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Disposition kinetics of amphotericin B in rats
Wu, Yuhong, M.S.
The University of Arizona, 1993
UMI
300 N. Zeeb Rd.
Ann Arbor, MI 48106
DISPOSITION KINETICS OF AMPHOTERICIN B IN RATS
by
YUHONG WU
A Thesis Submitted to the Faculty of the
DEPARTMENT OF PHARMACEUTICAL SCIENCES
In Partial Fulfillment of the Requirement
For the Degree of
MASTER OF SCIENCE
In the Graduate College
THE UNIVERSITY OF ARIZONA
19 9 3
2
STATEMENT BY AUTHOR
This thesis has been submitted in partial fulfillment of requirements for an
advanced degree at The University of Arizona and is deposited in the University
Library to be made available to borrowers under rules of the library.
Brief quotations from this thesis are allowable without special permission,
provided that accurate acknowledgement of source is made. Requests for permission
for extended quotation from or reproduction of this manuscript in whole or in part
may be granted by the head of the major department or the Dean of the Graduate
College when in his or her judgment the proposed use of the material is in the
interests of scholarship. In all other instances, however, permission must be obtained
from the author.
SIGNED:.
APPROVAL BY THESIS DIRECTOR
This thesis has been approved on the date shown below:
3-U-fS
Sherry Chow, Ph.D.
Assistant Professor of Pharmaceutical Sciences
Date
3
TO MY DEAR HUSBAND
ZHENGWEI ZHAO
4
ACKNOWLEDGEMENTS
The author wishes to express her sincere gratitude to her advisor, Dr. M.
Mayersohn, for providing guidance, inspiration, and continued support and
encouragement during the course of this research. His honest devotion, constructive
criticism, effective discussions and suggestions greatly helped the author to
accomplish this project.
The author
expresses
her sincere gratitude for
the advice,
help,
encouragement, and friendship of Dr. H. Chow.
Sincere appreciation is also due to Dr. S. H. Yalkowsky for serving as a
member of committee.
In addition, the author wish to sincerely thank all the individuals in the
pharmacokinetic group.
The author takes this chance to thank her parents for their love, support and
guidance though these years.
Also, this work would not have been possible without the encouragement,
understanding and overwhelming support from her dear husband.
5
TABLE OF CONTENTS
page
LIST OF ILLUSTRATIONS
8
LIST OF TABLE
10
ABSTRACT
11
CHAPTER
1.
2.
STATEMENT OF PROBLEM
12
1.1
Statement of Problem
13
1.2
Objectives
17
1.3
Organization
17
LITERATURE REVIEW
19
2.1
Chemistry
19
2.2
Stability
21
2.3
Pharmacology
22
2.3.1
Mechanisms of Action
22
2.3.2
Oxidation-Dependent Mechanism
23
2.4
2.5
Toxicity
24
2.4.1
Nephrotoxicity
25
2.4.2
Prevention of Toxicity
28
2.4.3
Liposomes
29
Pharmacokinetics
31
2.5.1
Human Studies
31
2.5.2
Animal Studies
35
6
TABLE OF CONTENTS
continued
page
2.6
3.
35
EXPERIMENTAL SECTION
39
3.1
Materials and Instruments
39
3.2
Pharmacokinetic Studies
41
3.2.1
Catheter
41
3.2.2
Animal Surgery
42
3.2.3
Pharmacokinetic Studies
43
3.3
Sampling
44
3.4
Preparation of Intravenous Solution
45
3.5
Sample Preparation
46
3.6
AmB Assay by HPLC
46
3.7
Calculations
47
3.8
Creatinine
50
3.8.1
Creatinine Assay
50
3.8.2
Creatinine Clearance
51
3.9
4.
Analysis Methods
Data Analysis
52
3.9.1
Model-Independent Method
52
3.9.2
Model-Dependent Method
57
3.9.3
Statistical Analysis
58
RESULTS
61
4.1
Analytical Method
61
4.2
Stability of AmB in Urine
62
7
TABLE OF CONTENTS.....continued
page
4.3 Pharmacokinetic Data
5. DISCUSSION
65
92
5.1
Discussion
92
5.2
Conclusions
98
5.3
Suggestions for Further Work
98
APPENDIX A
APPENDIX B
APPENDIX C
REFERENCE
Serum and Urine AmB Concentration
versus Time
100
Plot of Serum AmB Concentration
versus Time
108
Fitted Curves Generated by ADAPT II
116
124
8
LIST OF ILLUSTRATIONS
page
Figure 1.
Chemical Structure of AmB, Cholesterol and
Ergosterol
20
Figure 2.
Mechanism of Nephrotoxicity
27
Figure 3.
Three Compartment Model
33
Figure 4.
UV Absorption Spectrum of AmB
48
Figure 5.
