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Pharmacokinetics of sodium dichloroacetate after repetitive intravenous infusion

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Title:
Pharmacokinetics of sodium dichloroacetate after repetitive intravenous infusion
Creator:
Chu, Pei-i, 1959-
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English
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vi, 80 leaves : ill. ; 29 cm.

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Subjects / Keywords:
Blood ( jstor )
Diabetes ( jstor )
Dosage ( jstor )
Erythrocytes ( jstor )
Kidney dialysis ( jstor )
Lactates ( jstor )
Lactic acidosis ( jstor )
Plasmas ( jstor )
Sodium ( jstor )
Volunteerism ( jstor )
Acetic Acids -- metabolism ( mesh )
Dissertations, Academic -- Pharmacy -- UF ( mesh )
Infusions, Parenteral ( mesh )
Pharmacy thesis M.S.P ( mesh )
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bibliography ( marcgt )
non-fiction ( marcgt )

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Thesis:
Thesis (M.S.)--University of Florida, 1984.
Bibliography:
Bibliography: leaves 77-80.
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Pei-i Chu.

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Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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/PHARMACOKINETICS OF SODIUM DICHLOROACETATE
AFTER REPETITIVE INTRAVENOUS INFUSION/










BY

PEI-I CHU


A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF MASTER OF SCIENCE
IN PHARMACY


UNIVERSITY OF FLORIDA


1984




/PHARMACOKINETICS OF SODIUM DICHLOROACETATE
AFTER REPETITIVE INTRAVENOUS INFUSION
BY
PEI-I CHU
A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF MASTER OF SCIENCE
IN PHARMACY
UNIVERSITY OF FLORIDA
1984


ACKNOWLEDGEMENTS
The author wishes to thank her major professor, Dr.
Stephen H. Curry, for his strong support and guidance. His
wonderful personality has also been appreciated. The author
would also like to thank the committee members, Dr. Kamlesh
M. Thakker, Dr. Stephen H. Schulman, and Dr. Margaret 0.
James, for all of their advice and suggestions during the
preparation for this thesis. The author must not forget to
acknowledge the typist Lynn for typing her thesis and having
the patience in producing the final draft. Finally, with
much joy and gratitude, the author would like to give
special thanks to her parents. Without them, she would not
have been able to become what she is today.
n


TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS ii
ABSTRACT v
CHAPTER
I.INTRODUCTION 1
Dichloroacetic Acid 1
Lactic Acidosis 6
Known Kinetics of DCA 8
Technique for Assaying DCA 9
II.PURPOSE OF THIS RESEARCH 11
III.MATERIALS AND METHODS 12
Chemicals 12
Apparatus 13
Preparation of Reagents 13
Human Subjects 15
Assay of DCA 16
Study of the Effect of DCA Metabolites
(Oxalate, Glyoxalate, Glycolic Acid) to
DCA Assay 17
Study of DCA Binding Using a Dianorm
Equilibrium Dialyser 17
Red Blood Cell Localization Study and .
Partition Coefficient 20
Oxalate Assay 21
Creatinine Assay 21
i i i


IV.DATA ANALYSIS 23
Compartment Model 23
Elimination Rate Constant and Half Life .... 23
Volume of Distribution 24
Protein Binding 25
Red Blood Cell Localization and Partition
Coefficient 26
Area Under the Blood Level Curve 27
Statistical Analysis 28
V.RESULTS 29
Identity of DCA and DCA Assay 29
Concentrations in Plasma 30
Protein Binding 31
Red Cell Localization 31
VI.DISCUSSION 66
Binding and Red Cell Localization 66
Pharmacokinetic Analysis 68
APPENDIX 74
REFERENCES 77
BIOGRAPHICAL SKETCH 81
tv


Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree Master of Science
in Pharmacy
PHARMACOKINETICS OF SODIUM DICHLOROACETATE
AFTER REPETITIVE INTRAVENOUS INFUSION
By
Pei-I Chu
August 1934
Chairman: Dr. Stephen H. Curry
Major Department: Pharmacy
Twelve healthy volunteers each received one or two
series of five intravenous infusion doses of sodium
dichloroacetate (DCA) at two hour intervals. Dose levels
were 10 mg/kg (three subjects), 15 mg/kg (one subject), 25
mg/kg (five subjects), and 50 mg/kg (4 subjects)(one male
subject was studied twice). Plasma DCA showed the expected
rise and fall during and after each dose, with higher
levels induced by higher doses. Twenty-four hour urinary
oxalate excretion was also measured and there was a linear
relationship between urinary oxalate excretion and DCA dose.
Protein binding was found by equilibrium dialysis to be
constant up to a total DCA concentration of 300y g/ml.
Individual protein binding fractions ranged from 0.2 to 0.42
v


in nine volunteers at 100 yg/ml DCA concentration. There
was no significant difference in the fraction bound when
dialysis was at 25 degrees or 37 degrees for one hour. The
partition coefficient of DCA between red blood cells and
plasma water was constant within any one batch of blood up
to 1000 yg/ml DCA. Individual partition coefficients in nine
volunteers ranged from 0.17 to 0.78 at a whole blood DCA
concentration of 100y g/ml.
The plasma levels were analyzed by comparison with zero
order infusion/first order elimination pharmacokinetic
models. The mean half life values following the first,
second, third, fourth and fifth infusions were 90.0 min,
110.0 min, 188.4 min, 210.0 min, and 451.8 min.
respectively. The area under the curve of concentration
versus time to infinity was linearly related to dose. There
was no significant change in apparent volume of distribution
after each dosing. The mean apparent volume of distribution
of DCA was 0.3 1/kg.
The average rates of metabolism of DCA following each
infusion were 1187.4, 1089.1, 881.7, 950.2, and 803.6
mg/hour. Because of the large standard deviations on these
means, there were no significant differences among these
data except for between the second and the last infusions.
This might indicate enzyme poisoning during DCA metabolism.
More volunteers need to be studied to draw more definite
conclusions.
vi


CHAPTER I
INTRODUCTION
Dichloroacetic Acid
Dichloroacetic acid has been known for many years as an
escharotic, topical keratolytic and topical astringent
compound [1]. Dichloroacetic acid is a relatively strong
organic acid. Its dissociation constant is 5140 x 10(-5)
which lies between that of monochloroacetic acid 155 x
10(-5) and trichloroacetic acid 12100 x 10(-5) Chemically,
it can be obtained through the reaction of chloral hydrate
and aqueous sodium cyanide:
CUC CH0H + NaCN
4 I
0H H
boil Cl C C00H
i
Cl
The decomposition of dichloroacetic acid is thought to
occur through the following pathway:
Dichloroacetic Acid H20 (HO fe CHC00H -* 0HCC00H + H20
(glyoxalic acid)
Glyoxalic acid boil/alkalU C00H C00H
> + ,
ch2oh C00H
Glycol lie Acid Oxalic Acid
1


2
This decomposition involves dechlorination. This
reaction also occurs enzymatically. The enzymatic mechanism
for dechlorination of dichloroacetate is unknown, but [14 C]
oxalate is formed from [ 14C] dichloracetate by isolated rat
liver mitochondria [2], suggesting that the necessary
enzymes are in these organelles. Bacterial dehalogenases
that substitute hydroxyl groups for the chlorine of
dichloroacetate have been described [3], Dehalogenation of
dichloroacetate has not been demonstrated in mammalian
tissues other than liver.
Dichloracetate possesses systemic pharmacological
activity. The LDso orally in rats is 2.82g/kg. The effects
of DCA on intermediary metabolism have also been extensively
studied. Dichloroacetate reduces blood glucose
concentrations in both diabetic and fasted animals but not in
healthy, fed animals [4-7]. The possible mechanism is
thought to occur through the pathway shown in Fig. 1 [8].
Dichloroacetate accelerates pyruvate, lactate and
alanine oxidation by activation of pyruvate dehydrogenase
via direct inhibition of pyruvate dehydrogenase kinase.
Consequently, release of lactate and alanine from peripheral
tissues into circulation is reduced [9], and fewer
three-carbon precursors are available for hepatic glucose
synthesis.
In a previous report [10] it was shown that orally
administered sodium dichloroacetate, at a daily dose of 50
mg/kg, rapidly and significantly decreased plasma lactate


3
Lactate Metabolism
G1ucose
1
Alanine ^ Pyruvate ?=> Lactate
.a .' 1 > w w v.\Mitochondrial
Membrane
Pyruvate
Pyruvate DCA
-Dehydrogenase
Acetyl CoA
l
Lipid Krebs Cycle Ketone Bodies
Fig. 1. Probable Mechanism of DCA on Glucose Metabolism.


4
and alanine concentrations in maturity-onset diabetic
patients who had normal or slightly elevated basal plasma
lactate concentration. Other workers have shown that
intravenously administered DCA can prevent or even reverse
hyperlactatemia and lactic acidosis induced in animals by
phenformin administration [11-13], acute hepatitis [14],
functional hepatectomy [15-16], epinephrine infusion [17],
or exercise [18,19].
Dichloroacetate reportedly diminishes the
hyperlactatemia of endotoxin shock in rabbits [20,21] and
chickens [22]. Pretreatment of rabbits with dichloroacetate
reduces both the degree of hyperlactatemia and mortality due
to E. Coli endotoxin. Loubatieres et al. first reported that
dichloroacetate has no effect on the hyperlactatemia of
hypoxic dogs [23], but later found that lactate values
returned to normal more quickly in dichloracetate treated
animals, but only after normal oxgenation was restored.
Dichloroacetate diminishes lactate levels after intense
muscular work in dogs [23]. Dichloroacetate also increases
the time rats can swim before exhaustion, an effect which
may be explained by dichloroacetate accumulation in muscle
[24].
Irsigler et al. first used dichloroacete to treat human
phenformin-related lactic acidosis [25]. Their first two
patients were severely acidotic (pH<6.95) and did not
survive. Their third patient was only slightly acidotic
(pH=7.3) and administration of dichloroacetate, fluids,


5
glucose, insulin, furosemide, tris buffer and oxygen brought
about marked clinical improvement.
Stacpoole et al. [26] administered dichloroacetate to
thirteen patients with lactic acidosis of various causes.
The metabolic effects of dichloroacetate were evaluated in
eleven patients. Seven patients had a mean decrease in
lactate of 80 percent. Despite the improvement in the lactic
acidemia, all patients but one died of their underlying
disease.
Dichloroacetate has potiential toxicity when given for
prolonged periods. In rats, it is metabolized by the liver
to the potientially toxic metabolites, oxalate and
glyoxylate [2]. Chronic oral doses of DCA 50 mg/kg/day
for several weeks produced a reversible neuropathy in
animals and in one human subject [27]. In contrast, acute
and sub-acute intravenous toxicity studies of DCA in animals
at doses even greater than 50mg/kg/day showed no evidence
of toxicity. In the study of Wells et al. [28], at an
intravenous dose of 50mg/kg, one healthy subject complained
of drowsiness up to 24 hours after drug administration. The
greater potential toxicity of DCA administered orally than
intravenously may be due to the fact that more of the drug
is initially converted in the liver to toxic metabolites when
it is given orally, since a greater proportion of a single
drug dose reaches the liver when given by mouth than when
given by vein. Toxicological evaluation was necessary for
higher and longer DCA dosing.


6
Lactic Acidosis
Lactic acidosis is a disorder of intermediary
metabolism. It can be defined as a metabolic acidosis due
totally or in part to an accumulation of blood lactate and
accompanying hydrogen ion.
The accumulation of lactate in the blood has always
been recognized as a normal response to vigorous exercise
and as a manifestation of acute hypoxia secondary to shock
or asphyxia. In 1961, Huckabee called attention to the
occurrence of hyperlactatemia in a variety of clinical
circumstances lacking evidence of either circulatory failure
or hypoxemia [29,30]. Since then, a large and still growing
literature on this subject has appeared and some experts now
suspect that lactic acidosis may be the most frequent form
of acute metabolic acidosis.
Lactate is produced and consumed in all cells, but
there is net production or consumption only in certain
tissues. Lactate is normally added to the plasma by the
skin, gut, skeletal muscles, blood cells and brain at a rate
of 10 to 20 mmol per kilogram of body weight per day. The
net rate of production of lactate in all cells is determined
by the rate of formation of pyruvate (with which the lactate
is in equilibrium) and by the cytoplasmic redox potential,
which influences the equilibrium relation between lactate and
pyruvate. The formation of pyruvate, in turn, is determined


7
mainly by the factors regulating glycolysis. Lactic
acidosis results whenever the rate of production of lactate
exceeds the capacity of the lactate utilizing reactions. The
primary disturbance may be an increase in production or a
decrease in utilization, or most frequently, a combination
of the two. In any case, the result is a rise in the blood
level of lactate and pyruvate, a fall in plasma bicarbonate
and usually some reduction in blood pH. Most clinicians
establish the diagnosis of lactic acidosis with a blood
level greater than 5 meq/1.
By far the most common clinical form of lactic acidosis
is that accompanying severe acute circulatory or respiratory
failure, in which the sudden rise in blood lactate is a
biochemical sign of tissue hypoxia. Sometimes, the lactate
level begins to rise even before the clinical signs of shock
appear, but the ominous implications of a rapidly increasing
blood lactate in injured or bleeding patients and in those
with sepsis or acute cardiac failure are by now all too
familiar. Unless their circulatory failure can be rapidly
reversed most such patients will die with severe lactic
acidosis.
It is well established that the first and most
important step in treating lactic acidosis is to attempt to
correct the underlying cause (e.g., infection with
antibiotics, heart failure with pressor drugs). Beyond
this, all other methods remain controversial. Use of sodium
bicarbonate is standard practice. Raising the arterial pH


8
above 7.0 will, in theory, convert the splanchnic bed from
a lactate-producing to lactate-consuming site. But rapid
intravenous boluses of NaHC03 may produce acute
intra/extra-cellular potassium and calcium shifts which, in
addition to alkalosis, may be arrythmogenic, and
intracellular pH is a complex function of arterial pH, pC02
and intracellular bicarbonate concentration. Thus one cannot
rationalize correcting intracel1ular pH merely by decreasing
extracellular acidemia. Animal experiments involving NaHC03
administration to dogs with experimentally-induced lactic
acidosis does not increase survival or improve arterial pH
and may, in fact, worsen cardiac function [31]. A limited
number of controlled clinical studies exist, which show that
treatment with NaHC3 significantly reduces morbidity or
mortality in patients with lactic acidosis, compared to
patients receiving only general supportive care. Thus
treatment of lactic acidosis needs to be reconsidered with
the use of NaHC03.
Known Kinetics of DCA
Dichloroacetate plasma levels have been studied only in
human volunteers given single intravenous doses. Plasma drug
concentrations were assayed by gas chromatography. Evidence
has shown that peak plasma concentrations were linearly
related to the dose up to 30 mg/kg [28]. At higher doses
some subjects showed disproportionately higher


9
concentrations than predicted by the linear relation seen at
lower doses. Nonlinear disposition was also indicated by
the time course of plasma DCA concentrations following drug
administration. Evidence had been obtained of applicability
of a Michaelis-Menten elimination model to DCA plasma level
data above 100 yg/ml, while a first order elimination model
was applicable at lower levels, with a mean DCA half-life of
approximately 30 minutes. For the patient study [26], an
apparent increase in the apparent volume of distribution of
DCA with second and third doses was observed and also there
was an apparent slowing of elimination, also with second and
third doses. Lukas et al [32] also observed a decreased
intrinsic clearance and plasma clearance for the higher dose
infusion case and concluded "dose dependent pharmacokinetics
of DCA."
Technique for Assaying DCA
Dichloroacetate is conventionally assayed by gas
chromatography and detected by electron capture detector.
Gas chromatography is one type of partition chromatography.
The components of a vaporized sample are fractionated as a
consequence of partition between a mobile gaseous phase and
a stationary phase held in a column. The stationary phase
of the column can be either an adsorbent or a liquid
distributed in the form of a thin film on the surface of a
solid support. The solid support consists of porous inert


10
particles. In this study, Chromasorb 101 was used as the
packing material. Chromosorb 101 consists of porous
particles. At the end of the column, the individual
components emerged separated in time. They were then
detected and the detector signal was displayed on a
recorder. Here the electron capture detector was used.
The effluent from the column was passed over an
emmiter-nickel-63. An electron from the emitter caused
ionization of the carrier gas and the production of a burst
of electrons. The electron capturing species eluting from
the column reacted with the electrons to form ions, which
were swept from the cell. The net result was a reduction in
the number of electrons found at steady state or a drop in
the standing current.
Quantitative analysis was based upon a comparison of
either the height or the area of chromatographic peak of the
analyte with the standards. A carefully measured quantity
of TCA as an internal standard was introduced into each DCA
standard and sample, and the ratio of DCA to TCA peak height
served as the analytical parameter. The calibration curve
was linear up to 200 yg/ml and the coefficient of variation
varied from 2.5 to 4.03%.


