AN ABSTRACT OF THE THESIS OF
Edward H. Piepmeier, Jr. for the degree of Doctor of Philosophy in
Pharmacy presented on April 16, 1991.
Title: Formulation of an Oral Acetylsalicylic Acid Suspension and
Pharmacokinetics of Parenteral Thrombomodulin Analogues.
1"?
Abstract approved:
Redacted for Privacy
i
Mark Christensen, rn. v.
Sustained c ncentrations of active compound were maintained
in vitro and in vivo for an oral and a parenteral dosage form
respectively. The vehicle of a oral dosage form was modified and the
molecular structure of a parenteral dosage form was modified. An oral
dosage form was tested in vitro using dissolution apparatus. A
parenteral dosage form was tested in vivo using rats.
A new oral suspension dosage form for acetylsalicylic acid was
compared to two controlled release forms and two immediate release
dosage forms which are currently commercially available. A parenteral
thrombomodulin analogue conjugated to polyethylene glycol was
compared to the unconjugated thrombomodulin analogue. In each
case the goal was to maintain sustained concentrations of active
compound.
Formulation of an Oral Acetylsalicylic Acid Suspension
and Pharmacokinetics of Parenteral Thrombomodulin Analogues
by
Edward H. Piepmeier, Jr.
A THESIS
submitted to
Oregon State University
in partial fulfillment of
the requirements for the
degree of
Doctor of Philosophy
Completed April 16, 1991
Commencement June 1991
APPROVED:
4
,
.
_
Redacted for Privacy
P ofAsOr of Pharmacy in charge of major
,1
Redacted for Privacy
-
Dean of College of Pharmacy
Redacted for Privacy
Dean of
duate Scho
Date thesis presented
Presented by
April 16, 1991
Edward Piepmeier, Tr.
ACKNOWLEDGEMENTS
This thesis is dedicated to Heidi and Andrew who make my
work worthwhile. I wish to express my sincere gratitude to my major
professors, Dr. J. Mark Christensen and Dr. James Ayres for their
guidance, support and tireless proofreading. My sincere appreciation
goes to my parents for providing moral support to help me attain my
goals.
The research reported in this thesis was supported in part by a
grant from Berlex Biosciences. I would like to thank the people at
Berlex Biosciences, especially Dr. John Morser, Dr. David Light and
Susan Fink, for providing the thrombomodulin analogue and
conjugate, as well as for performing the activated protein C assay.
Finally I would like to thank my fellow graduate students for their help
and friendship.
TABLE OF CONTENTS
CHAPTER 1: THREE LAYERED POLYMER COATING OF
ASPIRIN IN A SUSPENSION DOSAGE FORM
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
CHAPTER 2: HIGH DOSE AND LOW DOSE
THROMBOMODULIN ANALOGUE
PHARMACOKINETICS IN RATS
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
CHAPTER 3: CONJUGATION OF THROMBOMODULIN
ANALOGUE WITH POLYETHYLENE GLYCOL
TO INCREASE THE HALF LIFE IN THE BLOOD
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
1
2
3
8
17
38
40
41
43
50
56
73
75
76
78
80
86
95
BIBLIOGRAPHY
97
APPENDIX
104
LIST OF FIGURES
Figure 1.1 Diagram Describing the Dissolution of an Aspirin
Formulation Including the Dissolution of Salicylic Acid
Present in the Formulation and the Degradation of ASA to
SA in Solution.
20
Figure 1.2 Milligrams of Acetylsalicylic Acid 0, and Salicylic
Acid , and Combination of Acetylsalicylic Acid and Salicylic
Acid U, Dissolved in Simulated Gastric Fluid and Simulated
Intestinal Fluid Versus Time From Genuine Bayer Aspirin,
Immediate Release Tablets.
23
Figure 1.3 Milligrams of Acetylsalicylic Acid 0, and Salicylic
Acid , and Combination of Acetylsalicylic Acid and Salicylic
Acid II, Dissolved in Simulated Gastric Fluid and Simulated
Intestinal Fluid Versus Time From Therapy Bayer Aspirin,
Enteric Coated Tablets.
24
Figure 1.4 Milligrams of Acetylsalicylic Acid 0 and Salicylic
Acid , and Combination of Acetylsalicylic Acid and Salicylic
Dissolved in Simulated Gastric Fluid and Simulated
Acid
Intestinal Fluid Versus Time From Measurin Timed Release
Aspirin, Eight Hour Release Tablets.
26
Figure 1.5 Milligrams of Acetylsalicylic Acid 0, and Salicylic
Acid , and Combination of Acetylsalicylic Acid and Salicylic
Acid U, Dissolved in Simulated Gastric Fluid and Simulated
Intestinal Fluid Versus Time From Eight Hour Bayer Timed
Release Aspirin, Eight Hour Release Tablets.
27
Figure 1.6 Milligrams of Acetylsalicylic Acid Cl, and Salicylic
Acid , and Combination of Acetylsalicylic Acid and Salicylic
Acid U, Dissolved in Simulated Gastric Fluid and Simulated
Intestinal Fluid Versus Time From Experimental Beads With
Triple Polymer Coating.
28
Figure 1.7 Milligrams of Acetylsalicylic Acid 0, and Salicylic
Acid , and Combination of Acetylsalicylic Acid and Salicylic
Acid U, Dissolved in Simulated Gastric Fluid and Simulated
Intestinal Fluid Versus Time From Experimental Beads With
Triple Polymer Coating, Incubated in Grape Jelly at 400C for
One Week.
29
Figure 1.8 Milligrams of Acetylsalicylic Acid 0, and Salicylic
Acid , and Combination of Acetylsalicylic Acid and Salicylic
Acid , Dissolved in Simulated Gastric Fluid and Simulated
Intestinal Fluid Versus Time From Experimental Beads With
Triple Polymer Coating, Incubated in Grape Jelly at 400C for
Two Weeks.
30
Figure 1.9 Milligrams of Acetylsalicylic Acid 0, and Salicylic
Acid , and Combination of Acetylsalicylic Acid and Salicylic
Acid II, Dissolved in Simulated Gastric Fluid and Simulated
Intestinal Fluid Versus Time From Experimental Beads With
Triple Polymer Coating, Incubated in Grape Jelly at 400C for
Six Weeks.
31
Figure 1.10 Percent of Total Acetylsalicylic Acid and Salicylic
Acid Dissolved in Simulated Gastric Fluid and Simulated
Intestinal Fluid Versus Time From Genuine Bayer Aspirin,
Immediate Release Tablets 0, and Therapy Bayer Aspirin,
33
Enteric Coated Tablets .
Figure 1.11 Percent of Total Acetylsalicylic Acid and Salicylic
Acid Dissolved in Simulated Gastric Fluid and Simulated
Intestinal Fluid Versus Time From Eight Hour Bayer Timed
Release Aspirin, Eight Hour Release Tablets 0, and Measurin
34
Timed Release Aspirin, Eight Hour Release Tablets .
Figure 1.12 Percent of Total Acetylsalicylic Acid and Salicylic
Acid Dissolved in Simulated Gastric Fluid and Simulated
Intestinal Fluid Versus Time From Experimental Beads
With Triple Polymer Coating, Incubated in Grape Jelly at
35
400C for Zero 0, One , Two 0, and Six Weeks II.
Figure 2.1 A schematic model showing the five domains in
the structure of endothelial thrombomodulin.
44
Figure 2.2 Schematic representation of the coagulation
system which forms crosslinked fibrin, and the fibrinolytic
system which breaks the crosslinked fibrin into fibrin
degradation products.
45
Figure 2.3 The fraction of initial protein C activation
versus time curves for ten rats receiving 10, 20, 50, 100,
or 200 micrograms of thrombomodulin with leucine
substituted for methionine at the 388th position.
57
Figure 2.4 Average fraction of initial radiolabel remaining
from 1251 thrombomodulin analogue for low dose , and
high dose administered.
59
Figure 2.5 Fraction of initial protein C activation 0
remaining over time and fraction of initial radiolabel
remaining over time for a rat which was administered 200
micrograms of thrombomodulin with leucine substituted
for methionine at the 388th position.
61
Figure 2.6 Fraction of radiolabel from 1251 labelled
67
thrombomodulin analogue which appears in the bile at
low doses 0, and high doses .
Figure 2.7 Fraction of radiolabel from 1251 labelled
69
thrombomodulin analogue found in tissues following
sacrifice of high dose rats.
Figure 3.1 Fraction of initial protein C activation remaining
over time for four doses of thrombomodulin analogue and
two different doses of PEG conjugate analogue.
89
Figure 3.2 Average fraction of initial radiolabel remaining
from 1251 thrombomodulin analogue for low dose and
high dose administered, and PEG conjugated
thrombomodulin analogue with 50% of initial activity 0
and 70% of initial activity O.
91
LIST OF APPENDIX FIGURES
Figure A.1 The fraction of initial radiolabel administered
remaining over time for radiolabelled Bovine Serum
Albumin 0, low dose radiolabelled thrombomodulin analogue with radiolabelled BSA , and high dose radiolabelled
thrombomodulin analogue with radiolabelled BSA E.
107
108
Figure A.2 This figure shows the result of step #1 when the
radiolabel vs. time curves shown in figure A.1 are used to
determine the fraction of radiolabelled bovine serum albumen
in each of the two thrombomodulin analogue BSA mixtures.
109
Figure A.3 The radiolabelled bovine serum albumen in the
high dose thrombomodulin analogue BSA mixture is calculated to be 33.7%. So the BSA curve is multiplied by 0.337 and
this resulting curve is subtracted from the thrombomodulin
analogue BSA mixture to obtain the radiolabel attributable
to the thrombomodulin analogue only 0.
110
Figure A.4 The radiolabelled bovine serum albumen in the
low dose thrombomodulin analogue BSA mixture is calculated to be 48.9%. So the BSA curve is multiplied by 0.489 and
this resulting curve is subtracted from the thrombomodulin
analogue BSA mixture to obtain the radiolabel attributable
to the thrombomodulin analogue only
Figure A.5 These curves represent the fraction of radiolabel
which is attributable only to thrombomodulin analogue
relative to the initial amount of radiolabel injected.
111
Figure A.6 The amount of radiolabel remaining due to
thrombomodulin analogue relative to the initial radiolabel
injected into the rat in the form of thrombomodulin
analogue.
112
Figure A.7 High dose and low dose pharmacokinetics of
thrombomodulin analogue in rats. The high dose and low
dose administered as pure thrombomodulin samples is
compared to the mathematically derived curves from the high
dose and low dose 0 samples administered with
radiolabelled BSA.
113
LIST OF TABLES
Table 1. 1 The percent of drug in beads which is
acetylsalicylic acid or salicylic acid is compared to what
would be expected with the coating procedures carried out
in this experiment.
10
Table 1.2 Percent of ASA and SA measured in each dosage
form relative to the total weight of each formulation.
12
Table 1.3 Percent of ASA and SA found in the bead and jelly
components of beads incubated in jelly at 400C over time
relative to the total amount of drug in each formulation.
13
Table 1.4 The rate constants for release of ASA as SA and
ASA and the rate constant for conversion of ASA to SA in
18
intestinal fluid in simulated gastric fluid and simulated
intestinal fluid.
Table 1.5 The time is the amount of time elapsed after
beginning dissolution. The dosage form is transferred to
the simulated intestinal fluid at 120 minutes. The actual
number of mg of ASA determined by HPLC assay in
simulated intestinal fluid is listed in the second column.
The amount of ASA which would be expected based on the
rates of ASA release and rates of ASA to SA conversion
found in Table 1.4 is calculated and shown in the third
column. The final column shows the percent of
theoretical release which was actually observed and the
coefficients of variation.
22
Table 2.1 The fraction of initial protein C activation over
time + standard deviations for rats receiving
thrombomodulin with leucine substituted for methionine
at the 388th position.
58
Table 2.2 Average fraction + standard deviations of initial
radiolabel remaining in the blood from 1251
thrombomodulin analogue for low dose and high dose.
60
Table 2.3 Fraction of thrombomodulin analogue remaining
as determined by 1251 radiolabel and activated protein C
assay for a rat which received 200ug of thrombomodulin
analogue with leucine substituted for methionine at the
388th position.
62
Table 2.4 Radioactivity half life times in minutes
63
following low dose and high dose of thrombomodulin
analogue as determined by monoexponential and
biexponential curve fitting. Monoexponential half lives
as determined by activated protein C assay.
Table 2.5 Fraction of initial radiolabel from 1251 labelled
68
thrombomodulin analogue which appears in the bile at
high doses and low doses. Each timepoint represents
data from up to four rats.
Table 2.6 Each tissue collected represents the following
percent of total body weight.
70
Table 2.7 Fraction of radiolabel from 1251 labelled
72
thrombomodulin analogue found in tissues following
sacrifice of high dose rats. Four rats were sacrificed one
hour apart at 1, 2, 3, and 4 hours.
Table 3.1 Half lives in minutes and areas under the curve
in mOD/minute versus minutes as determined by
protein C activity for thrombomodulin analogue with
methionine converted to leucine at the 388th position and
for the polyethylene glycol conjugate of the
thrombomodulin analogue.