Plot of Standard Curve of AmB in Serum
49
Figure 6.
The Plot of Relationship between Urine
Excretion Rate and Time
54
Figure 7.
Two Compartment Model Describing AmB
60
Figure 8.
Representative Chromatograms of AmB and
Internal Standard in Serum
63
Plot of Mean AmB Serum Concentration versus
Time for High Dose
76
Figure 10. Plot of Mean AmB Serum Concentration versus
Time for Medium Dose
77
Figure 11. Plot of Mean AmB Serum Concentration versus
Time for Low dose
78
Figure 12. Plot of Mean Steady-State Concentration
versus Dose
79
Figure 13. Plot of Half-life versus Dose
80
Figure 9.
Figure 14. Plot of Volume of Distribution versus Dose
..
81
Figure 15. Plot of Systemic Clearance versus Dose
82
Figure 16. Plot of Renal Clearance versus Dose
83
Figure 17. Plot of Renal Clearance versus AmB Serum
Concentration
84
9
LIST OF ILLUSTRATIONS
continued
page
Figure 18. Plot of Renal Clearance versus AmB
Steady-state Concentration
85
Figure 19. Plot of AmB Renal Clearance versus Time in
the Low, Medium, and High Dose Groups
86
Figure 20. Plot of Fraction of the Dose of AmB Excreted
into the Urine versus Dose
87
Figure 21. Plot of the Individual Creatinine Clearance
before and after AmB Treatment in the Low,
Medium and High Dose Groups
88
Figure 22. Plot of Comparison of Creatinine Clearance
before and after AmB dosing
89
Figure 23. Plot of Renal Clearance versus Creatinine
Clearance
90
Figure 24. Plot of Renal Clearance versus Urine Flow
...
91
10
LIST OF TABLES
page
TABLE 1.
TABLE 2.
Factors Responsible for the Increased
Prevalence of Fungal Infections
15
The Major Invasive Deep Fungal Infections
in Different Immunocompromised Patients
16
TABLE 3.
Pharmacokinetic Parameters of AmB in Humans
..
TABLE 4.
HPLC Methods Used to Analyze AmB
38
TABLE 5.
Intra-Day Assay Results of AmB
64
TABLE 6.
Inter-Day Assay Results of AmB
64
TABLE 7.
Stability of AmB in Urine
66
TABLE 8.
Summary of Pharmacokinetic Parameters
72
TABLE 9.
Creatinine Clearance
74
TABLE 10. Summary of Pharmacokinetic Parameters (ModelDependent Method)
34
75
11
ABSTRACT
Amphotericin B (AmB) is widely used for the treatment of systemic infections.
The current therapeutic regimens for this drug are complex because of limited
pharmacokinetic information. Male Sprague-Dawley rats were randomly divided into
three different dose groups, receiving 0.332, 0.838, 1.323 mg/kg of AmB. A two-step
infusion procedure was employed to rapidly achieve steady-state conditions.
Serum samples and urine samples were collected and assayed by HPLC. Halflife, volume of distribution (Varea) and systemic clearance (CLs) are (± SD): 953.2
± 300.6 min, 5.14 ± 1.53 L/kg and 3.72 ± 0.8 ml/min*kg, respectively. There are
no significant differences for any parameter as a function of dose. However, renal
clearance
decreases when the total infused dose increases.
We conclude that
volume of distribution and systemic clearance are linear over the dose range studied.
The dose-dependent renal clearance may be due to either the nephrotoxicity
associated with AmB or saturable secretion.
12
CHAPTER 1
STATEMENT OF PROBLEM
Amphotericin B (AmB), a polyene antibiotic, is a natural fermention product
produced by Streptomyces nodous ( Trejo et al., 1963) which was originally isolated
from soil from the Orinoco River region in Venezuela in 1953. Streptomyces nodous
co-produces two polyene antibiotics, the tetraene amphotericin A (AmA) and the
hapatene amphotericin B. Both antibiotics were found to possess a broad spectrum
of antifungal action (Steinberg et al., 1956), but AmA was not further developed.
AmB remains the drug of choice for most forms of deep-seated fungal
infections, despite its serious side effects and the introduction of newer anti-fungal
compounds.
Diseases caused by fungi are called mycoses or fungal infections, which range
from very common mild chronic infections affecting the skin, via deep cutaneous or
subcutaneous infections, to acute or chronic systemic infections. Systemic fungal
13
infections may occur in normal hosts, however, in immunocompromised or
physiologically compromised patients, these infections are a major cause of morbidity
and mortality (Gold et al., 1984; Bodey et al., 1984). For example, aspergillosis and
candidiasis are among the leading causes of deaths from infections in patients with
leukemia. A high incidence of fungal infections also occurs in people who receive
intensive chemotherapy, broad-spectrum antibiotics and have potential defects in
neutrophil functions (Table 1 and Table 2).