CHAPTER II
PURPOSE OF THIS RESEARCH
The purpose of this research is as follows.
(1 ) To confirm the identity of DCA.
(2) To evaluate the relationship between plasma levels of
DCA and dose.
(3) To characterize the apparent volume of distribution and
elimination rate constant on multiple dosing.
(4) To study the red blood cell localization and distribution
coefficient of DCA.
(5) To determine the protein binding of DCA at different dose
levels.
(6) To find out if the metabolites of DCA will interfere with
DCA analysis.
11


CHAPTER III
MATERIALS AND METHODS
Chemicals
Dichloroacetic acid was as the sodium salt obtained as
a gift from Tokyo Kasei Kogyo Company.
Trichloroacetic acid was purchased from Sigma Chemical
Company Lot 102F-0440, No. T-488 5.
Boron Trif1uororide-Methanol was purchased from Sigma
Chemical Company Lot No. 23F 7180, No. B-1252.
Benzene was purchased from Burdick & Jackson
Laboratories Inc, LC Grade Lot No. AD815.
Potassium monobasic phosphate was Analytical Grade
purchased from Mallinckrodt Inc., Lot WCPS. No. 7100.
Sodium chloride was also from Mallinckrodt Chemical
Works, Lot YGVB, No. 7581.
Sodium oxalate reagent grade was purchased from J.T.
Baker Chemical Company Lot No. 37256.
Sodium Glyoxylate was purchased from Sigma Chemical
Company Lot. 67C-5027, No. G-4502.
Glycolic acid was purchased from Sigma Chemical Company
Lot 99C-0243, No. G-1884.
Sodium phosphate dibasic reagent grade scientific
products distributed by Mallinckrodt, No. 7917-5.
Human plasma was obtained from pooled sources (blood
bank supplies) .
12


Dialysis membrane was purchased from Diachema AG, No.
13
10.15, diameter 64 nm, M.W. cut off 10,000.
Nitrogen gas used was from Liquid Air Corporation
(UN1066).
Apparatus
The gas chromatography system used was a Varian 3700
Model, with Ni Electron capture detector kit part no.
02-001972-00. The recorder was Model 9176 by Varian.
The centrifuge used was a Dynac II Centrifuge from Clay
Adams, Division of Becton, Dickinson and Company.
The pH meter used was Beckman Model 4500 digital pH
meter.
The rotator used was a Tube Rotator form Scientific
Equipment Products.
The Dianorm Equilibrium Dialyser used was from Diachema
A.G. (Ruschikon/Zurich). A Gilson pipetman was used for
injection. A Micro 2 cell type was used.
Preparation of Reagents
Isotonic Phosphate Buffer Solution
Isotonic buffer solution was made by adding 1.9g
potassium monobasic phosphate, 8.1g sodium dibasic phosphate
and 4.11g of sodium chloride to 1000ml volumetric flask and
adding deionized H 0 up to the mark. The pH was measured
with a Beckman pH meter as mentioned before.


14
Trichloroacetic Acid (TCA) Stock Solution and Working
Solution
The TCA stock solution was made by dissolving 250 mg of
TCA in 250 ml of deionized water. A one in ten dilution of
the stock solution (100y g/ml) was used as the internal
standard.
DCA Stock Solution and Standard Sample of Calibration Curve
The DCA stock solution was made by dissolving 100 mg
sodium dichloroacetate in 100 ml plasma. A series of
dilutions was made with a micropipette to give 20, 40, 60,
80, 100, 150, and 200 yg/ml solutions as standards for the
calibration curve.
Preparation of DCA Injection Solution
Two and one half grams of sodium dichloroacetate were
dissolved in 19 ml isotonic saline and 6 ml of sodium
phosphate injection solution (276 mg of monobasic sodium
phosphate monohydrate and 142 mg of dibasic sodium phosphate
per mililiter of H2O) to give a final concentration of 100
mg/ml. The solution was sterilized by autoclaving at 250
degrees for fifteen to twenty minutes after passage through
an 0.22 microfilter (Milipore). Samples of the injection
solution were assayed by gas chromatography. Limulus Lysate
Test was also performed to test for pyrogenicity.


Human Subjects
15
Seven male and five female healthy volunteers
participated. Ages ranged from 22 to 57 yrs (Table 1). One
male subject was studied twice, each time at a different
drug dose. Subjects were initially screened as outpatients
by means of medical history, physical examination, complete
blood count and routine chemical analysis of renal, hepatic
and coagulation function. They were then admitted as
inpatients on the morning of study to the Clinical Research
Center of Shands Hospital, University of Florida. The
subjects were fasted overnight. A small diameter catheter
was inserted into a large superficial vein in each forearm.
A heparin lock was attached to one catheter for blood
withdrawal, while the other catheter was kept open with a
slow saline infusion between DCA doses.
After the catheter placement, the first DCA infusion
was begun. Dichloroacetate in phosphate-buffered saline (pH
5.2) was diluted to a total volume of 50 ml and administered
over 30 minutes using a Harvard infusion pump. On each
occasion, the subject received five identical drug doses,
each spaced two hours apart. Repeated doses of 10 mg/kg DCA
were administered in the first three investigations. One
subject received 15 mg/kg doses. Five subjects, including
one of the first three, received 25 mg/kg doses. The last
four investigations were at the 50 mg/kg dose level. Blood
samples were obtained at 30 minute intervals for the first


16
8.5-9 hours, and subsequently at 3-4 hour intervals up to 24
hours after starting the first infusion. Two ml of blood
were collected in citrate-containing Vacutainer tubes for
DCA assay. The blood was stored in an ice bucket until
centrifuged. After centrifugation, the supernatant was
frozen at -20 degrees C until assayed. Immediately before
the first drug infusion, subjects voided and then commenced
a 24 hr urine collection for oxalate determination. This
project was approved by the applicable Institutional Review
Board (see Appendix). The subjects gave informed consent to
participate. An Initial New Drug certificate for the study
of DCA has been issued by the FDA to Dr. P.W. Stacpoole, the
physician in charge of the volunteers.
Assay of DCA
A sample (0.1 ml) of plasma (unknown or standard) was
placed in a Hypo-vial (sealable thick glass vial). If the
plasma concentration was too high, 0.1 ml of diluted plasma
was ued. Then 50 yl of a 100 yg/ml TCA solution and 2 ml of
14% boron trifluride in methanol were added. The vial was
closed with a rubber septum and then the septum was covered
with an aluminum cap and agitated briefly and heated in a
boiling water bath for ten minutes. The vial was then
cooled to room temperature. One mililiter of water and two
mililiters of benzene were then added to the vial through
the rubber septum. The vial was then rotated on the rotator


17
for two hours and then the vial was opened. The contents of
the vial were then poured into a test tube and centrifuged
at 2000 rpm for two minutes. Samples (2 y1) of the
supernatant (benzene layer) were then injected into the gas
chromatograph. The Chromosorb 101 column was 6 ft x 2mm
i.d. at a temperature of 180 degrees. The carrier gas was
nitrogen at a flow rate of 60 ml/min. The Ni-63 electron
capture detector was set at 300 degrees.
Study of the Effect of OCA Metabolites
(Oxalate, Glyoxalate, Glycolic Acid) to DCA Assay
Solutions were prepared containing 100 y g/ml DCA, with
1000, 100, and 10 yg/ml of oxalate, glyoxalate and glycolic
acid individually. Each sample was assayed in duplicate by
gas chromatography following the procedure described in the
DCA assay.
Study of DCA Binding Using
a Dianorm Equilibrium Dialyser
The Dianorm equilibrium dialyser consists of a set of
four racks of cell holders, each containing five dialysis
cells located between spring-loaded metal spacers to avoid
leakage. One rack of 5 cells was shown in Figure 2.
The cells are made of Teflon to minimize absorption of
the drug into the material of the Dialyser. The cells are
divided into two halves (half cell A and B, respectively)


18
Fig. 2. Diagram of Cell Unit of Dianorm Equilibrium
Dialyser
1 Driving Flange with Guide Rods
2. Teflon Cell BASE or Half-Cell A) 5 Pair in
3. Teflon Cell LID or Half-Cell B) Complete Stack'
4. Spring Loaded Cell Spacers, 6 in Complete Stack
5. Bearing Flange secured with 3 Knurled Nuts


19
which are separated by a semi-permeable membrane. The
solutions under study are injected into half cell. The
dialysis membranes used were previously soaked in deionized
water for 30 minutes. Each half cell was injected with
1.5ml of plasma or pH 7.4 isotonic buffer solution. For
consistency and convenience, the plasma was always put into
the thick half cell and buffer into the thin half cell. The
racks of the cells were rotated at 4 revolutions per minute.
To study the time to equilibrium, 100 yg/ml DCA was put
on buffer side and the cell was rotated. Samples were
withdrawn after 1, 2, 4, 8 and 16 hrs of dialysis. Both
buffer and plasma were assayed for DCA content. To study
whether DCA equilibrium was affected by adding it to either
the buffer side or the plasma side, a solution, (100 yg/ml)
of DCA was also put into the plasma side initially and
dialysed for 1, 2, 4, 8 and 16 hrs. After dialysis, the DCA
concentration on both sides were measured.
To study the effect of temperature on binding, dialysis
was performed at 37 degrees C and 25 degrees C in a water
bath. A solution (100 ug/ml) of DCA was added to the buffer
side and dialysed for one hour. The DCA concentrations on
both sides were assayed.
To study the effect of DCA concentration on protein
binding, DCA concentrations of 100, 150, 200, 250 and 300
ug/ml were dialyzed at 37 degrees C for one hour. 'The
sodium dichloroacetate was assayed on both sides
after dialysis.


20
Individual plasma samples were also dialyzed.
Diehloroacetic acid was added to blank plasma samples from
some of the subjects at a concentration of 100 yg/ml to
study the individual variability of protein binding. The
DCA concentrations on both sides after dialysis were assayed
and fraction of protein binding of DCA was calculated and
1 is ted.
Red Blood Cell Localization Study
and Partition Coefficient
Eighteen capped test tubes, each with 10 ml of whole
blood inside containing DCA at 100 yg/ml were put on a tube
rotator. Samples were withdrawn at 5, 10, 30, 60, 90, and
120 minutes to study the time to reach equilibrium. Three
tubes were used at each time point.
Centrifugation was at 2000 rpm for five minutes after
rotation. Plasma was separated from the red blood cells and
the plasma content of DCA was measured.
To study the effect of DCA concentration on red blood
cell localization, whole blood concentrations of 50, 100,
300, 600 and 1000 yg/ml DCA were prepared. These samples
were rotated for two hours to make sure equilibrium had been
reached. After centrifugation at 2000 rpm for 5 minutes,
separating plasma from the red blood cells, the plasma
content of DCA was assayed.


21
The partition coefficients were determined in the
following way. Fifteen capped test tubes, each with 5 ml
whole blood inside, were taken and washed with pH 7.4
isotonic buffer solution three times in order to remove all
the plasma protein. The washed blood cells were
reconstituted by adding pH 7.4 isotonic buffer solution to
give the original blood volume. This formed "pseudoblood."
Sodium dichloroacetate was added to the pseudoblood,
resulting, in triplicate, in whole "blood" concentrations of
50, 100, 300, 600 and 1000 yg/ml. After centrifuging at
2000 rpm and separating the buffer and red blood cells, the
DCA content in the buffer was assayed.
Oxalate Assay
Twenty four hour urine was collected and adjusted with
HC1 to a pH value less than 3, and mixed thoroughly. A 60
ml aliquot was submitted to the Clinical Chemistry
Laboratory in Shands Hospital of the J. Hillis Miller
Health Center, University of Florida. Analysis was done by
atomic absorption spectrophotometry [33].
Creatinine Assay
Twenty four hour urine was refrigerated until analysis.
The creatinine assay was carried out by the Clinical
Chemistry Laboratory in Shands Hospital. Creatinine


22
concentration was determined by the red color development on
reacting with picric acid [34].


CHAPTER IV
DATA ANALYSIS
Compartment Model
The concentration-time data for most drugs can be
analyzed with the use of compartmental models. The one
compartment model assumes the drug to be homogeneously
distributed throughout the body. The two compartment model
assumes a central compartment containing the blood volume
and the highly perfused tissues such as liver, kidneys and
lungs. The peripheral compartment then consists of the
poorly perfused tissues such as muscle and fat. Previous
studies [23,32] have shown that DCA follows a one compartment
distribution model in its distribution. So in this study, a
one compartment model was adopted.
Elimination Rate Constant and Half Life
The elimination rate constant assesses the speed of
drug elimination from the systemic circulation. It can be
calculated from the slope of the concentration-time profile.
If the drug follows first order kinetics, then the
elimination rate constant can be obtained by calculation of
the slope of the graph of natural logarithm of the
concentration versus time. If the drug follows a zero order
23


24
kinetics, then the elimination rate constant can be obtained
by calculating the slope of a concentration versus time
profile. In this study, the elimination rate constant of
each volunteer following termination of each infusion was
calculated in this way for both zero and first order
models and the goodness of fit (R2) was also calculated.
The elimination half-life (first order model) was also
calculated following termination of each infusion using the
equation:
t = 1n2 Eq. (1)
TFT
Volume of Distribution
Volume of distribution is not a real volume but a
hypothetical volume of body fluid that would be required for
the body content of the drug to distribute at the same
concentration as that found in the blood. Using the first
order rate constants, the apparent volume of distribution of
DCA was calculated using the formula:
-kel*t -kel*t
Cpt = ko [1-e ] + Cp e Eq. (2)
Vd*kel t-1
where:
Cpt is the peak plasma concentration following each
I.V. infusion,


25
ko
is the infusion rate,
Cp
t
is the time of infusion, in this work it is always
equal to 30 minutes or 0.5 hours),
is the plasma concentration prior to starting the
t-1
infusion.
If the plasma concentration was obtained after the first dose,
the Cp is equal to zero.
t-1
Protein Binding
Protein binding is the phenomenon occurring when a drug
combines with plasma protein or tissue protein to form a
reversible complex. Drug can be displaced from binding by
other compounds having higher affinity for the binding sites.
The pharmacologic effect is thought to be from the free drug
only. So the extent of binding is an important feature of
the drug, and the fraction of protein bound is defined:
f = fD-PI = fD-PI
Eq. (3)
m +[o-p]
tot free
free
In this study, drug concentration on the plasma side is
given by [D] + [D-P], and drug concentration in the buffer
side is the free drug concentration, since only free drug
passes the membrane. So:


26
f =
|Drug
concentration
in plasma
sidej
Drug
concentration
in plasma
si de|
Eq. (4)
-
|Drug
concentration
in buffer
side'
|Drug
concentration
in plasma
side|
Red Blood Cell Localization and Partition Coefficient
The purpose of studying red cell localization is to
find out the amount of DCA that is bound to red blood cells.
The partition coefficient can be defined as:
D = Drug Concentration in Erythrocyte Eq. (5)
Drug Concentration in Plasma Water
This is significant in at least two ways. First blood
is often hemolyzed on collection. Hemolysis will then cause
inflation of plasma levels. Second, red blood cell
localization can be a significant factor in calculation of
volume of distribution. Variations in red blood cell
localization can possibly cause variation in drug response.
The partition coefficient can also be used to calculate
the fraction of protein binding if the drug concentrations
in red cells and in the plasma are obtained [35], since:
D = { Cb f
?p*-a--f)*(i_H) xrry
1} (1-H) Eq. (6)
H


27
Multiplication of both sides by H, and rearrangement gives:
H*D = Cb (1-H)
tnrt
Eq. (7)
where H is the hematocrit which can be determined
experimentally, Cb stands for whole blood concentration
which can be determined before the experiment, Cp is the
plasma concentration after equilibrium with red cells, and f
is the calculated fraction of protein binding.
Area Under the Blood Level Curve
Area under the curve (AUC) is an estimate of the total
amount of drug absorbed for first order elimination. In
this study, it is calculated from:
AUC = AUC T +
o o Ke I
Eq. (8)
where AUCQ is the area under the curve from time zero to the
last sampling time T, which can be obtained by a trapezoidal
method, and Ct is the plasma concentration for the last
sample. Kel is the elimination rate constant of the drug
for each individual.


Statistical Analysis
28
The elimination rate constant was obtained by best fit
of the slope of linear regression using a T155-11 hand-held
calculator. The comparison of observed half-life, volume of
distribution, etc., of each individual after each dosing was
done by analyses of variance (ANOVA), the SAS statistical
program package, and Duncan's multiple range test [36]. A P
value of 0.05 was chosen as the standard of whether
treatment is of statistical difference or not. The
comparison of goodness of fit by using R? where R is
correlation coefficient for the linear model or the log.
linear model was done using student's t test.


CHAPTER V
RESULTS
Identity of DCA and DCA Assay
The DCA sample was shown to be of required identity and
purity first by NMR spectroscopy, which showed the CH bond
of the CHCI2 grouping (Fig. 3). The IR spectrum showed
bands consistant with C=0, COOH and C-Cl bonds (Fig. 4).
Elemental analysis gave a content which agreed with the
putative formula (Table 15). The gas chromatography of DCA
is shown in Fig. 5. The first peak is the methylester of
DCA on reaction of DCA with BF3 /methanol. That was proved
by injection of an authentic sample of the ester. The
second and also the last peak was the methylester of TCA.
Injection solutions typically assayed at 99.5% of the stated
concentration using the specific gas-chromatographic method
and both the DCA sample and the authentic ester sample as
standards. A typical calibration curve of peak height ratio
versus DCA concentration is presented in Fig. 6. The square
of the correlation coefficient for the relationship was
0.992. The relation was linear up to 200 yg/ml.
It has been known for some years that DCA is
metabolized to glyoxalic acid which is then converted to
oxalic acid. However, the present research was the first to
examine the relation of oxalic acid excretion to DCA dosing
29


30
(Fig. 7). There was no evidence in the literature to prove
that the putative DCA metabolites did not interfere with the
DCA assay. By adding various amounts (10, 100, 1000 yg/ml)
of oxalic, glyoxalic and glycolic acids separately to 100
yg/ml DCA sample, it was shown that there was no
relationship between contaminant and found DCA concentration
(Table 2). Besides, there were no interfering peaks on gas
chromatography.
Concentrations in Plasma
The concentration-time profiles in the volunteers
plasma are shown in Figs. 8-20. Concentrations showed the
expected rises during each infusion and the expected falls
following the end of each infusion, and a steady decline to
or towards zero after the first dose. There was, however, a
large degree of variation within the data. The area under
the curve for each set of data from zero to the 24 hour
point using the trapezoidal rule was calculated, and the
extrapolated plasma concentration to time infinity was used
to calculate the area under curve to infinity (Equation 8).
The correlation coefficient for a linear relationship
between area under curve to infinity and dose was 0.72,
(p<0.05).
Protein Binding
The time to reach equilibrium of protein binding was
one hour (Table 3). The effect of temperature on protein
binding was also studied. There was no significant
difference between 37 degrees C and 25 degrees C after


dialysis for one hour (Table 4). The DCA was added to both
plasma and buffer sides of the membrane separately, and
there was no significant difference in the results of the
two experiments (Tables 3 and 5). This means protein
binding of DCA was reversible. The study of the
relationship between protein binding and concentration
showed that binding was linear up to 300y g/ml (Table 6).
Red Cell Localization
It was shown that five minutes was enough for the
distribution of DCA into red blood cells to reach
equilibrium (Table 7). So a time of five minutes was used
for later experiments. It was shown that the partition
coefficient between red cells and plasma water was a
constant up to 1000yg/ml DCA whole blood concentration
(Table 8).