87
Table 3.2 The fraction of initial protein C activation over
time ± standard deviations for rats receiving
thrombomodulin with leucine substituted for methionine
at the 388th (M388L) position conjugated with PEG.
90
Table 3.3 Half lives in minutes, areas under the curve in
mOD/minute versus minutes, and mean residence
times in minutes (MRT) are calculated for each rat using
92
RSTRIP.
Table 3.4 Average fraction ± standard deviations of
initial radiolabel remaining in the blood from 1251
thrombomodulin analogue conjugated to PEG.
93
FORMULATION OF
AN ORAL ACETYLSALICYLIC ACID SUSPENSION
AND PHARMACOKINETICS OF
PARENTERAL THROMBOMODULIN ANALOGUES
CHAPTER 1
THREE LAYERED POLYMER COATING OF ASPIRIN IN A
SUSPENSION DOSAGE FORM
2
ABSTRACT
An experimental controlled release aspirin (ASA) suspension
dosage form was investigated wherein the ASA was coated with three
distinct polymer layers in "sandwich" fashion. The experimental
suspension and four commercially available aspirin products were
analyzed for acetylsalicylic acid and its primary degradation product,
salicylic acid. Dissolution profiles for one immediate release tablet,
one enteric coated tablet, two controlled release tablets, and the
experimental suspension were compared. The experimental
formulation releases all drug over eight hours. Salicylic acid release
from the experimental formulation was higher than from
commercially available solid dosage forms.
INTRODUCTION
Controlled release of acetylsalicylic acid (ASA) increases the
duration of action and decreases gastrointestinal irritation. A short
duration of action from immediate release tablets is due to rapid
elimination of ASA through degradation and metabolism in both the
gastrointestinal and vascular fluids. The distribution and elimination
half lives for ASA in the blood are 2.8 and 15 minutes respectively (1,2).
This elimination cannot be countered with increased doses of ASA.
When the gastrointestinal tract is exposed to high concentrations of
ASA, cell necrosis occurs.
Irritation occurs to the Gastrointestinal (GI) tract when rapid
dissolution or disintegration causes a large surface area of high
concentrations of ASA in liquid, semiliquid or solid forms to come in
contact with the GI tract (3). Rapid disintegration of immediate
release dosage forms causes high dissolution layer concentrations of
ASA with large surface areas of undissolved particles to come in
contact with the mucosal membrane of the stomach. Buffers minimize
the time for aspirin dissolution and the time the aspirin remains in
contact with the stomach and decrease bleeding compared to non
buffered tablets (4). An immediate release buffered tablet causes more
irritation to the stomach than microencapsulated tablets (3). Enteric
4
coating minimizes gastric irritation (5), however aspirin is absorbed
readily in the stomach and so release of low concentrations in the
stomach is beneficial if a rapid effect is desired. Additionally, enteric
coated tablets cause high concentrations of aspirin to come in contact
with the intestinal mucosa where it is degraded rapidly by esterase.
The esterase activity in the duodenum is ten times that of the stomach
(2).
ASA is rapidly metabolized and cleared following absorbtion. In
vascular fluids, ASA is deacetylated to form salicylic acid which is then
metabolized through the following routes: conjugation of salicylic acid
(SA) with glycine to form salicyluric acid, conjugation of SA with
glucuronic acid to form salicyl phenolic glucuronide and salicyl acyl
glucuronide, and oxidation of SA to gentisic acid. SA is also excreted
directly by the kidney (6). In vitro, acetylsalicylic acid degrades to
salicylic acid, salicylsalicylic acid, acetylsalicylsalicylicacid, acetylsalicylic
anhydrate, acetylsalicylsalicylsalicylic acid,
acetylsalicylsalicylsalicylsalicylic acid, salicylsalicylsalicylic acid, and
salicylsalicylsalicylsalicylic acid (7).
The primary degradation product is salicylic acid (SA) which was
monitored along with ASA in this study. Products containing ASA
lose much of their activity when ASA is degraded to SA. ASA, unlike
SA, has both antiinflammatory and antipyretic effects. ASA has greater
5
analgesic affects than SA (8). ASA is an inhibitor of cyclooxygenase.
PGE2, a precursor to cyclooxygenase, is inhibited by ASA but not by SA
(9). Aspirin tolerance in asthmatics occurs when SA binds to the
cyclooxygenase supplementary binding site. Therefore, SA inhibits
some activity of ASA.
Complete thromboxane inhibition can occur from the amount
of ASA released from one immediate release tablet per day but
collagen induced platelet aggregation can occur shortly after the ASA
levels decline. Induction of platelet aggregation increases the risk for
myocardial infarction the morning after an ASA tablet has been taken,
when ASA has not been present in the blood for hours (10).
By releasing ASA from a dosage form over a sustained period of
time, a sustained level of ASA may be maintained in the blood. The
controlled release property of the experimental dosage form has the
potential to reduce the risk of myocardial infarction associated with
immediate release dosage forms. Because the new formulation is a
suspension, it will pass through a nasal gastric tube and still provide
sustained release, unlike crushed solid dosage forms.
The only non-tablet, solid, sustained release product currently
available is Theo-Dur Sprinkle Sustained Action Capsules (ScheringPlough, New Jersey). The contents of the capsule, microencapsulated
6
theophylline beads, are intended to be sprinkled on or mixed in with
food prior to ingestion. If microencapsulated beads are chewed or
crushed, however, all sustained release properties are lost. The only
sustained release suspension currently available is the Pennkinetic
system (ion exchange system). Suspensions are typically messy to
administer and may not enjoy a high acceptance rate among
consumers. A suspension of very high viscosity, however, may be
easier to administer and therefore more acceptable. Geriatric patients
may have chewing or swallowing difficulties (11). In this case,
traditional oral dosage forms are not ideal and alternate drug delivery
systems are needed. Therefore a goal in the development of the
experimental dosage form was to formulate a dosage form that is easily
administered to and readily accepted by geriatric consumers.
The experimental product is a controlled release suspension
consisting of beads coated with three distinct membrane coats and
presuspended in a grape jelly type vehicle. In the experimental
product, the enteric coat prevents ASA release in the stomach, while
the controlled release polymer coat prevents high concentrations of
aspirin from being released upon entering the intestine. The stomach
will be protected from irritation and the aspirin continuously released
in the intestine. The purpose of this research is to track ASA and SA in
dissolution tests and to formulate and evaluate a sustained release
7
ASA suspension as well as commercially available ASA preparations.
8
MATERIALS AND METHODS
Nonpareil sugar beads (25 mesh) were coated with ASA to a final
mesh of 18-20. Polyvinylpyrrolidone and
hydroxypropylmethylcellulose were dissolved in 95% ethanol for use
as binders. Acetylsalicylic Acid (Sigma) was dissolved in the ethanol
solution, and loaded onto sugar beads at 300C. The ASA beads were
then coated with Aquacoat 7%, by weight theoretical, followed by
Eudragit 5%, followed by Aquacoat 7%.
The sugar cores were loaded with acetylsalicylic acid using a
STREA-1 laboratory scale spray coater (Aeromatic, Towaco, NJ)
equipped with a 6-inch Wurster column and Lab-Line/P.R.L. Hi-Speed
Fluid Bed Dryer (Lab-Line Instruments, Inc., Melrose Park, IL).
Ethanolic drug solution was applied to freely moving sugar beads
through a 1.2mm orifice diameter spray nozzle. Atomization air
pressure was 10-12 psi. A medium perforated plate was placed below
the nozzle of the Wurster column and fluidization air was applied
with a blower speed of 50-80% of capacity. The fluidization air kept the
beads moving freely throughout the drug loading. A peristaltic pump
(Minipuls 2, Gilson Medical Electronics, Middleton, WI) introduced
drug suspension into the coating chamber at a rate of 10m1/minute.
Freshly prepared acetylsalicylic acid beads were transferred to a sieve
9
and oven dried at 400C for one day to remove residual moisture. Dried
acetylsalicylic acid beads were then sieved and the ten mesh fraction
was coated with a triple layer of polymers, using the same spray coating
equipment. The drug load of acetylsalicylic acid and salicylic acid were
determined using high pressure liquid chromatography.
Two commercially available latex dispersions, Aquacoat and
Eudragit L-30D, were used for polymer film application. Coating
formulations containing these materials were applied only to 10 mesh
fractions of acetylsalicylic acid cores to minimize variability in release
behavior due to drug core size variations (12).
During the application of Aquacoat, bed temperature was kept at
400C and the blower speed and delivery rate varied to maintain a
fluidized circulation because of differences in the stickiness of the
developing film. Blower speed ranged from 50 to 100% and delivery
rate ranged from 0-10 milliliters per minute. An 0.8 millimeter orifice
diameter spray nozzle was used. Eudragit L-30D was applied under
identical conditions. After each coating, three samples of beads were
pulverized, dissolved and analyzed for drug content with high
pressure liquid chromatography. The expected and actual results
following each coating procedure are shown in Table 1.1. The beads
(16g) were mixed in with grape jelly (Danish Orchards, 30g) and
carbopol (BF Goodrich, 0.4g). The jelly was divided into jars containing
10
Table 1. 1 The percent of drug in beads which is acetylsalicylic acid or
salicylic acid is compared to what would be expected with the coating
procedures carried out in this experiment. Higher assayed values than
expected may be attributable to loss of beads during the coating
procedure and lower assayed values than expected may be attributable
to loss of coating material during the coating procedure. HPLC assay
was used until three values were obtained within 1% of each other.
BEADS
FIRST COAT
SECOND COAT
THIRD COAT
ASSAYED
92%
83
78
72
EXPECT ED
83%
77
73
69
11
1.6g of suspension equivalent to 556mg of beads or 400mg of ASA. All
commercial and experimental products were assayed for ASA and SA
content relative to their total formulation weight (Table 1.2). The
experimental beads were tested both in a suspension and out of
suspension (dry).
Stability studies were performed for the ASA suspension. The
jelly suspension was incubated at 400C. This temperature was chosen
to increase the rate of aging over the rate of aging found at room
temperature. The jelly and beads were separated and assayed using the
high pressure liquid chromatography method described below for ASA
and SA content after incubation for one, two, and six weeks (Table 1.3).
HIGH PRESSURE LIQUID CHROMATOGRAPHY
Acetylsalicylic acid and salicylic acid were measured
quantitatively in each sample by a modification of a previously
reported HPLC procedure (13). The mobile phase was prepared by
mixing acetonitrile, methanol and 0.1M Potassium Phosphate Dibasic
(10:34:56 by volume). The final pH was adjusted to 2.5 + 0.1 with
phosphoric acid. Solvent was filtered (0.4 microns) and degassed prior
to use. Flow rate was set to 1.0m1/minute. Effluent from the column
was monitored by UV absorption at 229nm. 3-hydroxybenzoic acid was
used as an internal standard. Peak height ratios were used for
12
Table 1.2 Percent of ASA and SA measured in each dosage form
relative to the total weight of each formulation. Each formulation is
within one standard deviation of the labelled value. All are analyzed
in triplicate.
DOSAGE FORM
Eight Hour
Immediate Release
Coated Beads
Measurin
Enteric Coat
*less than 0.25%
ASA% (SD)
89.5(0.815)
79.9(2.91)
72.0(1.66)
91.4(3.16)
73.7(8.80)
SA% (SD)
0.479(0.0199)
1.69(0.113)
2.19(0.0543)
0*
1.65(0.301)
13
Table 1.3 Percent of ASA and SA found in the bead and jelly
components of beads incubated in jelly at 400C over time relative to the
total amount of drug in each formulation. n=3
1week
ASA% mean(sd)
SA% mean(sd)
93.49(4.683)
1.240(1.157)
3.510(0.0543)
1.780(1.585)
jelly
60.46(2.697)
8.404(1.812)
6.475(0.812)
24.65(4.693)
beads
jelly
41.16(1.513)
22.15(6.978)
13.51(2.205)
23.18(6.330)
beads
jelly
2 weeks
6 weeks
beads
14
quantitation based on standard curves established on the same day. All
chromatographic separations were carried out at ambient temperature.
An HPLC solvent delivery system Model M-45, an
Automatic Injector Model 712-WISP, a 30cm X 3.9mm ID
microBondapak C18 Column, and a Model 441 Absorbance detector, all
from Waters Associates (Milford, Mass., U.S.A.), were used in this
study. A Shimadzu CR3A Integrator was used to record the peaks
measured at 229nm.
The United States Pharmacopeia dissolution Apparatus 2
(paddle) was used in all experiments. The paddle was rotated at
50rpm. Studies were carried out in simulated enzyme free gastric and
intestinal fluid. Simulated enzyme free gastric fluid was prepared by
dissolving 6g of NaC1 in 21 ml of concentrated HC1 and sufficient
deionized water to make 3L; the pH was adjusted to 1.4 ± 0.1 with
hydrochloric acid. Simulated enzyme free intestinal fluid was prepared
by dissolving 20.4g of potassium phosphate monobasic (EM Industries,
Inc., Gibbstown, N.J) in 750m1 of deionized water. To this was added
4.56g of NaOH and 1.2L of water. The pH was adjusted to 7.4 ± 0.1 with
2N NaOH before the volume was made up to 3L with water.