1.1 Statement of the Problem
The incidence of fungal infections has increased dramatically during the last
decade. Deep-seated mycoses are beginning to create serious problems for clinicians
working with certain populations of patients, which include patients that have cancer
or are immunocompromised or physiologically compromised. There have been a
number of recent surveys which provide an impression of the importance of fungal
infections in severely ill patients. For example, disseminated candidiasis is rapidly
becoming a major threat to cancer patients, especially those with hematologic
malignancies (Bodey et al., 1984). Recent data have indicated that over a 4-year
period, 11.9% of patients with acute leukemia suffered from fungal infections caused
by Candida sp. and torulopsis glabrata (Maksymiuk et al., 1984).
The need for effective antifungal therapies has been felt more and more acutely
with the emergence of the acquired immune deficiency syndrome (AIDS) and AIDS-
14
related complex (ARC), which are often associated with systemic fungal infections.
Recent data from the Centers for Disease Control reveal that between 100,000 and
200,000 Americans meet the criteria for AIDS and ARC, and that between 1 and 2
million healthy Americans have already been infected.
Of patients showing
antibodies to the human immune deficiency virus, according to estimates of the
Centers for Disease Control, between 5% and 25% will progress to AIDS and 25%
will develop one of the ARC diseases. As expected, a significant number of these
cases will be associated with various fungal infections.
One yeast-like fungus,
Cryptococcus neoformans is reported to be the fourth most common cause of lifethreatening infections in AIDS patients (Kovacs et al., 1985). Data from the Centers
for Disease Control for 3170 AIDS patients registered at the Centers between May
1983 and June 1984 indicate that a total of 58% of the reported cases of AIDS have
been associated with various mycoses (Chandler et al., 1985).
The main antifungal drugs available now belong to two families, the polyene
and azole groups. Although there are a number of antifungal drugs available, which
are used for the treatment of serious fungal infections, most of them have drawbacks
such as efficacy, adverse effects, and the development of resistance. AmB remains
the drug of choice for the treatment of systemic fungal infections (Graybill et al.
1983). Usage of this drug has become more prevalent during the past decade as a
result of a dramatic increase in the number of patients having defective immune
response.
The agent has been in clinical use for over 30 years,
yet the
15
Table 1
Factors Responsible for the Increased Prevalence
of Fungal Infections
AIDS and longer surviving AIDS patients
More aggressive cancer chemotherapy
Increase in transplantation operations
More aggressive intensive care medicine
New and use of more prosthetic devices
Broader spectrum antibiotic usage
Greater travel and exposure to foci of endemic mycoses
Increase in intravenous drug abuse
More parenteral nutrition and use of lipid suspension
Table 2
The Major Invasive Deep Fungal Infections in Different
Immunocompromised Patients
Candida
Aspergillus
neutropenia
neutropenia
AIDS
AIDS
lymphoma
lymphoma
infants
burns
transplantation
transplantation
corticos­
neonates
burns
corticosteroids
teroids
diabetes
corticos­
sarcoidosis
corticos­
teroids
teroids
chronic granu­
drug abuse
lomatous disease
TPN
a:
a
total parenteral nutrition
Cryptococcus
Histoplasma
17
of AmB remain poorly understood. The current therapeutic dosage regimens of
AmB are based on years of empirical evidence and anecdotal experience.
Furthermore, the use of AmB, is often limited by its acute and chronic toxic effects
such as renal impairment, hypokalemia and anemia (Butler et al.,1966). Because of
these adverse effects,
different clinicians attest to a variety of administration
methods and accompanying premedication in order to minimize or prevent the acute
reactions to AmB. Understanding the disposition kinetics and predicting toxicity as
a result of dosing becomes a particularly important issue in optimizing the
therapeutic use of this antifungal drug.
1.2 Objectives of Research
The major objectives of this thesis research are to:
(1) develop analytical methods in order to quantitate AmB in different
biological fluid samples.
(2) characterize the disposition kinetics of AmB and examine its association
with nephrotoxicity after administration of different doses of AmB to rats.
1.3 Organization of Thesis
This thesis is presented in five chapters.
The first chapter is a brief
introduction to this study. The second chapter gives a background knowledge and
a brief review of the literature of AmB stability, mechanisms of action, toxicity and
18
pharmacokinetics. Chapter three describes the experiments which were conducted.
Chapter four contains the results. Chapter five provides the discussion, conclusions
and recommendations for further research.
