N.M. 14.6 4.4 12.8 4.2 8.6 0.25 Minutes M. 0.7 0.61 3.2 —2 « 6 4.6
120 N.M. 13.6 5.7 15.5 1.65 13.85 0,12 Minutes M. 0.8 0.92 1.65 -0.73 2.0 150 N.M. 12.6 5.8 14.6 4.0 10.6 0.32 Minutes M. 1.0 1.7 2.1 —0.4 2.1
180 N.M. 11.7 5.5 12.8 3.6 9.2 0.31 Minutes M. 1.6 1.7 2.0 -0.3 1.25
,*See the text. N.M. = Nonmetabolite. M. = Metabolite.
H ë TABLE 22 RENAL CLEARANCE STUDIES ON DHQ, DOG 2
Nature Plasma Expected Amount In Expected of the Cone. G.F.R. Total Amt. Urine Amount Clearance Time Drug mcg/ml ml/mln/kg Filtered* mcg/mln/kg Reabsorbed* ml/mln/kg 60 N.M. 4.0 6.3 5.0 1.9 3.1 0.47 Minutes M. 0.2 0.26 3.3 -3.0 16.5
90 N.M. 3.2 5.8 3.7 1.4 2.3 0.44 Minutes M. 0.4 0.5 3.1 -2,6 7.7
120 N.M. 2.3 5.0 2.3 1.1 1.2 0.47 Minutes M. 0.7 0.7 2.0 -1.3 2.8
150 N.M. 2.1 5.6 2.3 1.2 1.1 0.57 Minutes M. 0.5 0.6 2.1 -1.5 4.2 180 N.M. 1.8 5.0 1.8 0.76 1.1 0.42 Minutes M. 0.5 0.5 1.8 -1.3 3.6 240 N.M. 1.5 4.8 1.4 1.2 0.2 0.8 Minutes M. 0.6 0.5 0.8 -0,3 1.3 300 N.M. 1.5 5.3 1.5 1.1 0.4 0.73 Minutes M. 0.5 0.5 0.9 -0.4 1.8 360 N.M. 1.4 5.2 1.4 1.0 0.4 0.71 Minutes M. 0.4 0.4 0.06 0.34 0.15 *See the text. N.M. = Nonmetabolite. M. = Metabolite. H H H TABLE 23
RENAL CLEARANCE STUDIES ON DHQ, DOG 3
Nature Plasma Expected Amount in Expected of the Cone, G.P.R. Total Amt. Urine Amount Clearance Time Drug mcg/ml ml/min/kg Filtered* mcg/min/kg Reabsorbed* ml/minAg
60 N.M. 3.8 4,4 3.3 4,5 -1.2 1,2 Minutes M. 0.52 0.45 8,3 -7.8 15.9 90 N.M. 3.7 3.9 2.8 3.9 -1.1 1.1 Minutes M, 0.5 0.38 8,2 -7.8 16.4 120 N,M. 3.3 4,1 2.7 3.3 —0*6 1.0 Minutes M, 0,6 0,5 8.3 -7.8 13.8 150 N.M. 3.1 4,3 2,6 2,8 -0,2 0.9 Minutes M, 0.5 0,4 7.8 -7.4 15.6 180 N.M. 3.0 4.9 2.9 2.6 0.3 0.86 Minutes M, 0.30 0.5 6,4 -5.9 21;3
*8ee the text.
N.M. = Nonmetabolite,
M. = Metabolite, H îo TABLE 24
RENAL CLEARANCE STUDIES ON DHQ, DOG #4
Nature Plasma Expected Amount in Expected of the Cone. G.P.R. Total Amt. Urine Amount Clearance Time Drug mcg/ml ml/min/kg Filtered* mcg/min/kg Reabsorbed* m l/ m i n A s
60 N.M. 1.6 4.3 1.4 2.4 —1 .0 1.5 Minutes M. 2.0 1.7 2.2 -0.5 1.1
120 N.M. 1.5 4.0 1.2 1.9 -0 .7 1.3 Minutes M. 1.6 1.3 2.5 -1.2 1.6
180 N.M. 1.5 5.2 1.6 1.7 -0.1 1.1 Minutes M. 1.5 1.6 2.3 -0.7 1.5
*See the text.
N.M. = Nonmetabolite,
M. = Metabolite.
H G TABLE 25
REKAL CLEARANCE STUDIES ON DHQ, DOG 5
Nature Plasma Expected Amount in Expected of the Cone. G.P.R. Total Amt. Urine Amount Clearance Time Drug mcg/ml ml/min/kg Filtered* mcg/min/kg Reabsorbed* ml/min/kg
60 Minutes N.M. 2.3 9.0 4.1 3.9 o.a 1.7 1 M. 1.6 2.8 3.6 —0 .8 2.2
120 Minutes N.M. 1.7 9.7 3.2 3.5 -0.2 2.0 2 M. 1.7 3.2 3.3 -0.1 1.9
180 Minutes N.M. 1*7 6.8 2.3 2*5 -0.2 1*5 3 M. 1.2 1.6 3.3 -1.7 2.7
*See the text, N.M. = Nonmetabolite. M. = Metabolite.
% TABLE 26
RENAL CLEARANCE STUDIES ON DHQ, DOG 6
Nature Plasma Expected Amount in Expected of the Cone. G.F.R. Total Amt. Urine Amount Clearance Time Drug mcg/ml ml/min/kg Filtered* mcg/min/kg Reabsorbed* ml/min/kg
60 Minutes N.M. 2.4 5.8 2.7 2.9 —0.2 1.2 1 M. 1.9 2.2 4.2 -2.2 2.2
120 Minutes N.M. 2.1 6.5 2.7 3.6 -0.9 1.7 2 M. 1.5 1.9 5.4 -2.5 3.6
180 Minutes N.M. 1.7 11.5 3.9 3.0 0.9 1.8
3 M. 1.3 .... 4.0 -1.1 3.1 *See the text. N.M. = Nonmetabolite. M. = Metabolite.
H H UJ TABLE 27
RENAL CLEARANCE STUDIES ON DHQ, DOG 7
Nature Plasma Expected Amount in Expected of the Cone, G.P.R. Total Amt. Urine Amount Clearance Time Drug mcg/ml ml/min/kg Filtered* mcg/min/kg Reabsorbed* ml/min/kg 60 N.M. 0.39 5.5 0.42 .018 o.4o 0.05 Minutes M. 0.1 7 0.18 .007 0.173 0,04 90 N.M. 0 .3 1 4.7 0.2 8 .011 0 .2 7 0.03 Minutes M. 0.18 0.1 6 .013 0.15 0.07
120 N.M. 0 .2 8 4.9 0.28 .008 0.2 7 0.03 Minutes M. 0.12 0.11 .015 0.09 0.12
150 N.M. 0 .2 7 5.2 0.28 .005 0.2 7 0.02 Minutes M. 0.08 0.08 .012 0 .0 7 0.1 5
180 N.M. 0.20 5.2 0.20 .008 0.19 0.025 Minutes M. 0.07 0 .0 7 .014 0.06 0.2
*See the text.
N.M. = Nonmetabolite.
M. - Metabolite.
H S TABLE 28
CUMULATIVE RENAL EXCRETION OF DHQ DURING 6 HOURS
0-1 Hour 0—2 Hours 0-3 Hours 0-4 Hours 0-5 Hours 0-6 Hours Per Cent Fer Cent Per Cent Per Cent t*er Cent Per Cent Amt. of Total Amt. of Total Amt. of Total Amt, of Total Amt. of Total Amt. of Total No. (mgs) Dose (mgs) Dose (mgs) Dose (mgs) Dose (mgs) Dose (mgs) Dose 1 6.6o 6.9 10.08 9.2 13.78 12.6 2 10.09 8.5 18.50 15.6 25.45 21.5
3 6.8o 7.4 14.3 15.7 20.4 22.4 2 3 .8 2 6 .1 2 7 .2 29.9 2 9 .6 32.5
4 1.25 1.4 10.85 11.9 14.75 16.2 17.95 19.7 20.95 2 3 .0 24.4 26 .8
5 3.98 4.2 5.68 6.0 8.0 8.4 11.4 12.0 14.2 14.9 17.0 17.9
6 6.8 6.8 12.9 12.9 16.4 16.4 16.8 16 .8 17.2 17.2 17.5 17.5
Mean 5.9 5.8 12.0 11.8 16.4 16.2 17.4 18.6 1 9 .8 21.2 22.1 23 .6 S.D. +2.5 +3.7 +5.2 +6.0 +6.6 +7.2 118
in these experiments* The individual results on biliary clearance are shown in Table 29. An average of 4.1 percent of the total dose was excreted in the bile within six hours.
Comparison of renal and biliary excretion.— A
comparative rate of cumulative renal and biliary excretion
is represented graphically in Figure 17, The average
percent of the total dose for both renal and biliary
excretion is shown on the ordinate and time on the
abscissa. The curves for both renal and biliary excretion
show a change in the slope at the end of three hours.
Metabolite concentrations of DHQ in Various tissues and Biological fluids
Tissues.— Metabolite determinations were made on
liver, kidney and heart tissues at the end of 64 minutes.
The calculations for the percent of the drug metabolized
in the tissues and fluids were performed as follows:
T.A, " Total amount in micrograms or milligrams or total radio-activity (DFM) in the sample
T.E. - Total amount in micrograms or milligrams or total radio-activity (DFM) in the extract, representing unchanged amount of the drug in the sample.
M.B (metabolite) ■ T.A. - T.E.