Dissolution was performed in quadruplicate for each dosage
form. Three of the same dosage form were analyzed on the same day
and a fourth was analyzed on a different day to insure that day to day
15
variability was not significant. The dosage form was added to 900m1 of
gastric fluid at 37 + 0.50C in a water bath. This temperature was
maintained throughout the study.
All dosage forms were tested first in simulated gastric fluid and
then changed to simulated intestinal fluid after 2 hours to simulate
passage of the residual dosage form from the stomach into the upper
intestine. The simulated intestinal fluid was prepared fresh and
brought to 370C just prior to the actual dissolution test. Transfer of
dosage forms from gastric to intestinal fluid was carried out by filtering
the gastric fluid and then placing the filter paper in 900m1 of simulated
intestinal fluid in the vessel. Rotation of the paddle then resumed for
the duration of the test. The change in fluids took 10 to 15 minutes.
Fifty microliter samples were taken at 15, 30, 60, 90, 120 (before transfer
to intestinal fluid), 180, 240, 300, 360, 720, and 1440 minutes. Samples,
50u1, were run on HPLC within one hour following sampling using 3hydroxybenzoic acid, 1mg/ml, 950u1, as an internal standard. When
drug concentrations were low, equal volumes of sample and internal
standard were combined and analyzed. Standard curves were run for
both methods of analysis. The first five samples were taken from the
simulated gastric fluid and the remaining samples were taken from the
simulated intestinal fluid. The following dosage forms were purchased
commercially; Measurin Timed Release Aspirin (Winthrop, 650mg),
16
Therapy Bayer Aspirin (Bayer, 325mg), Genuine Bayer Aspirin (Bayer,
325mg), and Eight Hour Bayer Timed Release Aspirin (Bayer, 650mg).
Quattro was used to calculate peak height ratios and conversion of peak
height ratios to concentration in mg/ml. Standard deviations and
regression of all linear fits were also calculated using Quattro. First
order linear release rates and conversion rates were assumed due to the
high solubility of ASA, 1g/100m1 at 370C. The highest content of ASA
in a formulation was 650mg and the dissolution beaker contained
900m1.
17
RESULTS AND DISCUSSION
Each of the commercial formulations in this experiment were
tested for ASA and SA content (Table 1.2). In each case the labelled
content was within one standard deviation of the assayed amount. All
commercial products met USP requirements. The experimental
formulation did not meet USP requirements for an extended release
aspirin formulation. USP requires that immediate release aspirin
formulations be between 90% and 110% of the labeled dose and that
both delayed release and extended release aspirin formulations be
between 95% and 105% of the labelled dose. The ASA and SA content
of the experimental formulation changed while it incubated in the jelly
suspension at 400C (Table 1.3).
The dissolution rate constants are calculated from the amount of
drug undissolved at each timepoint (Table 1.4). The dissolution rate
constants in simulated intestinal fluid are calculated based on the
undissolved drug which remains after the simulated gastric fluid is
removed. After all of the ASA has been released from the tablet as
ASA or SA then it is possible to calculate the rate constant for
conversion from ASA to SA. This value is 0.021hour-1 based on
calculations from immediate release and enteric coated tablets. The
other formulations continued to release ASA over most of the
sampling period and so accurate ASA to SA conversion values were
18
Table 1.4 The rate constants (hour-I) for release of ASA as SA and ASA
and the rate constant for conversion of ASA to SA in intestinal fluid
in simulated gastric fluid and simulated intestinal fluid. Standard
deviations are shown in parentheses.
Gastric Fluid
total release
Immediate
Enteric
8-hour
Measurin
Dry Beads
One Week
Two Weeks
Six Weeks
1.1(0.11)
b
0.32(0.045)
0.45(0.033)
0.034(0.0041)
0.052(0.0027)
0.097(0.071)
0.13(0.016)
Intestinal Fluid
SA conversion
total release
0.11a
1.2(0.16)
0.42(0.070)
0.048(0.0024)
0.067(0.0074)
0.062(0.00064)
0.23(0.019)
0.30(0.073)
0.020(0.0017)
0.021(0.00062)
0.016(0.0016)c
alower limit (calculated based on total amount released in < one hour)
bless than 0.5% total dose released
c some ASA continues to be released and so this value is an estimate
19
not obtainable. The model depicted in Figure 1.1 was used to calculated
the concentration of ASA and SA in solution. The conversion of SA to
ASA was assumed to be negligible. The conversion of ASA to SA was
negligible in simulated gastric fluid at low pH (1.5), and 0.021hourl in
simulated intestinal fluid. The release of SA from the dosage forms
was very small and assumed to be complete after two hours in gastric
fluid. The rate constants that were calculated were based on the total
release of ASA and SA in both simulated gastric fluid and simulated
intestinal fluid. In addition the rate constant for conversion of ASA to
SA in intestinal fluid was used to compare estimated ASA
concentrations in the intestinal fluid with actual concentrations in
intestinal fluid for the controlled release suspension. The equation
used in simulated gastric fluid to determine the amount of dose
remaining to be released was C.Ae-ki ( 0 A represents the total amount
of drug remaining to be dissolved in gastric fluid, k1 represents the rate
constant found in Table 1.4 and t represents the time in hours. The
equation used to determine the amount of drug remaining to be
released in simulated intestinal fluid was C =Be-k2(t) - De-k3(t). B
represents the total amount of drug which remains to be dissolved in
simulated intestinal fluid D represents the total amount of ASA
degraded to SA in simulated intestinal fluid, k2 and k3 represent rate
constants found in Table 1.4 and t represents to the time in hours. This
20
Figure 1.1 Diagram Describing the Dissolution of an AspirM (ASA)
Formulation Including the Dissolution of Salicylic Acid (SA) Present in
the Formulation and the Degradation of ASA to SA in Solution. Kl,
K2, and K3 are the rate constants which are listed in Table 1.4.
ASA
solid
K1
ASA
dissolved
K3
SA
solid
SA
dissolved
21
fit gave coefficients of variation of less than 10% for all samples (Table
1.5).
The immediate release tablets release 260mg of ASA as ASA and
8.7mg of ASA as SA in the first two hours (Figure 1.2). This is 89% of
the drug released in the gastric fluid, without much of the ASA being
hydrolyzed to SA. The remaining ASA is released within the first
hour in the intestinal fluid. The SA content does not increase
noticeably post 120 minutes in the intestinal fluid because only 11% of
the ASA remains in the dissolution flask after disposing of the ASA
which was dissolved in the gastric fluid. The remaining ASA degrades
to SA with a half life of 33 hours in simulated intestinal fluid. The half
life of approximately 33 hours for degradation of ASA to SA in
simulated intestinal fluid was calculated for enteric coated tablets and
Bayer eight hour controlled release tablets (Table 1.4).
The enteric coated tablets do not release any ASA until after they
are transferred into the intestinal fluid (Figure 1.3). In intestinal fluid
(pH 7.4) nearly all of the ASA dissolves in the first hour. The ASA
then degrades to SA over the next 22 hours until SA almost equals
ASA in concentration. This conversion of ASA to SA will not be as
great in vivo in the gastrointestinal tract as in in vitro intestinal fluid
since the dissolved ASA would be absorbed.
For the measurin formulation 58% of drug was dissolved in the
22
Table 1.5 The time is the amount of time elapsed after beginning
dissolution. The dosage form is transferred to the simulated intestinal
fluid at 120 minutes. The actual number of mg of ASA determined by
HPLC assay in simulated intestinal fluid is listed in the second column.
The amount of ASA which would be expected based on the rates of
ASA release and rates of ASA to SA conversion found in Table 1.4 is
calculated and shown in the third column. The final column shows
the percent of theoretical release which was actually observed and the
coefficients of variation.
DRY BEADS
time ASA ACTUAL
63
240
86
360
139
480
167
540
183
720
208
840
252
1080
215
1440
CALCULATED
72.5
99.1
151
174
179
202
244
208
% THEORETICAL
86.9
86.8
92.1
96.0
102
103
103
103
CV=6.23
BEADS INCUBATED IN SUSPENSION FOR ONE WEEK
CALCULATED % THEORETICAL
time ASA ACTUAL
89.7
100
89.7
240
300
360
420
540
720
1080
1440
104
117
129
144
170
171
156
114
156
140
157
179
184
177
91.2
75.0
92.1
91.7
95.0
92.9
88.1
CV=6.92
BEADS INCUBATED IN SUSPENSION FOR TWO WEEKS
CALCULATED % THEORETICAL
ASA ACTUAL
time
240
300
360
480
1440
125
148
159
202
149
89.8
116
132
182
124
139
128
120
111
120
CV=8.50
BEADS INCUBATED IN SUSPENSION FOR SIX WEEKS
CALCULATED % THEORETICAL
time ASA ACTUAL
240
300
360
420
720
1080
1440
197
233
244
251
230
203
176
186
223
237
246
228
209
186
106
104
103
102
101
97.1
94.6
CV=3.94
23
Figure 1.2 Milligrams of Acetylsalicylic Acid El, and Salicylic Acid ,
and Combination of Acetylsalicylic Acid and Salicylic Acid III,
Dissolved in Simulated Gastric Fluid (0-2 hours) and Simulated
Intestinal Fluid (2-24 hours) Versus Time From Genuine Bayer
Aspirin, Immediate Release Tablets.
400 -
300 -
-.0- ASA
---*- SA
200 -
ASA & SA
100 -
416.1r.-1"--r-1
0
240
480
I
720
TIME(MINUTES)
960
1
1200
1 440
24
Figure 1.3 Milligrams of Acetylsalicylic Acid CI, and Salicylic Acid ,
and Combination of Acetylsalicylic Acid and Salicylic Acid W
Dissolved in Simulated Gastric Fluid (0-2 hours) and Simulated
Intestinal Fluid (2-24 hours) Versus Time From Therapy Bayer Aspirin,
Enteric Coated Tablets.
400
ASA
SA
ASA+SA
0
240
480
720
TIME (MINUTES)
960
1200
1440
25
first two hours in gastric fluid but the remaining drug did not dissolve
entirely until after an additional 46 hours in intestinal fluid (Figure
1.4). The SA increases along with the ASA content in intestinal fluid
due to ASA to SA conversion.
The Bayer Eight Hour tablets released 44% of drug in the first
two hours with the remainder dissolving over the next six hours
(Figure 1.5). The salicylic acid increased slowly over the twelve hours.
After 24 hours, 78% of the ASA from the dry experimental dosage form
is released (Figure 1.6). There is a gradual increase in SA, in solution,
comparable to that seen in measurin. 93% of the drug was released
after 48 hours total elapsed time. The experimental dosage form
(beads) incubated in jelly for one week release 58% of total after 12
hours and 69% of total drug after 24 hours (Figure 1.7). The beads
incubated in jelly for two weeks released 80% of drug after twelve
hours with the remainder being released within 24 hours (Figure 1.8).
After six weeks of incubation the beads release the entire amount of
drug within eight hours (Figure 1.9). The drug released as SA
increased, the longer the beads were incubated in jelly (Table 1.3).
The percent of total labelled salicylate released over time curves
show release from each formulation without regard for ASA
degradation (Figures 9-11). The enteric and immediate release dosage
forms have released their entire contents within one hour of reaching
26
Figure 1.4 Milligrams of Acetylsalicylic Acid 0 and Salicylic Acid 11,
and Combination of Acetylsalicylic Acid and Salicylic Acid III,
Dissolved in Simulated Gastric Fluid (0-2 hours) and Simulated
Intestinal Fluid (2-24 hours) Versus Time From Measurin Timed
Release Aspirin, Eight Hour Release Tablets.
700
600
0 500
w
ta
0-- ASA
1,-- SA
< 400
w
1
w
cc 300
ASA+SA
0
2 200
100
0
0
1000
2000
TIME (MINUTES)
3000
27
Figure 1.5 Milligrams of Acetylsalicylic Acid 0, and Salicylic Acid 0,
and Combination of Acetylsalicylic Acid and Salicylic Acid MI,
Dissolved in Simulated Gastric Fluid (0-2 hours) and Simulated
Intestinal Fluid (2-24 hours) Versus Time From Eight Hour Bayer
Timed Release Aspirin, Eight Hour Release Tablets.
700
600
w
500 -
<
400 w
ASA
-4-- SA
-J
cc
300 -
ASA+SA
0
2 200 100 0
240
480
720
960
TIME (MINUTES)
1200
1440
28
Figure 1.6 Milligrams of Acetylsalicylic Acid 0, and Salicylic Acid ,
and Combination of Acetylsalicylic Acid and Salicylic Acid III,
Dissolved in Simulated Gastric Fluid (0-2 hours) and Simulated
Intestinal Fluid (2-24 hours) Versus Time From Experimental Beads
With Triple Polymer Coating.