8.0
7.0
6.0
5.0
4.0
PPM ( 6 )
3.0
2.0
_l I
1.0 0
3. NMR Spectroscopy of Sodium Dichloroacetate in D20. Sweep offset was 300 HZ. (5 PPM)
GJ
ro


Fig. 4. IR Spectrum of Sodium Dichloroacetate.
co
CO


34
Blank Plasma
Fig. 5. Gas Chromatogram of
Dichloroacetate and
the Internal Standard
Trichloroacetic Acid,
both as Methyl
Esters.
Plasma
DCA


Peak Height Ratio
35
Concentration (p g/ml)
Fig.1 6. Calibration Graph for Dichloroacetate in
Plasma. Y = 0.07 + 0.0085x, r2 = 0.992,
where Y is the Peak Height Ratio and x is
the concentration.


0xcil3.tc Excietion (mg 0x3la.tc/mg Crctininc)
Fig. 7. Graph of 24 hours Urinary Oxalate Excretion
vs. Dose in Eleven Subjects Given Various
Doses of DCA. Y = 29 + 3.47x, R = 0.67,
where Y is mg oxalate/mg Creatinine,
x is the Dose of DCA. Subject #4 was not
Included because the Dose was in Doubt.


AUC (mg-h/ml)
Fig. 8. Graph of Area Under the Curve of Plasma
Dichloroacetate Concentration vs. Time
against Dose in Eleven Subjects given
Various Doses. Y = 0.]6x 0.24, R =
0.73, where Y is the Area Under the Curve
in mg-h/ml and x is the Dose in mg/kg.
Subject #4 was in Doubt.


Concent rotion(ug/m|)
Fig. 9. Plasma Concentration Time Profile for Subject No. 1 after 10 mg/kg
IV Infusion. "Si Stands for Infusion Period.


250
200
150
100
50
0
Fig. 10. Plasma Concentration Time Profile for Subject No. 2 after 10 mg/kg
IV Infusion. " Stands for Infusion Period.
CO
CD


Fig. 11. Plasma Concentration Time Profile for Subject No. 3a after 10 mg/kg
IV Infusion. "! Stands for Infusion Period.
-p
o


Fig. 12. Plasma Concentration Time Profile for Subject No. 4 after 15 mg/kg
IV Infusion. Stands for Infusion Period.


160
140
120
100
80
60
40
20
0
Time (hr)
. 13. Plasma Concentration Time Profile for Subject No. 5 after 25 mg/kg
IV Infusion. "!! Stands for Infusion Period.
-P*
no


Concentration(ug/m1)
160
140
Fig. 14. Plasma Concentration Time Profile for Subject No. 6 after 25 mg/kg
IV Infusion. | Stands for Infusion Period.


160
Fig. 15. Plasma Concentration Time Profile for Subject No. 7 after 25 mg/kg
IV Infusion. ¡ Stands for Infusion Period.


200T
Fig. 16. Plasma Concentration Time Profile for Subject No. 8 after 25 mg/kg
IV Infusion. "|j Stands for Infusion Period. ^
cn


160
140
120
100
80
60
40
20
-*
O'
ig. 17. Plasma Concentration Time Profile for Subject No. 3b after 25 mg/kg
IV Infusion. j | Stands for Infusion Period.


Fig. 18. Plasma Concentration Time Profile for Subject No. 9 after 50 mg/kg
IV Infusion. Stands for Infusion Period.


1000
800
600
400
200
O'
ig. 19. Plasma Concentration Time Profile for Subject No. 10 after 50 mg/kg
IV Infusion. j j Stands for Infusion Period.
4=
00


Concentration (ig/ml)
Fig. 20. Plasma Concentration Time Profile for Subject No.11 after 50 mg/kg
IV Infusion. Stands for Infusion Period.


Fig. 21. Plasma Concentration Time Profile for Subject No. 12 after 50 mg/kg
IV Infusion. "jj Stands for Infusion Period.


51
TABLE 1. Demographic Data.
Patient No.
Sex
Age
Wt
(kg)
Hematocrit
[%)
Dose
(mg/kg)
1
M
25
74.8
44.4
10
2
M
22
76.0
49.0
10
3a
M
42
72.4
39.2
10
4
F
23
61.4
41.3
15
5
M
24
80.6
45.5
25
6
M
24
67.8
42.6
25
7
M
33
73.3
42.8
25
8
F
37
56.8
39.9
25
3b
M
42
72.4
39.2
25
9
M
57
101.4
47.5
50
10
F
45
57.6
41.5
50
11
F
27
65.0
44.1
50
12
F
27
58.0
40.9
50


52
TABLE 2. Determination of the Interference of DCA
Metabolite in DCA Assay. 100 yg/ml DCA was
Initially Added to Three Different Concentrations
of DCA Metabolite.
y g/ml DCA.
Results were Expressed as
DCA Plus:
1000 y g/ml
100 yg/ml
10 ^ g/ml
metabolite
metabolite
metabolite
Metabolite:
Oxalate
103.6
99.9
102.3
104.8
99.9
102.3
Glycolate
97.5
105.9
101.1
101.1
97.5
98.9
Glycoxylate
110.8
103.6
104.8
97.5
107.2
105.9


53
TABLE 3. Determination of Time to Reach Dialysis Equilibrium
Using Dianorm Equilibrium Dialyser. Dichloro-
acetate 100 yg/ml was Initially Added to the Plasma
Side.
Assayed
Concentrations

of DCA on
Both
Sides were
1
2
Time (hr)
4
8
16
Plasma
Concentration
55.1
49.9
54.2
54.6
58.1
(yg/ml)
53.4
50.8
62.9
57.9
53.6
Buffer
Concentration
35.8
33.4
38.4
36.0
41.2
(yg/ml)
36.2
36.2
33.7
36.2
31.6


54
TABLE 4. Studies of the Effect of Temperature on Protein
Binding. Dichloroacetate (100 y g/ml) Was
Initially Added to Buffer Side. Both Buffer and
Plasma Side were Assayed After Dialysis, "f"
Stands for Fraction of Protein Binding.
37
C
25
C
PI asma
Side
Cone.
(y g/ml)
Buffer
Side
Cone.
(y g/ml)
f
Plasma
Side
Cone.
(y g/ml)
Buffer
Side
Cone.
(yg/ml)
f
Dialysis
57.1
45.4
0.20
59.5
43.9
0.26
After
One
57.8
42.4
0.27
58.9
45.1
0.23
Hour
55.8
41.4
0.26
53.3
45.5
0.15
AVE
56.9
43.1
0.24
57.2
44.8
0.21
SE
0.57
1.21
0.02
1.96
0.46
0.03


55
TABLE 5. Determination of Time to Reach Dialysis
Equilibrium. Dich1oroacetate (100 y g/ml) was
Initially Added to the Buffer Side. Concentrations
of DCA on Both Sides were Assayed.
1
Time
2
(hr)
4
8
16
Plasma
58.2
59.4
52.6
52.9
53.4
(y g/ml )
47.4
52.2
46.3
55.0
47.1
Buffer
35.5
39.2
35.6
33.8
37.4
(ug/ml)
34.0
33.5
33.8
39.8
30.9


TABLE 6. Studies of Protein Binding of DCA at Different Concentrations. Sodium Dichloroacetate was Assayed
on both Buffer and Plasma Side. Y = 0.59X -2.82, R = 0.0996, Y stands for Average Plasma Side
Concentration After Dialysis. X stands for Initial DCA Concentration. Y' = 2.54 + 0.38X, R = 0.998,
Y' stands for Average Buffer Side Concentration After Dialysis.
Original DCA
Concentration
Added to
Buffer Side
(pg/ml)
100
150
200
250
300
P
b
f
P
b
f
P
b
f
P
b
f
P
b
f
55.3
37.6
0.32
78.7
66.0
0.16
95.1
61.6
0.35
159.9
112.6
0.25
160.2
108.1
0.33
56.6
38.3
0.32
85.6
61.9
0.28
126.1
84.8 0.33
177.8
98.5
0.45
164.3
116.4
0.29
53.0
38.3
0.28
68.2
58.5
0.14
141.9
80.0
0.44
128.4
97.3
0.24
173.5
107.0
0.38
56.5
40.1
0.29
91.2
66.4
0.27
110.3
76.5
0.30
140.1
95.5
0.30
178.1
116.8 0.34
109.80
80.0
0.27
Mean
55.4
38.6
0.30
81.0
63.0
0.21
116.5
76.6
0.33
149.3
100.9
0.31
169.4
112.1
0.34
SE
0.09
0.55
0.01
4.3
1.9 (
3.03
8.05
3.90
0.03
10.5
3.9
0.07
4.1
2.6
0.02
(_n
cr>


57
TABLE 7. Determination of Time to Reach Equilibrium for Red
Cell Localization. Dichioroacetate (100yg/ml)
was Initially Added to the Whole Blood. Plasma
Concentration was Assayed. Blood was Obtained
from Volunteer 3a.
5
10
Time 1
30
[min)
60
90
120
140.7
130.6
131.6
131.6
130.1
133.9
Concentraton
135.2
139.9
125.9
120.9
127.0
120.5
(y g/ml)
143.0
132.2
136.1
134.0
127.9
136.2
130.8
129.1
145.3
127.9
124.2
115.3
140.7
126.4
140.7
129.4
130.3
146.6
138.8
135.3
135.9
123.4
135.5
152.7
Mean
138.2
132.2
135.9
127.9
129.2
134.2
SE
1.8
2.0
2.8
2.0
1.6
5.9


58
TABLE 8. Partition Coefficient (P) of DCA Between Red Blood
Cells and Isotonic Buffer up to 1000 yg/ml Whole
Pseudoblood Concentration. Blood was Obtained From
Subject 3a.
DCA Concentration ,
y g/ml
50
100
300
600
1000
0.19
0.43
0.46
0.22
0.39
0.32
0.39
0.35
0.58
0.44
p
0.47
0.46
0.39
0.73
0.45
0.41
0.33
0.48
0.79
0.41
0.23
0.32
0.52
0.45
0.42
0.47
0.52
0.36
0.71
0.42
Mean
0.35
0.41
0.42
0.58
0.42
SE
0.05
0.03
0.03
0.09
0.01


59
TABLE 9. Protein Binding (f), Red Blood Cell/Plasma Ratio
(RBC/P), and Partition Coefficient for Red Blood
Cell/Plasma Water Distribution in Nine Volunteers.
Da'ta were Obtained at 100 yg/ml Initial DCA
Concentration.
3a
4
Subject
5 7
No.
8
9
10
11
12
f
0.30
0.39
0.33 0.32
0.20
0.35
0.42
0.40
0.20
RBC/P
0.08
0.47
0.20 0.06
0.32
0.42
0.17
0.47
0.57
Partition
Coefficient
0.17
0.78
0.42 0.36
0.42
0.57
0.29
0.66
0.62


60
2
TABLE 10. Sodium Dichloroacetate in Plasma: Calculated R and kel Values
for Repeated I.V. Infusions in Different Volunteers, Using a
First Order Kinetics Model.
Infusion
Number
I
II
III
IV
V
kel
R2
kel
R2
kel
R2
kel
R2
kel
R2
Volunteer
1
1.54
0.72
0.92
0.94
0.38
0.94
0.25
0.89
0.25
0.89
2
0.01
0.06
0.56
1.00
0.07
0.55
0.06
0.11
0.03
0.85
3a
2.09
0.98
2.45
0.96
1.90
0.93
1.13
0.89
1.10
1.00
4
0.13
0.74
0.02
0.02
0.18
0.63
0.01
0.02
0.26
0.84
5
0.74
0.92
0.30
0.70
0.17
0.83
0.20
0.97
0.26
0.88
6
0.90
0.97
0.36
0.48
1.56
0.66
0.18
0.92
0.23
0.97
7
0.47
0.80
0.35
0.90
0.05
0.01
0.16
0.70
0.12
0.95
8
2.76
0.90
0.62
0.99
0.20
0.74
0.20
0.70
0.03
0.84
3b
0.41
0.81
0.31
0.94
0.21
0.91
0.22
0.98
0.20
0.98
9
0.37
0.97
0.31
0.99
0.18
0.29
0.30
0.76
0.14
0.98
10
0.25
0.55
0.19
0.55
0.12
0.87
0.29
0.87
0.10
0.87
11
0.14
0.08
0.45
0.90
0.11
0.29
0.03
0.19
0.07
0.86
12
0.41
0.08
0.45
0.90
0.11
0.29
0.03
0.19
0.07
0.86
Mean
0.98
0.71
0.64
0.80
0.50
0.63
0.30
0.68
0.22
0.91
SE
0.27
0.09
0.05
0.08
0.19
0.08
0.08
0.09
0.08
0.02


61
TABLE 11. Calculated DCA Volume of Distribution (1/kg) Using
a First Order Kinetic Model Applied to Repeated IV
Infusion Data in 11 Subjects. Subject 4 was not
Included Because the Dose was not Certained. R <
0.81 Was Not Listed (Expressed in
No. of Infusion
I
II
III IV
V
Volunteer
1
0.13
0.31
0.32
0.28
0.35
3a
0.20
0.10
0.11
0.19
0.25
5
0.25
0.29
0.43
0.34
0.49
6
0.36
--
0.25
0.20
0.6
7
0.37
-1.18
-14.2
8
0.47
0.28
0.28
0.37
0.27
3b
0.24
0.45
0.43
0.39
0.39
9
0.24
0.23
0.27
0.25
10
--
0.49
0.16
0.09
11
0.23
--
0.22
12
0.23
0.30
--
0.42
0.22
AVE
0.28
0.27
0.30
0.29
0.31
SE
0.03
0.04
0.04
0.03
0.05
Negative data was not included in average.
Subject 2 was not included because data was not available


62
TABLE 12. Calculated Half Life for Volunteer After Each
Dose. R Value Smaller Than 0.81 in Line Fit was
not Included (Expressed in
No. of Infusion
I
II
III IV
V
Volunteer
1
0.45
0.75
1.82
2.77
1.87
2
1.24
23.10
3a
0.33
0.28
0.36
0.61
0.63
4
5.5
2.67
5
0.94
2.31
4.10
3.46
2.66
6
0.77
--
3.85
3.01
7
1.47
1.98
4.33
5.77
8
0.25
1.15
3.46
3.46
23.1
3b
1.69
2.24
3.3
3.15
3.46
9
1.87
2.24
2.31
4.95
10
--

5.78
2.39
6.93
11
--
1.54
--
9.9
12
1.69
4.62
--
8.66
9.9
AVE
1.50
1.84
3.14
3.50
7.53
SE
0.46
0.36
0.66
0.63
2.07


63
TABLE 13. Dichloroacetate in Plasma: Rates of Metabolism
of Each Subject After Repeated IV Infusions
at 2-Hour Intervals (Numbered Consecutively
I-V). Subject 2 was not Included Because
Data Was Not Available. R < 0.81 was not
Listed (Expressed in
Interval
I
II
III
IV
V
Subject
1
829.2
901.7
334.5
273.9
383.0
3a
953.3
1037.7
874.6
634.2
706.8
4
110.2
1522.2
5
1259.9
738.4
677.6
805.1
1460.2
6
1230.2
2266.0
241.9
1029.2
7
766.1

8
3271.4
757.3
293.3
523.7
82.9
9
1730.6
2020.7
2987.2
1400.1
10
--
1004.8
1478.4
474.5
11
1621.4
--
466.6
12
1046.7
678.8
--
781.5
506.1
AVE
1187.4
1089.1
881.7
950.2
803.6
SE
267.6
169.5
250.9
282.2
146.8
* Data was not available


64
2
TABLE 14. Sodium Di chioroacetate in Plasma: Calculated R and Rate
Constants for Repeated I.V. Infusion in 12 Volunteers,
Using a Zero Order Kinetics Model.
Infusion
Number
I
II
III
IV
V
kel
R2
kel
R2
kel
R2
kel
R2
kel
R2
Volunteer
1
+3.07
0.71
19.8
0.86
10.7
0.97
10.6
0.86
5.56
0.95
2
1.30
0.25
55.5
0.99
10.0
0.51
9.1
0.11
4.80
0.78
3a
+18.9
0.75
35.4
0.70
33.6
0.78
21.5
0.81
4.63
0.73
4
+16.6
0.75
3.42
0.01
42.8
0.70
1.72
0.05
10.5
0.84
5
+39.4
0.95
24.2
0.82
17.7
0.83
25.4
0.97
9.81
0.97
6
+15.8
0.66
15.4
0.47
15.6
0.99
15.5
0.93
8.10
0.95
7
-38.8
0.83
-24.4
0.86
6.34
0.02
-21.4
0.70
5.68
0.85
8
+29.1
0.96
48.6
0.95
15.0
0.73
21.3
0.71
4.58
0.78
3b
+28.4
0.77
24.0
0.95
20.3
0.93
24.6
0.99
9.11
0.93
9
+55.2
0.98
70.3
0.997
49.2
0.33
84.4
0.77
22.0
0.97
10
+48.6
0.57
38.2
0.52
30.6
0.87
114.8
0.84
37.7
0.72
11
+12.5
0.04
86.1
0.93
27.4
0.29
11.1
0.22
21.2
0.80
12
+59.6
0.77
35.2
0.99
81.0
0.63
29.6
0.79
23.8
0.84
Average
29.7
0.69
38.01
0.77
27.7
0.66
30.8
0.67
12.5
0.85
SE
4.9
0.08
6.86
0.080
5.7
0.08
9.72
0.09
2.56
0.03
Negative kels were not included in average.