Percent M.B (percent of the drug metabolized) -
M.B. X 100 T.A. TABLE 29
CUMULATIVE BILIARY EXCRETION OP DHQ DURING 6 HOURS
0-;L Hour 0-2 Hours 0-3 Hours 0-4 Hours 0-5 Hours 0-6 Hours ter dent ter dent Per Cent Per Cent Per Cent Per Cent Amt, of Total Amt. of Total Amt. of Total Amt. of Total Amt. , of Total Amt. of Total No. (mgs) Dose (mgs) Dose (mgs) Dose (mgs) Dose (mgs) Dose (mgs) Dose
1 0.88 0.96 1.81 2.0 2.4l 2.6 2.84 3.1 3.26 3.6 3.43 3.8
2 0.49 0.51 1.3 1.36 1.80 1.9 2.1 2.2 2.4 2.5 2.5 2.6
3 1.5 1.5 3.3 3.3 4.5 5.2 5.2 6.0 6.0 6.3 6.3 6.3
Mean 0,95 0.96 2,1 2.2 2.9 3.3 3.3 3.7 3.8 4.1 4.0 4.1 S.D. +0.3 +1.0 +1.T +1.9 +1.9 +1.8
H VÛ 120
Figure 17,— Excretion of DHQ in urine and bile for 6 hours. Cumulative urinary excretion is taken from the mean of 6 experiments and cumulative biliary excretion is taken from an average of 3 experiments. 121
25 X- ^ CUMULATIVE-RENAL EXCRETION
O O CUMULATIVE-BILIARY EXCRETION
2 0 - (/)
10 - <
LU U cr LiJ Q_
0 3 HOURS 122
Table 30 represents the individual data on the determination of the metabolites concentrations of the drug in liver, kidney and heart at the end of 64 minutes.
The liver shows the highest percentage of the drug metabolized, ranging from 34.0 to 43.0 percent (mean 38.1 percent). In the kidney the percent metabolized varies from 24.0 to 30.0 (mean 28.1) and in the heart it varies from 20.0 to 25.4 (mean 22.6).
Biological fluids.— The metabolites concentrations in plasma, urine and bile at varying time intervals were determined.
Table 31 shows the percent of drug metabolised in plasma at one-half, one and three hours after admini stration of the drug in individual experiments. There is a marked individual variation in experiment 1 to 3 and the percentage of the drug metabolised is much lower than in the experiments 4 to 6, where the percent of drug metabolised varies from 40.0 to 55.0 percent during one and three hours.
Table 32 shows the concentration of drug metabolites in urine samples of individual experiments at one half, one and three hours after drug administration.
The percent of drug metabolised increased with time. An average of 38.5 percent of total drug was metabolised 123
TABLE 30
CONCENTRATION OF METABOLITES OP DHQ IN VARIOUS TISSUES AT THE END OF 64 MINUTES iîaïQe of the Tissue Experiment T.A. M.B. Per Cent
Liver 1 3978.3 1714.0 43.0 2 3195.4 1109.6 34.7 3 3308.8 1155.7 34.9 4 11657.7 6691.6 4 0 .0 Mean 3 8 .1 S.D. +4 .0 Kidney 1 5184.8 1494.1 2 8 .8 2 2314.4 694.8 30.0
3 3682.2 890.6 2 4 .2 4 6280.5 1859.2 29.6 Mean 2 8 .1 S.D. +2 .6 Heart 1 2395.0 564,6 23.5 2 1693.6 431.9 25.4 3 1760.0 358.3 2 0 .3 4 2164.5 495.5 21 .2 Mean 2 2 .6 S.D. +2 .3 T.A. = Total radioactivity (DPM/ml) M.B. = Radioactivity in the aliquot of metabolite (DPM/ml) Per cent M.B. = r.x; ^ 100 TABLE 31
CONCENTRATION OP METABOLITES OP DHQ IN PLASMA
1/2 Hour 1 Hour 3 Hours Per Cent Per Cent Per Cent Experiment T.A. M.B. M.B. T.A. M.B. M.B. T.A. M.B. M.B. 1 15.9 0.2 1.2 15.8 0.2 1.3 13.3 1.6 12.1
2 4.3 0.3 6.9 4.2 0.2 4.8 1.8 0.4 22.2
3 4.0 0.4 10.0 3.6 0.52 14.5 4.0 0.52 13.0
4 — « —— —— 3.6 2.0 55.5 3.1 1.6 51.6
5 - — 3.9 1.6 4i.o 2.9 1.2 41.3
6 — — — — — - 4.3 1.9 44.1 3.0 1.3 43.3
T.A. = Total amount of the drug (mcg/ml)
M.B. = Amount of the metabolite (mcg/ml)
Per cent M.B, = x 100 TABLE 32
CONCENTRATION OF METABOLITES OF DHQ IN URINE
1/2 Hour 1 Hour 3 Hours Per Cent iPer Cent Per Cent Experiment T.A. M.B. M.B. T.A. M.B. M.B.T.A. M.B. M.B.
1 12.6 2.7 21.4 7.8 2.3 29.5 5.6 2.0 35.7
2 7.8 3.3 42.3 5.3 3.3 62.2 2.6 1.8 69.2
3 15.3 6.6 43.1 12.8 8.3 64.8 9.0 6.4 71.1
4 9.5 3.5 37.0 4.6 2.2 48.0 4.0 2.3 57.5
5 11.3 3.8 34.0 7.5 3.6 48.0 5.8 3.3 57.0
6 6.9 3.7 53.6 7.1 4.2 59.1 7.0 4.0 57.1
Mean 38.5 51.9 57.9 S.D. +10.6 _ £ 3 . 0 +12.6 T.A. = Amount of total drug in urine (mcg/min/kg). M.B. = Amount of metabolite in urine (mcg/min/kg).
Per cent M.B. = x 1 0 0 T.A. 126 within 30 minutes post-injection and thereafter, the average percent of drug metabolised was 51.9 and 57.9 during one and three hours respectively.
The concentration of biliary metabolites for the three experiments is shown in Table 33. An average of
75.3 percent of total drug was metabolized within 30 minutes and thereafter, it was 79.9 and 39.3 percent during one, three and six hours respectively. TABIiE 33
CONCENTRATION OP METABOLITES OF DHQ IN BILE
1/2 Hour 1 Hour 3 Hours 6 Hours Per Cent Per Cent Per Cent Per Cent No. T.A. M.B. M.B. T.A. M.B. M.B. T.A. M.B. M.B.T.A. M.B. M.B.
1 311.1 233.0 74.9 578.2 461.5 79.8 286.3 214.9 75.0 176.9 95.5 53.8
2 77.5 54.0 70.0 408.5 322.0 78.9 773.1 587.2 75.8 64.0 22.0 34.3
3 500.0 406.1 81.2 1019.4 825.8 81.0 1823.1 1622.6 89.0 365.1 113.1 30.9
Mean 75.3 79.9 79 .9 39.3 S.D. +5.6 +1.0 +7.8 +12.5
T.A, = Total micrograms of drug excreted in bile.
M.B, = Total micrograms of metabolites excreted in bile.
Per Cent M.B, = x 100 TlAV
to CHAPTER IV
DISCUSSION
Experimental techniques
The experimental arrangement for this study is shown in Figure 3.
Cannulation.— The polyethylene tubes were inserted for approximately 5 cm in the femoral artery and femoral vein. This permitted the rapid withdrawal of a relatively large volume of blood and enabled the simultaneous withdrawal of two samples.
Catheterization of coronary sinus is comparatively difficult and if it is not properly done, may lead to serious errors in the interpretation of data. Rayford and
Greg (58) described a technique for chronic catheterization of the coronary sinus in dogs after puncturing the wall of the coronary sinus. They inserted a specially designed polyvinyl catheter, fitted with a stainless steel obturator and a mattress suture was placed superficially in the wall of the coronary sinus. With the aid of an assistant, a slight tension was applied to the ends of the opposing loop.
128 129
In this way, the wall of the coronary sinus was elevated, to facilitate the entrance of the catheter. When the catheter had been Installed the mattress suture was tightened and thereby the catheter was tied in position.
. The disadvantages of this technique were that it was laborious and bleeding from the sinus opening before
the insertion of the cannula was frequent. In this study
after trying different techniques, the following was
found to be the easiest method to insure successful
cannulation of the sinus. An 18 gauge Rochester needle was
inserted into the posterior end of the right atrial
appendage, parallel to the inferior vena cava and then
directed into the coronary sinus. The caudal end of the
plastic tubing in the Rochester needle had been previously
perforated with small holes. This facilitated and
minimized the chances of any delay in withdrawal of the
samples. The position of the catheter in the coronary
sinus was always ascertained by palpating the length of
Rochester needle through the wall of the coronary sinus
and also by direct observation after opening the heart at
the end of the experiment.
The collection of bile from the gall bladder
required a surgical procedure. The major difficulties
encountered were: (1) guiding the catheter from the bile
duct into the gall bladder and (2) the continuous and 130 complete collection of bile, Wheeler and Ramos (85) and
Fitz and Brooks (22, 23) cannulated the bile duct with a
Thomas cannula through the ampulla of Valter by way of an opening in the duodenum. Recently Marshall et al, (54) modified this technique by using a special type of plastic tubing (silicone rubber tubing) without applying any lig ature on the bile duct.
To prevent bile leakage and thereby obtain quantitative recovery of DHQ in the bile, the gall Gladder was cannulated either indirectly by guiding the catheter through the bile duct or directly by puncturing the gall bladder. The distal end of the biliary catheter was perforated with small holes to enable complete evacuation
of the gall bladder. To minimize surgical shock, 5
percent dextrose was continuously infused intravenously
at the rate of 2 drops/min/kg. After cannulating the
gall bladder, the incision was closed surgically.