400
G
300 -
w
ASA
10-- SA
_1 200 -
ASA+SA
cc
0
2
100 -
0
240
480
720
960
TIME(MINUTES)
1200
1 440
29
Figure 1.7 Milligrams of Acetylsalicylic Acid 0, and Salicylic Acid III,
and Combination of Acetylsalicylic Acid and Salicylic Acid III,
Dissolved in Simulated Gastric Fluid (0-2 hours) and Simulated
Intestinal Fluid (2-24 hours) Versus Time From Experimental Beads
With Triple Polymer Coating, Incubated in Grape Jelly at 400C for One
Week.
350
300 -
0 ASA
250 w
Cl)
<I
200 -
w
12""".....,1
1
ul
150 ce
10-- SA
(5
2 100 50 0
0
240
i
I
I
1
480
720
960
1200
TIME (MINUTES)
1 440
ASA+SA
30
Figure 1.8 Milligrams of Acetylsalicylic Acid CI, and Salicylic Acid ,
and Combination of Acetylsalicylic Acid and Salicylic Acid II,
Dissolved in Simulated Gastric Fluid (0-2 hours) and Simulated
Intestinal Fluid (2-24 hours) Versus Time From Experimental Beads
With Triple Polymer Coating, Incubated in Grape Jelly at 400C for Two
Weeks.
400
300
-0-- ASA
---e- SA
200
ASA+SA
100
0
0
240
480
720
960
TIME (MINUTES)
1200
1 440
31
Figure 1.9 Milligrams of Acetylsalicylic Acid 0, and Salicylic Acid 0,
and Combination of Acetylsalicylic Acid and Salicylic Acid IS,
Dissolved in Simulated Gastric Fluid (0-2 hours) and Simulated
Intestinal Fluid (2-24 hours) Versus Time From Experimental Beads
With Triple Polymer Coating, Incubated in Grape Jelly at 400C for Six
Weeks.
500
-0- ASA
--*-- SA
ASA&SA
.
0
1
240
.
1
480
-
I
1
720
960
TIME (MINUTES)
.
1
1200
1440
32
the intestinal fluid (Figure 1.10). The dissolution of Bayer eight hour
tablets give release rates of 0.32 hour-1 in simulated gastric fluid and
0.42 hour-1 in simulated intestinal fluid. The dissolution of measurin
displayed much less consistent release rates 0.45-1 in gastric fluid and
0.048 hour-1 in intestinal fluid (Figure 1.11). The experimental
formulation approximates a zero order release over the first eight
hours or longer depending on the amount of time it has been aged in
jelly although first order release rate constants provided better fits
(Figure 1.12, Table 1.5).
For the experimental formulation, the outer coat protects the
enteric coat from becoming damaged while in suspension (11). The
enteric coat prevents rapid release in the stomach and the inner coat
prevents rapid release in the intestine. This dosage form provides
controlled release comparable to the two commercially available solid
dosage forms. Chemical degradation of acetylsalicylic acid beads
suspended in jelly is retarded, but not sufficiently to allow the beads to
be used commercially. For the experimental formulation, 8% of the
ASA is released in the first two hours in gastric fluid, which is an
improvement in bioavailability to the stomach over enteric coated
tablets. ASA is readily absorbed in the stomach compared to the
intestines (2). This 8% release may provide immediate beneficial
effects of ASA while minimizing the effects of high ASA
33
Figure 1.10 Percent of Total Acetylsalicylic Acid and Salicylic Acid
Dissolved in Simulated Gastric Fluid (0-2 hours) and Simulated
Intestinal Fluid (2-24 hours) Versus Time From Genuine Bayer
Aspirin, Immediate Release Tablets 0, and Therapy Bayer Aspirin,
Enteric Coated Tablets .
-0--
IMMEDIATE
-*-- ENTERIC
i
0
240
1
i
480
720
960
TIME (MINUTES)
i
I
1200
1440
34
Figure 1.11 Percent of Total Acetylsalicylic Acid and Salicylic Acid
Dissolved in Simulated Gastric Fluid (0-2 hours) and Simulated
Intestinal Fluid (2-24 hours) Versus Time From Eight Hour Bayer
Timed Release Aspirin, Eight Hour Release Tablets 0, and Measurin
Timed Release Aspirin, Eight Hour Release Tablets .
120
-0- 8 HOUR
-*-- MEASURIN
240
480
720
960
TIME (MINUTES)
1200
1440
35
Figure 1.12 Percent of Total Acetylsalicylic Acid and Salicylic Acid
Dissolved in Simulated Gastric Fluid (0-2 hours) and Simulated
Intestinal Fluid (2-24 hours) Versus Time From Experimental Beads
With Triple Polymer Coating, Incubated in Grape Jelly at 400C for Zero
0, One , Two 0, and Six Weeks U.
120 100 -
80 DRY BEADS
ONE WEEK
60 -
TWO WEEKS
SIX WEEKS
I
0
240
.I.I .1.1.
480
720
TIME (MINUTES)
960
1200
1440
36
concentrations in the stomach. The problem of ASA to SA conversion
must be addressed in future work to develop a sustained release
suspension of ASA.
The amount of drug dissolved in gastric fluid is added to the
dissolution data in the intestinal fluid.
Dissolved ASA in simulated
gastric fluid is not transferred to the simulated intestinal fluid. Since
the degradation of ASA to SA is not recorded from the gastric fluid
after two hours, there is no apparent SA formation for the immediate
release tablets which do not release much ASA after two hours. The
controlled release formulations which release most of the ASA in the
first two hours have little SA formation compared to the enteric coated
formulation. The enteric coated tablets show a large amount of SA
formation in intestinal fluid as the ASA remains in the intestinal fluid
throughout sampling. This may mimic in vivo ASA and SA
concentrations in the GI tract since ASA is absorbed rapidly in the
stomach. ASA is absorbed relatively slower in the intestines where
ASA is degraded to SA at the high intestinal pH. As stated previously
some of the ASA would be absorbed in the intestines and so there may
not be as much SA present in the intestines as there is in the intestinal
fluid during dissolution.
The percent of ASA released as SA increases for the
experimental formulation, the longer it incubates (40C, 1-6 weeks) in its
37
jelly suspension (Table 1.3). The rate of release from the jelly
suspension also increases as the suspension is incubated over longer
periods of time (Table 1.4). Future formulations may minimize the
amount of SA formation in the beads and transfer of the drug into the
jelly through improvements in decreasing the permeability of the
enteric coat.
38
REFERENCES
Rowland, M., and S. Riegelman. Pharmacokinetics of
acetylsalicylic acid and salicylic acid after intravenous administration in
man. The Journal of Pharmaceutical Sciences 57(8):1313-1319 (1968).
(1)
Kergueris, M. F., Bourin, M., Larousse, C., Lasserre, M. P., and A.
Ortega. Effects of administration route on pharmacokinetics of aspirin
in the rabbit. Methods and Findings in Experimental and Clinical
Pharmacology 10(3):171-175 (1988).
(2)
(3)
Alcorn, G., Lucker, P. W., Procaccini, R. L., and W. A. Ritschel. In
vivo-in vitro correlations with various aspirin formulations. Methods
and Findings in Experimental Clinical Pharmacology 9(11):761-767
(1987).
Leonards, J., and G. Levy. Biopharmaceutical aspects of aspirininduced gastrointestinal blood loss in man. The Journal of
(4)
Pharmaceutical Sciences 58(10):1277-1279 (1969).
(5)
Frenkel, E. P., McCall, M. S., Douglass, C. C., and S. Eisenberg.
Fecal blood loss following aspirin and coated aspirin microspherule
administration. The Journal of Clinical Pharmacology Nov-Dec:347-351
(1968).
(6)
Day, R., Furst, D. E., Dromgoole, S. H., and H. E. Paulus. Changes
in salicylate serum concentrations and metabolism during chronic
dosing in normal volunteers. Biopharmaceutics & Drug Disposition
9:273-283 (1988).
Verstraeten, A., Roets, E., and J Hoogmartens. Quantitative
determination by high-performance liquid chromatography of
acetylsalicylic acid and related substances in tablets. Journal of
(7)
Chromatography 388:201-216 (1987).
Bell, S., M. Berdick, M. and W. Holliday. Drug blood levels as
(8)
indices in evaluation of a sustained-release aspirin. The Journal of
New Drugs Sep-Oct:284-293 (1966).
Tashjian, Jr., A. H., Voelkel. E. F., Lazzaro, M., Goad, D., Bosma,
T., and L. Levine. Tumor necrosis factor-alpha (cachectin) stimulates
(9)
bone resorption in mouse calvaria via a prostaglandin-mediated
mechanism. Endocrinology 120(5):2029-2036 (1987).
39
Anti lla, M., P. Kahela, and P. Uotila. The absorption of
acetylsalicylic acid from an enteric-coated formulation and the
inhibition of thromboxane formation. International Journal of Clinical
(10)
Pharmacology, Therapy and Toxicology 26(2):88-92 (1988).
Mansell, L. New acetaminophen products: formulation
development and bioavailability. Ph.D. Thesis, Oregon State
(11)
University, 11-12 (1990).
Hossain, M. and J. W. Ayres. Variables that influence coat
integrity in a laboratory spray coater. Pharmaceutical Technology Oct:72(12)
82 (1990).
Aarons, L., Hopkins, K., Rowland, M., Brossel, S., and J. F.
Thiercelin. Route of administration and sex differences in the
pharmacokinetics of aspirin, administered as its lysine salt.
Pharmaceutical Research 6(8):660-666 (1989).
(13)
40
FORMULATION OF
AN ORAL ACETYLSALICYLIC ACID SUSPENSION
AND PHARMACOKINETICS OF
PARENTERAL THROMBOMODULIN ANALOGUES
CHAPTER 2
HIGH DOSE AND LOW DOSE THROMBOMODULIN ANALOGUE
PHARMACOKINETICS IN RATS
41
ABSTRACT
Pharmacokinetics were determined for a thrombomodulin
analogue produced in insect cells through recombinant DNA
technology. Anticoagulant activity of the thrombomodulin analogue
is a result of inactivation of thrombin in the presence of protein C,
protein S, and phospholipid surfaces. The activity of thrombomodulin
analogue was measured by the amount of protein C which is activated
through an activated protein C assay (APCA). The elimination half life
for thrombomodulin analogue activity as determined by APCA ranged
from 2.5 minutes to 5.2 minutes for low dose, and high dose
respectively. Thrombomodulin analogue pharmacokinetics were also
followed using 1251 radiolabel. The curves obtain by following
radiolabel were best fit by a biexponential equation. The half life as
determined by radiolabel for the initial elimination phase following
intravenous administration of 1251 labelled thrombomodulin analogue
in rats was 1.5+0.76 and 2.1+0.48 minutes, for low dose and high dose
respectively and the 1251 terminal elimination phase tin was
approximately 360+130 minutes. Rat bile and urine samples were
collected along with the following tissues; heart, lungs, liver, kidney,
spleen, brain, and muscle. Less than 1% of the radiolabel appeared in
the urine. Radio label in bile indicates that vesicular mediated
42
transcellular transport is occurring. The peak bile concentration of
radiolabel occurs at 30 to 45 minutes. Approximately 10% of the
radioactivity appeared in the bile. Less than 10% of the radioactivity is
found in the remaining tissues which were sampled.
43
INTRODUCTION
Thrombomodulin (TM) exists in a soluble form which circulates
through the vasculature, and an insoluble form which remains on the
vascular endothelium. The entire amino acid sequence of endothelial
TM has been determined (1). The structure of soluble TM is unknown
although it is presumed to be a cleaved form of endothelial TM (Figure
2.1) (2). TM has been purified from rabbit and human lung, as well as
rabbit, bovine, and human placenta (3,4). Human TM has been
expressed in COS-1 cells (4). It was initially proposed that there is a
difference in activity of rabbit TM (5), bovine TM (6) and human TM
(7). The difference in activity may be due to difficulties in removing
high molecular weight cationic compounds during purification (8).
TM is an endogenous protein which has both anticoagulant and
fibrinolytic activity (Figure 2.2). This activity of thrombomodulin is
carried out in part by its ability to activate protein C. The fibrinolytic
activity of TM is due to activated protein C's direct enzymatic action
which lyses fibrin clots. There are three anticoagulant activities of TM;
protein C activation, inhibition of thrombin indirectly through
antithrombin III, and direct inhibition of thrombin. TM forms a 1:1
complex with thrombin which activates protein C a thousand times
faster than thrombin alone (9). TM activates protein C in the presence
44
Figure 2.1 A schematic model showing the five domains in the
structure of endothelial thrombomodulin. The amino terminus is
followed by a domain which is not homologous with any other known
protein. The second domain consists of six repeating epithelial growth
factor (EGF) like sequences. The third domain consist of a region with
sites for potential glycosylation. The final domain prior to the carboxy
terminus is a lipophilic transmembrane domain.