19
CHAPTER 2
LITERATURE REVIEW
2.1 Chemistry
The chemical structure of AmB was elucidated in 1970 and is shown in Figure
1 (Mechlinski et al.,1970). The molecule consists of a large macrolide lactone ring
with 37 carbon atoms. One side of the macrolide ring is composed of a rigid
lipophilic chain of seven conjugated double bonds and on the opposite side of the
ring there is a similar number of hydroxyl groups. The molecule is amphipathic and
this feature of its structure is believed to be important in its biological mechanisms
of action. The macrolide ring also contains a six-membered ketalic ring and the
amino sugar mycosamine is bonded to the ring through an a -glycosidic linkage. Two
functional groups,
carboxylic acid and amino,
form the basis for chemical
AMPHOTERICIN B
OH
OH
O
COOH
NH2
CHOLESTEROL
ERGOSTEROL
Figure 1. Chemical structure of amphotericin
B, cholesterol and ergosterol.
21
modification of AmB.
Crystalline AmB is insoluble in water
and
most solvents except
dimethylsulfoxide (DMSO). The commercial product of AmB, Fungizone, is a
mixture of AmB, deoxycholate and sodium phosphate buffer.
The detergent
deoxycholate produces an aqueous micellar suspension rather than a solution of the
drug for parenteral administration.
When diluted in a 5% glucose injection,
Fungizone forms a colloidal dispersion suitable for intravenous injection. However,
the presence of salts such as sodium or potassium chloride or acidic pH leads to
aggregation of the colloid (Barrriere, 1990).
2.2 Stability
AmB has an extended unsaturated chromophore and the presence of a variety
of functional groups which make it sensitive to diverse chemical and physical attack.
When exposed to extremes of pH, light and heat, AmB exhibits poor stability and
reduction of bioactivity (Waksman et al., 1965). Products resulting from degradation
are usually biologically inactive.
AmB was very stable ( < 5% loss) in human serum at -20°C for up to 14 days,
with only a 10% loss of drug after 6 months. But loss of AmB exceeded 60% after
six months in DMSO solution (Edmonds et al, 1989).
AmB is administered intravenously to patients due to its poor gastrointestinal
absorption. Kintzel studied the stability of AmB and found that in 5% dextrose
22
solution it was stable at concentrations of 0.92,1.2 and 1.4 mg/ml when stored at 6°C
and 25°C for up to 36 hours (Kintzel et al., 1991). No precipitation, turbidity, gas
formation or color change was observed.
2.3 Pharmacology
AmB shows a high order of in vitro activity against many species of fungi.
Histoplasma capsulatum, Coccidiodes immitis, Candida species, Blastomycse
dermatiaidis, Cryptococcus neoformans, Aspergillus fumigatus have been shown to be
inhibited in vitro by concentrations of AmB ranging from 0.03 to 1.0 mcg/ml (
Shadomy et al. 1978). AmB has no effect on bacteria and viruses.
2.3.1 Mechanisms of Action
The major mechanism of action of AmB is attributed to its ability to bind to
the fungal cells. AmB can bind more strongly to ergosterol, the main component of
the fungal cell membrane, than to cholesterol, the main component of the
mammalian cell membrane.
This binding results in an increase in membrane
permeability that produces loss of protons and cations from the cell. The clinical
usefulness of AmB is due to its binding to ergosterol.
However, its toxicity is
associated with its binding to cholesterol.
The binding mechanism is based on the AmB structure. There are two types
of interactions between AmB and ergosterol or cholesterol, specific binding and
23
nonspecific binding (Herve et al., 1989). The hydrogen bonds, specific binding, are
formed between hydroxyl groups of the sterol and the carboxyl groups of the AmB
molecule. This binding is strengthened by participation of the amino group of the
amino sugar side chain of the AmB molecule (see Figure 1) (Brajtburge et al., 1990).
The second type of interaction, nonspecific binding, involves the rigid chain of the
seven conjugated double bonds of AmB and the whole sterol molecule (see Figure
2) (Herve et al., 1989).
Herve and coworkers also conclude that the flat shape of
most ergosterol conformers having the double bond located at C-22 of the alkyl side
chain was responsible for the greater sensitivity to AmB of ergosterol- containing
membranes compared with the sensitivity to AmB of cholesterol-containing
membranes.
2.3.2 Oxidation-Dependent Mechanism
The action of AmB on fungal cells is believed to involve more than one
mechanism (Brajtburg et al, 1990). It has been reported that the lethal effects of
higher concentrations of AmB on Candida albicans are not a simple consequence of
its membrane-permeabilizing effects, but involves oxidative damage to the cell
(Sokol-Anderson et al., 1986 a,b).
AmB induces a cascade of oxidative reactions and causes the formation of free
radicals during its auto-oxidation process (Lamy-Freund et al., 1985). These radicals
may have several different effects on cells, such as stimulating the immune system.
24
AmB markedly augmented the polymorphonuclear leukocyte immunoglobulin Gmediated ingestion of opsonized sheep erythrocytes; this effect was inhibited by
superoxide dismutase (Gresham et al., 1988). The experiments done by Brajtburg
indicate that hemolysis of erythrocytes and cell death may be inhibited by
extracellular catalase and potentiated in the presence of ascorbic acid and other
prooxidants (Brajtburg et al., 1989).