Electrode assembly,— Different types of electrodes
have been employed for measurement of the conduction
velocities of the ventricular wall excitatory process. The
unipolar electrode assembly has been employed in dogs (44)
and many other animals, including man (45), however, the
results were inaccurate according to the criteria laid
down by Sodi Pallares (66) and Veyrat (75). In these
studies the intrinsic deflections were not synchronous 131 with the arrival of the excitatory wave at the electrode.
Bipolar electrodes provide a more accurate way of measuring the conduction time, and also for interpreting the potentials and contour of the electrical impulse (19, 61,
62). Recently, Crocker and Smith (11) studied ventricular activation processes in cattle by using a five paired bipolar electrode assembly. With this bipolar electrode assembly system, the arrival of the excitatory impulse at the inter-electrodal midpoint could be accurately timed.
In this study a multipolar electrode assembly consisting of fourteen differential bipolar electrodes and a pair of stimulating electrodes (Figure 4) was used. Further the inter-electrodal distances designed in this electrode assembly were 0.5 mm as distances greater than 1 mm lead to unreliable results (60). Proper impedance matching with the recording apparatus made possible the recording of high frequencies without wave distortion.
Intracellular microelectrodes (51) have also been employed for recording transmembrane potentials in single cardiac muscle fibers. Ihe intracellular electrode is not influenced by the activity of the fibers adjacent to the one penetrated (59). This type of electrode was believed to be too specialized and not applicable to this study. Furthermore, no specific knowledge of the direction from which the penetrated cell is excited could be gained. 132
In a close bipolar recording system, various factors, such as the accumulation of clots at the electrode roycardium interface, greatly influence the recording* The presence of a clot could lead to serious changes in the recording of an action potential which ultimately results in errors in the interpretation of the data. An abrasion of the myocardium by the rough surface of the electrode is probably an other important factor affecting the recording. In an attempt to prevent this, only electrodes with smooth surfaces were used.
Effect of dihydroquinidine on myocardial conduction volocity
The conduction of the excitation wave in the various parts of the heart involves both the ventricular muscle fiber and also the specialized conducting tissue, the Purkinje fiber. The conduction times were determined from electrograms obtained from an electrically driven heart in anesthetized dogs using the techniques of Swain
(69) and Hamlin et al. (31). Specially designed bipolar electrode assembly which is best suited for such studies was employed.
Various mathematical models (logarithmic, exponential, and linear) were fitted by means of a computer to this derived data on conduction time versus electrodal distance in millimeters at several points from the 133 stimulus. In addition electrodal distances were grouped in several paired sets and then subjected to the analysis.
The paired distance groups tested were 2 to 9 mm and 10 to 15 mm; 2 to 10 mm and 11 to 15 mm; and 2 to 11 and 12 to 15 mm. The best fit was assumed to be that model which showed the least sum of squared residuals. It consisted of a set of two linear models for each of the various post-injection time intervals and the control.
These were derived from 2 to 9 mm and 10 to 15 mm away from the stimulus, representing first and second linear models respectively. The mathematical model for this system is Y - a + bx, in which T is equal to the con duction time in milliseconds, x is the electrodal distance from the stimulus, a is the y intercept, and b is the slope of the line, representing the conduction, time for each millimeter of distance. The conduction velocity in millimeters per second was derived by dividing 1000 by the values of b for each post-injection time period.
The conduction of the excitation wave is slower
in the first linear model as compared to the second linear model. It is proposed that the first linear model
represents the conduction through myocardium, and the
second model represents conduction through both myocardium,
and Purkinje fibers. 134
In the past several workers have also proposed that the conduction time between the electrodes close to stimulus is relatively slower in comparison to the electrodes at a greater distance from the stimulus (31,
44, 45).
In this study, the mean calculated control con duction velocity was 223.2 mm per second, which was the same (230 mm/sec) as mentioned by Hamlin et al. (31), but lower than 880 mm/sec as reported by Schaffer and
Trautwein (60). Earlier, Lewis (44) estimated that regular myocardial fibers in the dog's ventricle conduct at a rate of 300 to 500 mm per second.
The magnitude and duration of the effect of DHQ can be visualized in Figures 6, 7 and Table 8. It has been ascertained that DHQ produced an appreciable prolongation of conduction time. THie effect of the drug was manifest within 30 seconds after its administration, when the myocardial conduction velocity was reduced to
23.9 percent from the control. This demonstration of the known dromotropic effect of the drug indicates that DHQ is quite potent and also serves as proof that the technique employed in this study was adequate.
The maximum effect of the drug was seen one minute after its administration. At this time the conduction velocity was reduced by 38.9 percent from the control. 135
However, there was a greater coefficient of variation during this time* The effect of the drug was slightly diminished after one minute and from two to sixteen minutes post injection period, the conduction velocity was reduced by 33.6 to 25.4 percent from the control. The intensity of the effect of the drug during 32 to 64 minutes was approximately one half of the peak effect i.e. the conduction velocity at this time was reduced by approxi mately 18 percent from the control. There were comparatively less individual variations in the effect of
DHQ during 16 to 32 minutes.
The information on the relationship between the intensity of DHQ effect on myocardial conduction velocity at various time intervals after its administration is not available and for that of quinidine is very limited.
Quinidine, in a total dose of 300 milligrams (approximately
30 mg/kg) administered intravenously to dogs, has been reported to produce its peak effect on conduction velocity within five minutes with a reduction of 29 percent from the control (31). Swain and Weidner (69) studies on conduction velocity using heart lung preparations also indicated that quinidine produced prolongation of the conduction time. However, they did not report the percent changes in the velocity. 136
From the existing information, an attempt could be made to compare the potency of dihydroquinidine with that of quinidine. From the information obtained in this study, DHQ can be assessed to be approximately three times more potent in reducing the conduction velocity as compared to the reported work on quinidine by Hamlin et al. (31).
DHQ produced its peak effect on myocardial conduction velocity by reducing it 38.9 percent from the control with a dose of 10 mg/kg, whereas the peak effect by quinidine
(31) was produced by the administration of approximately
30 mg/kg body weight. In addition, the peak effect with
DHQ was produced within one minute after its administration in comparison to quinidine where it was reported to be up to five minutes after injection of the drug. Furthermore the maximum effect of DHQ on reducing the conduction velocity was 33 percent greater than quinidine.
The effect of quinidine on the individual ventri cular and Purkinje fibers in calves, sheep guinea pigs and dogs with the use of intracellular microelectrodes is available from the works of Weidman (78), Johnson (37) and Hoffman (29). Their results indicate that quinidine decreased the amplitude and rate of rise of the action potential in both Purkinje and ventricular muscle fibers.
In comparison to dihydroquinidine, the cardiac glycosides like ouabain and strophanthin have little or 137 no action on myocardial conduction, but conduction through
Purkinje fiber is slowed, lliis information was obtained from Hoe and Mendez (55) and Swain and Wwidner's (69) work who used a similar type surface electrode as employed in this study. If the assumption that the second linear model derived from 10 to 16 mm electrodal distance from the stimulus represents conduction through muscle fibers and Purkinje fibers is true, it is suggested by this study that dihydroquinidine may have suppressed the conduction through Purkinje fibers.
The value of dihydroquinidine as an antifibril- latory agent lies in the fact that it increases the refractory period, but its tendency to slow conduction places it also in a disadvantageous position. When
conduction time is prolonged relatively more than the refractory period, the chances for ventricular fibrillation are increased due to the initiation of the secondary
impulse before the primary ones have completed the circuit.
Correlation between the effect of DHQ on myocardial conduction velocity and its concentration in the heart
In an attempt to analyze the pharmacological
activity of DHQ quantitatively, its pharmacological action was compared with its concentration in the heart at that
time. In the past, several attempts have been made to 138 correlate the therapeutic effects of quinidine with the peripheral venous concentration of the drug (67, 68, 77) but no definite correlation could be established. There were discrepancies between the Intensity of the cardiac effects and the quinidine plasma level, that is, the plasma levels of the drug decreased faster than the intensity of cardiac effects.
The data on the effect of DHQ on myocardial con duction velocity at different times as a measure of its pharmacological activity is given in Table 8, however the cardiac concentration of the drug present during different times was not studied except at the end of 64 minutes.
However, from the data available, on the concentration of the drug in arterial and coronary vascular systems and the difference between these two concentrations, an estimate of the net flux of the drug from the capillaries into the heart at different times (Figure 13) may be determined.
In an attempt to correlate the above information, it is then obvious that the maximum effect of DHQ on myocardial conduction velocity occurred within two minutes when it was reduced to 39 to 33,6 percent from the control, and at this time the movement of the drug from the capillaries into the heart was also maximal. 139
The effect of the drug on conduction velocity following two minutes was decreased from its peak effect, however, during four to 16 minutes period it remained relatively constant as represented by 29.3 to 25.4 percent reduction from the control conduction velocity. The estimated net influx during this period was also appreci ably reduced, especially following four minutes and after that it was minimal and approximately approached the estimated efflux. During the equilibration period, it is assumed that the amount of the drug localized in the myocardium is fairly constant, which correlates with the consistency of the pharmacological activity.