NH2
i
(
Domains
)
)
)
(
(
)
H---'
71L--)
c-0---:
NH2-terminal
EGF-like structures
r---*--;
0-glycosylation site-rich
transmembrane
cytoplasmic
COOH
(Suzuki, 1987)
45
Figure 2.2 Schematic representation of the coagulation system which
forms crosslinked fibrin, and the fibrinolytic system which breaks the
crosslinked fibrin into fibrin degradation products. Thrombomodulin
acts on the extrinsic pathway, the Xa-Va complex, and thrombin to
exhibit anticoagulant activity. Thrombomodulin acts directly on
crosslinked fibrin to exhibit fibrinolytic activity.
extrinsic
intrinsic
XIII
Val
thrombin
prothrombin
fibrinogen
fibrin monomers
Xllta
fib in polymers
Ca"1
fibrin degradation
products
crosslinked
fibrin
plasminogen
plasmin--a2-anti-plasmin
plasminogen
activator
inhibitor
plasminogen activator
(Bertina, 1988)
46
of a phospholipid surface, Ca2+, and protein S, providing anticoagulant
activity by degrading factors Va and VIIIa. Therefore the TM-thrombin
complex does not inhibit clot formation outside blood vessels, while it
maintains blood fluidity through anticoagulant and fibrinolytic activity
within blood vessels where it is in the presence of cofactor proteins (2).
Sustained levels of TM are beneficial to continuously prevent
clotting within the vasculature. Unlike other anticoagulants, TM's
activity is decreased outside of the vasculature where clotting may be
necessary to prevent blood loss. TM provides prophylactic
anticoagulant activity while it lyses clots. TM's continuous fibrinolytic
and anticoagulant activity in the vasculature is desired since the
blood's ability to coagulate is not entirely inhibited by TM and the risk
of hemorrhage may be minimized relative to dosing with other
anticoagulants.
Coagulation problems are a leading cause of acute morbidity.
Abnormalities in coagulation can be a result of obstetrical conditions,
infections, neoplastic diseases, tissue trauma, and congenital defects.
Two classes of drugs are commonly used for coagulation therapy;
anticoagulants such as heparin and warfarin which prevent clotting,
and fibrinolytic agents such as tissue plasminogen activator and
streptokinase which lyse clots. Thrombomodulin provides a novel
approach to coagulation therapy because it has both anticoagulant and
47
fibrinolytic activity.
Endogenous TM may not be sufficient to regulate the
coagulation and fibrinolytic system. Purified TM may be used to
diagnose and correct for abnormal levels of endogenous protein C and
TM. Protein C abnormalities in a patient may be detected by use of
anticoagulant activation with thrombin, TM, or Protac (snake venom).
Patients showing lack of anticoagulant activation have an increased
risk of thrombosis after surgical procedures (10).
TM is also currently being investigated to counteract coagulation
problems which occur as a result of tissue trauma. TM may be useful
to prevent blood clot formation when used in conjunction with
heparin in cardiac bypass surgery (11). Human TM inhibits coagulation
by a different mechanism than heparin. Heparin only activates
antithrombin III to inhibit thrombin and does not completely inhibit
the formation of thrombin. Protein C, protein S, and TM levels
decrease during cardiopulmonary bypass surgery. Increase in thrombin
is responsible for depletion of the TM induced protein C anticoagulant
activity. This decrease promotes clotting. TM and tissue plasminogen
activator are both absent in obstructed coronary artery bypass grafts (12).
TM is suppressed in bovine aortic endothelium when it is under
hypoxic conditions (14). Endothelial cell injury, depletion due to
excessive deposition of thrombin, or local proteolysis following
48
endothelial cell damage all may cause a decrease in TM and tissue
plasminogen activator levels. TM's activation of protein C is an
important regulator of the coagulofibrinolytic system. Presence of
excess TM is less likely to result in adverse affects because the presence
of other cofactors and enzymes in the coagulation cascade regulate the
activity of TM.
The active portion of TM consists of six loops, each with a
sequence similar to epithelial growth factor (EGF) (Figure 2.1). The
fourth, fifth, and six loops of the EGF like structures of TM were
suggested to be the minimally active fragment for anticoagulant
activity (9). However the same authors show that the first, second, and
third loops are also as active as native TM. Therefore the six similar
EGF like loops appear to have similar activity.
The TM analogue used in this study exhibits anticoagulant and
fibrinolytic activity by the same mechanism as native TM as
determined by activated protein C assay (APCA). The TM analogue is
structurally different than native TM in three ways (Figure 2.1). The
TM analogue (M388L) is made with leucine substituted for methionine
at the 388th position to prevent loss of activity which occurs if
methionine is oxidized. M388L is missing the lipophilic
transmembrane portion that attaches native endothelial
thrombomodulin to the vasculature. Finally, glycosylation of M388L is
49
different than native TM because M388L is produced through
recombinant techniques.
The purpose of this research was to evaluate M388L
pharmacokinetics including determination of the routes of
distribution and elimination for M388L. This is accomplished through
1251 radiolabelling and the use of APCA. Understanding the
pharmacokinetics of this M388L will increase the therapeutic options
available for prevention of thrombosis and for dealing with emboli.
50
MATERIALS AND METHODS
PREPARATION OF THROMBOMODULIN.
Human thrombomodulin (TM) analogue (M388L) with leucine
substituted for methionine at the 388th amino acid position was
prepared using an insect cell line to prevent oxidation and loss of
activity. Since the recombinant M388L was produced in insect cells,
glycosylation of the protein is different than native human
thrombomodulin. M388L is missing the lipophilic portion which is
found in native endothelial TM. M388L was analyzed by gel
electrophoresis to ensure that there were no contaminating
compounds.
RADIOIODINATION OF THROMBOMODULIN.
M388L was radiolabelled with 1251 to give a specific activity of 2 to
lOuCi/ug. Purity was determined by polyacrylamide gel
electrophoresis. Pharmacological activity was measured as the ability
of M388L to activate Protein C as determined by the activated protein C
assay described later.
ANIMAL EXPERIMENTS.
Male Sprague Dawley Rats 280-300g, 65-75 days old, were
51
maintained on Wayne Rodent Blocks (Harlan Sprague Dawley). The
rats were anesthetized with an interperitoneal (IP) injection of sodium
pentobarbital, 6-7.5mg/100g body weight. Maintenance doses were
given IP, 1mg/100g body weight, as needed to insure full anesthesia
during all procedures. Euthanasia was by exsanguination without the
rat ever regaining consciousness. Surgery began each morning
between 9AM and 12PM. A polyethylene cannula (PE-10, Clay Adams)
was inserted into the bile duct through a 3-4cm abdominal incision.
The incision was then clamped and kept moist throughout the
procedure with gauze soaked in normal saline. Another cannula (PE50) was placed in the right femoral artery. One hundred units of
heparin were injected into rats where protein C activation by M388L
was not measured. Non-radiolabelled M388L was sampled without the
use of heparin. A rapid 0.1m1 burst of saline into the cannula was
usually all that was necessary to clear the cannula. The cannula was
gradually inserted further into the artery to break clotting when
necessary in unheparinized rats where protein C activation by M388L
was measured. The rat was placed in a supine position on a heating
pad under a heating lamp to maintain 380C body temperature. The
M388L dose was administered through the arterial cannula followed by
1m1 of 0.9% saline.
M388L was injected into four rats at a low dose (0.2ug/rat) or at a
52
high dose (10Oughat). The M388L was stored in phosphate buffered
saline with 0.1% bovine serum albumin. For low dose samples, 0.2ug
of radiolabelled M388L was diluted with 0.5m1 0.9% saline immediately
prior to injection. For high dose an additional 10Oug of unlabeled
M388L (1mg/m1) was added to 0.2ug of radioactive M388L immediately
prior to injection. For samples where protein C activity of M388L was
measured, 500, 250, 100, 50, 25, or bug total M388L was injected was
injected into four rats for each dose. Bile was collected continuously
and blood was sampled at -1, 1, 2, 4, 5, 6,10, 20, 30, 45, 60, 75, 90, 105, 120,
150, 180, 210, and 240 minutes for all doses. The volume of blood
sampled was replaced with 0.9% normal saline.
Upon completion of bile and blood collection the rat was
exsanguinated. One rat each was sacrificed at one, two, three, or four
hours of elapsed time after dosing M388L. Urine and tissue samples
were collected upon completion of the 1-4 hour study period. Liver
and kidneys were weighed and portions of each were collected for
sampling. Spleen, heart, lung, brain, and urine were collected in their
entirety and a small weighed portion of thigh muscle was sampled.
Sacrificing animals one hour apart allowed observation of distribution
of radiolabel in tissues over time.
53
ASSAY METHOD, RADIOLABELLED THROMBOMODULIN
1251 radioactivity in blood, bile, urine, and tissues was measured
using an automated gamma counter (Beckman Gamma 4000, 74.9%
efficiency). Each sample was counted for one minute. Counts per
minute for each sample were compared to counts per minute for the
initial M388L dose injected, after subtracting the background in each
case. This resulting fraction of the dose was then corrected for the
weight of the tissue or fluid sample. Radio labelled bovine serum
albumin (BSA) was found in early M388L preparations and
pharmacokinetic curves obtained from these radiolabelled M388L
samples were mathematically corrected for radiolabelled BSA
(Appendix). These data are compared to M388L pharmacokinetics
determined from doses free of BSA. BSA presence was necessary in
M388L solutions to prevent adsorption of M388L onto the container
walls. In early studies, BSA was inadvertently radiolabelled along with
the M388L. Later, M388L was radiolabelled separately, and gel
electrophoresis was used to show that M388L is the only source of
radiolabel in all M388L pharmacokinetics reported outside of the
Appendix.
54
THROMBOMODULIN ACTIVITY (ACTIVATED PROTEIN C ASSAY).
Heparin was not administered to rats providing samples for
activated protein C assay (APCA) because heparin interferes with
measurement of thrombomodulin activity through the APCA. Blood
samples were diluted 1:20 in phosphate buffered saline containing 0.5%
bovine serum albumin and centrifuged immediately following
collection. Supernatant liquids of blood and the entire bile samples
were frozen in dry ice acetone and packed in dry ice for later analysis.
Activation of protein C by M388L is measured according to the
following assay. Thrombin and protein C are dissolved in 20mM Tris
HCL, 0.1M NaC1, 2.5mM CaC12, and BSA (5mg/m1) at pH 7.4. Twenty
microliters each of sample, thrombin, 3nM (Sigma), and protein C,
0.5uM (Genzyme) are mixed and incubated for 60 minutes at 37°C.
Hirudin, 20u1, 0.16units/m1 (American Diagnostica) is then added and
the mixture is incubated at 37°C for 15 minutes to stop the protein C
activation. Chromogenic substrate 52266,100u1, 1.0mM (Kabi Vitrum)
is then added and absorbance measured every sixty seconds at 405nm
for 15 minutes. The change in absorbance per minute is proportional
to the concentration of activated protein C. Standard protein C
activation rates are determined from a single batch of insect cell line
M388L (64units/m1) manufactured on site.
55
CALCULATIONS
The number of micrograms of each dose administered was
determined by APCA. The activity was compared to the activity per
microgram of a single batch of insect cell line M388L. The fraction of
radioactivity or protein C activity at each timepoint is compared to the
activity of the dose injected. The activity of the dose injected, in each
rat, is determined by taking a small sample of blood from each rat prior
to dosing and mixing the blood with an amount of M388L which is
proportional to the entire dose administered in 20m1 of blood. The
activity of the single batch of insect cell line M388L in each rats blood
was not determined and so the amount of M388L remaining in the
blood over time is not able to be calculated. Background radioactivity
and protein C activity for each rat's serum is subtracted from the
activity of that rat's samples. The parameters for protein C activity
pharmacokinetics of the thrombomodulin analogue were calculated
using PCNONLIN (Modified Newton-Gauss method).
56
RESULTS AND DISCUSSION
The pharmacokinetics of M388L are evaluated via the 1251 label
pharmacokinetics and the ability of M388L to activate protein C. The
volume of blood in each rat is estimated at approximately 20m1 based
on body weight. This volume is used to calculate the total radiolabel
remaining in blood over time.
The pharmacokinetics of M388L's protein C activation over time
(Figure 2.3, Table 2.1) at five doses in the blood (10, 20, 50, 100, and
200ug) can be compared to radiolabelled thrombomodulin analogue
over time (Figure 2.4, Table 2.2). Figure 2.5 and Table 2.3 show the
APCA activity relative to the counts per minute of 1251 in the blood
after M388L was administered to a single rat. 1251 radioactivity
clearance from rat plasma lags the clearance of activation of protein C
after 5-10 minutes. This may be due to radiolabel becoming detached
from M388L or the M388L becoming inactivated.
The M388L half lives as determined by following the radiolabel
or protein C activation assay of the initial elimination phase and the
terminal elimination phase are listed in Table 2.4. There is a single
exponential fit for the M388L's profile as assayed by protein C
activation. After 60 minutes the activity approaches 1% of the initial
activity (Figure 2.3, Table 2.1). The M388L's ability to cause protein C
57
Figure 2.3 The fraction of initial protein C activation versus time
curves for ten rats receiving 10, 20, 50, 100, or 200 micrograms of
thrombomodulin with leucine substituted for methionine at the 388th
position. Protein C activation before dosing is subtracted from protein
C activation after dosing and the differences are used in calculating the
fraction of initial protein C activation at each timepoint.