65
TABLE 15. Identity of Sodium Dich1oroacetate by Elemental
Analysis.
ELEMENT
FOUND
THEORY
C
15.71
15.8
H
1.39
1.3
0
21.1
Cl
46.35
46.7
Na
15.1


CHAPTER VI
DISCUSSION
Binding and Red Cell Localization
In this research, attention was particularly focussed
on plasma concentrations of dichloroacetate. A major factor
in interpreting plasma concentrations of drugs is bindg of
the drugs to plasma protein. This was studied for
dichloroacetate using the Dianorm apparatus. Preliminary
experiments concerned factors such as time to reach
equilibrium.
Times to reach dialysis equilibrium are different for
individual ligands. Also, the dialysis time increases with
decreasing temperature, increasing dialysis volume,
increasing thickness of membrane, and increasing molecular
weight of ligand.
The Dianorm apparatus is designed for optimal geometry
and a high concentration gradient is maintained across the
membrane. In this research, slow rotation was used (4 rpm)
to make sure mixing was thorough. The thin wall of the
cell assured rapid heat exchange between water bath and cell
content. By using this apparatus DCA equilibrium was
reached within one hour (Table 3). There was no significant
difference for equilibrium time between 25 degrees C and 37
degrees C.
Binding of drugs to plasma protein may have a marked
effect on the distribution and pharmacologic effects of a
66


67
drug, and on the rate at which it is eliminated from the
body. This is because distribution of drugs to body tissues
takes place from the blood compartment. Also, it is the
free drug concentration which equilibrates with the body
tissues. The fraction of drug in the plasma bound to plasma
protein is not immediately available for distribution into
the extravascular space or for certain modes of elimination.
Because of this, the binding of dichloroacetate to plasma
proteins from individual subjects was studied. The fraction
bound at a whole plasma dichloroacetate concentration of 100
ug/ml varied from 0.2 to 0.42 (Table 9). At this level,
protein binding is generally considered to be of no great
clinical significance.
Several drugs enter the erythrocyte quite rapidly and
others do so at a slower rate. The extent of penetration
can often be correlated with the solvent/water partition
coefficients of these drugs highly lipid soluble drugs
show greater accumulation in the erythrocytes.
Dichloroacetate is a highly water soluble compound, so the
predicted partition coefficient between erythrocytes and
water is very low. This was proved to be so by the present
experiments. It has been suggested that determination of the
RBC/plasma concentration ratio may serve as a simple and
rapid technique for the indirect measurement of plasma
protein binding. This would be applicable in the
large-scale screening of abnormal plasma binding in routine
clinical blood samples, thereby facilitating


68
individualization of drug therapy. However, in our case,
the RBC/plasma concentration ratio did not show very large
variation (Table 9).
The calculated fractions of DCA protein binding in two
subjects using equation 7 were 0.60 (subject 3a) and 0.59
(subject 5). Each of these figures is a mean of six
determinations. The standard deviation in subject 3a was
0.23. These means are higher than the means for fraction
bound obtained by equilibrium dialysis. However, they were
obtained by using the mean partition coefficient and the
standard deviation of the partition coefficient was not
taken into consideration. It has been shown in the
literature that buffer systems, ionic strength and pH value
will affect binding properties [37,38,39]. It is necessary
to conduct further investigations concerning the various
factors affecting DCA protein binding. But from the
clinical point of view, 0.3 versus 0.60 was not of
physiological significance.
Pharmacokinetic Analysis
The decay of DCA concentrations in plasma was examined
following termination of each infusion. In each patient,
the four available concentration-time points from dose I to
IV and five or six available points following the last
infusion were compared with a linear model (concentration
versus time) and then with a logarithmic concentration versus


69
time model. The mean values of (where R is the
correlation coefficient) for the relationship using both
models are listed in Table 10 and Table 14. Although the
goodness of fit was comparable for the two models, the zero
order model was rejected for two reasons. First, other
investigators have shown first order kinetics to apply at
the level of this data seen in this work after the initial
doses. There was a trend that more data points gave better
R2 (where R is the correlation coefficient) for the first
order than zero order model. This was found by comparing
the R? for the data after the fifth doses using first order
or zero order models. The first order model is
significantly better than the zero order model at p=0.1
level. Second, the putative zero order slopes were
calculated to range from 4.58ug/ml/h to 114.8 yg/ml/h. That
means the output rate would be very close or even greater
than input rate, which is inconceivable.
Using only the data for which R was greater than 0.81,
which is the lower limit for significance of a four-point
correlation, the mean half-life calculation was found to be
90.0 min for first dosing, 110.4 min, 188.4 min, 210.0 min,
and 451.8 min for 2nd, 3rd, 4th, and 5th infusions (Table
12). The mean half-life rose steadily from the first dose
to the fifth dose, and the differences are statistically
different (p<0.005).
Data of this kind could have several mechanistic
implications. For example, first order decay kinetics are a


70
special case of Michaelis-Menten (MM) kinetics, applicable
when the substrate concentration (S) is low. In this work S
was clearly low enough for the first order simplification of
the MM model to apply, even when S increased from the first
to the fifth dose. However, it can be argued that the data
should have shown gradual deviation from the first order
case at higher S values if Kn and Vmax had remained
constant. Since this devation did not occur, it may be that
changes in either Kn or Vmax occurred as dosage progressed,
but with retention of applicability of the first order
model. Mechanisms for such a change could include slowing of
dichloroacetate metabolism following enzyme poisoning by the
substrate, and/or product inhibition. Definition of the
mechanism must await suitable further studies, including in
vitro metabolism studies, and development of zero order
input/first order output pharmacokinetic models appropriate
to intermittent intravenous infusion dosing. Additionally,
models for zero order input Michaelis-Menten output will be
needed.
The calculated rates of metabolism after each dose are
listed in Table 13. These rates were obtained by multiplying
plasma DCA concentrations by volumes of distribution and
rate constants of elimination. There was a small trend
towards a reduced rate of metabolism with time. Statistics
showed that the rate of metabolism after the fifth dose was
significantly less than that after the second dosing
(p<0.05). These calculations were conducted because three


71
possible observations could have been made:
(1) an increase in the rate of metabolism with time
and/or level might have indicated adherence to first order
or Michaelis-Menten kinetics, with a tendency towards
saturation of the enzyme concerned (the normal for enzyme
kinetics);
(2) a confirmed decrease in the rate of metabolism
would have indicated a reduction in enzyme activity; and
(3) no change would have been equivocal. While failing
to confirm a decrease, the calculations do rule out an
increase. There is support for the theory that DCA
metabolism involves enzyme poisoning or product inhibition
but more experiments are needed to confirm this idea.
The applications of the term "volume of distribution"
are many, including those to dosage regimen calculation of
drugs based on the characteristics of an individual patient.
It is possible to observe different plasma concentration
values in different individuals after the administration of
the same dose because of the differences in the volume of
distribution. That is the rationale for the calculation of
dosage adjustment according to body weight.
No evidence was obtained for a volume of distribution
change with repeated dosing (Table 11). This observation
disagreed with earlier observations in patients [26].
However, all of the patients in the other study were in shock
at the time of commencing DCA infusion, and a rise in blood
pressure, possibly caused by a positive inotropic effect of


72
DCA occurred. This is being investigated further, but it is
not to be expected to be seen in healthy volunteers, who
showed no cardiovascular changes when treated with DCA.
Obviously, a change in the DCA volume of distribution might
well be linked to cardiac output and related cardiovascular
status.


APPENDIX


74
UNIVERSITY OF FLORIDA J. HILLIS MILLER HEALTH CENTER BOXJ-14
Vice President for Health Affair* Gainesville, Florida zip 32610
HEALTH CENTER INSTITUTIONAL REVIEW BOARD MAY 11, 1983
TO:
FROM:
SUBJECT:
Peter W. Stacpoole, M. D., Ph.D.
Assistant Professor, College of Medicine
B. Joe Wilder, M. D., J-l*
Chairman, Institutional Review Board
Approval of the Institutional Review Board
Project # 133-83 entitled Clinical Pharmacology of*
Sodium Dichloroacetate (DCA)."
uW
The Institutional Review Board has recommended the approval of your protocol,
identified above, for a period of 12 months, and has determined that
human subjects will be at risk.
Approval of your research project is, therefore, granted until 5/11/84
By the end of this period you will be asked to inform the Board on the status
of your project. If this has not been completed, you may request renewed
approval at that time.
You are reminded that a change in protocol in this project requires its resub
mission to the Board. Also, the principle investigator must report to the Chair
of the Institutional Review Board promptly, and in writing, any unanticipated
problems involving risks to the subjects or others, such as adverse reactions
to biological drugs, radio-isotopes or to medical devices.
If it is anticipated that VA patients will be included in this project, or if
the project is to be conducted in part on VA premises or performed by any VA
employee during VA-compensated time, final approval should be obtained by appli
cation to the Veterans Administration Hospital Research Office.
By a copy of this memorandum the chairman of your department is reminded that
he is responsible for being informed concerning research projects involving
human subjects in his department. He should review the protocols of such
investigations as often as he thinks is necessary to insure that the experiment
is being conducted in compliance with our institution and with DHHS regulations.
cc: James E. McGuigan, M. D. co-PIs
B. Joe Wilder, M. D. JStephen H. Curry, Ph.D.
Thomas G. Baumgartner, M. Ed., Pharm.D. T. G. Baumgartner, Pharm.D.
William C. Thomas, Jr., M. D.
Clinical Research Center
Becky Stevens, Ph.D.
Collogo of Medicino College of Nursing o College of Phormocy o Cotlogo of Health Related Profossions Coliogo of Dontisfry
Collogo of Veterinary Medicine o Shonds Teaching Hospital and Clinics a Votorons Administration Hospital
KOUAU (MlOVMCMr O anon TUN IT Y / A m*MA T V t ACTION CMNUOTKO


75
oA
HEALTH CENTER INSTITUTIONAL REVIEW BOARD
A-M
UNIVERSITY OF FLORIDA
Vice
resident for Health Affairs
J. HILLIS MILLER HEALTH CENTER BOX J-14
Gainesville, Florida zip 32610
May 11, 1983
Peter W. Stacpoole, M. D., Ph.D.
Assistant Professor
College of Medicine
Department of Medicine
Box J-226
SUBJECT: Protocol #133-83, Clinical Pharmacology of
Sodium Dichloroacetate (DCA)
Dear Dr. Stacpoole:
Your protocol, referenced above, was reviewed at our
meeting this date.
It was the decision of the Committee that this study can be
approved if the following condition is met:
In the three (3) Informed Consent Forms, clearly state
that there is remote possibility of added risks because
of the use of this drug. (state what these are).- If
there are none, state so.
You will find enclosed the Approval Letter for this study.
Please send four copies (4) each of the three (3) revised
Informed Consent Forms to this office (J-14) at your earliest
convenience, since approval was contingent upon this condition
being met.
We are glad to be of service to you in this capacity, and we
appreciate your cooperation in this matter.
aw*, mk
B. Joe Wilder, M. D.
Chairman
BJW/ddo
continued
Coll* ge of Mdicin College of Nursing e College of Pharmacy e College of Health Related Professions e College of Dentistry
College of Veterinary Medicine e Shonds Teaching Hospital and Clinics e Veterans Administration Hospital
EQUAL employment opportunity/a ppipmatiye action employer


Page 2 May 11, 1983
Protocol #133-83
P. W. Stacpoole, M. D.,
Ph.D.
cc: "St ephen H. Curry, Ph.D.,
co-PI
Thomas G. Baumgartner, M.Ed.,
Pharm.D., co-PI
File
Enclosure: Approval Letter (#133-83)


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80
(35) Garrett, E.R., and Lambert, H.J., "Pharmacokinetics of
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J. Pharm. Pharmacol. 33:179-182, 1981.


BIOGRAPHICAL SKETCH
Pei-I Chu was born in Cang Shang, Taiwan, Republic of
China, on November 29, 1959. She graduated from the
Department of Pharmacy of National Taiwan University in
June, 1981, and passed the National Pharmacy board in July
that year. She worked as a teaching assistant in the
Department of Biochemistry in the College of Medicine of
National Taiwan University from 1981 to 1982. She joined
the graduate program in the College of Pharmacy, University
of Florida, in September, 1982, to work towards the degree
of Master of Science in Pharmacy with a major in
pharmaceutics, under the guidance of Dr. Stephen H. Curry.


I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of M-5-t-er of Science,
in Pharmacy. ( U ,,
N,(j^
Stephen H. Curry^ Chairman
Professor of Pharmacy
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,as
a dissertation for the degree of Mas-ter^of Scien^t.
iin Pharmacy. / / ^ ^'
Stephen H. S^hulman
Professor of Pharmacy
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Maste/ of Science,
in Pharmacy.
71 jUt
Kamlesh M. Thakker
Assistant Professor of
Pharmacy
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adeq.uate, in scope and quality, as
a dissertation for the degree of Master of Science
in Pharmacy.
Margaret 0.
J
James
Assistant Professor of
Medicinal Chemistry


This Thesis was submitted
of Pharmacy and to the Graduate
fulfillment of the requirements
in Pharmacy.
August, 1984
to the Graduate Faculty of the College
School, and was accepted as partial
for the degree of Master
sOuZ'
Dean,
Science
/tC(/ Pharmacy
Dean for Graduate Studies and
Research


Full Text
/PHARMACOKINETICS OF SODIUM DICHLOROACETATE
AFTER REPETITIVE INTRAVENOUS INFUSION
BY
PEI-I CHU
A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF MASTER OF SCIENCE
IN PHARMACY
UNIVERSITY OF FLORIDA
1984

ACKNOWLEDGEMENTS
The author wishes to thank her major professor, Dr.
Stephen H. Curry, for his strong support and guidance. His
wonderful personality has also been appreciated. The author
would also like to thank the committee members, Dr. Kamlesh
M. Thakker, Dr. Stephen H. Schulman, and Dr. Margaret 0.
James, for all of their advice and suggestions during the
preparation for this thesis. The author must not forget to
acknowledge the typist Lynn for typing her thesis and having
the patience in producing the final draft. Finally, with
much joy and gratitude, the author would like to give
special thanks to her parents. Without them, she would not
have been able to become what she is today.
n

TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS ii
ABSTRACT v
CHAPTER
I.INTRODUCTION 1
Dichloroacetic Acid 1
Lactic Acidosis 6
Known Kinetics of DCA 8
Technique for Assaying DCA 9
II.PURPOSE OF THIS RESEARCH 11
III.MATERIALS AND METHODS 12
Chemicals 12
Apparatus 13
Preparation of Reagents 13
Human Subjects 15
Assay of DCA 16
Study of the Effect of DCA Metabolites
(Oxalate, Glyoxalate, Glycolic Acid) to
DCA Assay 17
Study of DCA Binding Using a Dianorm
Equilibrium Dialyser 17
Red Blood Cell Localization Study and .
Partition Coefficient 20
Oxalate Assay 21
Creatinine Assay 21
i i i

IV.DATA ANALYSIS 23
Compartment Model 23
Elimination Rate Constant and Half Life .... 23
Volume of Distribution 24
Protein Binding 25
Red Blood Cell Localization and Partition
Coefficient 26
Area Under the Blood Level Curve 27
Statistical Analysis 28
V.RESULTS 29
Identity of DCA and DCA Assay 29
Concentrations in Plasma 30
Protein Binding 31
Red Cell Localization 31
VI.DISCUSSION 66
Binding and Red Cell Localization 66
Pharmacokinetic Analysis 68
APPENDIX 74
REFERENCES 77
BIOGRAPHICAL SKETCH 81
tv

Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree Master of Science
in Pharmacy
PHARMACOKINETICS OF SODIUM DICHLOROACETATE
AFTER REPETITIVE INTRAVENOUS INFUSION
By
Pei-I Chu
August 1934
Chairman: Dr. Stephen H. Curry
Major Department: Pharmacy
Twelve healthy volunteers each received one or two
series of five intravenous infusion doses of sodium
dichloroacetate (DCA) at two hour intervals. Dose levels
were 10 mg/kg (three subjects), 15 mg/kg (one subject), 25
mg/kg (five subjects), and 50 mg/kg (4 subjects)(one male
subject was studied twice). Plasma DCA showed the expected
rise and fall during and after each dose, with higher
levels induced by higher doses. Twenty-four hour urinary
oxalate excretion was also measured and there was a linear
relationship between urinary oxalate excretion and DCA dose.
Protein binding was found by equilibrium dialysis to be
constant up to a total DCA concentration of 300y g/ml.
Individual protein binding fractions ranged from 0.2 to 0.42
v

in nine volunteers at 100 yg/ml DCA concentration. There
was no significant difference in the fraction bound when
dialysis was at 25 degrees or 37 degrees for one hour. The
partition coefficient of DCA between red blood cells and
plasma water was constant within any one batch of blood up
to 1000 yg/ml DCA. Individual partition coefficients in nine
volunteers ranged from 0.17 to 0.78 at a whole blood DCA
concentration of 100y g/ml.
The plasma levels were analyzed by comparison with zero
order infusion/first order elimination pharmacokinetic
models. The mean half life values following the first,
second, third, fourth and fifth infusions were 90.0 min,
110.0 min, 188.4 min, 210.0 min, and 451.8 min.
respectively. The area under the curve of concentration
versus time to infinity was linearly related to dose. There
was no significant change in apparent volume of distribution
after each dosing. The mean apparent volume of distribution
of DCA was 0.3 1/kg.
The average rates of metabolism of DCA following each
infusion were 1187.4, 1089.1, 881.7, 950.2, and 803.6
mg/hour. Because of the large standard deviations on these
means, there were no significant differences among these
data except for between the second and the last infusions.
This might indicate enzyme poisoning during DCA metabolism.
More volunteers need to be studied to draw more definite
conclusions.
vi