In contrast, extracellular scavengers and
prooxidants did not affect prelytic or prelethel AmB- induced K+ leakage from cells.
This type of damage by AmB is not linked to cell membrane permeability and is not
dependent on the kind of sterol.
2.4 Toxicity
Despite the development of alternative antifungal agents, AmB remains the
primary drug for the treatment of life-threatening fungal infections. However, the
usage of AmB is limited by its side effects, acute and chronic toxicities. Infusionrelated acute toxicities have included fever, chills, nausea, vomiting, anorexia,
headache, bronchospasm, hypotension and anaphylaxis.
Chronic toxicities are
nephrotoxicity, bone marrow suppression and renal losses of potassium and
magnesium. Clements investigated 50 patients receiving I.V. infusion of AmB with
a mean cumulative dose of 582 mg. Acute toxicities accompanying infusion occurred
in 66% of all patients. Nephrotoxicity occurred in 60% of patients and baseline
serum creatinine values in these patients rose by a mean of 1.55 mg/dL, with
25
increases in creatinine ranging from 0.2 to 5.2 mg/dL (Clements et al., 1990).
In an attempt to reduce the incidence of toxicity and to increase patient
acceptance of therapy, special protocols for infusion of AmB and management of
toxicities have evolved. Premedication before infusion is one of the methods used
for controlling acute toxicities. Diphenhydramine, acetaminophen, hydrocortisone,
and meperidine are suggested to be potentially useful premedications, but there are
few reports documenting the efficacy of these agents (Winn et al., 1963). In addition
to the use of premedication, standard guidelines for the administration of AmB have
been developed and incorporated into standard clinical practice. AmB is usually
administered a test dose of 1 mg diluted in 100-250 ml of 5 % dextrose in water and
infused over 1-2 hours ( Graybill et al., 1983 ). If infusion-related toxicities occur,
infusion rates are often slowed further or therapy may be interrupted temporarily
(Bannett et al., 1985). However, toxicities continue to occur with usage of AmB
despite current methods of infusion and premedication.
2.4.1 Nephrotoxicity
Nephrotoxicity has been regarded as one of the most serious adverse effects
to AmB therapy. Early reports indicated that as many as 80 to 90% of patients who
received the drug developed some degree of renal dysfunction (Butler et al., 1964).
The clinical presentation of nephrotoxicity includes azotemia, renal tubular acidosis,
impaired concentrating ability, and renal sodium, potassium and magnesium wasting,
26
with consequent dehydration, hypokalemia and hypomagnesemia. The mechanism
of amphotericin B associated nephrotoxicity is poorly understood. Amphotericin B
causes renal damage by several postulated mechanisms (Said et al., 1980). Figure
2 illustrates the potential pathways by which AmB induces nephrotoxicity. The main
mechanism may involve effects on membrane permeability, an extension of the
mechanism of antifungal activity on the cells of the host. As stated in the previous
section, AmB damages cell walls of fungal and cell membranes of the host by binding
to a sterol, ergosterol of the fungal cell and cholesterol of mammalian cells. This
binding action creates pores, increasing the permeability of the membrane which
results in leakage of cellular constituents and electrolytes followed by cell lysis and
death (Sabra et al., 1990). In addition to the alterations in the permeability of the
luminal membranes of the tubular epithelium (Andreoli et al., 1973), modifying renal
blood flow (RBF) and glomerular filtration rate (GFR) acutely by increased solute
delivery to the macula densa and activating the tubuloglomerular feedback (TGF)
mechanism is also a possible explanation (Cheng et al., 1982; Gerkins et al., 1980).
Tubuloglomerular feedback, a regulatory mechanism coordinating and regulating the
function of both proximal and distal tubules, is manifested as an immediate
constriction of afferent arterioles evoked by increased amounts of monovalent ions
at the macula densa region of the distal renal tubules (Wright et al., 1977;
Schnermann et al., 1975).
The end result is ischemic damage to the kidney.
Impaired urinary concentrating ability and distal renal tubular acidosis with secondary
AMPHOTERICIN B
SOLUTE PERMEABILITY
N«CI UPTAKE
MACULA DENSA
ACTIVATION
TUBULO-GLOMERULAR
FEEDBACK
A
|GLOMERULAR
T FILTRATION
RATE
1
I RENAL BLOOD FLOW
-•
iRENAL VASCULAR
T
RESISTANCE
L RENAL BLOOD FLOW
RENAL VASCULAR
RESISTANCE
OXYGEN DELIVERY m.T.A.L.
AMPHOTERICIN B
HYPOXIC INJURY m.T.A.L.