The fifty percent sudden drop (18 percent reduction in the conduction velocity from control) in the drug activity beyond 16 minutes is not clearly understood, especially when this is during the equilibration period and the concentration of the drug localized should be uniform. The inconsistency in the pharmacological activity as compared to the earlier period may, however, be that although the receptor sites are fully saturated with the drug, the molecule may not be active due to the metabolism of the drug. The metabolite determination study also indicates that approximately 23 percent of the drug localized in the heart at the end of 64 minutes was present as metabolites. The poor antiarrhythmic value of the 140 metabolites of quinidine and other antiarrhythmic drugs has been reported previously (10, 52).
Distribution of DHQ in various body compartments
Disappearance from the plasma.--Most of the previous work on the distribution of anti-fibrillatory drugs was done with quinidine. Wegria and Boyle (77) in their studies on dogs reported that the maximum concentration of quinidine in plasma after oral admini stration of 50 mg quinidine sulphate per kg body weight was reached within three hours. Following this peak, the concentration in the plasma declined sharply in 6 hours. This was followed by a slower rate of disappear ance during the next 10-14 hours. Further, it was reported by them that the arterial and venous difference was insignificant after a few hours.
As a result of intravenous administration of quinidine in humans and other species, it is known that the peak levels in venous samples are attained within a few minutes, subsiding in 5 to 10 minutes (67, 68, 81,
82).
In this study an attempt was made to determine simultaneously the arterial,venous and coronary sinus plasma concentrations of dihydroquinidine after its intravenous administration. Dihydroquinidine was found 141 to leave the blood vascular system rapidly during the first few minutes, however, the rate of disappearance did show a fairly wide variation. The values were expressed both in mcg/ml and percent of the total dose per 100 ml of plasma in order to compare the results of this study with quinidine studies. The peak concentrations in the arterial, venous and coronary sinus samples were reached in one half minute and they were 27,3 mcg/ml, 9.5 mcg/ml and 12,4 mcg/ml, respectively. The early rate of decline in the first four minutes was very fast in arterial samples. Compared to this, the fall in concentrations of venous and coronary sinus samples was less rapid. A plateau in arterial, venous and coronary sinus concentrations was attained at approximately the same time (16 to 32 minutes after administration) (Figure 12). The concentrations in venous samples, however, remained lower than arterial and coronary sinus throughout.
By comparing these results with the disappearance rate of quinidine by Schlerlis et al. (63) in arterial, venous and coronary sinus samples, some interesting differential features are observed. At the dosage level of 10 mg/kg of quinidine, the peak arterial levels ranged from 4,6 to 22,6 mcg/ml which were comparatively lower than
DHQ as reported in the present study (16,7 to 39.5 mcg/ml).
The most striking difference from Schlerlies's et al. was 142 that In their study, the coronary sinus levels were greater than arterial or venous during the first ten minutes, The higher concentration in coronary sinus as compared to arterial samples has been explained by Schlerlis et al.
(63) upon the hypothesis that the initially bound quinidine with myocardium was subsequently washed out by the perfusing blood. In this study the concentration in arterial samples always remained higher than coronary sinus samples during the early phase (Figure 12)•
Following a plateau period of 32 minutes, there was very little difference in arterial and coronary sinus plasma levels. However, the venous levels always remained lover than arterial and coronary sinus levels. This is in con trast to Scherlis et al. work where the quinidine levels for arterial, venous and coronary sinus were similar at the end of thirty minutes. The application of Hodges Sign
Test at a significance level of 1 percent, also showed
the median of A (arterial concentration)to be greater
than the median of V (venous concentration) and median of
C (coronary sinus concentration) to be greater than the median of V for all times except 64 minutes. However, the median of A was greater than the median of C only at 1,
2, 32 and 64 minutes time intervals.
The over all lowered values of quinidine in
arterial, venous and coronary sinus samples as compared 143 to the present study may be attributed to the sensitivity of analyzing techniques and individual variations. The radiometric assay method used in this study is more sensitive than the fluorometric techniques (47) used by Scherlis et al.
In comparison to the venous plasma disappearance rate of in cat (86) it can be ascertained that venous concentration at different time intervals (as expressed by mcg/ml) was approximately eight-fold in this study. For instance, at 2 minutes, the concentration of
DHQ-H^ in cat was reported to be 0.87 mcg/ml, whereas in this study it was found to be 6.6 mcg/ml. This difference may be attributed to the higher dose of DHQ used as the concentration in the blood is dose dependent. However, when the concentration in the plasma is expressed in per cent of the total dose, the proportion remaining in the blood becomes lower in this study as compared to the cat.
For example, it was 0.6 percent of the total dose in this study as compared to 8.7 percent in the cat study. This may be explained on the assumption that as a result of higher total dose, the diffusion rate from the blood is also increased and consequently the proportion of the drug remaining in the blood in relation to the total dose becomes less. 144
Among the various mathematical models (linear, exponential and logarithmic) used to relate the disappear ance of the arterial, venous and coronary sinus plasma concentrations against the time, the best fit vas the log function. This is represented by the formula log of Y = a + bx, where Y is the concentration in arterial, venous and coronary sinus samples, a is the Y intercept, b is the slope of the line and x is the time. As an example of this model, the disappearance rate for arterial, venous and coronary sinus samples has been shown
graphically in Figures 9 to 11* From these graphs two
linear models marked A and B were derived for each.
The biological half life (T 1/2) values from the
fast component for arterial, venous and coronary sinus
samples were approximately 1,4 minutes, 1.6 minutes and
2.6 minutes respectively. The T 1/2 of the slow component, was 187 minutes for the arterial, 232 minutes for the venous and 140 minutes for coronary sinus. No definite
explanation for the significance of the fast and slow
components in these graphs could be given, but it may be
assumed that the fast component for the arterial, venous
and coronary sinus vascular system may depict equilibration
between the vascular system and various body tissues.
The slow component may represent the rate at which the drug is excreted from the body by various channels. 145
Okita (56), Heilman (28) and Zilversroith (90) also derived biological half lives in a similar wayrand made such pharmacological assumptions as in this study. Biological half life calculated for the earlier sinus samples is longer, as compared to venous and arterial samples. This may imply that the equilibrating rate between heart and blood is slower. Whitney (86) found the biological half life for the fast component of the venous curve for DHQ in the cat to be 2.5 minutes.
As pointed out earlier, there were fairly wide individual variations in the plasma concentrations, es pecially in the early samples. Similar individual variations in the concentration of quinidine plasma levels have also been reported by Weis and Hatcher, (82) and
Scherlis et al. (63), In this study, an exceptionally high value in arterial, venous and coronary sinus plasma concentrations were seen in experiment 5. These values were significantly higher than other experiments at 1 percent level according to Hodges Sign Test. The reason for this exceptionally high value could not be determined.
For this reason, these values were not taken into consideration for finding the mean values. It is inter esting to note that the general pattern of decline in plasma concentration in experiment 5 was similar to that seen in other experiments. 146
To estimate the probable extraction rate of DHQ by the heart and in other body tissues, the arterial- coronary sinus and arterial-venous differences in concentration were determined and are shown in Figure 13.
The initial arterial-coronary sinus and arterial-venous difference (extraction rate) at 1 minute is the highest, which could very well depict the early rapid clearance of the drug from the blood by equilibration with the tissues. Thereafter, the extraction rate is slowed, which is comparatively much slower in the heart as compared to other tissues as depicted by arterial-venous and arterial- coronary sinus difference. The arterial-venous difference declined very gradually after two minutes, a fact suggest ing that tissues were continuously extracting the drug from the circulation. The extraction rate in the heart beyond 2 minutes remains nearly the same, but at four minutes the arterial-coronary sinus difference shows a negative value, which suggests that the efflux from the heart was greater than the influx at this time.
Tissue distribution
The distribution of DHQ in various tissues of the dog was studied at the end of 64 minutes and also at the end of six hours in order to determine the rate of decline in their concentrations. In experiment 5 (Table 15), by 147
the Hodge's Sign Test, the plasma concentrations were significantly higher at the 1 percent level, but the tissue
concentrations were not significantly different at this
level from the other four experiments. However, in experiment 5, tissue to plasma ratios were significantly different at the 1 percent level. Accordingly tissue
concentrations were grouped together for all the five
experiments and tissue to plasma ratios were grouped for
experiments 1 to 4 only.
For the purpose of discussion and comparison,
tissues were grouped into three physiological groups. In
order to find several homogeneous subsets among various
tissues and their tissue to plasma ratios, Duncan's
Multiple Range Test was applied at a significance level
of 5 percent. This test was applied for the first four
experiments only, because of significant difference in
the tissue to plasma ratio in experiment 5. Group 1
represented cardiac, skeletal, and smooth muscles. Among
various chambers of the heart, the concentrations in
micrograms per gram of tissue in the left auricle, right
auricle, left ventricle, and right ventricle were 13.6,
13.3, 16.1 and 16.3 respectively (Table 16). These
concentrations were not significantly different from each
other. Group 2 consisted of the excretory organs (liver,
kidney and lung) and spleen. The concentrations (mcg/gm) 148 in the kidney, lung, liver and spleen were 34.0, 33.5,
29.1 and 25.9 respectively (Table 16). According to
Duncan's Multiple Range Test the concentrations in the kidney and lung (Homogeneous group) are not significantly different from each other similarly, the concentrations in the liver and the spleen, forming another homogeneous group, are not significantly different from each other.
The concentrations of Group 3, consisting of the endocrine glands (adrenal, thyroid and ovary) and the brain, are 22.7, 5.8, 10.2 and 4.6 mcg/gm, respectively.
The concentration in the adrenal was the highest in the endocrine-brain group and was not significantly different from those of the liver and spleen in the excretory group.
The concentrations in the ovary, thyroid and brain did not differ significantly from each other at 64 minutes.