CHAPTER I
INTRODUCTION
Dichloroacetic Acid
Dichloroacetic acid has been known for many years as an
escharotic, topical keratolytic and topical astringent
compound [1]. Dichloroacetic acid is a relatively strong
organic acid. Its dissociation constant is 5140 x 10(-5)
which lies between that of monochloroacetic acid 155 x
10(-5) and trichloroacetic acid 12100 x 10(-5) . Chemically,
it can be obtained through the reaction of chloral hydrate
and aqueous sodium cyanide:
CUC - CH0H + NaCN
J i
0H H
boil Cl - C - C00H
â–º i
Cl
The decomposition of dichloroacetic acid is thought to
occur through the following pathway:
Dichloroacetic Acid H20 (HO fe CHC00H -* 0HCC00H + H20
(glyoxalic acid)
Glyoxalic acid boil/alkalU C00H , C00H
> I + I
ch2oh C00H
Glycol lie Acid Oxalic Acid
1

2
This decomposition involves dechlorination. This
reaction also occurs enzymatically. The enzymatic mechanism
for dechlorination of dichloroacetate is unknown, but [14 C]
oxalate is formed from [ 14C] dichloracetate by isolated rat
liver mitochondria [2], suggesting that the necessary
enzymes are in these organelles. Bacterial dehalogenases
that substitute hydroxyl groups for the chlorine of
dichloroacetate have been described [3], Dehalogenation of
dichloroacetate has not been demonstrated in mammalian
tissues other than liver.
Dichloracetate possesses systemic pharmacological
activity. The LDso orally in rats is 2.82g/kg. The effects
of DCA on intermediary metabolism have also been extensively
studied. Dichloroacetate reduces blood glucose
concentrations in both diabetic and fasted animals but not in
healthy, fed animals [4-7]. The possible mechanism is
thought to occur through the pathway shown in Fig. 1 [8].
Dichloroacetate accelerates pyruvate, lactate and
alanine oxidation by activation of pyruvate dehydrogenase
via direct inhibition of pyruvate dehydrogenase kinase.
Consequently, release of lactate and alanine from peripheral
tissues into circulation is reduced [9], and fewer
three-carbon precursors are available for hepatic glucose
synthesis.
In a previous report [10] it was shown that orally
administered sodium dichloroacetate, at a daily dose of 50
mg/kg, rapidly and significantly decreased plasma lactate

3
Lactate Metabolism
G1ucose
1
Alanine ^ Pyruvate ?=> Lactate
.a .' 1 > w w v.\Mitochondrial
T Membrane
Pyruvate
Pyruvate DCA
-Dehydrogenase
Acetyl CoA
l
Lipid Krebs Cycle Ketone Bodies
Fig. 1. Probable Mechanism of DCA on Glucose Metabolism.

4
and alanine concentrations in maturity-onset diabetic
patients who had normal or slightly elevated basal plasma
lactate concentration. Other workers have shown that
intravenously administered DCA can prevent or even reverse
hyperlactatemia and lactic acidosis induced in animals by
phenformin administration [11-13], acute hepatitis [14],
functional hepatectomy [15-16], epinephrine infusion [17],
or exercise [18,19].
Dichloroacetate reportedly diminishes the
hyperlactatemia of endotoxin shock in rabbits [20,21] and
chickens [22]. Pretreatment of rabbits with dichloroacetate
reduces both the degree of hyperlactatemia and mortality due
to E. Coli endotoxin. Loubatieres et al. first reported that
dichloroacetate has no effect on the hyperlactatemia of
hypoxic dogs [23], but later found that lactate values
returned to normal more quickly in dichloracetate treated
animals, but only after normal oxgenation was restored.
Dichloroacetate diminishes lactate levels after intense
muscular work in dogs [23]. Dichloroacetate also increases
the time rats can swim before exhaustion, an effect which
may be explained by dichloroacetate accumulation in muscle
[24].
Irsigler et al. first used dichloroacete to treat human
phenformin-related lactic acidosis [25]. Their first two
patients were severely acidotic (pH<6.95) and did not
survive. Their third patient was only slightly acidotic
(pH=7.3) and administration of dichloroacetate, fluids,

5
glucose, insulin, furosemide, tris buffer and oxygen brought
about marked clinical improvement.
Stacpoole et al. [26] administered dichloroacetate to
thirteen patients with lactic acidosis of various causes.
The metabolic effects of dichloroacetate were evaluated in
eleven patients. Seven patients had a mean decrease in
lactate of 80 percent. Despite the improvement in the lactic
acidemia, all patients but one died of their underlying
disease.
Dichloroacetate has potiential toxicity when given for
prolonged periods. In rats, it is metabolized by the liver
to the potientially toxic metabolites, oxalate and
glyoxylate [2]. Chronic oral doses of DCA 50 mg/kg/day
for several weeks produced a reversible neuropathy in
animals and in one human subject [27]. In contrast, acute
and sub-acute intravenous toxicity studies of DCA in animals
at doses even greater than 50mg/kg/day showed no evidence
of toxicity. In the study of Wells et al. [28], at an
intravenous dose of 50mg/kg, one healthy subject complained
of drowsiness up to 24 hours after drug administration. The
greater potential toxicity of DCA administered orally than
intravenously may be due to the fact that more of the drug
is initially converted in the liver to toxic metabolites when
it is given orally, since a greater proportion of a single
drug dose reaches the liver when given by mouth than when
given by vein. Toxicological evaluation was necessary for
higher and longer DCA dosing.

6
Lactic Acidosis
Lactic acidosis is a disorder of intermediary
metabolism. It can be defined as a metabolic acidosis due
totally or in part to an accumulation of blood lactate and
accompanying hydrogen ion.
The accumulation of lactate in the blood has always
been recognized as a normal response to vigorous exercise
and as a manifestation of acute hypoxia secondary to shock
or asphyxia. In 1961, Huckabee called attention to the
occurrence of hyperlactatemia in a variety of clinical
circumstances lacking evidence of either circulatory failure
or hypoxemia [29,30]. Since then, a large and still growing
literature on this subject has appeared and some experts now
suspect that lactic acidosis may be the most frequent form
of acute metabolic acidosis.
Lactate is produced and consumed in all cells, but
there is net production or consumption only in certain
tissues. Lactate is normally added to the plasma by the
skin, gut, skeletal muscles, blood cells and brain at a rate
of 10 to 20 mmol per kilogram of body weight per day. The
net rate of production of lactate in all cells is determined
by the rate of formation of pyruvate (with which the lactate
is in equilibrium) and by the cytoplasmic redox potential,
which influences the equilibrium relation between lactate and
pyruvate. The formation of pyruvate, in turn, is determined

7
mainly by the factors regulating glycolysis. Lactic
acidosis results whenever the rate of production of lactate
exceeds the capacity of the lactate utilizing reactions. The
primary disturbance may be an increase in production or a
decrease in utilization, or most frequently, a combination
of the two. In any case, the result is a rise in the blood
level of lactate and pyruvate, a fall in plasma bicarbonate
and usually some reduction in blood pH. Most clinicians
establish the diagnosis of lactic acidosis with a blood
level greater than 5 meq/1.
By far the most common clinical form of lactic acidosis
is that accompanying severe acute circulatory or respiratory
failure, in which the sudden rise in blood lactate is a
biochemical sign of tissue hypoxia. Sometimes, the lactate
level begins to rise even before the clinical signs of shock
appear, but the ominous implications of a rapidly increasing
blood lactate in injured or bleeding patients and in those
with sepsis or acute cardiac failure are by now all too
familiar. Unless their circulatory failure can be rapidly
reversed most such patients will die with severe lactic
acidosis.
It is well established that the first and most
important step in treating lactic acidosis is to attempt to
correct the underlying cause (e.g., infection with
antibiotics, heart failure with pressor drugs). Beyond
this, all other methods remain controversial. Use of sodium
bicarbonate is standard practice. Raising the arterial pH

8
above 7.0 will, in theory, convert the splanchnic bed from
a lactate-producing to lactate-consuming site. But rapid
intravenous boluses of NaHC03 may produce acute
intra/extra-cellular potassium and calcium shifts which, in
addition to alkalosis, may be arrythmogenic, and
intracellular pH is a complex function of arterial pH, pC02
and intracellular bicarbonate concentration. Thus one cannot
rationalize correcting intracel1ular pH merely by decreasing
extracellular acidemia. Animal experiments involving NaHC03
administration to dogs with experimentally-induced lactic
acidosis does not increase survival or improve arterial pH
and may, in fact, worsen cardiac function [31]. A limited
number of controlled clinical studies exist, which show that
treatment with NaHCÜ3 significantly reduces morbidity or
mortality in patients with lactic acidosis, compared to
patients receiving only general supportive care. Thus
treatment of lactic acidosis needs to be reconsidered with
the use of NaHC03.
Known Kinetics of DCA
Dichloroacetate plasma levels have been studied only in
human volunteers given single intravenous doses. Plasma drug
concentrations were assayed by gas chromatography. Evidence
has shown that peak plasma concentrations were linearly
related to the dose up to 30 mg/kg [28]. At higher doses
some subjects showed disproportionately higher

9
concentrations than predicted by the linear relation seen at
lower doses. Nonlinear disposition was also indicated by
the time course of plasma DCA concentrations following drug
administration. Evidence had been obtained of applicability
of a Michaelis-Menten elimination model to DCA plasma level
data above 100 yg/ml, while a first order elimination model
was applicable at lower levels, with a mean DCA half-life of
approximately 30 minutes. For the patient study [26], an
apparent increase in the apparent volume of distribution of
DCA with second and third doses was observed and also there
was an apparent slowing of elimination, also with second and
third doses. Lukas et al. [32] also observed a decreased
intrinsic clearance and plasma clearance for the higher dose
infusion case and concluded "dose dependent pharmacokinetics
of DCA."
Technique for Assaying DCA
Dichloroacetate is conventionally assayed by gas
chromatography and detected by electron capture detector.
Gas chromatography is one type of partition chromatography.
The components of a vaporized sample are fractionated as a
consequence of partition between a mobile gaseous phase and
a stationary phase held in a column. The stationary phase
of the column can be either an adsorbent or a liquid
distributed in the form of a thin film on the surface of a
solid support. The solid support consists of porous inert

10
particles. In this study, Chromasorb 101 was used as the
packing material. Chromosorb 101 consists of porous
particles. At the end of the column, the individual
components emerged separated in time. They were then
detected and the detector signal was displayed on a
recorder. Here the electron capture detector was used.
The effluent from the column was passed over an
emmiter-nickel-63. An electron from the emitter caused
ionization of the carrier gas and the production of a burst
of electrons. The electron capturing species eluting from
the column reacted with the electrons to form ions, which
were swept from the cell. The net result was a reduction in
the number of electrons found at steady state or a drop in
the standing current.
Quantitative analysis was based upon a comparison of
either the height or the area of chromatographic peak of the
analyte with the standards. A carefully measured quantity
of TCA as an internal standard was introduced into each DCA
standard and sample, and the ratio of DCA to TCA peak height
served as the analytical parameter. The calibration curve
was linear up to 200 yg/ml and the coefficient of variation
varied from 2.5 to 4.03%.

CHAPTER II
PURPOSE OF THIS RESEARCH
The purpose of this research is as follows.
(1 ) To confirm the identity of DCA.
(2) To evaluate the relationship between plasma levels of
DCA and dose.
(3) To characterize the apparent volume of distribution and
elimination rate constant on multiple dosing.
(4) To study the red blood cell localization and distribution
coefficient of DCA.
(5) To determine the protein binding of DCA at different dose
levels.
(6) To find out if the metabolites of DCA will interfere with
DCA analysis.
11

CHAPTER III
MATERIALS AND METHODS
Chemicals
Dichloroacetic acid was as the sodium salt obtained as
a gift from Tokyo Kasei Kogyo Company.
Trichloroacetic acid was purchased from Sigma Chemical
Company Lot 102F-0440, No. T-488 5.
Boron Trif1uororide-Methanol was purchased from Sigma
Chemical Company Lot No. 23F - 7180, No. B-1252.
Benzene was purchased from Burdick & Jackson
Laboratories Inc, LC Grade Lot No. AD815.
Potassium monobasic phosphate was Analytical Grade
purchased from Mallinckrodt Inc., Lot WCPS. No. 7100.
Sodium chloride was also from Mallinckrodt Chemical
Works, Lot YGVB, No. 7581.
Sodium oxalate reagent grade was purchased from J.T.
Baker Chemical Company Lot No. 37256.
Sodium Glyoxylate was purchased from Sigma Chemical
Company Lot. 67C-5027, No. G-4502.
Glycolic acid was purchased from Sigma Chemical Company
Lot 99C-0243, No. G-1884.
Sodium phosphate dibasic reagent grade scientific
products distributed by Mallinckrodt, No. 7917-5.
Human plasma was obtained from pooled sources (blood
bank supplies).
12

Dialysis membrane was purchased from Diachema AG, No.
13
10.15, diameter 64 nm, M.W. cut off 10,000.
Nitrogen gas used was from Liquid Air Corporation
(UN1066).
Apparatus
The gas chromatography system used was a Varian 3700
Model, with Ni Electron capture detector kit part no.
02-001972-00. The recorder was Model 9176 by Varian.
The centrifuge used was a Dynac II Centrifuge from Clay
Adams, Division of Becton, Dickinson and Company.
The pH meter used was Beckman Model 4500 digital pH
meter.
The rotator used was a Tube Rotator form Scientific
Equipment Products.
The Dianorm Equilibrium Dialyser used was from Diachema
A.G. (Ruschikon/Zurich). A Gilson pipetman was used for
injection. A Micro 2 cell type was used.
Preparation of Reagents
Isotonic Phosphate Buffer Solution
Isotonic buffer solution was made by adding 1.9g
potassium monobasic phosphate, 8.1g sodium dibasic phosphate
and 4.11g of sodium chloride to 1000ml volumetric flask and
adding deionized H 0 up to the mark. The pH was measured
with a Beckman pH meter as mentioned before.

14
Trichloroacetic Acid (TCA) Stock Solution and Working
Solution
The TCA stock solution was made by dissolving 250 mg of
TCA in 250 ml of deionized water. A one in ten dilution of
the stock solution (100y g/ml) was used as the internal
standard.
DCA Stock Solution and Standard Sample of Calibration Curve
The DCA stock solution was made by dissolving 100 mg
sodium dichloroacetate in 100 ml plasma. A series of
dilutions was made with a micropipette to give 20, 40, 60,
80, 100, 150, and 200 yg/ml solutions as standards for the
calibration curve.
Preparation of DCA Injection Solution
Two and one half grams of sodium dichloroacetate were
dissolved in 19 ml isotonic saline and 6 ml of sodium
phosphate injection solution (276 mg of monobasic sodium
phosphate monohydrate and 142 mg of dibasic sodium phosphate
per mililiter of H2O) to give a final concentration of 100
mg/ml. The solution was sterilized by autoclaving at 250
degrees for fifteen to twenty minutes after passage through
an 0.22 microfilter (Milipore). Samples of the injection
solution were assayed by gas chromatography. Limulus Lysate
Test was also performed to test for pyrogenicity.