CELL PERMEABILITY
OXYGEN DEMAND
I ACTIVE TRANSPORT
I
ELECTROLYTES
Figure 2.
Potential pathways by which
amphotericin B causes alterations in renal
cell permeability; initial alterations in
renal hemodynamics (A) and renal tubular cell
injury (B) in the medullary limb of the thick
ascending limb of the loop of Henle.
28
potassium wasting are associated with these effects (Bhathena et al., 1978).
The responsiveness of TGF seems to be influenced by sodium ion
concentration. Salt loading can block TGF and reduce the nephrotoxicity. Stein
reported that administration of sodium, 90 meq/day, during each day of amphotericin
B therapy is effective in decreasing nephrotoxicity associated with amphotericin B
(Stein et al., 1989).
Mannitol, an osmotic diuretic, prevents amphotericin B
nephrotoxicity by causing vasodilation in the renal tubules, maintaining GFR, and
promoting flushing of the tubules.
Other drugs such as furosemide and
aminophylline are reported to reduce the toxicity (Gerkens et al., 1980).
2.4.2 Prevention of Toxicity
Several approaches have been developed over the past few years in an effort
to overcome the toxicities associated with the clinical use of AmB and these include:
(1) alternative antifungal agents have been developed; (2) derivatives of AmB have
been synthesized and studied; (3) the combination of AmB with other drugs has been
used to treat the fungal infections; and (4) AmB was incorporated into lipid vesicles
in an effort to increase antifungal activity and decrease toxicity.
The main antifungal drugs belong to two families: polyene and azole (Medoff
et al.,1983). The polyene family include three main products: nystatin, amphotericin
B and natamycin. Of these, only amphotericin B can be used parenterally and in the
treatment of systemic fungal infections (Bindschadler et al., 1969). The development
29
of the azole antifungal class has meant that there are alterative drugs for the
treatment of systemic fungal infections (Fromtling et al., 1988). Among the large
number of azole antifungal drugs , four drugs -
miconazole, ketoconazole,
fluconazole and itraconazole - are currently available for the treatment of systemic
fungal infections.
However, most have drawbacks in terms of efficacy, adverse
effects and the development of resistance.
Derivatives of AmB have been synthesized to study the antifungal activity and
toxicity. The free carboxylic acid group of AmB has formed the basis for the
preparation of ester derivatives and the free amino group has been used to prepare
N-acyl, N-methylated, N-glycosylated and N-guanidine derivatives of amphotericin B
(Schaffner et al, 1987).
Ester derivatives of AmB were active in vitro and in vivo.
Also, they were less nephrotoxic and better tolerated than amphotericin B. However,
clinical testing was discontinued following reports of progressive neurological
dysfunction and diffuse white matter degeneration in patients receiving prolonged
high-dose treatment with the methyl ester (Ellis et al., 1982). Most other derivatives
were both less toxic and active than amphotericin B (Schaffner et al., 1987). None
has achieved clinical importance.
2.4.3 Liposomes
Another alternative approach is to use liposomes as a carrier of amphotericin
B.
Liposomes are microscopic vesicles consisting of concentrically arranged
30
phospholipid bilayers (Bangham et al., 1974; Szoka et al., 1980). These multilamellar
vesicles display a prominent lipid compartment and a minor aqueous compartment
formed by the intralayer spaces. Since AmB is highly lipophilic with a general
structure consisting of a mycosamine group and a series of seven double bonds, which
makes AmB compatible with incorporation into a phospholipid bilayer.
The
rationale for the work on AmB liposomes was that the targets of liposomes include
organs such as liver and spleen that are rich in macrophage and which are also
frequent targets of systemic fungal infections. This consideration made liposome
AmB development attractive. Besides liposomes, other drug-lipid formulations such
as colloidal sulfate also have been developed to reduce the toxicity of amphotericin
B since 1982. In 1983, Lopez-Berestein et al. first used multilamellar amphotericin
B
liposomes
composed
of
dimyristoylphosphatidylcholine,
dimyristoyl-
phosphatidylglycerol and amphotericin B (molar ratio 7 : 3 :1) to treat mice infected
with a lethal dose of Candida albicans.
All animals given one dose of AmB-
containing liposomes (4 mg/kg) survived for longer than 42 days while all animals
in the untreated control group died within eight days after infection (Lopez-Berestein
et al., 1983). In addition, liposomes have been used as a vehicle for AmB in the
treatment of cancer patients with fungal infections (Lopez-Berestein et al., 1987).
Patients tolerated liposome AmB much better than AmB in the form of Fungizone.
Increased doses could be administered, and a significant clinical improvement was
observed in some patients. The mechanism of action of liposome AmB in improving
31
the therapeutic index of AmB has not been completely elucidated.