M388L bug
M388L bug
-CI-
M388L 2Oug
M388L 2Oug
M388L 5Oug
M388L 5Oug
M388L 10Oug
M388L 10Oug
M388L 100ug
M388L 200ug
17.
z .001
0
20
40
60
80
TIME (MINUTES)
100
120
140
58
Table 2.1 The fraction of initial protein C activation over time +
standard deviations for rats receiving thrombomodulin with leucine
substituted for methionine at the 388th position (M388L).
Time
(minutes)
0
200ug
n=1
10Oug
5Oug
2Oug
n=3
n=2
n=2
10ug
n=2
1.00
1.00
1.00
1.00
1.00
2
5
0.54
7
10
Dose Administered
0.096
15
0.91+0.33
0.78+0.09
0.71+0.08
0.72+0.12
0.65+0.11
0.60+0.09
0.54+0.00
0.65+0.09
0.51+0.16
0.40+0.04
0.36+0.02
0.37+0.04
0.24+0.19
0.33+0.01
0.28+0.04
0.21+0.07
0.28+0.07
0.30+0.09
0.16+0.07
0.17+0.11
20
0.033
0.11+0.06
0.15+0.01
0.16+0.09
0.05+0.00
30
0.015
0.03+0.02
0.04+0.02
0.04+0.00
0.05+0.00
45
0.008
0.01+0.01
0.02+0.02
0.03
0.05+0.00
60
0.006
0.01+0.00
0.01+0.01
0.01
0.05+0.00
59
Figure 2.4 Average fraction of initial radiolabel remaining from 1251
thrombomodulin analogue for low dose (0.2ug) , and high dose
administered. (n=4 for each dose)
(10Oug)
1
CO
LL
OJ
Z-
LOW DOSE
17:a
CC
HIGH DOSE
O0
- -I
.<
UCC
.1
0
10
20
30
TIME(MINUTES)
40
50
60
60
Table 2.2 Average fraction + standard deviations of initial radiolabel
remaining in the blood from 1251 thrombomodulin analogue (M388L)
for low dose and high dose.
Time
(minutes)
2
Dose Administered
High Dose (10Oug)
n=4
0.62+0.10
Low Dose (0.2ug)
n=4
0.42+0.08
5
0.32+0.03
0.21+0.03
10
0.16+0.03
0.12+0.02
20
0.15+0.01
0.15+0.01
30
0.15+0.01
0.14+0.01
45
0.14+0.01
0.12+0.01
60
0.13+0.01
0.12+0.01
61
Figure 2.5 Fraction of initial protein C activation CI remaining over
time and fraction of initial radiolabel remaining over time for a rat
which was administered 200 micrograms of thrombomodulin with
leucine substituted for methionine at the 388th position.
CI
.
l
i
20
40
-
protein C activation
radiolabel
1
1
60
80
TIME(MINUTES)
.
1
100
120
62
Table 2.3 Fraction of thrombomodulin analogue (M388L) remaining as
determined by 1251 radiolabel and activated protein C assay (APCA) for a
rat which received 200ug of thrombomodulin analogue with leucine
substituted for methionine at the 388th position.
Time
(minutes)
1251 Radio label
APCA
0
1.00
1.00
5
0.46
0.54
10
0.20
0.10
20
0.15
0.03
30
0.15
0.01
45
0.13
0.01
60
0.11
0.01
90
0.09
0.00
120
0.10
0.01
63
Table 2.4 Radioactivity (1251) half life times in minutes following low
dose (0.2ug, n=4) and high dose (10Oug, n=4) of thrombomodulin
analogue as determined by monoexponential and biexponential curve
fitting. Monoexponential half lives as determined by activated protein
C assay (APCA). The APCA half lives are the range. Standard
deviations are given in parentheses. Calculated using RSTRIP.
MONOEXPONENTIAL
(minutes)
1251
HIGH
LOW
APCA HIGH
LOW
Average
33(12)
39(10)
5.2
2.5
3.3(0.80)
BIEXPONENTIAL
(minutes)
2.1(0.48)
1.5(0.76)
700(140)
360(130)
*
*
*
*
64
activity fluctuates between 0.5% and 1% of the initial activation for
hours after the dose is administered. The standard deviation for most
points include the baseline however the difference between the protein
C activation and baseline is statistically significant (p<0.01) when
performing a rank sum test. For the radiolabelled low dose M388L, the
half life of the initial elimination phase was 1.5+0.76 minutes and the
half life of the terminal elimination phase was 360+130 minutes when
fitted to a biexponential curve (r2=0.987). For the radiolabelled high
dose TM analogue the initial elimination phase was 2.1+0.48 minutes
and the terminal elimination phase was 700+140 minutes when fitted
to a biexponential curve (r2=0.998). The radioactivity elimination half
lives vary greatly among rats, 220 to 530 minutes for low dose rats, and
410 to 870 minutes for high dose rats. The radioactivity is different at 2
minutes (p<0.01) and at 5 minutes (p<0.05) for low dose and high dose
radiolabelled M388L. There is no significant difference between low
dose and high dose pharmacokinetics of radioactivity for points after 60
minutes (p<0.5). The M388L half life as assayed by protein C activation
also increases slightly with increased TM analogue concentration
administered. This curve is best fit by a single exponential. The
elimination half life of TM analogue as assayed by protein C activation
is 2.5 minutes for 10ug administered and increases gradually with
increasing dose administered to 5.2 minutes for 10Oug with the average
65
r2 value equal to 0.979. These initial elimination phase half lives are
shorter than those reported by Erlich (3) who administered
thrombomodulin to rabbits. Erlich reported a 18 minute half life for
the radiolabel and 20 and 12 minutes half lives for the thrombin
clotting time and activated partial thromboplastin time respectively.
This discrepancy may be due to the different number of timepoints
sampled by Erlich, 5 and 30 minutes compared to 1, 2, 4, 5, 6, 10, 20, and
30 minutes sampled in this experiment. Alternatively this difference
may be due to use of rabbits instead of rats or a difference in the TM
used. Erlich administered 200ug/kg which would be equivalent to
6Oug for the rats used in this experiment. The elimination phase half
lives are approximately the same for M388L in rats (360 minutes) and
thrombomodulin in rabbits (385 minutes) when measuring radiolabel.
When measuring TM analogue by activation of protein C it is difficult
to detect a decrease in activity in rats after 60 minutes post dose.
The half life of mouse tissue thrombomodulin in mice is nine
minutes. The elimination in mice was largely due to hepatic clearance
as indicated by its initial distribution in the liver and then
accumulation in the intestine (14).
Figure 2.4 and Table 2.2 show that 80-90% of circulating
radiolabel in the blood has disappeared after 10 minutes. There is an
initial elimination phase with a one to two minute half life followed
66
by a long terminal elimination phase with a half live of 360 to 700
minutes for the radiolabel. The terminal elimination of low dose
M388L is the same as elimination of high dose M388L (the r squared
value increases when combining the two elimination phases) even
though the terminal elimination rates when calculated for individual
rats vary from 220 to 880 minutes. This variation in calculated
elimination half life is due to the low number of timepoints between
one and four hours and the lack of sampling after four hours.
Figure 2.6 and Table 2.5 show that peak bile concentration of
radiolabelled M388L occurs at 30-45 minutes. This time to peak is
consistent with vesicular mediated transcellular transport (VMTT) in
the liver (15). The observed rapid alpha elimination phase is partially
due to VMTT clearing the radiolabel on the first pass. This is also
consistent with the levels of radiolabel found in the liver (Figure 2.7).
The only non passive way for the liver to clear the radiolabel more
rapidly is by receptor mediated transport (13). Each point on the bile
curve represents the fraction of the initial dose released in bile per
hour. Only 30% of M388L radioactivity was accounted for in the blood,
bile, urine and tissues sampled after the first 60 minutes.
The weights of tissue collected relative to body weight are in
Table 2.6. The tissues collected include the liver, spleen, kidney, brain,
heart, lung, brain, and muscle. Approximately 10% of the radiolabel is
67
Figure 2.6 Fraction of radiolabel from 1251 labelled thrombomodulin
analogue which appears in the bile at low doses (0.2ug) 0, and high
doses (1 0Oug) O. Each point represents data from up to four rats.
0.02
0.01 -
0.00
0
1
1
30
60
1
1
120
150
1
90
TIME(MINUTES)
180
68
Table 2.5 Fraction of initial radiolabel from 1251 labelled
thrombomodulin analogue (M388L) which appears in the bile at high
doses (10Oug) and low doses (0.2ug). Each timepoint represents data
from up to four rats.
Time
(minutes)
Dose Administered
High Dose (10Oug) Low Dose (0.2ug)
5
0.0014+0.0002
0.0015+0.0005
15
0.0089+0.0013
0.0090+0.0053
25
0.0158+0.0025
0.0154+0.0027
37
0.0163+0.0025
0.0148+0.0018
52
0.0141+0.0027
0.0183+0.0108
75
0.0120+0.0012
0.0109+0.0002
97
0.0112+0.0009
112
0.0098+0.0004
127
0.0093+0.0002
142
0.0088+0.0000
157
0.0086+0.0000
172
0.0084+0.0005
69
Figure 2.7 Fraction of radiolabel from 1251 labelled thrombomodulin
analogue found in tissues following sacrifice of high dose rats (10Oug).
Four rats were sacrificed one hour apart at 1, 2, 3, or 4 hours.
0.05
-J
w
u-
0
0.04
co
-1
Z 0 0.03
0g
0
cc
cc
I
u- <
E
60 minute
M 120 minute
M 180 minute
ta
240 minute
0.02
0.01
0.00
liver kidney spleen heart
TIME(MI MUTES)
lung
brain
urine muscle
70
Table 2.6 Each tissue collected represents the following percent of total
body weight.
TISSUES COLLECTED
TISSUE
% BODY WEIGHT*
LIVER
KIDNEY
SPLEEN
HEART
LUNG
2.3 3.6
0.63 - 0.89
0.13 0.20
0.26 - 0.37
0.27 0.40
0.32 0.43
BRAIN
*tissue weight divided by total body weight of rat
71
recovered in the tissues, 5% is removed while sampling blood, less
than 10% is found in the bile, 1% is found in the urine, and 10%
remains in the blood after two to four hours. Nearly two thirds of the
radiolabel remains in the unsampled tissues of the rat. Results from
the tissues sampled over time are shown in Figure 2.7 and Table 2.7.
M388L is rapidly eliminated within the first ten minutes after IV
administration. Future research may enable TM analogues to become
clinically useful by modification of the molecule to increase the
duration of protein C activation.
72
Table 2.7 Fraction of radiolabel from 1251 labelled thrombomodulin
analogue (M388L) found in tissues following sacrifice of high dose rats
(10Oug). Four rats were sacrificed one hour apart at 1, 2, 3, and 4 hours.
Time of sacrifice
Tissue
1 hour
2 hours
3 hours
4 hours
liver
0.048
0.041
0.025
0.031
kidney
0.026
0.016
0.013
0.013
spleen
0.003
0.002
0.001
0.001
heart
0.002
0.002
0.001
0.001
lung
0.004
0.004
0.003
0.003
brain
0.001
0.001
0.000
0.000
urine
0.000
0.001
0.008
0.009
muscle
0.001
0.001
0.001
0.001
73
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Honda, G., and S. Yamamoto. Structure and expression of human
(1)
thrombomodulin, a thrombin receptor on endothelium acting as a
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Dittman, W., and P. Majerus. Structure and function of
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(2)
Erlich, H., Esmon, N., and N. Bang. In vivo behavior of
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(3)
injection into rabbits. T. Lab. Clin. Med. 115(2):182-189 (1990).
Zushi, M., Gomi, K., Yamamoto, Maruyama, I., Hayashi, T., and
(4)
K. Suzuki. The last three consecutive epidermal growth factor-like
structures of human thrombomodulin comprise the minimum
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264(18):10351-10353 (1989).
(5)
Bourin, M., Oh lin, A. K., Lane, D. A., Stenflo, J., and U. Lindahl.
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determinants on thrombin for fibrinogen and thrombomodulin. The
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Esposito, C., Gerlach, H., Brett, J. Stern, D., and H. Vlassara.
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(8)
Preissner, K. T., Koyama, T., Muller, D. Tschopp, J., and G.
Muller-Berghaus. Domain structure of the endothelial cell receptor
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Gomi, K., Zushi, M., Honda, G., Kawahara, S., Matsuzaki, 0.,
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Yada, I., Yuasa, H., Kusagawa, M., and K. Deguchi. The role of the
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protein C-thrombomodulin system in physiologic anticoagulation
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(12) Brody, J., N. Pickering, and G. Fink. Abnormalities of
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75
FORMULATION OF
AN ORAL ACETYLSALICYLIC ACID SUSPENSION
AND PHARMACOKINETICS OF
PARENTERAL THROMBOMODULIN ANALOGUES
CHAPTER 3
CONJUGATION OF THROMBOMODULIN ANALOGUE WITH
POLYETHYLENE GLYCOL TO INCREASE THE HALF LIFE IN THE
BLOOD
76
ABSTRACT
Thrombomodulin analogue activities were monitored by
measuring radiolabel 1251 and activation of protein C in the blood.