Human Subjects
15
Seven male and five female healthy volunteers
participated. Ages ranged from 22 to 57 yrs (Table 1). One
male subject was studied twice, each time at a different
drug dose. Subjects were initially screened as outpatients
by means of medical history, physical examination, complete
blood count and routine chemical analysis of renal, hepatic
and coagulation function. They were then admitted as
inpatients on the morning of study to the Clinical Research
Center of Shands Hospital, University of Florida. The
subjects were fasted overnight. A small diameter catheter
was inserted into a large superficial vein in each forearm.
A heparin lock was attached to one catheter for blood
withdrawal, while the other catheter was kept open with a
slow saline infusion between DCA doses.
After the catheter placement, the first DCA infusion
was begun. Dichloroacetate in phosphate-buffered saline (pH
5.2) was diluted to a total volume of 50 ml and administered
over 30 minutes using a Harvard infusion pump. On each
occasion, the subject received five identical drug doses,
each spaced two hours apart. Repeated doses of 10 mg/kg DCA
were administered in the first three investigations. One
subject received 15 mg/kg doses. Five subjects, including
one of the first three, received 25 mg/kg doses. The last
four investigations were at the 50 mg/kg dose level. Blood
samples were obtained at 30 minute intervals for the first

16
8.5-9 hours, and subsequently at 3-4 hour intervals up to 24
hours after starting the first infusion. Two ml of blood
were collected in citrate-containing Vacutainer tubes for
DCA assay. The blood was stored in an ice bucket until
centrifuged. After centrifugation, the supernatant was
frozen at -20 degrees C until assayed. Immediately before
the first drug infusion, subjects voided and then commenced
a 24 hr urine collection for oxalate determination. This
project was approved by the applicable Institutional Review
Board (see Appendix). The subjects gave informed consent to
participate. An Initial New Drug certificate for the study
of DCA has been issued by the FDA to Dr. P.W. Stacpoole, the
physician in charge of the volunteers.
Assay of DCA
A sample (0.1 ml) of plasma (unknown or standard) was
placed in a Hypo-vial (sealable thick glass vial). If the
plasma concentration was too high, 0.1 ml of diluted plasma
was ued. Then 50 yl of a 100 yg/ml TCA solution and 2 ml of
14% boron trifluride in methanol were added. The vial was
closed with a rubber septum and then the septum was covered
with an aluminum cap and agitated briefly and heated in a
boiling water bath for ten minutes. The vial was then
cooled to room temperature. One mililiter of water and two
mililiters of benzene were then added to the vial through
the rubber septum. The vial was then rotated on the rotator

17
for two hours and then the vial was opened. The contents of
the vial were then poured into a test tube and centrifuged
at 2000 rpm for two minutes. Samples (2 y1) of the
supernatant (benzene layer) were then injected into the gas
chromatograph. The Chromosorb 101 column was 6 ft x 2mm
i.d. at a temperature of 180 degrees. The carrier gas was
nitrogen at a flow rate of 60 ml/min. The Ni-63 electron
capture detector was set at 300 degrees.
Study of the Effect of OCA Metabolites
(Oxalate, Glyoxalate, Glycolic Acid) to DCA Assay
Solutions were prepared containing 100 y g/ml DCA, with
1000, 100, and 10 yg/ml of oxalate, glyoxalate and glycolic
acid individually. Each sample was assayed in duplicate by
gas chromatography following the procedure described in the
DCA assay.
Study of DCA Binding Using
a Dianorm Equilibrium Dialyser
The Dianorm equilibrium dialyser consists of a set of
four racks of cell holders, each containing five dialysis
cells located between spring-loaded metal spacers to avoid
leakage. One rack of 5 cells was shown in Figure 2.
The cells are made of Teflon to minimize absorption of
the drug into the material of the Dialyser. The cells are
divided into two halves (half cell A and B, respectively)

18
Fig. 2. Diagram of Cell Unit of Dianorm Equilibrium
Dialyser
1 . Driving Flange with Guide Rods
2. Teflon Cell BASE or Half-Cell A) 5 Pair in
3. Teflon Cell LID or Half-Cell B) Complete Stack'
4. Spring Loaded Cell Spacers, 6 in Complete Stack
5. Bearing Flange secured with 3 Knurled Nuts

19
which are separated by a semi-permeable membrane. The
solutions under study are injected into half cell. The
dialysis membranes used were previously soaked in deionized
water for 30 minutes. Each half cell was injected with
1.5ml of plasma or pH 7.4 isotonic buffer solution. For
consistency and convenience, the plasma was always put into
the thick half cell and buffer into the thin half cell. The
racks of the cells were rotated at 4 revolutions per minute.
To study the time to equilibrium, 100 yg/ml DCA was put
on buffer side and the cell was rotated. Samples were
withdrawn after 1, 2, 4, 8 and 16 hrs of dialysis. Both
buffer and plasma were assayed for DCA content. To study
whether DCA equilibrium was affected by adding it to either
the buffer side or the plasma side, a solution, (100 yg/ml)
of DCA was also put into the plasma side initially and
dialysed for 1, 2, 4, 8 and 16 hrs. After dialysis, the DCA
concentration on both sides were measured.
To study the effect of temperature on binding, dialysis
was performed at 37 degrees C and 25 degrees C in a water
bath. A solution (100 ug/ml) of DCA was added to the buffer
side and dialysed for one hour. The DCA concentrations on
both sides were assayed.
To study the effect of DCA concentration on protein
binding, DCA concentrations of 100, 150, 200, 250 and 300
ug/ml were dialyzed at 37 degrees C for one hour. 'The
sodium dichloroacetate was assayed on both sides
after dialysis.

20
Individual plasma samples were also dialyzed.
Diehloroacetic acid was added to blank plasma samples from
some of the subjects at a concentration of 100 yg/ml to
study the individual variability of protein binding. The
DCA concentrations on both sides after dialysis were assayed
and fraction of protein binding of DCA was calculated and
1 is ted.
Red Blood Cell Localization Study
and Partition Coefficient
Eighteen capped test tubes, each with 10 ml of whole
blood inside containing DCA at 100 yg/ml were put on a tube
rotator. Samples were withdrawn at 5, 10, 30, 60, 90, and
120 minutes to study the time to reach equilibrium. Three
tubes were used at each time point.
Centrifugation was at 2000 rpm for five minutes after
rotation. Plasma was separated from the red blood cells and
the plasma content of DCA was measured.
To study the effect of DCA concentration on red blood
cell localization, whole blood concentrations of 50, 100,
300, 600 and 1000 yg/ml DCA were prepared. These samples
were rotated for two hours to make sure equilibrium had been
reached. After centrifugation at 2000 rpm for 5 minutes,
separating plasma from the red blood cells, the plasma
content of DCA was assayed.

21
The partition coefficients were determined in the
following way. Fifteen capped test tubes, each with 5 ml
whole blood inside, were taken and washed with pH 7.4
isotonic buffer solution three times in order to remove all
the plasma protein. The washed blood cells were
reconstituted by adding pH 7.4 isotonic buffer solution to
give the original blood volume. This formed "pseudoblood."
Sodium dichloroacetate was added to the pseudoblood,
resulting, in triplicate, in whole "blood" concentrations of
50, 100, 300, 600 and 1000 yg/ml. After centrifuging at
2000 rpm and separating the buffer and red blood cells, the
DCA content in the buffer was assayed.
Oxalate Assay
Twenty four hour urine was collected and adjusted with
HC1 to a pH value less than 3, and mixed thoroughly. A 60
ml aliquot was submitted to the Clinical Chemistry
Laboratory in Shands Hospital of the J. Hillis Miller
Health Center, University of Florida. Analysis was done by
atomic absorption spectrophotometry [33].
Creatinine Assay
Twenty four hour urine was refrigerated until analysis.
The creatinine assay was carried out by the Clinical
Chemistry Laboratory in Shands Hospital. Creatinine

22
concentration was determined by the red color development on
reacting with picric acid [34].

CHAPTER IV
DATA ANALYSIS
Compartment Model
The concentration-time data for most drugs can be
analyzed with the use of compartmental models. The one
compartment model assumes the drug to be homogeneously
distributed throughout the body. The two compartment model
assumes a central compartment containing the blood volume
and the highly perfused tissues such as liver, kidneys and
lungs. The peripheral compartment then consists of the
poorly perfused tissues such as muscle and fat. Previous
studies [23,32] have shown that DCA follows a one compartment
distribution model in its distribution. So in this study, a
one compartment model was adopted.
Elimination Rate Constant and Half Life
The elimination rate constant assesses the speed of
drug elimination from the systemic circulation. It can be
calculated from the slope of the concentration-time profile.
If the drug follows first order kinetics, then the
elimination rate constant can be obtained by calculation of
the slope of the graph of natural logarithm of the
concentration versus time. If the drug follows a zero order
23

24
kinetics, then the elimination rate constant can be obtained
by calculating the slope of a concentration versus time
profile. In this study, the elimination rate constant of
each volunteer following termination of each infusion was
calculated in this way for both zero and first order
models and the goodness of fit (R2) was also calculated.
The elimination half-life (first order model) was also
calculated following termination of each infusion using the
equation:
1 n 2
t
Eq. (1)
FéT
Volume of Distribution
Volume of distribution is not a real volume but a
hypothetical volume of body fluid that would be required for
the body content of the drug to distribute at the same
concentration as that found in the blood. Using the first
order rate constants, the apparent volume of distribution of
DCA was calculated using the formula:
-kel*t
-kel*t
Cpt = ko [1-e
Vd*kel
] + Cp
t-1
* e
Eq. (2)
where:
Cpt
is the peak plasma concentration following each
I.V. infusion,

25
ko
is the infusion rate,
Cp
t
is the time of infusion, in this work it is always
equal to 30 minutes or 0.5 hours),
is the plasma concentration prior to starting the
t-1
infusion.
If the plasma concentration was obtained after the first dose,
the Cp is equal to zero.
t-1
Protein Binding
Protein binding is the phenomenon occurring when a drug
combines with plasma protein or tissue protein to form a
reversible complex. Drug can be displaced from binding by
other compounds having higher affinity for the binding sites.
The pharmacologic effect is thought to be from the free drug
only. So the extent of binding is an important feature of
the drug, and the fraction of protein bound is defined:
f = fD-PI = fD-PI
Eq. (3)
m TffJ HR
tot free
free
In this study, drug concentration on the plasma side is
given by [D] + [D-P], and drug concentration in the buffer
side is the free drug concentration, since only free drug
passes the membrane. So:

26
f =
|Drug
concentration
in plasma
sidej
Drug
concentration
in plasma
si de|
Eq. (4)
-
|Drug
concentration
in buffer
side'
|Drug
concentration
in plasma
side|
Red Blood Cell Localization and Partition Coefficient
The purpose of studying red cell localization is to
find out the amount of DCA that is bound to red blood cells.
The partition coefficient can be defined as:
D = Drug Concentration in Erythrocyte Eq. (5)
Drug Concentration in Plasma Water
This is significant in at least two ways. First blood
is often hemolyzed on collection. Hemolysis will then cause
inflation of plasma levels. Second, red blood cell
localization can be a significant factor in calculation of
volume of distribution. Variations in red blood cell
localization can possibly cause variation in drug response.
The partition coefficient can also be used to calculate
the fraction of protein binding if the drug concentrations
in red cells and in the plasma are obtained [35], since:
D = { Cb f
TTp*-a_-f)*(i-H) TT^ry
1} * (l-H) Eq. (6)
H

27
Multiplication of both sides by H, and rearrangement gives:
H*D = Cb - (1-H)
t^Tt
Eq. (7)
where H is the hematocrit which can be determined
experimentally, Cb stands for whole blood concentration
which can be determined before the experiment, Cp is the
plasma concentration after equilibrium with red cells, and f
is the calculated fraction of protein binding.
Area Under the Blood Level Curve
Area under the curve (AUC) is an estimate of the total
amount of drug absorbed for first order elimination. In
this study, it is calculated from:
AUC” = AUC T +
o o Ke I
Eq. (8)
where AUCQ is the area under the curve from time zero to the
last sampling time T, which can be obtained by a trapezoidal
method, and Ct is the plasma concentration for the last
sample. Kel is the elimination rate constant of the drug
for each individual.

Statistical Analysis
28
The elimination rate constant was obtained by best fit
of the slope of linear regression using a T155-11 hand-held
calculator. The comparison of observed half-life, volume of
distribution, etc., of each individual after each dosing was
done by analyses of variance (ANOVA), the SAS statistical
program package, and Duncan's multiple range test [36]. A P
value of 0.05 was chosen as the standard of whether
treatment is of statistical difference or not. The
comparison of goodness of fit by using R? where R is
correlation coefficient for the linear model or the log.
linear model was done using student's t test.

CHAPTER V
RESULTS
Identity of DCA and DCA Assay
The DCA sample was shown to be of required identity and
purity first by NMR spectroscopy, which showed the CH bond
of the CHCI2 grouping (Fig. 3). The IR spectrum showed
bands consistant with C=0, COOH and C-Cl bonds (Fig. 4).
Elemental analysis gave a content which agreed with the
putative formula (Table 15). The gas chromatography of DCA
is shown in Fig. 5. The first peak is the methylester of
DCA on reaction of DCA with BF3 /methanol. That was proved
by injection of an authentic sample of the ester. The
second and also the last peak was the methylester of TCA.
Injection solutions typically assayed at 99.5% of the stated
concentration using the specific gas-chromatographic method
and both the DCA sample and the authentic ester sample as
standards. A typical calibration curve of peak height ratio
versus DCA concentration is presented in Fig. 6. The square
of the correlation coefficient for the relationship was
0.992. The relation was linear up to 200 yg/ml.
It has been known for some years that DCA is
metabolized to glyoxalic acid which is then converted to
oxalic acid. However, the present research was the first to
examine the relation of oxalic acid excretion to DCA dosing
29

30
(Fig. 7). There was no evidence in the literature to prove
that the putative DCA metabolites did not interfere with the
DCA assay. By adding various amounts (10, 100, 1000 yg/ml)
of oxalic, glyoxalic and glycolic acids separately to 100
yg/ml DCA sample, it was shown that there was no
relationship between contaminant and found DCA concentration
(Table 2). Besides, there were no interfering peaks on gas
chromatography.
Concentrations in Plasma
The concentration-time profiles in the volunteers
plasma are shown in Figs. 8-20. Concentrations showed the
expected rises during each infusion and the expected falls
following the end of each infusion, and a steady decline to
or towards zero after the first dose. There was, however, a
large degree of variation within the data. The area under
the curve for each set of data from zero to the 24 hour
point using the trapezoidal rule was calculated, and the
extrapolated plasma concentration to time infinity was used
to calculate the area under curve to infinity (Equation 8).
The correlation coefficient for a linear relationship
between area under curve to infinity and dose was 0.72,
(p<0.05).
Protein Binding
The time to reach equilibrium of protein binding was
one hour (Table 3). The effect of temperature on protein
binding was also studied. There was no significant
difference between 37 degrees C and 25 degrees C after

dialysis for one hour (Table 4). The DCA was added to both
plasma and buffer sides of the membrane separately, and
there was no significant difference in the results of the
two experiments (Tables 3 and 5). This means protein
binding of DCA was reversible. The study of the
relationship between protein binding and concentration
showed that binding was linear up to 300y g/ml (Table 6).
Red Cell Localization
It was shown that five minutes was enough for the
distribution of DCA into red blood cells to reach
equilibrium (Table 7). So a time of five minutes was used
for later experiments. It was shown that the partition
coefficient between red cells and plasma water was a
constant up to 1000yg/ml DCA whole blood concentration
(Table 8).

8.0
7.0
6.0
5.0
4.0
PPM ( 6 )
3.0
2.0
_l I
1.0 0
3. NMR Spectroscopy of Sodium Dichloroacetate in D20. Sweep offset was 300 HZ. (5 PPM)
GJ
ro

Fig. 4. IR Spectrum of Sodium Dichloroacetate.
co
CO

34
Blank Plasma
Fig. 5. Gas Chromatogram of
Dichloroacetate and
the Internal Standard
Trichloroacetic Acid,
both as Methyl
Esters.
Plasma
DCA

Peak Height Ratio
35
Concentration (p g/ml)
Fig.' 6. Calibration Graph for Dichloroacetate in
Plasma. Y = 0.07 + 0.0085x, r2 = 0.992,
where Y is the Peak Height Ratio and x is
the concentration.

0xcil3.tc Excietion (mg 0x3la.te/mg Crctininc)
Fig. 7. Graph of 24 hours Urinary Oxalate Excretion
vs. Dose in Eleven Subjects Given Various
Doses of DCA. Y = 29 + 3.47x, R = 0.67,
where Y is mg • oxalate/mg Creatinine,
x is the Dose of DCA. Subject #4 was not
Included because the Dose was in Doubt.

AUC (mg-h/ml)
Fig. 8. Graph of Area Under the Curve of Plasma
Dichloroacetate Concentration vs. Time
against Dose in Eleven Subjects given
Various Doses. Y = 0.]6x - 0.24, R =
0.73, where Y is the Area Under the Curve
in mg-h/ml and x is the Dose in mg/kg.
Subject #4 was in Doubt.

Concent rotion(ug/m|)
Fig. 9. Plasma Concentration Time Profile for Subject No. 1 after 10 mg/kg
IV Infusion. ";j " Stands for Infusion Period.

250
200
150
100
50
0
Fig. 10. Plasma Concentration Time Profile for Subject No. 2 after 10 mg/kg
IV Infusion. " " Stands for Infusion Period.
CO
CD

Fig. 11. Plasma Concentration Time Profile for Subject No. 3a after 10 mg/kg
IV Infusion. " Stands for Infusion Period.
-F*
O

Fig. 12. Plasma Concentration Time Profile for Subject No. 4 after 15 mg/kg
IV Infusion. " Stands for Infusion Period.

160
140
120
100
80
60
40
20
0
Time (hr)
. 13. Plasma Concentration Time Profile for Subject No. 5 after 25 mg/kg
IV Infusion. "!! " Stands for Infusion Period.
-P*
no

Concentration(ug/m1)
160
140
Fig. 14. Plasma Concentration Time Profile for Subject No. 6 after 25 mg/kg
IV Infusion. " • | " Stands for Infusion Period.

160
Fig. 15. Plasma Concentration Time Profile for Subject No. 7 after 25 mg/kg
IV Infusion. " ! ¡ " Stands for Infusion Period.

200T
Fig. 16. Plasma Concentration Time Profile for Subject No. 8 after 25 mg/kg
IV Infusion. "|j " Stands for Infusion Period. ^
cn

160
140
120
100
80
60
40
20
■t»
O'
ig. 17. Plasma Concentration Time Profile for Subject No. 3b after 25 mg/kg
IV Infusion. " j | " Stands for Infusion Period.

Fig. 18. Plasma Concentration Time Profile for Subject No. 9 after 50 mg/kg
IV Infusion. " { • " Stands for Infusion Period.

1000
800
600
400
200
O'
ig. 19. Plasma Concentration Time Profile for Subject No. 10 after 50 mg/kg
IV Infusion. " j j " Stands for Infusion Period.
4=»
00

Concentration (¿ig/ml)
Fig. 20. Plasma Concentration Time Profile for Subject No.11 after 50 mg/kg
IV Infusion. " \ \ " Stands for Infusion Period.