2.5 Pharmacokinetics
After nearly 30 years of use, AmB remains the mainstay of therapy for
systemic fungal infections. The pharmacokinetics of AmB are not well known. The
available information on pharmacokinetics and metabolism is too inadequate to
provide a rational basis for designing less toxic but efficacious dosage regimens.
Dosage regimens are often dictated by toxicity rather than by the findings of
controlled clinical studies. Decisions on selecting daily dosage, total dosage, duration
of therapy, and whether or not to use AmB are often determined by the clinical
experience of the physician in treating fungal infections.
Adult patients exhibit a large volume of distribution, 3 to 4 L/kg, and a long
terminal elimination-phase half-life, which ranges from 11 to 16 days (Atkinson et al.,
1978). No metabolites of AmB have yet been identified in in vivo and in vitro studies
(Christiansen et al., 1985). Renal and biliary clearances were found to account for
10-15% of the systemic clearance depending on the species used in the investigation.
2.5.1 Human Studies
The pharmacokinetics of AmB have been described in two patients (Atkinson
et al., 1978) (Table 3). In these patients, the distribution kinetics of AmB were
described by a three-compartment model, a central compartment and quickly and
32
slowly equilibrating compartments illustrated in Figure 3. The elimination phase
half-life of AmB is approximately 15 days due to the slow return of AmB from the
slowly equilibrating peripheral compartment.
The volume of this peripheral
compartment accounts for 80% of the total distribution volume of 4 L/kg, yet its
intercompartment clearance, 9 ml/min, is only 10% that of the rapidly equilibrating
peripheral compartment. AmB concentration in the slowly equilibrating peripheral
compartment is higher than serum concentration when therapy is stopped. Renal
excretion is a relatively minor pathway for the elimination of AmB, accounting for
only 3% of total elimination.
Studies in children demonstrated a higher clearance and shorter half-life
(Benson et al., 1989). The total clearance, apparent volume of distribution, and
elimination half-life were 0.46 ml/min* kg, 0.76 L/kg, 18.1 h, respectively.
In
addition, they found a strong inverse correlation between patient's age and clearance
of AmB. Two implications of this finding are possible. First, younger children with
high clearance perhaps receive inadequate dosages of AmB for the treatment of
fungal infection. Second, older children are being exposed to excessive doses of
AmB. However, no correlations exist between changes in serum creatinine and
patient age, clearance, total dosage, or duration of therapy.
The distribution of AmB in tissues was obtained at autopsy from eight patients
(Christiansen et al., 1985). The highest concentrations of AmB were found in the
liver; in one patient the amount of AmB in the liver was 41% of the total dose
Renal
Elimination
Non-Renai
Elimination
Figure
3.
Pharmacokinetic
model
of
amphotericin B distribution and elimination.
After intravenous injection into the central
compartment (Vc), amphotericin B distributes
into fast-(Vf) and slow-(Vs) equilibrating
peripheral compartments.
The rate of this
distribution
is governed
by the
intercompartmental clearances Qf and Qs ( from
Atkinson and Bennett, 1978 ).
Table 3
Ref.
Pharmacokinetic Parameters of AmB in Humans
No.patients
CL
(ml/min*kg)
Atkinson
adult
et al.
2
Chabot
et al.b
Stark
et al.
Koren
et al.
Benson
et al
adult
Varea
T1/2
(L/kg)
(h)
a
0.43 ± 0.08
3.99 ± 0.40
0.17
3.8
17
3.72 ± 3.91
3.10 ± 2.32
17.7 ± 17.6
0.43 ± 0.08
0.38 ± 0.02
9.93 ± 1.50
0.46 ± 0.20
0.76 ± 0.52
18.1 ± 6.65
14
children
9
children
13
children
9
T1/2 not reported, terminal T1/2 was 15 days
standard deviations not reported
35
administered. High concentrations were also found in the spleen, kidney and lung.
In total, these tissues account for 20-50% of the total amount recovered. The heart
contains measurable concentration, as do fat and muscle, but amounts are much
lower.
2.5.2 Animal Studies
Craven et al. (1979) studied the kinetics of AmB in dogs. They used the dog
as an animal model with bile duct cannulation and found that excretion of AmB in
the bile accounted for 3% of the dose within 10 days, while excretion in the urine
was prolonged and greater (21%). The half-life of AmB in dogs is 4 days, the
volume of distribution is 6.7 L/kg and systemic clarence is about 0.73 ml/min»kg.
The distribution of AmB in tissues was determined by Niki et al. (1990) in rats
that were treated with daily intraperitoneal doses for 7 days. Rats were sacrificed
24 h after the last dose. The highest concentrations of AmB were found in the
spleen and liver. Low concentrations were detected in serum.
2.6 Analytical Methods
A variety of microbiological assays have been available to measure the activity
of AmB.