Thrombomodulin analogue exhibits a rapid initial elimination phase
of 1-2 minutes when following the radiolabel of 1251 labelled
thrombomodulin analogue, and 5-7 minutes when following protein
C activation.
The thrombomodulin analogue was conjugated to polyethylene
glycol (PEG). The PEG conjugation resulted in conjugates with three
different activities relative to the unconjugated thrombomodulin
analogue. The different change in activity may be due to PEG blocking
the active site of the thrombomodulin analogue. The difference in
activities between the thrombomodulin analogue conjugates may be
due to different amounts of PEG conjugation. Two PEG conjugates
with 50% and 70% of initial thrombomodulin analogue activity were
monitored using radiolabel. The conjugate with the larger decrease in
activity relative to unconjugated thrombomodulin analogue exhibited
the longer half life. For the PEG conjugate with 70% of initial
thrombomodulin activity, as determined by 1251, had a half life for the
initial elimination phase of 3.76+1.96 and the terminal elimination
77
phase half life was 90.7+28.0. For the PEG conjugate with 50% of initial
thrombomodulin activity, as determined by 1251, had a half life for the
initial elimination phase of 3.45+1.79 and the terminal elimination
phase half life was 139+33.2. The elimination of the conjugate with
50% of thrombomodulin analogue activity was best fit by a single
exponential with a half life of 60+1.1 and 80+6.2 minutes as
determined by protein C activation assay for the two doses
administered, 2Oug and 5Oug respectively.
78
INTRODUCTION
Polyethylene glycol conjugation has been used successfully to
increase the half lives of many proteins. The conjugation of PEG to
proteins has the additional benefit of decreasing the immunogenicity
of proteins however a decrease in the activity in many proteins is also
observed. Bovine liver catalase (1), superoxide dismutase, lactoferrin
and alpha macrogloblin (2), urate oxidase (3), adenosine deaminase (4),
interleukin 2 (5), alpha proteinase inhibitor (6), and superoxide
dismutase (7) have apparent increased plasma concentrations over
time when conjugated with polyethylene glycol compared to that of
the native protein. Decreased immunogenicity was demonstrated for
urate oxidase (3), adenosine deaminase (4), bovine liver catalase (1),
phenylalanine ammonia lyase (8), bovine liver arginase (9), bovine
serum albumin (10), and superoxide dismutase (7) when conjugated to
polyethylene glycol. Decreased activity was reported for urate oxidase
(3), interleukin 2 (5), and alpha proteinase inhibitor (6) when
conjugated with PEG. Sufficient activity remains with some PEG
conjugates so that the protein remains clinically effective (7,8).
A successful method of increasing half life of thrombomodulin
analogue was found by conjugation of polyethylene glycol (PEG) to the
analogue at lysine, tyrosine and the terminal amine positions. Two
79
different radiolabelled PEG linked thrombomodulin analogue (PEGM388L) samples with different unquantified numbers of PEG
attachment and quantified anticoagulant activity were tested. One PEG
conjugate was monitored through its activation of protein C.
The purpose of this research was to evaluate PEG-M388L
pharmacokinetics to detemine if clearance of M388L could be decreased
through conjugation with PEG. Sustained protein C activity in the
blood will increase the duration of fibrinolytic and anticoagulant
activity thereby decreasing thrombosis. By increasing the half life in
the blood of a M388L through conjugation with PEG a
thrombomodulin analogue may be found that is therapeutically more
useful than co-administration of both an anticoagulant and a
fibrinolytic agent.
80
MATERIALS AND METHODS
PREPARATION OF THROMBOMODULIN.
Human thrombomodulin (TM) analogue (M388L) with leucine
substituted for methionine at the 388th amino acid position was
prepared using an insect cell line to prevent oxidation and loss of
activity. Since the recombinant M388L was produced in insect cells,
glycosylation of the protein is different than native human
thrombomodulin. M388L is missing the lipophilic portion which is
found in native endothelial TM. M388L was analyzed by gel
electrophoresis to ensure that there were no contaminating
compounds.
PREPARATION OF THROMBOMODULIN CONJUGATES
M388L was conjugated to polyethylene glycol by incubation of
the TM analogue with paranitrophenol polyethylene glycol (Sigma,
4000MW). Two conjugates were used one conjugate had 50% of M388L
activity (PEG-M388L-50) and the other conjugate had 70% of M388L
activity (PEG-M388L-70).
RADIOIODINATION OF THROMBOMODULIN.
Thrombomodulin analogue was radiolabelled with 1-125 to give
81
a measurable specific activity of at least ten times background at
1/10,000th of the dose. Purity was determined by gel electrophoresis.
Pharmacological activity was measured by the ability of the
thrombomodulin in the sample to activate Protein C as determined by
the activated protein C assay described later.
ANIMAL EXPERIMENTS
Male Sprague Dawley Rats 280-300g, 65-75 days old, were
maintained on Wayne Rodent Blocks (Harlan Sprague Dawley). The
rats were anesthetized with an interperitoneal (IP) injection of sodium
pentobarbital, 6-7.5mg/100g body weight. Maintenance doses were
given, 1mg/100g body weight, as needed to insure full anesthesia
during all procedures. Euthanasia was by exsanguination without the
rat ever regaining consciousness. Surgery began each morning
between 9AM and 12PM. A polyethylene cannula (PE-10, Clay Adams)
was inserted into the bile duct. Another cannula (PE-50) was placed in
the right femoral artery. One hundred units of heparin were injected
into rats when protein C activation by M388L or PEG-M388L was not
measured. Non-radiolabelled blood samples of the M388L or PEG-
M388L were taken from animals without the use of heparin. A rapid
0.1m1 burst of saline into the cannula was usually all that was
necessary to clear the cannula. The cannula was gradually inserted
82
further into the artery to break clotting when necessary in
unheparinized rats where M388L or PEG-M388L was measured by
protein C activation. The rat was placed in a supine position on a
heating pad under a heating lamp to maintain 380C body temperature.
The M388L or PEG-M388L dose was administered through the arterial
cannula followed by 1m1 of 0.9% saline.
M388L or PEG-M388L were injected into four rats at a low dose
(0.2ug) or at a high dose (10Oug). The M388L or PEG-M388L was stored
in phosphate buffered saline with 0.1% bovine serum albumin. For
low dose samples, 0.2ug of radiolabelled M388L or PEG-M388L was
diluted with 0.5m1 0.9% saline immediately prior to injection. For
high dose an additional 10Oug of unlabeled M388L or PEG-M388L was
added immediately prior to injection. For samples where protein C
activity of M388L or PEG-M388L was measured, 100, 50, 20, or 10ug total
M388L or PEG-M388L was injected. For the protein C activation assay
(APCA) two rats were used for each of four doses. Bile was collected
continuously and blood was sampled at -1, 1, 2, 4, 5, 6, 10, 20, 30, 45, 60,
75, 90, 105, 120, 150, 180, 210, and 240 minutes for all doses. The
volume of blood sampled was replaced with 0.9% normal saline.
Upon completion of bile and blood collection the rat was
exsanguinated. One rat each was sacrificed at one, two, three, or four
hours of elapsed time for each M388L or PEG-M388L dosing. Urine and
83
tissue samples were collected upon completion of the 1-4 hour study
period. Liver and kidneys were weighed and portions of each were
collected for sampling. Spleen, heart, lung, brain, and urine were
collected in their entirety and a small portion of thigh muscle was
sampled. Sacrificing animals one hour apart allowed observation of
distribution of radiolabel in tissues over time.
ASSAY METHOD FOR RADIOLABELLED THROMBOMODULIN
1251 radioactivity in blood, bile, urine, and tissues were measured
using an automated gamma counter (Beckman Gamma 4000, 74.9%
efficiency). Each sample was counted for one minute. Counts per
minute for each sample were compared to counts per minute for the
initial M388L or PEG-M388L dose injected, after subtracting the
background in each case. This resulting fraction of the dose was then
corrected for the different weight of the tissue or fluid sample.
THROMBOMODULIN ACTIVITY (ACTIVATED PROTEIN C ASSAY)
Heparin was not administered to rats providing samples for the
activated protein C assay (APCA) because of heparin interference with
the measurement of thrombomodulin (TM) activity through the
APCA activation. Blood samples were diluted 1:20 in phosphate
buffered saline containing 0.5% bovine serum albumin and
84
centrifuged immediately following collection. Supernatant liquids of
blood and the entire bile samples were frozen in dry ice acetone and
packed in dry ice for later analysis. Activation of protein C by M388L or
PEG-M388L is measured according to the following APCA. Thrombin
and protein C are dissolved in 20mM Tris HCL, 0.1M NaC1, 2.5mM
CaC12, and BSA (5mg/m1) at pH 7.4. Twenty microliters each of
sample, thrombin, 3nM (Sigma), and protein C, 0.5uM (Genzyme) are
mixed and incubated for 60 minutes at 37°C. Hirudin, 20u1,
0.16units/m1 (American Diagnostica) is then added and the mixture is
incubated at 37°C for 15 minutes to stop the protein C activation.
Chromogenic substrate S2266,100u1, 1.0mM (Kabi Vitrum) is then
added and absorbance measured every sixty seconds at 405nm for 15
minutes. The change in absorbance per minute is proportional to the
concentration of activated protein C. The results are reported in
change in mOD per minute as well as the fraction of the change in
initial mOD per minute over time. Standard protein C activation rates
are determined from a single batch of insect cell line M388L
(64units /ml) manufactured on site.
CALCULATIONS
The number of micrograms of each dose administered was
determined by APCA. The activity was compared to the activity per
85
microgram of a single batch of insect cell line M388L. The fraction of
radioactivity or protein C activity at each timepoint is compared to the
activity of the dose injected. The activity of the dose injected is
confirmed by taking a small sample of blood from each rat prior to
dosing and mixing the blood with an amount of M388L or PEG-M388L
which is proportional to the entire dose administered in 20m1 of blood.
The activity of the single batch of insect cell line M388L in each rats
blood was not determined and so the amount of M388L PEG-M388L
remaining in the blood over time is not able to be calculated.
Background radioactivity and protein C activity for each rat's serum is
subtracted from the activity of that rat's samples. The parameters for
protein C activity pharmacokinetics of the thrombomodulin analogue
were calculated using PCNONLIN (Modified Newton-Gauss method).
86
RESULTS AND DISCUSSION
Paranitrophenol polyethylene glycol 4000 was conjugated to
M388L produced in insect cells. Conjugation of PEG to M388L may
occur with the lysine and tyrosine residues as well as the amino
terminus of proteins. The seven tyrosine residues found in soluble
thrombomodulin are located at the 288 and 296 positions of the second
loop, at the 334 and 337 position of the third loop, at the 358 and 368
position of the fourth loop and at the 413 position of the fifth loop.
There are no tyrosine residues in the first or sixth loop. Following
optimization of the procedure for conjugation of the PEG, 30-50% of
the M388L activity is lost. Treatment of the PEG conjugated M388L
with hydroxylamine to remove the PEG groups restored the
anticoagulant activity, demonstrating that no irreversible damage to
the active fragment occurred. The conjugate which retained 70%
activity (PEG-M388L-70) was used in radiolabel studies only. The
conjugate which retained 50% activity (PEG-M388L-50) was followed
both with 1251 radiolabel and activated protein C assay (APCA).
The half lives for M388L and PEG-M388L are listed in Table 3.1.
PEG-M388L-50 was administered in four replicates of two doses and the
M388L was administered in two replicates of four doses. The half lives
decrease with decreasing dose. Thrombomodulin activity may
87
Table 3.1 Half lives in minutes (T1/2) and areas under the curve in
mOD/minute versus minutes (AUC) as determined by protein C
activity for thrombomodulin analogue with methionine converted to
leucine at the 388th position and for the polyethylene glycol conjugate
of the thrombomodulin analogue. Standard deviations are in
parentheses. The parameters were calculated using PCNONLIN
(Modified Newton-Gauss Method).
M388L
PEGM388L-50
T1/2
10
7.21(0.242)
6.79(1.09)
5.68(0.294)
5.39(0.0241)
AUC*
200(8)
80(3)
40(3)
20(1)
50
20
82.5(6.22)
61.2(1.06)
4000(400)
2000(100)
DOSE
100ug
50
20
*based on mOD/min which is proportional to activity
88
measured by the activation of protein C through use of an APCA. The
mOD/minute values are proportional to activation of protein C.
Activated protein C cleaves a substrate used in the assay to cause a
change in absorbance which provides mOD/minute values which are
proportional to the concentration of activated protein C. Figure 3.1
shows the average values at each time point for each dose of M388L
and PEG-M388L administered. Table 3.2 shows the average values at
each timepoint for each dose of PEG-M388L and Table 2.1 shows the
average values at each timepoint for each dose of M388L.
Figure 3.2 shows the fraction of the initial radiolabel remaining
in the blood over time for 1251 radiolabelled M388L and PEG-M388L.