All the tissues showed tissue to plasma ratios of greater than one at 64 minutes, except brain and skeletal muscle which were 0.81 and 0.96 respectively. The homogeneous sets obtained by the Duncan Test and then ranked in order of their magnitude are: set 1 - (largest T — ) kidney; set 2 - lung, liver and spleen; set 3 — liver, P spleen, and adrenal; set 4 - adrenal, right ventricle, and left ventricle; set 5 - right ventricle, left ventricle, right auricle, left auricle, and ovary ; set 6 - (smallest T — ) other tissues. 149
The application of Duncan's Multiple Range Test on 6 hours distribution studies showed that lung showed significantly higher tissue to plasma ratio and the kidney and liver forming the second homogeneous subset, showed the next significantly higher tissue to plasma ratio. The thyroid showed the least tissue to plasma ratio. The tissue to plasma ratios among cardiac tissues were not significantly different from each other.
The concentrations and tissue to plasma ratio in all groups dropped from a higher level at 64 minutes to a lower value at 6 hours (Figures 14, 15, 16). However, there were some individual differential features among various tissues on the manner of their decline in concen tration and tissue to plasma ratio with the time. Among tissues in the excretory group, spleen showed the greatest decline in concentration and in tissue to plasma ratio, followed by the kidney and then the liver. The lung concentration did not change to an appreciable extent. In group three the concentration in the thyroid also did not decrease much at the end of 6 hours. These observations tend to show that the concentrations of the drug in the lung and thyroid are retained for a longer time than other tissues.
In comparing the DHQ-E^ tissue distribution studies in cat (Whitney, 86) at one hour to the present study, it 150 can be ascertained that in general, the concentrations in the various tissues as expressed in mcg/100 gm of tissue were very low in the study on the cat. It may be due to the fact that the doses of DHQ used in the cat studies were much lower than those employed in the present study on the dog. One cannot discount the possibility of a species difference. However, from a comparative qualita tive analysis point of view, Whitney (86) found that the highest concentration was observed in the liver followed by lung, ventricle, kidney and other tissues, which were quite contrary to the findings in the present study where the highest concentration was in the kidney followed by lung, liver and other organs. The concentration of the drug in the ventricles of cat was unusually high and varied over a wide range. Further, the concentration of the drug in the thyroid also ranked among the tissues localizing higher concentration in the cat study, as compared to the present study, where its concentration was relatively lower. However, the persistency of the I drug localization in thyroid for a long time was common in both the studies. From the cat study, liver was con sidered to be the route of excretion in comparison to the present study, where the channel of excretion was primarily the kidney. 151
From the distribution studies of quinidine in dogs (77) following oral administration in varying doses
(10 to 50 mg/kg), it was seen that localization rate in tissues was mostly dose dependent. The rate of decline of concentration in the tissues was relatively rapid up to
6 hours and thereafter, from 10-14 hours there was a reduced rate of decline. The concentration in the kidney was the highest among the various tissues analyzed. The rate of decrease as based on the initial concentration in various tissues like heart and kiendy was similar to this study. However, comparison of their distribution data at the end of 6 hours after oral administration of 10 mg/kg show that the concentrations in kidney and left ventricle were higher than found in this study. This may be due to the fact that oral administration leads to slow absorption and consequently gradual increase in the total concen tration of the drug in tissues.
Hiatt and Quinn (34) studied the distribution of quinidine in dogs after attaining a plateau in the plasma concentration by constant infusion of the drug. Their
results also indicated relatively lower binding capacity of the alkaloid in brain and skeletal muscle in comparison
to the higher binding capacity in the glandular tissue.
The highest concentration was in liver, followed by lung
and kidney. The thyroid samples showed a higher concen
tration than observed in this study. 152
The distribution patterns of various cinchona alkaloids in birds is different than that in mammals.
Kelsey et al. (40) studied distribution of quinidine in various tissues in rabbits, chicken and ducks at 10 minutes, 1 hour and 4 hours interval after the intravenous injec tion of 10 mg/kg. The rabbit showed the highest concentration in the lungs and least in the liver. However, the concentration of the drug was higher in kidney than in liver which was similar to the present study. The next highest concentration to the lungs was in the spleen, which was quite high in comparison to this study. On the other hand, the distribution of quinidine in chicken and duck was highest in the liver and least in the kidney.
It is important to emphasize that the quinidine in plasma is bound to the plasma protein to the extent of 80 percent (7, 8) and thus, the diffusable fraction in the interstitial fluid, which is free to enter the cells may be less than 20 percent of the plasma concentration.
The localization of the alkaloids in the tissue represents some sort of association with the intracellular structures. Studies on subcellular distribution of DHQ could possibly give a satisfactory solution for this problem. Earlier, Weiss and Hatcher (82) suggested the binding of the drug in lung was in the capillary endo thelium, but no satisfactory proof is available. Another 153 possibility of binding of the drug with mast cells in
certain tissues has been proposed.
Metabolite determination
The early studies on the excretion of various
cinchona alkloids in man have shown that a large percent
of the drug was excreted as metabolites (25, 70), The major site of metabolism is considered to be the liver.
In the rabbit; the metabolism has been attributed to the
oxidation of the drug by an enzyme present in the liver
(41) and in human studies, this enzyme was found to be an
aldehyde oxidase (9).
The metabolic products of quinidine were reported
to be hydrolysis of either quinuclidine ring or quinoline
ring or both (4), and the two major metabolites are mono-
hydroxy-non phenolic and monohydroxy-phenolic quinidine
metabolites (Figure 2). These metabolic products have
been shown to possess a reduced antiarrhythmic activity
as compared to the unchanged drug (10).
In this study, the unchanged DHQ molecule was
extracted from various tissues (liver, kidney) and fluids
(plasma, urine and bile). The non-extractable radio
activity was assumed to represent the metabolite portion.
According to Brodie (5), this procedure slightly
underestimates the quantity of metabolite due to its
partition coeficient between the alkalinized sample and 154 dlchlorethane. As far as the further isolation of the various metabolites was concerned, it was beyond the scope of this study. In this study only the measurement of the radioactivity of the extracted sample was taken into consideration. From this preliminary study, useful information quantitating the extent of metabolites in various tissues and fluids has been obtained and from this the need for further isolation of various metabolites is indicated.
The determination of metabolites in various tissues at the end of 64 minutes after the administration of DHQ indicated that the liver had a higher portion of metabo lite than the kidney and heart (Table 30), The average percent of the total drug that was metabolized in the liver, kidney and heart were 38.1, 28.1 and 22.6 percent respectively.
These results are in agreement with those reported by Conn and Luchi (9) as a result of the administration of tritiated quinidine in rabbit and dog. It was found that metabolites constituted approximately 50 percent of the drug in liver and 30 to 35 percent in other tissues like kidney, heart and skeletal muscle.
The early reports on the determination of metabolites of various cinchona alkaloids in birds and mammals also indicated that liver was primarily the organ 155 concerned in the metabolism (40)« In comparison to quinidine, quinine was more readily metabolized both in in vitro and in in vivo studies in the tissues of rat and rabbit. However, in birds (chicken, turkey, pigeon, duck and goose) the metabolites of quinidine were greater than quinine. Furthermore, the rate of metabolite formation for both quinine and quinidine in ducks was slower than chicken.
The metabolite determination from the plasma samples at different time intervals (Table 31) showed marked individual variation, especially in experiments
1 to 3 where the percent of drug that was metabolized was much lower than the other three experiments (4 to 6 ).
In these experiments during the first thirty minutes after the administration of the drug, 1 to .10 percent of the total concentration present in the plasma was as metabolites and thereafter, the rate of metabolite formation increased, which ranged from 12,0 to 22.2 percent of the total concentration in the plasma at the end of three hours. However, in experiments 4 to 6 , the percent of the metabolites present were much greater and there were less individual variations. The amount of meta bolites in these experiments at the end of 1 hour ranged from 41.0 to 55.0 and at the end of 3 hours, it ranged from 41.0 to 51.6 percent of the total drug. 156
From the studies on the determination of the metabolite in urine and bile, it was found that the percent
of the total drug which was present as metabolite was twice
as great in the bile as in the urine. However, the excretion rate of the drug (metabolite plus non-metabolite)
in urine was approximately six fold to that of bile. The
higher rate of biliary metabolites to that of urinary are
in conformity with the higher percent of the metabolite
present in the liver in comparison to the kidney. The
rate of metabolite in the kidneys increased with the time
and it was higher at the end of 3 hours (57.9 percent) than
earlier, when it was 38.5 percent and 51.9 percent at the
end of 30 and 60 minutes, respectively. In contrast to
this, the metabolite concentration in bile was quite
constant for three hours (75.0 to 80.0 percent) and later
it was reduced to an average of 39.3 percent at the end
of six hours.
Conn and Luchi (9) reported that 50-80 percent of
the quinidine present in the urine of dog, rabbit and man
was metabolized, which are quite in agreement with the
findings of this study.
From the data on cumulative renal and biliary
excretion and also on their rate of metabolite formation
in this study, it can be ascertained that 3.1 percent of
the total dose (5.8 percent) excreted in one hour and 9.4 157 percent of the total dose (16.2 percent) excreted in three hours in urine were metabolites. In biliary excretion,
0.7 percent of the total dose (0.96 percent) in the first hour, 2.6 percent of the total dose (3.2 percent) in three hours and 1.3 percent of the total dose (4.1 percent) at the end of six hours were metabolites.