Fig. 21. Plasma Concentration Time Profile for Subject No. 12 after 50 mg/kg
IV Infusion. " \ \ " Stands for Infusion Period.

51
TABLE 1. Demographic Data.
Patient No.
Sex
Age
Wt
(kg)
Hematocrit
(%)
Dose
(mg/kg)
1
M
25
74.8
44.4
10
2
M
22
76.0
49.0
10
3a
M
42
72.4
39.2
10
4
F
23
61.4
41.3
15
5
M
24
80.6
45.5
25
6
M
24
67.8
42.6
25
7
M
33
73.3
42.8
25
8
F
37
56.8
39.9
25
3b
M
42
72.4
39.2
25
9
M
57
101.4
47.5
50
10
F
45
57.6
41.5
50
11
F
27
65.0
44.1
50
12
F
27
58.0
40.9
50

52
TABLE 2. Determination of the Interference of DCA
Metabolite in DCA Assay. lOOyg/ml DCA was
Initially Added to Three Different Concentrations
of DCA Metabolite.
y g/ml DCA.
Results were Expressed as
DCA Plus:
1000 y g/ml
E
CD
o
o
rH
10 ^ g/ml
metabolite
metabolite
metabolite
Metabolite:
Oxalate
103.6
99.9
102.3
104.8
99.9
102.3
Glycolate
97.5
105.9
101.1
101.1
97.5
98.9
Glycoxylate
110.8
103.6
104.8
97.5
107.2
105.9

53
TABLE 3. Determination of Time to Reach Dialysis Equilibrium
Using Dianorm Equilibrium Dialyser. Dichloro-
acetate 100 yg/ml was Initially Added to the Plasma
Side.
Assayed
Concentrations
•
of DCA on
Both
Sides were
1
2
Time (hr)
4
8
16
Plasma
Concentration
55.1
49.9
54.2
54.6
58.1
(yg/ml)
53.4
50.8
62.9
57.9
53.6
Buffer
Concentration
35.8
33.4
38.4
36.0
41.2
(yg/ml)
36.2
36.2
33.7
36.2
31.6

54
TABLE 4. Studies of the Effect of Temperature on Protein
Binding. Dichloroacetate (100 y g/ml) Was
Initially Added to Buffer Side. Both Buffer and
Plasma Side were Assayed After Dialysis, "f"
Stands for Fraction of Protein Binding.
37
C
25
C
PI asma
Side
Cone.
(y g/ml)
Buffer
Side
Cone.
(y g/ml)
f
Plasma
Side
Cone.
(y g/ml)
Buffer
Side
Cone.
(yg/ml)
f
Dialysis
57.1
45.4
0.20
59.5
43.9
0.26
After
One
57.8
42.4
0.27
58.9
45.1
0.23
Hour
55.8
41.4
0.26
53.3
45.5
0.15
AVE
56.9
43.1
0.24
57.2
44.8
0.21
SE
0.57
1.21
0.02
1.96
0.46
0.03

55
TABLE 5. Determination of Time to Reach Dialysis
Equilibrium. Dich1oroacetate (100 y g/ml) was
Initially Added to the Buffer Side. Concentrations
of DCA on Both Sides were Assayed.
1
Time
2
(hr)
4
8
16
Plasma
58.2
59.4
52.6
52.9
53.4
(pg/ml)
47.4
52.2
46.3
55.0
47.1
Buffer
35.5
39.2
35.6
33.8
37.4
(yg/ml)
34.0
33.5
33.8
39.8
30.9

TABLE 6. Studies of Protein Binding of DCA at Different Concentrations. Sodium Dichloroacetate was Assayed
on both Buffer and Plasma Side. Y = 0.59X -2.82, R = 0.0996, Y stands for Average Plasma Side
Concentration After Dialysis. X stands for Initial DCA Concentration. Y' = 2.54 + 0.38X, R = 0.998,
Y' stands for Average Buffer Side Concentration After Dialysis.
Original DCA
Concentration
Added to
Buffer Side
(pg/ml)
100
150
200
250
300
P
b
f
P
b
f
P
b
f
P
b
f
P
b
f
55.3
37.6
0.32
78.7
66.0
0.16
95.1
61.6
0.35
159.9
112.6
0.25
160.2
108.1
0.33
56.6
38.3
0.32
85.6
61.9
0.28
126.1
84.8 0.33
177.8
98.5
0.45
164.3
116.4
0.29
53.0
38.3
0.28
68.2
58.5
0.14
141.9
80.0
0.44
128.4
97.3
0.24
173.5
107.0
0.38
56.5
40.1
0.29
91.2
66.4
0.27
110.3
76.5
0.30
140.1
95.5
0.30
178.1
116.8 0.34
109.80
80.0
0.27
Mean
55.4
38.6
0.30
81.0
63.0
0.21
116.5
76.6
0.33
149.3
100.9
0.31
169.4
112.1
0.34
SE
0.09
0.55
0.01
4.3
1.9 (
3.03
8.05
3.90
0.03
10.5
3.9
0.07
4.1
2.6
0.02
cn
cr>

57
TABLE 7. Determination of Time to Reach Equilibrium for Red
Cell Localization. Dichioroacetate (100yg/ml)
was Initially Added to the Whole Blood. Plasma
Concentration was Assayed. Blood was Obtained
from Volunteer 3a.
5
10
Time l
30
[min)
60
90
120
140.7
130.6
131.6
131.6
130.1
133.9
Concentraton
135.2
139.9
125.9
120.9
127.0
120.5
(y g/ml)
143.0
132.2
136.1
134.0
127.9
136.2
130.8
129.1
145.3
127.9
124.2
115.3
140.7
126.4
140.7
129.4
130.3
146.6
138.8
135.3
135.9
123.4
135.5
152.7
Mean
138.2
132.2
135.9
127.9
129.2
134.2
SE
1.8
2.0
2.8
2.0
1.6
5.9

58
TABLE 8. Partition Coefficient (P) of DCA Between Red Blood
Cells and Isotonic Buffer up to 1000 yg/ml Whole
Pseudoblood Concentration. Blood was Obtained From
Subject 3a.
DCA Concentration ,
n g/mi
50
100
300
600
1000
0.19
0.43
0.46
0.22
0.39
0.32
0.39
0.35
0.58
0.44
p
0.47
0.46
0.39
0.73
0.45
0.41
0.33
0.48
0.79
0.41
0.23
0.32
0.52
0.45
0.42
0.47
0.52
0.36
0.71
0.42
Mean
0.35
0.41
0.42
0.58
0.42
SE
0.05
0.03
0.03
0.09
0.01

59
TABLE 9. Protein Binding (f), Red Blood Cell/Plasma Ratio
(RBC/P), and Partition Coefficient for Red Blood
Cell/Plasma Water Distribution in Nine Volunteers.
Da'ta were Obtained at 100 yg/ml Initial DCA
Concentration.
3a
4
Subject
5 7
No.
8
9
10
11
12
f
0.30
0.39
0.33 0.32
0.20
0.35
0.42
0.40
0.20
RBC/P
0.08
0.47
0.20 0.06
0.32
0.42
0.17
0.47
0.57
Partition
Coefficient
0.17
0.78
0.42 0.36
0.42
0.57
0.29
0.66
0.62

60
2
TABLE 10. Sodium Dichloroacetate in Plasma: Calculated R and kel Values
for Repeated I.V. Infusions in Different Volunteers, Using a
First Order Kinetics Model.
Infusion
Number
I
II
III
IV
V
kel
R2
kel
R2
kel
R2
kel
R2
kel
R2
Volunteer
1
1.54
0.72
0.92
0.94
0.38
0.94
0.25
0.89
0.25
0.89
2
0.01
0.06
0.56
1.00
0.07
0.55
0.06
0.11
0.03
0.85
3a
2.09
0.98
2.45
0.96
1.90
0.93
1.13
0.89
1.10
1.00
4
0.13
0.74
0.02
0.02
0.18
0.63
0.01
0.02
0.26
0.84
5
0.74
0.92
0.30
0.70
0.17
0.83
0.20
0.97
0.26
0.88
6
0.90
0.97
0.36
0.48
1.56
0.66
0.18
0.92
0.23
0.97
7
0.47
0.80
0.35
0.90
0.05
0.01
0.16
0.70
0.12
0.95
8
2.76
0.90
0.62
0.99
0.20
0.74
0.20
0.70
0.03
0.84
3b
0.41
0.81
0.31
0.94
0.21
0.91
0.22
0.98
0.20
0.98
9
0.37
0.97
0.31
0.99
0.18
0.29
0.30
0.76
0.14
0.98
10
0.25
0.55
0.19
0.55
0.12
0.87
0.29
0.87
0.10
0.87
11
0.14
0.08
0.45
0.90
0.11
0.29
0.03
0.19
0.07
0.86
12
0.41
0.08
0.45
0.90
0.11
0.29
0.03
0.19
0.07
0.86
Mean
0.98
0.71
0.64
0.80
0.50
0.63
0.30
0.68
0.22
0.91
SE
0.27
0.09
0.05
0.08
0.19
0.08
0.08
0.09
0.08
0.02

61
TABLE 11. Calculated DCA Volume of Distribution (1/kg) Using
a First Order Kinetic Model Applied to Repeated IV
Infusion Data in 11 Subjects. Subject 4 was not
Included Because the Dose was not Certained. R <
0.81 Was Not Listed (Expressed in
No. of Infusion
I
II
III IV
V
Volunteer
1
0.13
0.31
0.32
0.28
0.35
3a
0.20
0.10
0.11
0.19
0.25
5
0.25
0.29
0.43
0.34
0.49
6
0.36
--
0.25
0.20
0.6
7
0.37
-1.18
-14.2
8
0.47
0.28
0.28
0.37
0.27
3b
0.24
0.45
0.43
0.39
0.39
9
0.24
0.23
0.27
0.25
10
--
0.49
0.16
0.09
11
0.23
--
0.22
12
0.23
0.30
--
0.42
0.22
AVE
0.28
0.27
0.30
0.29
0.31
SE
0.03
0.04
0.04
0.03
0.05
Negative data was not included in average.
Subject 2 was not included because data was not available

62
TABLE 12. Calculated Half Life for Volunteer After Each
Dose. R Value Smaller Than 0.81 in Line Fit was
not Included (Expressed in
No. of Infusion
I
II
III IV
V
Volunteer
1
0.45
0.75
1.82
2.77
1.87
2
1.24
23.10
3a
0.33
0.28
0.36
0.61
0.63
4
5.5
2.67
5
0.94
2.31
4.10
3.46
2.66
6
0.77
--
3.85
3.01
7
1.47
1.98
4.33
5.77
8
0.25
1.15
3.46
3.46
23.1
3b
1.69
2.24
3.3
3.15
3.46
9
1.87
2.24
2.31
4.95
10
--
—
5.78
2.39
6.93
11
--
1.54
--
9.9
12
1.69
4.62
--
8.66
9.9
AVE
1.50
1.84
3.14
3.50
7.53
SE
0.46
0.36
0.66
0.63
2.07

63
TABLE 13. Dichloroacetate in Plasma: Rates of Metabolism
of Each Subject After Repeated IV Infusions
at 2-Hour Intervals (Numbered Consecutively
I-V). Subject 2 was not Included Because
Data Was Not Available. R < 0.81 was not
Listed (Expressed in
Interval
I
II
III
IV
V
Subject
1
829.2
901.7
334.5
273.9
383.0
3a
953.3
1037.7
874.6
634.2
706.8
4
110.2
1522.2
5
1259.9
738.4
677.6
805.1
1460.2
6
1230.2
2266.0
241.9
1029.2
7
766.1
★
8
3271.4
757.3
293.3
523.7
82.9
9
1730.6
2020.7
2987.2
1400.1
10
--
1004.8
1478.4
474.5
11
1621.4
--
466.6
12
1046.7
678.8
--
781.5
506.1
AVE
1187.4
1089.1
881.7
950.2
803.6
SE
267.6
169.5
250.9
282.2
146.8
* Data was not available

64
2
TABLE 14. Sodium Di chioroacetate in Plasma: Calculated R and Rate
Constants for Repeated I.V. Infusion in 12 Volunteers,
Using a Zero Order Kinetics Model.
Infusion
Number
I
II
III
IV
V
kel
R2
kel
R2
kel
R2
kel
R2
kel
R2
Volunteer
1
+3.07
0.71
19.8
0.86
10.7
0.97
10.6
0.86
5.56
0.95
2
1.30
0.25
55.5
0.99
10.0
0.51
9.1
0.11
4.80
0.78
3a
+18.9
0.75
35.4
0.70
33.6
0.78
21.5
0.81
4.63
0.73
4
+16.6
0.75
3.42
0.01
42.8
0.70
1.72
0.05
10.5
0.84
5
+39.4
0.95
24.2
0.82
17.7
0.83
25.4
0.97
9.81
0.97
6
+15.8
0.66
15.4
0.47
15.6
0.99
15.5
0.93
8.10
0.95
7
-38.8
0.83
-24.4
0.86
6.34
0.02
-21.4
0.70
5.68
0.85
8
+29.1
0.96
48.6
0.95
15.0
0.73
21.3
0.71
4.58
0.78
3b
+28.4
0.77
24.0
0.95
20.3
0.93
24.6
0.99
9.11
0.93
9
+55.2
0.98
70.3
0.997
49.2
0.33
84.4
0.77
22.0
0.97
10
+48.6
0.57
38.2
0.52
30.6
0.87
114.8
0.84
37.7
0.72
11
+12.5
0.04
86.1
0.93
27.4
0.29
11.1
0.22
21.2
0.80
12
+59.6
0.77
35.2
0.99
81.0
0.63
29.6
0.79
23.8
0.84
Average
29.7
0.69
38.01
0.77
27.7
0.66
30.8
0.67
12.5
0.85
SE
4.9
0.08
6.86
0.080
5.7
0.08
9.72
0.09
2.56
0.03
Negative kels were not included in average.

65
TABLE 15. Identity of Sodium Dich1oroacetate by Elemental
Analysis.
ELEMENT
FOUND
THEORY
C
15.71
15.8
H
1.39
1.3
0
21.1
Cl
46.35
46.7
Na
15.1

CHAPTER VI
DISCUSSION
Binding and Red Cell Localization
In this research, attention was particularly focussed
on plasma concentrations of dichloroacetate. A major factor
in interpreting plasma concentrations of drugs is bindg of
the drugs to plasma protein. This was studied for
dichloroacetate using the Dianorm apparatus. Preliminary
experiments concerned factors such as time to reach
equilibrium.
Times to reach dialysis equilibrium are different for
individual ligands. Also, the dialysis time increases with
decreasing temperature, increasing dialysis volume,
increasing thickness of membrane, and increasing molecular
weight of ligand.
The Dianorm apparatus is designed for optimal geometry
and a high concentration gradient is maintained across the
membrane. In this research, slow rotation was used (4 rpm)
to make sure mixing was thorough. The thin wall of the
cell assured rapid heat exchange between water bath and cell
content. By using this apparatus DCA equilibrium was
reached within one hour (Table 3). There was no significant
difference for equilibrium time between 25 degrees C and 37
degrees C.
Binding of drugs to plasma protein may have a marked
effect on the distribution and pharmacologic effects of a
66

67
drug, and on the rate at which it is eliminated from the
body. This is because distribution of drugs to body tissues
takes place from the blood compartment. Also, it is the
free drug concentration which equilibrates with the body
tissues. The fraction of drug in the plasma bound to plasma
protein is not immediately available for distribution into
the extravascular space or for certain modes of elimination.
Because of this, the binding of dichloroacetate to plasma
proteins from individual subjects was studied. The fraction
bound at a whole plasma dichloroacetate concentration of 100
ug/ml varied from 0.2 to 0.42 (Table 9). At this level,
protein binding is generally considered to be of no great
clinical significance.
Several drugs enter the erythrocyte quite rapidly and
others do so at a slower rate. The extent of penetration
can often be correlated with the solvent/water partition
coefficients of these drugs - highly lipid soluble drugs
show greater accumulation in the erythrocytes.
Dichloroacetate is a highly water soluble compound, so the
predicted partition coefficient between erythrocytes and
water is very low. This was proved to be so by the present
experiments. It has been suggested that determination of the
RBC/plasma concentration ratio may serve as a simple and
rapid technique for the indirect measurement of plasma
protein binding. This would be applicable in the
large-scale screening of abnormal plasma binding in routine
clinical blood samples, thereby facilitating

68
individualization of drug therapy. However, in our case,
the RBC/plasma concentration ratio did not show very large
variation (Table 9).
The calculated fractions of DCA protein binding in two
subjects using equation 7 were 0.60 (subject 3a) and 0.59
(subject 5). Each of these figures is a mean of six
determinations. The standard deviation in subject 3a was
0.23. These means are higher than the means for fraction
bound obtained by equilibrium dialysis. However, they were
obtained by using the mean partition coefficient and the
standard deviation of the partition coefficient was not
taken into consideration. It has been shown in the
literature that buffer systems, ionic strength and pH value
will affect binding properties [37,38,39]. It is necessary
to conduct further investigations concerning the various
factors affecting DCA protein binding. But from the
clinical point of view, 0.3 versus 0.60 was not of
physiological significance.
Pharmacokinetic Analysis
The decay of DCA concentrations in plasma was examined
following termination of each infusion. In each patient,
the four available concentration-time points from dose I to
IV and five or six available points following the last
infusion were compared with a linear model (concentration
versus time) and then with a logarithmic concentration versus