The parameters calculated from radiolabelled M388L and PEG-M388L
are shown in Table 3.3. The data used to calculate the parameters in
Table 3.3 are given in Table 3.4 and Table 2.2. The radiolabel in the
blood over time curve is best fit by a biexponential equation. The
terminal elimination phase of the biexponential equation does not
represent active M388L in the case of unconjugated M388L as shown by
the APCA. This terminal elimination phase levels off at
approximately 10% of the initial injected radiolabel. The terminal
elimination phase of the PEG-M388L remains above 50% of the initial
radiolabel injected for over four hours. This terminal elimination
phase is reflected in the APCAs.
89
Figure 3.1 Fraction of initial protein C activation remaining over time
for four doses of thrombomodulin analogue (n=2) and two different
doses of PEG conjugate analogue (n=4). Thrombomodulin analogue is
administered at 10ug 0, 2Oug , 5Oug II, and 10Oug 0. PEG conjugated
thrombomodulin analogue is administered at 2Oug , and 5Oug A.
100
PEG 2OUG
PEG 5OUG
M388L 1OUG
M388L 2OUG
M388L 5OUG
M388L 100UG
.01
0
20
40
60
TIME (MINUTES)
80
90
Table 3.2 The fraction of initial protein C activation over time +
standard deviations for rats receiving thrombomodulin with leucine
substituted for methionine at the 388th (M388L) position conjugated
with PEG. The conjugate retained 50% of the initial M388L activity.
(n=4)
Time
(minutes)
Dose Administered
1
5Oug
0.910+0.127
2Oug
0.809+0.128
2
0.807+0.121
0.948+0.073
4
0.627+0.280
0.784+0.205
5
0.575+0.208
0.853+0.202
6
0.723+0.179
0.711+0.370
10
0.742+0.211
1.007+0.417
20
0.677+0.231
0.784+0.345
30
0.758+0.416
0.558+0.218
45
0.571+0.132
0.438+0.184
60
0.468+0.141
0.498+0.232
75
0.458+0.096
0.342+0.191
90
0.388+0.223
0.312+0.222
105
0.457+0.110
0.369+0.130
120
0.308+0.158
0.259+0.071
91
Figure 3.2 Average fraction of initial radiolabel remaining from 1251
thrombomodulin analogue for low dose (0.2ug) and high dose
administered, and PEG conjugated thrombomodulin
(1 0Oug)
analogue with 50% of initial activity 0 and 70% of initial activity a
(n=4 for each dose)
10?
0 peg#1
0 peg#2
111 LOW DOSE
HIGH DOSE
.01
0
I
I
30
60
f"""-120 150 180 210 240
1
90
time (minutes)
1
1
92
Table 3.3 Half lives in minutes (T1/2), areas under the curve in
mOD/minute versus minutes (AUC), and mean residence times in
minutes (MRT) are calculated for each rat using RSTRIP.
Radio labelled low (0.2ug) and high (10Oug) doses of thrombomodulin
analogue, and radiolabelled PEG conjugates of the thrombomodulin
analogue are depicted. All values are calculated based on the amount
of 125I radiolabel remaining over time. Standard deviations for the
average values are given in parentheses.
PARAMETERS CALCULATED USING RSTRIP (biexponential)
PEG
50%
activity*
Average
PEG
70%
activity*
Average
M388L
LOW
Average
M388L
HIGH
Average
T1(1 /2)
T2(1/2)
AUC
MRT
2.59
5.85
1.69
3.66
3.45(1.79)
97.7
140
138
100
200
100
200
100
200
200
300
0.737
58.4
78.2
135
91.4
90.7(28.0)
60
60
80
60
70
100
200
100
3.61
6.08
4.63
3.76(1.96)
179
139(33.2)
0.735
1.24
1.25
2.55
1.5(0.76)
2.80
1.84
1.75
2.02
2.1(0.48)
* relative to unconjugated thrombomodulin analogue
93
Table 3.4 Average fraction + standard deviations of initial radiolabel
remaining in the blood from 1251 thrombomodulin analogue (M388L)
conjugated to PEG. One conjugate had 50% of M388L activity (PEGM388L-50) and the other conjugate had 70% of M388L activity (PEGM388L-70).
Time
(minutes)
PEG-M388L-50
n=4
PEG-M388L-70
n=4.
2
0.957+0.087
1.254+0.710
5
0.841+0.064
0.676+0.043
10
0.756+0.056
0.524+0.031
20
0.712+0.039
0.449+0.039
30
0.647+0.038
0.381+0.028
45
0.592+0.041
0.327+0.023
60
0.543+0.034
0.290+0.028
90
0.480+0.035
0.224+0.030
120
0.416+0.042
0.190+0.024
180
0.358+0.040
0.149+0.047
240
0.335
0.099
94
The area under the curves (AUC) are measured in
mOD/minute versus minute rather than concentration versus time as
determined by protein C activation assay. For both M388L and PEG-
M388L, AUC are linear with respect to dose. This indicates that linear
pharmacokinetics are occurring for the doses administered. The AUC
data as determined by APCA for the PEG-M388L are higher for
conjugates which exhibit lower activity per unit mass relative to the
M388L. The decrease in activity is more than offset by the increase in
half life in the blood as shown by Figure 3.1. Thus a 2Oug or 5Oug dose
of PEG-M388L maintained higher amounts of protein C activation, as
measured by APCA, over time than the respective doses of
unconjugated M388L. AUC is calculated for 1251 radiolabel which
reflects concentration in terms of the fraction of the radiolabel
remaining. The values obtained for the AUC indicate that linear
pharmacokinetics are occurring for the doses administered and that
higher values are obtained for the conjugated TM analogue.
Conjugation of TM analogue with polyethylene glycol
increases the half life of TM analogue in the blood of rats. Further
research is necessary to find the amount of conjugation which
increases the half life and does not decrease the activity.
95
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APPENDIX
104
APPENDIX
In early experiments radiolabelling thrombomodulin (TM)
analogue resulted in some of the BSA in the sample also being
radiolabelled. Radio labelled BSA content was detected, but not
quantified using gel electrophoresis. In these cases radiolabelled BSA
present in each of the TM analogue samples was calculated based on
radioactivity in the blood 90 minutes post dosing. It is assumed that no
radiolabel from the TM analogue remains in the blood 90 minutes
after dosing. The radioactivity contributed by BSA in early
experiments was subtracted assuming linear pharmacokinetics and no
1251 TM analogue remaining after 90 minutes. The resulting curve,
Figure 2.6, gives a good approximation of the alpha distribution phase
of pure TM analogue but the beta elimination phase is not accurate
because the assumption that no radiolabel from TM analogue remains
after 90 minutes is false. The low dose elimination curve gives
negative values after 2 minutes due to error in estimation of the
radiolabelled BSA in the dose. The adequacy of this method was
determined by administering TM analogue without the presence of
radiolabelled BSA as determined by gel electrophoresis.
The calculated pharmacokinetic curves of TM analogue when
administered with radiolabelled BSA are similar to that for pure TM
analogue (Figure 2.6). The underestimation in calculated values is due
to the 10% radiolabel that remains in the pure TM curve.
CORRECTION PROCEDURE
Radio labelled thrombomodulin containing radiolabelled BSA and
pure radiolabelled BSA samples were injected according to the same
protocol as the radiolabelled thrombomodulin to determine the
elimination rates of thrombomodulin with BSA versus pure BSA in
rats. This was necessary to determine how radiolabelled BSA affects
the radiolabel versus time curves for radiolabelled thrombomodulin.
105
To correct for bovine serum albumin interference the following
procedure is used:
Assumptions
BSA is assumed to be eliminated at a rate independent of dose. There
is a systematic error if there is thrombomodulin (TM) analogue
remaining after 60 minutes since that TM analogue is assumed to be
BSA. Therefore if the half life of the TM analogue is too long then it
appears that the half life of the TM analogue is shorter than it actually
is. The half life of the TM analogue is at least as long as the calculated
half life found by this correction method.
The fraction of administered sample in the blood was calculated as
described above for all rats. Calculations are performed on the average
data from four rats for each of the doses administered. Data from the
low dose M388L, high dose M388L, rats were individually analyzed as
follows:
Step1
The value at each timepoint (fraction of dose administered in 20m1 of
blood based on average counts per minute data) was divided by the
value at the corresponding timepoint for the 100% BSA.
Step 2
The resulting fraction did not become constant until after ninety
minutes. The average of the resulting fractions between ninety and
two hundred and forty minutes was calculated (average of four points).
This average fraction is the fraction of BSA in the data set of interest.
Step 3
The timepoints in the 100% BSA data set are multiplied by the fraction
of BSA in the data point of interest and then these numbers are
subtracted from the corresponding time points in the data set to be
analyzed. This resulting value is the fraction of total radioactivity at a
particular timepoint which is attributable only to thrombomodulin
analogue.
Step 4
The fraction of BSA is subtracted from one (1.0) to give the fraction of
thrombomodulin in the sample.
Step 5
The fraction of initial thrombomodulin which this represents is then
calculated as follows. The fraction of radioactivity attributable to
106
thrombomodulin analogue is divided by the fraction of
thrombomodulin analogue in the sample.
107
Figure A.1 The fraction of initial radiolabel administered remaining
over time for radiolabelled Bovine Serum Albumin (BSA) -0, low dose
radiolabelled thrombomodulin analogue with radiolabelled BSA ,
and high dose radiolabelled thrombomodulin analogue with
radiolabelled BSA M. These are how the curves appear before step 1 of
the mathematical manipulation.
0.5
w 0.4
u.
,..,
0.3 -
-0- BSA
Oa<
<
LL
LOW DOSE
HIGH DOSE
0.2
C.)
<
i=
2. 0.1
0.0
0
30
60
90
120 150 180 210 240
TIME (MINUTES)
108
Figure A.2 This figure shows the result of step #1 when the radiolabel
vs. time curves shown in figure A.1 are used to determine the fraction
of radiolabelled bovine serum albumen (BSA) in each of the two
thrombomodulin analogue BSA mixtures. The mixture curves are
divided by the BSA curve and the average of the points in the boxed
area are used to calculate the percent of BSA in each of the two
mixtures. Step 2 is performed based on these curves.
1.0
-0- LOW DOSE/BSA
0.8
HIGH DOSE/BSA
0.6
Z
0.4
<
E
0.2
0
H
0
0
100
200
TIME (MINUTES)
300
109
Figure A.3 The radiolabelled bovine serum albumen (BSA) in the
high dose thrombomodulin analogue BSA mixture is calculated to be
33.7%. So the BSA curve is multiplied by 0.337 and this resulting curve
is subtracted from the thrombomodulin analogue BSA mixture to
obtain the radiolabel attributable to the thrombomodulin analogue
only 0. Step 3 is depicted in this figure.
0.35
-J
U.
CO
O -J 0.25 -
go--
HIGH DOSE
0---
BSA*0.337
HIGH DOSE - (BSA*0.337)
Z 0
O0
17:
O CC 0.15
cc -I
LI-
I
0.05 -
-0.05
0
30
60
90
III
1.
120 1 50 180 2 1 0 240
TIME (MINUTES)
110
Figure A.4 The radiolabelled bovine serum albumen (BSA) in the low
dose thrombomodulin analogue BSA mixture is calculated to be 48.9%.
So the BSA curve is multiplied by 0.489 and this resulting curve is
subtracted from the thrombomodulin analogue BSA mixture to
obtain the radiolabel attributable to the thrombomodulin analogue
only O. Step 3 is depicted in this figure.
-0.05
I
0
30
60
I
90
I
120 150 180 210 240
TIME (MINUTES)
111
Figure A.5 These curves represent the fraction of radiolabel which is
attributable only to thrombomodulin analogue relative to the initial
amount of radiolabel injected. These curves are found as the
calculated difference in figure 3a and 3b.
0.3
LOW DOSE
HIGH DOSE
m 0.2 LL <
O -I
z2
O e
P.
0.1 -
0 cc
cc - -I
<
F.:
0.0 --
-0.1
0
30
TIME (MINUTES)
6'0
90
112
Figure A.6 The amount of radiolabel remaining due to
thrombomodulin analogue relative to the initial radiolabel injected
into the rat in the form of thrombomodulin analogue. Step 5 is
depicted in this figure.
w
xI-
0.4
I'Ll
D
0 (.5
...1 I.4
P
0
_1
0.3
IP-- LOW DOSE
HIGH DOSE
U9 Z4C
Z c.)
=<
u_ < z
O
rn
O
_1
za0
I= LLI
0
2
o co
C
< co
cc
400
3 CC
4CCX
I--
0.2
0.1
0.0 -0.1
.
0
30
TIME (MINUTES)
60
90
113
Figure A.7 High dose (10Oug) and low dose (0.2ug) pharmacokinetics of
thrombomodulin analogue in rats. The high dose and Iciw dose
administered as pure thrombomodulin samples is compared to the
mathematically derived curves from the high dose and low dose 0
samples administered with radiolabelled BSA. The fraction of
radiolabel in the low dose sample which is attributable to BSA is 49%
and the fraction of radiolabel in the high dose sample which is
attributable to BSA is 34%.
0.8
low dose
high dose
co
w 0.6 u. <
O1
zo 2a
low dose containing BSA
high dose containing BSA
0.4 -
cc<
szt
cc -1
u.
0.2 -
z
I
0.0
10
I
I
20
30
TIME(MINUTES)
4
40
50
60