All these observations tend to indicate that liver is primarily the organ concerned in the biotransformation of the drug and, consequently, the quantity of metabolites in the bile is higher than in the urine. Furthermore, heart shows relatively lower degree of metabolite, the factor leading to greater concentration of the unchanged drug in this organ and ultimately responsible for the pharmacological activity of DHQ.
Excretion
The information available on the excretion of quinidine is limited and the mechanism of renal excretion is still unknown. In this study, renal and biliary excretion of DHQ were investigated by means of clearance techniques. The measurement of urinary and biliary excretion demonstrated that DHQ was primarily excreted by the urine in dog. From the preliminary information on urinary excretion of the drug in distribution experiment (Table 19) it is obvious that a very minute amount (0.05 percent) of 158 the total dose was excreted in the first five minutes and, thereafter, the rate of excretion was slowly increased.
The rate of urinary excretion increased from 16 to 30 minutes and following that became even more rapid. At the end of 30 minutes, 1.92 percent of the total dose was excreted and at the end of 64 minutes, an average of 3.3 percent of the total dose had been excreted. However, it must be pointed out that in this trial, the bladder was not completely evacuated as in the clearance studies and as such, the total amount estimated at various excretion times may be less.
More exact information on the cumulative renal excretion of the drug is available from the data obtained in the renal and biliary clearance experiments (Table 28).
The total amount of the drug excreted at different time intervals varied markedly within the individual. An average of 5.8 percent of the total dose was excreted at the end of one hour and subsequent to that, the rate of excretion supplemented almost equally for another two hours, and at the end of three hours, it was an average of
16.2 percent of the total dose. Following three hours, increments in excretion were much slower and at the end
of six hours, it was an average of 23.6 percent of the total dose. 159
The total excretion of the drug in the bile was approximately one-sixth of the total renal excretion.
However, the rate of excretion though much smaller showed a similar pattern to that of renal excretion. An average of 0.9 percent (Table 29) of the total dose was excreted in the bile at the end of one hour in comparison to urine, where 5.8 percent of the total dose was excreted at the end of one hour. Following this, the biliary excretion rate increased for another two hours, but with smaller increments in comparison to urinary excretion. At the end of three hours, an average of 3.2 percent of the total dose was excreted in the bile in comparison to 16.2 percent in urine. After three hours, less than one percent of the total dose was excreted for another three hours, thus making the cumulative excretion after six hours, 4.1 per cent of the total dose.
The over all comparative pattern of both renal and biliary excretion rate can be visualized from Figure 17, where upper and lower curves represent cumulative renal and biliary excretion respectively. The change in both the curves is at the same time (3 hours) which shows the change in the total amount excreted. The early part of both the curves represent a higher rate of excretion in comparison to the later part where the rate of excretion becomes lower. 160
These observations tend to show that the early rapid disappearance rate of the drug from plasma with a half life of 1.6 minutes is not a function of early excretion of the drug, since the amount excreted in the first five minutes is extremely low. However, the later part of the disappearance process, with a biological half life of about 5 hours appears to be accounted for primarily by excretion as evident from data on urinary and bilary excretion.
DHQ-H® excretiozT studies in cat (8 6 ) showed a higher amount of the drug excreted in feces as compared to urine, which is in contrast to the present study. This suggests a species variation between the cat and dog in route of excretion of DHQ-H^. It was found that in the cat average total amount of excretion over a 23 day period was 67.3 percent of the original dose and the amount excreted in feces was almost twice that of urine. Further, it was observed that 91 percent of the total amount excreted in urine and feces occurred within 72 hours following administration. The amount of excretion after
72 hours was less than 0.3 percent per day of the total dose.
In human beings, the main route of excretion of
quinidine, as with other cinchona alkaloids, has been
reported to be in the urine (16), Sokolow and Edgar (67) 161 found that after oral administration of quinidine, maximum blood levels were reached in approximately two hours and detectable levels persisted in the blood for 72 hours after the last dose of quinidine. The rate of excretion of the drug in urine was related to the height of the blood level and they reported that during the first day, an average of 4,8 percent of the first dose was excreted in the urine in twelve hours and 10,5 percent of this dose was excreted in the urine by 24 hours. In one patient, who was orally medicated with 0,4 gm of quinidine every
four hours (for 24 hours), it was found that the 6 percent of the first total 24 hour dose was excreted in the same period, followed by 11 percent and 20 percent in 48 hours and 72 hours, respectively. These observations show that urinary excretion of quinidine is a slower process after
oral administration in comparison to the present study
in dogs, where an average of 23,6 percent of the total
dose was excreted in the first six hours after the intra venous administration of DHQ, This is probably a result
of time-concentration curve.
The excretion rate among various cardiac glycosides
varies. Digitoxin, in comparison to digoxin and ouabain,
has been found to be more cumulative (24) but the channel
of excretion for all these cardiac glycosides is chiefly
through kidney in both man and dog (18, 32, 52). It 162 was found that 70 percent of the total Intravenously injected digoxin dose appeared in the urine of human subject in 5 days, compared to 56 percent excreted in the urine of dogs for a total period of four and one half days.
However, the excretion of these glycosides in bile in both man and dog was less, but more in dogs (17 percent of the dose) than in man (12 percent of the dose). This is markedly different from the excretion of ouabain in rats, which occurs mainly in bile (13).
From these findings, it could be concluded that the route of excretion for both DHQ and cardiac glycosides is more by the kidney than the liver. The excretion of
DHQ by the kidney is well related by the higher concen tration found in this organ as compared to the liver.
Similarly, the higher concentration of tritiated digoxin in the kidney (32) also relates to its route of excretion by the kidney.
Renal clearance
The mechanism of renal excretion of DHQ was determined by studies on renal clearance. Since quinidine binds approximately 80 percent with the plasma albumin
(7, 8 ), it is obvious that the entire concentration of DHQ seen in the plasma can not be filtered through glumerulus 163 filtration and accordingly it was considered leasable to rectify the amount filtered on the basis of the 20 percent filtration of the unbound drug in kidney. Thps the rectified data on the total amount filtered and also the amount reabsorbed was shown as expected amount in these columns.
The analysis of the data on renal clearance in seven dogs (Table 21 to 27) showed that the clearance rate for the metabolite portion of DHQ was greater than the non-metabolite portion and there was evidence for both tubular reabsorption and tubular secretion of the drug by the kidney. The rate and the extent of renal clearance, reabsorption, secretion and the total amount excreted varied with the individuals. Consequently, the individual experiments are discussed separately.
In dog #1, the plasma concentrations for the non metabolite were quite high (11.7 to 15.6 mcg/ml) in comparison to the other dogs. Such high concentrations were also seen in one of the distribution experiments and this was accounted for as individual variation. As a result of greater concentration of the non-metabolite in the plasma, the amount filtered was increased, which in turn resulted in the reabsorption (8.6 to 13.85 mcg/min/kg) of the drug. On the other hand, the amount of metabolite in the urine is greater than the amount filtered, thus 164 indicating tubular secretion. Tubular secretion is also suggested by the fact that the clearance rate for the metabolite (1.25 to 11.5) was greater than non-metabolite <0.12 to 0.35).
In dog #2, where clearance tests were performed for 6 hours, it was found that the clearance rate for metabolite (3.6 to 16.5)was greater than non-metabolite
(0.42 to 0.57) for three hours; thereafter, the clearance rate for metabolite was reduced. There was both reabsorption for the non-metabolite and secretion for the metabolite, however the amount of reabsorption for non-metabolite was lesser than in dog # 1 , which could be explained that the amount of the drug filtered in this dog at different time intervals was lesser in comparison to dog # 1 .
In dog #3 the clearance rate for metabolite (13,8 to 21.3) was also greater than non-metabolite (0.86 to 1.2) and both the clearance rates were comparatively greater than the other two dogs. This difference is probably due to the higher rate of excretion and metabolite concen tration in this dog as compared to the other two dogs.
Consequently this fact may also be responsible for the evidence of secretion for both non-metabolite and metabolite in dog #3. 165 In dogs #4 to 6 the renal clearance for both non- metabolite and metabolite were run at an hourly interval in comparison to 30 minutes in dogs #1 to 3. In these experiments the clearance rate for non-metabolite (1.1 to
1.9) was nevertheless, greater in comparison to dogs #1 to
3 (0.12 to 1.2). Although the clearance rate for metabolite was greater (1.1 to 3,6) than non-metabolite (1.1 to 1.9) in these experiments, yet quantitatively it was lesser than in dogs #1 to 3. Furthermore, there was evidence for secretion for both the non-metabolite and the metabolite, contrary to the evidence of secretion for metabolites only
in dogs #1 to 3. These differences can be explained due to lower concentration of the drug in the plasma, higher
rate of metabolite formation in both plasma and urine,
and higher rate of urinary excretion in comparison to dogs #1 to 3,
In dog #7, where comparatively much lower dose of
DHQ was administered, it is evident from the results that
practically the whole amount filtered was reabsorbed and very small quantity excreted in the urine.
From these observations it may be concluded that
the clearance rate for DHQ was related indirectly to the
concentration in the plasma. The extent of tubular
reabsorption or secretion depended on the amount filtered. 166
In comparing the mechanism of renal excretion of cardiac glycosides it can be seen from the studies made by Towbin et al. (72) on the renal excretion of tritiated digoxin in dogs by using stop-flo* and clearance techniques that the filtered digoxin was reabsorbed by the proximal tubules and there was no evidence for tubular secretion of the drug. The clearance rate per kidney was 10 ml/min, which is higher in comparison to DHQ. SUMMARY
The effect of 10 mg/kg of dlhydronulnidine (administered intravenously) on myocardial conduction velocity was determined in the dog with a specially designed fourteen paired bipolar electrode assembly.