69
time model. The mean values of (where R is the
correlation coefficient) for the relationship using both
models are listed in Table 10 and Table 14. Although the
goodness of fit was comparable for the two models, the zero
order model was rejected for two reasons. First, other
investigators have shown first order kinetics to apply at
the level of this data seen in this work after the initial
doses. There was a trend that more data points gave better
R2 (where R is the correlation coefficient) for the first
order than zero order model. This was found by comparing
the R? for the data after the fifth doses using first order
or zero order models. The first order model is
significantly better than the zero order model at p=0.1
level. Second, the putative zero order slopes were
calculated to range from 4.58ug/ml/h to 114.8 yg/ml/h. That
means the output rate would be very close or even greater
than input rate, which is inconceivable.
Using only the data for which R was greater than 0.81,
which is the lower limit for significance of a four-point
correlation, the mean half-life calculation was found to be
90.0 min for first dosing, 110.4 min, 188.4 min, 210.0 min,
and 451.8 min for 2nd, 3rd, 4th, and 5th infusions (Table
12). The mean half-life rose steadily from the first dose
to the fifth dose, and the differences are statistically
different (p<0.005).
Data of this kind could have several mechanistic
implications. For example, first order decay kinetics are a

70
special case of Michaelis-Menten (MM) kinetics, applicable
when the substrate concentration (S) is low. In this work S
was clearly low enough for the first order simplification of
the MM model to apply, even when S increased from the first
to the fifth dose. However, it can be argued that the data
should have shown gradual deviation from the first order
case at higher S values if Kn and Vmax had remained
constant. Since this devation did not occur, it may be that
changes in either Kn or Vmax occurred as dosage progressed,
but with retention of applicability of the first order
model. Mechanisms for such a change could include slowing of
dichloroacetate metabolism following enzyme poisoning by the
substrate, and/or product inhibition. Definition of the
mechanism must await suitable further studies, including in
vitro metabolism studies, and development of zero order
input/first order output pharmacokinetic models appropriate
to intermittent intravenous infusion dosing. Additionally,
models for zero order input Michaelis-Menten output will be
needed.
The calculated rates of metabolism after each dose are
listed in Table 13. These rates were obtained by multiplying
plasma DCA concentrations by volumes of distribution and
rate constants of elimination. There was a small trend
towards a reduced rate of metabolism with time. Statistics
showed that the rate of metabolism after the fifth dose was
significantly less than that after the second dosing
(p<0.05). These calculations were conducted because three

71
possible observations could have been made:
(1) an increase in the rate of metabolism with time
and/or level might have indicated adherence to first order
or Michaelis-Menten kinetics, with a tendency towards
saturation of the enzyme concerned (the normal for enzyme
kinetics);
(2) a confirmed decrease in the rate of metabolism
would have indicated a reduction in enzyme activity; and
(3) no change would have been equivocal. While failing
to confirm a decrease, the calculations do rule out an
increase. There is support for the theory that DCA
metabolism involves enzyme poisoning or product inhibition
but more experiments are needed to confirm this idea.
The applications of the term "volume of distribution"
are many, including those to dosage regimen calculation of
drugs based on the characteristics of an individual patient.
It is possible to observe different plasma concentration
values in different individuals after the administration of
the same dose because of the differences in the volume of
distribution. That is the rationale for the calculation of
dosage adjustment according to body weight.
No evidence was obtained for a volume of distribution
change with repeated dosing (Table 11). This observation
disagreed with earlier observations in patients [26].
However, all of the patients in the other study were in shock
at the time of commencing DCA infusion, and a rise in blood
pressure, possibly caused by a positive inotropic effect of

72
DCA occurred. This is being investigated further, but it is
not to be expected to be seen in healthy volunteers, who
showed no cardiovascular changes when treated with DCA.
Obviously, a change in the DCA volume of distribution might
well be linked to cardiac output and related cardiovascular
status.

APPENDIX

74
UNIVERSITY OF FLORIDA . J. HILLIS MILLER HEALTH CENTER BOXJ-14
Vice President for Health Affairs Gainesville, Florida • zip 32610
HEALTH CENTER INSTITUTIONAL REVIEW BOARD MAY 11, 1983
TO:
FROM:
SUBJECT:
Peter W. Stacpoole, M. D., Ph.D.
Assistant Professor, College of Medicine
B. Joe Wilder, M. D., J-l*
Chairman, Institutional Review Board
Approval of the Institutional Review Board
Project # 133-83 entitled ” Clinical Pharmacology of*
Sodium Dichloroacetate (DCA)."
uW
The Institutional Review Board has recommended the approval of your protocol,
identified above, for a period of 12 months, and has determined that
human subjects will be at risk.
Approval of your research project is, therefore, granted until 5/11/84
By the end of this period you will be asked to inform the Board on the status
of your project. If this has not been completed, you may request renewed
approval at that time.
You are reminded that a change in protocol in this project requires its resub¬
mission to the Board. Also, the principle investigator must report to the Chair
of the Institutional Review Board promptly, and in writing, any unanticipated
problems involving risks to the subjects or others, such as adverse reactions
to biological drugs, radio-isotopes or to medical devices.
If it is anticipated that VA patients will be included in this project, or if
the project is to be conducted in part on VA premises or performed by any VA
employee during VA-compensated time, final approval should be obtained by appli¬
cation to the Veterans Administration Hospital Research Office.
By a copy of this memorandum the chairman of your department is reminded that
he is responsible for being informed concerning research projects involving
human subjects in his department. He should review the protocols of such
investigations as often as he thinks is necessary to insure that the experiment
is being conducted in compliance with our institution and with DHHS regulations.
cc: James E. McGuigan, M. D. co-PIs
B. Joe Wilder, M. D. JStephen H. Curry, Ph.D.
Thomas G. Baumgartner, M. Ed., Pharm.D. T. G. Baumgartner, Pharm.D.
William C. Thomas, Jr., M. D.
Clinical Research Center
Becky Stevens, Ph.D.
Collogo of Modicino • Collogo of Nursing • Collogo of Phormocy o Collogo of Hoolth Rolotod Professions • Collogo of Dontisfry
Collogo of Votorinory Modicino o Shands Teaching Hospital and Clinics • Votorons Administration Hospital
’ KOUAU KM»lOVMCMr o noon TUNIT V / A rriSMA T» Vt ACTION CMNiovcn

75
ev^
HEALTH CENTER INSTITUTIONAL REVIEW BOARD
• * %* /,/
UNIVERSITY OF FLORIDA
Vice
’resident for Health Affairs
J. HILLIS MILLER HEALTH CENTER . BOX J-14
Gainesville, Florida • zip 32610
May 11, 1983
Peter W. Stacpoole, M. D., Ph.D.
Assistant Professor
College of Medicine
Department of Medicine
Box J-226
SUBJECT: Protocol #133-83, Clinical Pharmacology of
Sodium Dichloroacetate (DCA)
Dear Dr. Stacpoole:
Your protocol, referenced above, was reviewed at our
meeting this date.
It was the decision of the Committee that this study can be
approved if the following condition is met:
In the three (3) Informed Consent Forms, clearly state
that there is remote possibility of added risks because
of the use of this drug. (state what these are).-» If
there are none, state so.
You will find enclosed the Approval Letter for this study.
Please send four copies (4) each of the three (3) revised
Informed Consent Forms to this office (J-14) at your earliest
convenience, since approval was contingent upon this condition
being met.
We are glad to be of service to you in this capacity, and we
appreciate your cooperation in this matter.
SaA
B. Joe Wilder, M. D.
Chairman
BJW/ddo
continued
Coll. ge of Mtdiein* • College of Nursing e College of Pharmacy e College of Health Related Professions e College of Dentistry
College of Veterinary Medicine e Shonds Teaching Hospital and Clinics e Veterans Administration Hospital
EQUAL employment opportunity/a fpipmative action employer

Page 2 - May 11, 1983
Protocol #133-83
P. W. Stacpoole, M. D.,
Ph.D.
cc: ‘"St ephen H. Curry, Ph.D.,
co-PI
Thomas G. Baumgartner, M.Ed.,
Pharm.D., co-PI
File
Enclosure: Approval Letter (#133-83)

REFERENCES
(1) Windhol, M., Budavari, S., Stroumtsos, L.Y., and Fertig,
M.N., Merck Index, Merck & Co., Inc., Rahway, N.J., Ninth Edition, 1976.
(2) Demaugre, F., Cepanec, C., and Leroux, J.P., "Characterization
of Oxalate as a Catabolite of Dichloroacetate Responsible for the
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Cells." Biochem. Biophys. Res. Commun. 85:1180-1185, 1978.
(3) Little, M., and Williams, P.A., "A Bacterial Halidohydrolase:
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(4) Eichner, H.G., Stacpoole, P.W., and Forsham, P.H., "The
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(5) Lorini, M., and Ciman, M., "Hypoglycaemic Action of
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(6) Stacpooke, P.W., and Felts, J.M., "Diisopropyl ammonium
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(7) Eichner, H.L., Stacpoole, P.W., and Forsham, P.H.,
"Treatment of Streptozotocin Diabetes with Diisopropyl ammonium
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(8) Whitehouse, S., and Randle, P.J., "Activation of Pyruvate
Dehydrogenase in Perfused Rat Heart by Dichloroacetate." Biochem.
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(9) Goodman, M.N., Ruderman, N.B., and Aoki T.T., "Glucose and
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(10)Stacpoole, P.W., Moore, G.W., and Komhauser, D.M.,
"Metabolic Effects of Dichloroacetate in Patients with Diabetes
Millitus and Hyperlipoproteinemia." N. Enql. J. Med. 298:
526-630, 1978.
77

78
(11) Holloway, P.A.H., and Alberti, K.G.M.M., "Reversal
of Phenformin-Induced Hyperlactaemia by Dichloroacetate in
Normal and Diabetic Rats." Piabetologia 11:250-251, 1975.
(12) Holloway, P.A.H., and Alberti, K.G.M.M.,
"Phenformin-Induced Lactic Acidosis: Prevention by
Dichloroacetate." Clin. Sc i. Mol. Med. 50:33, 1976.
(13) Arieff, A.I., Leach, W., and Lazarowitz, V.,
"Treatment of Experimental Lactic Acidosis with
Dichloroacetate (DCA)." Clin. Res. 26:410A, 1978.
(14) Johnson, G.A.H., and Alberti, K.G.M.M., "The
Metabolic Effects of Sodium Dichloroacetate in Experimental
Hepatitis in the Rat." Biochem. Soc. Trans. 5:1387-1388,
1977 .
(15) Park, R., Leach, W. , and Arieff, A.,
"Dichloroacetate (DCA) Prevents Hyperlactatemia Following
Functional Hepatectomy (HPX)." Clinical Research 27-.48A,
1979.
(16) Park, R., Leach, W. , and Arieff, A.,
"Dichloroacetate (DCA) Decreases Extrahepatic Lactate
Production in Diabetic Dogs." Clin. Res. 27:374A, 1979.
(17) Loubatieres, A.L., Ribes, G., and Valette, G.,
"Pharmacological Agents and Acute Experimental
Hyperlactataemia in the Dog." Br. J. Pharmacol. 58:429p,
1976.
(18) Zambraski, E.J., and Merrill, G.F.,
"Dichloroacetate Sodium (DCA) Decreases Plasma Lactic Acid
(LA) Observed During Exercise in Dogs." Clin. Res. 27:380A,
1979.
(19) Schneider, S.H., Komanicky, P.M., and Goodman,
M.N., "Enhancement of Exercise Performance by Dichloroacetate
(DCA) in Rats." Diabetes 28(Suppl.2):61, 1979.
(20) Merrill, G.F., Rosolowsky, M., and Young, M.A.,
"The Influence of Dichloroacetate Sodium on the
Hyperlactacidemia of Endotoxin Shock." Fed. Proc., Fed. Am.
Soc. Exp. Biol. 38:1192, 1979.
(21) Merrill, G.F., and Rosolowsky, M., "Effect of
Dichloroacetate Sodium on the Lactacidemia of Experimental
Endotoxin Shock." Circ. Shock 7:13-21, 1980.
(22) Grover, G.J., Scanes, C.G., and Merrill, G.F.,
"Effects of Dichloroacetate Sodium on Circulating Levels of
Corticosterone, Glucose and Lactate during Endotoxemia in the

Domestic Fowl Gall us domesticus." Fed. Proc., Fed. Am. Soc.
Exp, Biol. 38:1123, 979.
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(23) Loubatieres, A.U., Ribes, G., and Valette, G.,
"Pharmacological Agents and Acute Experimental Hyperlactatemia
in the Dog." Br. J. Pharmacol. 58: 429, 1976.
(24) Schneider, S.H., Komanicky, P.M., and Goodman, M.N.,
"Enhancement of Exercise Performance by Dichloroacetate (DCA) in
Rat." Diabetes 28(Suppl 2):360, 1979.
(25) Irsigler, K., Brandle, J., Kaspar, L., Kritz, H., Lageder, H.,
and Regal, H., "Treatment of Biguanide-Induced Lactic Acidosis with
Dichloroacetate: 3 Case Histories." Arzneim-Forsch 29(1):555-558,
1979.
(26) Stacpoole, P.W., Harman, E.M., Curry, S.H., Baumgartner, T.G.,
and Misbin, R.I., "Treatment of Lactic Acidosis with Dichloroacetate."
N. Engl. J. Med. 309:390-396, 1983.
(27) Stacpoole, P.W., Moore, G.W., and Kornhauser, D.M.,
"Toxicity of Chronic Dichloroacetate." N. Engl. J. Med. 300:372,
1979.
(28) Wells, P.G., Moore, G.W., Rabin, D., Wilkinson, G.R.,
Oates, J.A., and Stacpoole, P.W., "Metabolic Effects and Pharmacokinetics
of Intravenously Administered Dichloroacetate in Humans." Diabetoloqia
19:109-113, 1980.
(29) Idem: "Abnormal Resting Blood Lactate II. Lactic Acidosis."
Am. J. Med. 30:840-848, 1961.
(30) Huckabee, W.E., "Abnormal Resting Blood Lactate I. The
Significance of Hyperlactatemia in Hospitalized Patients." Am. J.
Med. 30:833-839, 1961.
(31) Park, R., and Arieff, A., "Treatment of Lactic Acidosis
with Dichloroacetate in Dogs." J. Clin. Inves. 90:853-862, 1982.
(32) Lukas, G., Vyas, K.H., Brindle, S.D., LeSher, A’.R., and
Wagner, W.E. Jr., "Biological Disposition of Sodium Dichloroacetate
in Animals and Humans after Intravenous Administration." J. Pharm.
Sci. 69(4):419-421, 1980.
(33) Henry, R.J., Cannon, D.C., and Winkelman, J., Clinical
Chemistry, Principles and Technics, 2nd edition. New York, Harper
and Row, 1349, 1974.
(34) Israel, D., and Henry, J.B., Clinical Diagnosis by Laboratory
Methods, Philadelphia, W.B. Saunders Company, .88, 1974.

80
(35) Garrett, E.R., and Lambert, H.J., "Pharmacokinetics of
Trichloroethanol and Metabolites and Trichloroethanol and Metabolites
and Interconversions among Variously Referenced Pharmacokinetic
Parameters.11 J. Pharm. Sci. 62(4):550-572, 1973.
(36) Barr, A.J., Goodnight, J.H., Sail, J.P., and Helwig, J.T.,
1976. A User's Guide to SAS 76. Raleigh, N.C.; SAS Institute, Inc.
(37) Fleitman, J., and Perrin, J.H., "The Effects of pH, Calcium
and Chloride Ions on the Binding of Benoxaprofen to Human Serum
Albumin, Circular Dichroic and Dialysis Measurements." Int. J. Pharm.
11:227-236, 1982.
(38) Klotz, I.M., and Urquhart, J.M., "The Binding of Organic Ions
by Protein and Buffer Effects." J. Phy. Coll. Chem. 53:100-109, 1949.
(39) Henry, J.K., Dunlop, A.Q., Mitchell, S.N., Turner, P., and
Adams, P., "A Model for the pH Dependence of Drug Protein Binding."
J. Pharm. Pharmacol. 33:179-182, 1981.

BIOGRAPHICAL SKETCH
Pei-I Chu was born in Cang Shang, Taiwan, Republic of
China, on November 29, 1959. She graduated from the
Department of Pharmacy of National Taiwan University in
June, 1981, and passed the National Pharmacy board in July
that year. She worked as a teaching assistant in the
Department of Biochemistry in the College of Medicine of
National Taiwan University from 1981 to 1982. She joined
the graduate program in the College of Pharmacy, University
of Florida, in September, 1982, to work towards the degree
of Master of Science in Pharmacy with a major in
pharmaceutics, under the guidance of Dr. Stephen H. Curry.

I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of M^-s-t-er of Science,
in Pharmacy. ( U
l;
rf
Stephen H. Curry,' Chairman
Professor of Pharmacy
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,as
a dissertation for the degree of
¡in Pharmacy.
Professor of Pharmacy
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Maste/ of Science,
in Pharmacy.
71 jUt
Kamlesh M. Thakker
Assistant Professor of
Pharmacy
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adeq.uate, in scope and quality, as
a dissertation for the degree of Master of Science
in Pharmacy.
X
irJt 0.
Jr J
Margaret 0. James
Assistant Professor of
Medicinal Chemistry

This Thesis was submitted
of Pharmacy and to the Graduate
fulfillment of the requirements
in Pharmacy.
August, 1984
to the Graduate Faculty of the College
School, and was accepted as partial
for the degree of Master
sOuZ'
Dean,
Science
Pharmacy
Dean for Graduate Studies and
Research

UNIVERSITY OF FLORIDA



UNIVERSITY OF FLORIDA



PAGE 1

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