Tritium labeled dihydroquinidine was used to study the distribution, excretion and amount of metabolism of dihydroquinidine. —
The effect of the drug on conduction velocity was observed for 64 minutes. The maximum effect was seen within two minutes after the injection of the drug, when the velocity was reduced by 39 to 33.6 percent of normal. This effect disappeared with 29,3 to 25.4 percent
of reduction in conduction velocity seen during four to
sixteen minutes and 18 percent reduction in the conduction velocity during 32 to 64 minute post-injection.
Dihydroquinidine was assessed to be approximately
three times more potent in reducing the conduction velocity
than quinidine. This is based on the fact that the dose of DHQ required in this study to produce a similar response
as that obtained by other workers with quinidine was
approximately one-third as great with DHQ as with quinidine.
167 168
The plasma level studies indicated that the peak levels of 27.3 mcg/ml, 9,5 mcg/ml and 12,7 mcg/ml in arterial, venous and coronary sinus respectively were reached within one-half minute. The rate of decline in these vascular systems is described in two phases. The early rapid phase showed a biological half life of 1,4, 1.6 and 2.6 minutes in arterial, venous and coronary sinus plasma respectively. The biological half life calculated from the slow component for the arterial, venous and coronary sinus plasma was 187, 232 and 140 minutes respectively, A plateau in arterial, venous and coronary plasma was attained 16 to 32 minutes post-injection.
Following a plateau period, there was no significant difference in arterial and coronary sinus plasma levels.
However, the venous plasma levels always remained lower than arterial and coronary plasma.
The distribution of dihydroquinidine in tissues at the end of 64 minutes showed that the highest tissue to plasma ratio of 8.4 was in the kidney, followed by
7.6 in lung, and 6,0 in spleen. Among various muscular tissues, ventricles showed a higher tissue to plasma ratio of 3.6 in comparison to 1.6 and 0.8 in smooth and skeletal muscles, respectively. Among endocrine tissue, adrenal gland showed a higher tissue to plasma ratio than the ovary or thyroid, being 5,2, 2,4, and 1,0, respectively. 169
tissue to plasma ratio decreased over the interval from 64 minutes to six hours in all tissue # The decrease was greatest in the spleen and kidney and least in the lung and thyroid.
The determination of metabolites from various tissues and fluid indicated that a higher percent of the drug in the liver was present as metabolite than in the kidney or heart. The percent of the drug present as metabolites in the bile were approximately two to three fold of urine.
The cumulative biliary excretion of the drug was one-sixth that of renal excretion and it was only 4.1 percent of the total dose at the end of six hours. The renal clearance rate of the metabolite was approximately
2 to 10 fold greater than the nonmetabolite. BIBLIOGRAPHY
170 171
1. Armitage, A. K, : The influence of potassium concen tration on the action of quinidine and of some antimalarial substances on cardiac muscle* Brit* J, Pharmacol., 12: 74-78, 1957
2* Beckman,R.: Treatment in General Practice, W* B* Saunders Co., Philadelphia, 1934*
3. Bray, G. A*: A simple efficient liquid scintillator for counting aqueous solutions in a liquid scintillation counter* Anal* Biochem*, 1: 279-285, 1960*
4* Brodie, B. B*, Baer, J* E* and Craig, L* C«: Metabolic products of the cinchona alkaloids in human urine. J* Biol. Chero., 188: 567-581, 1951*
5. Brodie, B. B« and Undenfriend, S.: The estimation of basic organic compounds and a technique for the appraisal of specificity* J* Biol* Chem* 158: 705-714, 1945*
6 * Claus, Edward P. and Tyler, V*E, Jr.: Pharmacognosy, 5th ed*, 290-294, Lea and Febiger, Philadelphia, 1965*
7* Conn, H* L., Jr* and Luchi, R. J* : Ionic influences on quinidine albumin interaction* J* Pharmacol* Exp, Ther., 133: 76-83, 1960*
8 * Conn, H, L*, Jr* and Luchi, R. J*: Some quantitative aspects of the binding of quinidine and related quinidine compounds by human serum albumin. J* Clin* Invest*, 40: 3509-3516, 1961* -
9* Conn, H* L*, Jr. and Luchi, R* J*,: Cellular basis for regulation of the circulation by digitalis and quinidine* Circ* Res* 15, Supplement II, 153-168, 1964*
10* Conn, H* L., Jr*: Quinidine as an antiarrhythmlc agent* In Advances in Cardiopulm* Dis*, Edited by A, L* uanyai, and ti* L* uordon, xearoook Publishers, Chicago, 1964*
11, Crocker, H* D* and Smith, C* R * : The ventricular activation process in cattle* Ann. N.Y* Acad* Sci*, 127: 179-194, 1965* 172
12. Castiglloni, Arturo: A history of medicine, translated from the Italian. Edited by Krumbhar, E. B,, 2nd ed., A. A. Knopf, N. Y., 1947.
13. Cox, S., Roxburgh, G. and Wright, S. S.: The metabolism of ouabain in the rat, J. Fharm. and Pharmacol., 11: 535-539, 1959.
14. Delevett, Allen R. and Poindexter, C. A.; Plasma concentrations of quinidine with particular refer ence to therapeutically effective levels in two cases of paroxysmal nodal tachycardia. Amer. Heart J., 32; 697-703, 1946.
15. DiPalma, J. R. and Lambert, J. J.: Importance of the methoxy group in antifibrillatory compounds. Science, 107: 66-6 8 , 1948.
16. DiPalma, J. R.: Drill's Pharmacology in Medicine, 3rd ed., McGraw-Hill Book Co., H. Y., 1966.
17. Ditlefrsen, E. M, L.: Quinidine concentration in blood and excretion in urine following paranteral administration as related to congestive heart failure. Acta. Med. Scand., 159: 105-109, 1957.
18. Doherty, J. E. and Perkins, W. H.: Studies with tritiated digoxin in human subjects after intra venous administration. Amer. Heart J. 63: 528-536, 1962.
19. Durrer, D. and Van der Tweel, L. H,: Spread of activation in the left ventricular wall of the dog. Amer. Heart J., 46: 683-691, 1953.
20. Ferrer, M. I. et al.: Some effects of quinidine sulphate on ïhè heart and circulation in man. Amer. Heart J. 36: 816-837, 1948.
21. Frey, W ,: Weiters Erfaheungen mitchindin bei absoluter Herthunregelmasigkeit, Klin. Wschr., 55: 849-853, 1918.
22. Fitz, M. E., and Brooks, F. P.: The control of bile secretion in the unanesthetized dog. Fed. Proc., 21: 262, 1962.
23. Fitz, M. E., and Brooks, F. P.: Control of bile flow in the cholecystecomized dog. Amer. J. Physiol., 204: 825-828, 1963. 173 24. Goodman, Louis S. and Gilman, Alfred.: The Pharma cological Basis of Therapeutics. The Macmillan Co., N.Ÿ., 3rd ed,';”T96B”;
25. Hagg, H. B., Larson, P. S. and Schwartz, J.J.: The effect of urinary pH on the elimination of quinidine in man. J. Pharmacol* Exp. Ther., 79: 136-139, 1943.
26. Haggid, A. W.: Fundamental errors in the early history of cinchona. Bull. Hist. Med. 10: 417-466, 1941.
27. Han, J. and Hoe, G. K.: Non-uniform recovery of excitability in ventracular muscle. Circ. Res., 14: 44-60, 1964.
28. Heilman, Leon, Rosenfeld, R. S. and Gallagher, T. F.i Cholesterol synthesis from C^^ acetate in man. J. Clin. Invest., 33: 142-149, 1954.
29. Hoffman, B. F . : The action of quinidine and procaine amide on single fibers of dog ventricle and specialized conducting system. Anais Acad. Brasil Cienc. 29: 365-368, 1958.
30. Holland V. C.: A possible mechanism of action of quinidine. Amer. J, Physiol. 190: 492-494, 1957.
31. Hamlin, R. L. et al.: Effect of PLV-2, bradykinin, quinidine and vasopressin on myocardial conduction velocity in dogs. Amer. J. Vet. Res. 25: 1717-1723, 1964.
32. Harrison, C. E. et al.: The distribution and excretion of tritiated substances in experimental animals following the administration of digoxin-H*. J. Lab. Clin. Med. 87: 764-777, 1966.
33. Heidelberger, Michael and Jacobs, Walter A . : Syntheses in the cinchona series. I. The simpler cinchona alkaloids and their dihydro derivatives. J. Amer. Chem. Soc. 41: 826, 1919.
34. Hiatt, E. P. and Quinn, G. P.: The distribution of quinine, quinidine, cinchonine and cinchonidine in fluids and tissues of dogs. J, Pharmacol. Exp. Ther., 83: 101-105, 1945.
35. James, T. N. and Nadeau, R. A . : The mechanism of action of quinidine on the sinus node studied by direct perfusion through its artery. Amer. Heart J., 67: 804-811, 1964. 174
36. Jenkinsy Glenn et al.: The Chemistry of Organic Medicinal Products, 4th éd., John Wiley and sons, inc., V., 1947.
37. Johnson, E. A.: The effect Of quinidine, procaine amide and pyrilamine on the membrane resting and action potential of guinea pig ventricular muscule fiber. J. Pharmacol. Exp. Ther. 117: 237-244, 1956.
38. Kalmansohn, R. W. and Sampson, J. J.: Studies of plasma quinidine content, I. Relation to single dose administration by three routes. Circulation, 1: 564-568, 1950.
39. Kalmansohn, R. W. and Sampson, J. J.: Studies of plasma quinidine content. II. Relation to toxic manifestations and therapeutic effect. Circulation, 1: 569-575, 1950.
40. Kelsey, F. S., Oldham, F. K. and Ceiling, M. K. : The metabolism of quinine and quinidine in birds and mammals. J. Pharmacol. Exp. Ther. 85: 170-175, 1945.
41. Kaox, W. E.: The quinine-oxidizing enzyme and liver aldehyde oxidase. J. Biol. Chem. 163: 699-711, 1946.
42. Korns, H, H.: An experimental and clinical study of quinidine sulphate. Arch. Intern. Med., (Chicago) 31: 36-55, 1923.
43. Levy, R . : Alterations in the cardiac mechanism after administration of quinidine to patients with auricular fibrillation. Proc. Soc. Exp. Biol. Med., 19: 88-91, 1921.
44. Lewis, T. and Rothschild, M . : The excitatory process in the dog's heart. II. The ventricle., Phil. Trans. Boy. Soc. London; Ser. B, 206: 181-182, 1915.
45. Lewis, T.: Interpretations of the initial phase of the electrocardiogram with special reference to the theory of "limited potential differences." Arch. Intern. Med. 30: 269-285, 1922.
46. Lewis, T.: The value of quinidine in cases of auricular fibrillation and methods of studying the clinical reactions. Amer. J. Med. Sci., 163: 781- 794, 1922. 175 47. Linenthal, A. J., Ulick, 8. and Patterson, L. A . : Flurometrlc measurement of plasma quinidine and its correlation with cardiac effects in man., J. Clin. Invest. CAbst.) 26: 1188, 1947.
48. Linenthal, A. J,: The use of quinidine in the treatment of cardiac arrhythmias. Mod. Cone. Cardiov. Dis. 24: 299, 1955.
49. Loevinger, 0. R. and Berman, M , : Efficiency criteria in radioactivity counting. Nucleonics, 9: 26-39 (July) 1951.
50. Luchi, R. J., Helwig, J. and Conn, H. L., Jr.: Quinidine toxicity and its treatment. Amer. Heart J. 65: 340-348, 1963.
51. Ling, G. and Gerard, R. W. : The normal membrane potential of frog saritorius fibers. J. Cell. Comp. Physiol., 34: 383-391, 1949.
52. Hark, L. C. et al.; The physiological disposition and cardiac effects of procaine amide. J. Pharmacol. Exp. Ther. 102: 5-15, 1951.
53. Marks, B.H., Dutta, S., Guathier, J. and Elliott, D . : Distribution in plasma, uptake by the heart and excretion of ouabain-H* in human subjects, J. Phar macol. Exp. Ther. 145: 351-356, 1964.
54. Marshall, R.W., Moreno, O.M. and Brodie, D.A.: Chronic bile duct cannulation in the dog. J. Appl. Physiol. 16: 1191-1192, 1964.
55. Moe, G. K. and Mendez, R . : The action of several cardiac glycosides on conduction velocity and ventricular excitability in the dog heart. Circulation 4; 729-734, 1951.
56. Œita, Geroge T., et al.: Blood level studies of C^^ digitoxin in human subjects with cardiac failure. J. Pharmacol. Exp. Ther. 113: 376-382, 1955.
57. Rabe, P.: Ber. deutsch. chem. Geo. 55: 522, 1922 cited by Wilson, C; O. and Griswold, 0. Textbook of Organic Medicinal and Pharmaceutical Chemistry. 4th ed. J. B. Lippincott Co., Philadelphia, 1962, 278. 176
58. Rayford, C. R., Huvos, A. and Gregg, D. B.: Chronic catheterization of the coronary sinus., Proc. Soc. Exp. Biol. Med., 113: 876-879, 1963,
59. Ruch, T, C. and Patton, H. D. : Philology and Biophysics, W. B. Saunders Co., Philadelphia, 18th ed., 1960.
60. Schaefer, H. and Trautwein, W.: Uber die elenentaren elektrischen prozesse im Herzmuakel und ihre Rolle fur eine neue Theorie des elektrokardiograms., Pfluger's Arch. Ges. Kiysiol, 251: 417-448, 1949.
61. Schaefer, H.: The general order of excitation and recovery, Ann, N, Y. Acad. Sci,, 65: 743-767, 1957.
62. Scher, A. H. and Young, A. C.: Ventricular depolar ization and the genesis of the QRS., Ann N.Y. Acad. Sci., 65: 768-778, 1957,
63. Scherlis, L., Gonzalez, L. F. and Bessman, 8 . P.: Quinidine, arterial, venous, coronary sinus and myocardial concentrations. J. Clin. Invest. 40: 60-65, 1961.
64. Scott, C. C., Anderson, R. C, and Chen, K. K.: Comparison of the pharmacological action of quinidine and dihydroquinidine. J. Pharmacol. Exp. Ther. 84: 184-188, 1945.
65. Smith, Homer W.: Principles of Renal Physiology. Oxford Univer. Press, New Vork, 1956.
6 6 . Sodi-Pallares, Bisteni, D. A., Medrano, G. A. and Cisneros, F. ; "Hie activation of the free left ventricular wall in the dog's heart. Amer. Heart J. 49: 587-602, 1955.
67. Sokolow, Maurice and Edgar, A. L.: Blood quinidine concentrations as a guide in the treatment of cardiac arrhythmias. Circulation,1: 576-592, 1950.
6 8 . Sokolow, M. and Ball, R. E.: Factors influencing conversion of chronic atrial fibrillation with special reference to quinidine concentration. Circulation, 14: 568-583, 1956. 177 69. Swain, H, H. and Weldner, C. L.: A study of substances which alter intraventricular conduction in the isolated dog heart. J. Pharmacol. Exp. Ther., 120: 137-146, 1957.
70. Taggart, J. V. et al.; Studies on the chemotherapy of the human malarias• III. The Physiological dispo sition and antimalarial activity of the cinchona alkaloid. In symposium on malaria. J. Clin. Invest. 27; 3 part 2: 80-86, 1948.
71. Takern, Higuchi and Hansen, E. B . ; Pharmaceutical Analysis, Interscience Publishers, Hew York, London, T^SST.--
72. Towbin, S. J., Doherty, J. E. and Ferrell, C. B: Renal excretion of tritiated digoxin. Clin. Res., 12: 72, 1964 (Atost.).
73. VanDongen, K. and Sanches, A. J. R . : The Action of some quinine derivatives on fibrillation of the heart. Arch. Int. Pharmacodyn., 55: 52-60, 1937
74. Vaughan, William: The mode of action of quinidine on isolated rabbit atria interpretted from intracellular potential records, Brit. J. Pharmacol. 13: 276-287, 1958.
75. Veyrat, H . : La Composition des potentiels électriques recueillis en derivation unipolaire a la surface du coeur., Helv. Hiysiol. Pharmacol. Acta, 11: 395-422, 1953.
76. Watanabe, Y., Drelfus, L. S. and Lickoff, W . : Electrophysiologic antagonism and synergism of K and antiarrhythmlc agents. Amer. J. Cardiol., 12: 702-710, 1963.
77. Wegrla, R. and Boyle, M. N.: Correlation between the effect of quinidine sulphate on the heart and its concentration in blood plasma. Amer. J. Med. 4: 373-382, 1948.
78. Weidmann, S.: Effect of calcium ions and local anesthetics on electrical properties of Purkinje fibers. J. Physiol. (London) 129: 568-582, 1955. 79. Weisman, 8 . A . : Studies on the time required for the elimination of quinidine from the heart and other organs. Amer. Heart J. 20: 21-33, 1940. 178
80. Weisman, S. A,: Quinidine, pure quinidine and hydroquinidine• Amer, Heart J, 24: 545-549, 1942.
81. Weisman, S. A.: Review and evaluation of quinidine therapy for auricular fibrillation, J, A.M.A., 152: 496-499, 1953.
82. Weiss, Soma and Hatcher, R. A . : Studies on quinidine, J. Pharmacol. Exp. Iher., 30: 335-345, 1927.
83. Wenckebach, K. F.: Cinchona derivatives in the treatment of heart disorders., J.A.M.A,, 81: 472-474, 1923.
84. Westf T. C. and Amory, D, W.: Single fiber recording of the effect of quinidine at atrial pacemaker sites in the isolated right atrium of the rabbit, J. Pharmacol. Exp. Ther., 130: 183-193, 1960.
85. Wheeler, H. O. and Ramos, 0. L . : Determination of the flow and composition of bile in the unanesthetized dog during constant infusions of sodium taurocholate, J. Clin. Invest. 39: 161-170, 1960.
8 6 . Whitney, R. A.: Distribution and excretion of tritium labeled dihydroquinidine. Master's dissertation. College of Veterinary Medicine, Ohio State University, 1965.
87. Willus, F. A. and Keys, T. E.: A remarkably early reference to the use of cinchona in cardiac arrhythmias, Proc. Staff Meetings, Mayo Clin., 17: 294-296, 1942,
8 8 . Wilson, Charles 0. and Gisvold, Ole: Textbook of Médicinal and Pharmaceutical Chemistry, 4th ed., J. B. Lippincott Co., Philadelphia, i»62.
89. Wolferth, C. C.: Depression of cardiac conductivity during quinidine therapy., J.A.M.A., 80: 1289-1291, 1923.
90. Zilversmith, D. B. , Entenman, C. and Fishier, M. C . : The turnover rate of phospholipids in the plasma of the dog as measured with radioactive phosphorus., J. Gen. Physiol. 26: 333-340, 1943.