Most bioassays involve either serial tube dilution or agar diffusion with a
variety of indicator organisms, including Paecilomyces varioti, Candida tropicalis and
Saccharomyces Cerevisiae (Bannatyne et al., 1977; Bindschadler et al., 1969).
36
Bioassay requires 16-36 hour of incubation. In addition, natural antifungal activity
in the blood of normal individuals and other antifungal reagents may interfere with
the assay. Wide variations exist in the accuracy, precision, sensitivity and specificity
of the bioassay which make it difficult to compare data from different publications.
Radiometric bioassay for AmB (Drazin et al., 1976; Hopfer et al., 1977) was
relatively sensitive, accurate and precise. This method measured AmB based on the
estimation of potassium or rubidium ions. The addition of amphotericin B to a cell
suspension leads to the leakage of ions from the cells, in proportion to the
concentration of drug present. However, this method requires expensive equipment
or facilities for handing radioactive compounds. Furthermore, the reagents are costly
and have a relatively short shelf-life (Drazin et al., 1976), and the test results are
affected by other antifungal agents (Hopfer et al., 1977).
A reverse-phase high performance liquid chromatographic (HPLC) method
was first applied by Mechlinski (Mechlinski et al., 1974) for quality control analysis
of the components of different classes of polyene macrolides. Subsequently, NillsonEhle et al. (1977) used HPLC to measure AmB in biological fluids.
The
chromatographic separation was performed using a reverse phase C18 column and
eluent monitored at 405 nm. The limit of detection of this method was 0.02 ug/ml
compared with 0.05-0.1 ug/ml in the microbiological method (Green et al., 1965;
Bindschadler et al., 1969). HPLC methods have been widely used and Table 4
summarizes the available HPLC methods. HPLC has proven to offer faster, more
37
accurate and
more reproducible results compared
pharmacokinetic studies and routine clinical use.
to bioassay for both
In addition,
HPLC offers
improved sensitivity and specificity and is easier to standardize than bioassay.
Table 4
HPLC Methods Used to Assay AmB
Ref.
Kobayashi et al.
Column
I.S.a
C18
Mobile Phase
acetate buffer, MeOH,
acetonitrile, THF
Mayhew et al.
C18
p-nitrophenol
b
Granich et al.
C18
ANN
Bach et al.
C18
diperse yellow
acetonitrile, EDTA
acetonitrile, EDTA
acetonitrile, MeOH
42 dye
a
: I.S. represent internal standard
b
: ANN represent l-amino-4-nitronaphthalene
EDTA
39
CHAPTER 3
EXPERIMENTAL SECTION
3.1 Materials and Instruments
The materials used in the study were:
(1) Amphotericin B:
Fungizone ( each vial contains 50 mg AmB, 41 mg
deoxycholate and 20.2 mg sodium phosphate, Lot # C0217A/43730, E.R. Squibb
& Sons, Princeton, NJ) was obtained commercially. Pure AmB powder was supplied
by E.R. Squibb & Sons (Batch # 20-914-59718)
A stock solution of AmB (550 ug/ml) was prepared by dissolving AmB in
methanol and DMSO (1:1, v/v) in a volumetric bottle covered with aluminum foil
and stored at -20°C. Sonication was used to assist the dissolution of AmB in the
solvent.
40
(2) Internal standard:
l-Amino-4-nitronaphthaIene (ANN) was purchased from
Aldrich Chemical Co. (Milwaukee, Wisconsin) and was used as the internal standard
(I.S.) in the HPLC assay. A stock solution of internal standard (170 ug/ml) was
prepared by dissolving ANN in methanol and stored at -4°C.
(3) Creatinine assay kit: Creatinine colorimetric assay kits were purchased from
Sigma Chemical Co. (St. Louis, MO) and used for measurement of creatinine
concentrations in serum and urine samples.
(4) Reagents used for animal study: Sterile 0.9% sodium chloride solution (Kendall
McGaw Laboratories, Inc.), ethyl ether (Fisher Scientific) and 5% dextrose injection
(Baxter Healthcare Corporation) were used in the surgery and for intravenous
infusion.
(5) Other chemical reagents: Dimethyl sulfoxide (DMSO) was used for preparing
the AmB stock solution. KH2P04 (Aldrich Chemical Co.), Na2HP04 (Fisher Scientific
Inc.) were used for preparing buffer solutions. Methanol (Fisher Scientific Inc.) and
acetonitrile (Fisher Scientific Inc.) are HPLC grade and used in the mobile phase.
The instruments used in the study were:
(1) HPLC system: A high pressure liquid chromatograph system (a single piston
pump, Model 110 B Beckman Associates) was used with a reversed-phase Nova Pak®
C18 (3.9 x 150 mm, particle size 5/*m, Waters, Diversion of Millipore) stainless steel
column. The effluent was monitored at 405 nm using a fixed wavelength UV-visible
41
absorbance detector (Model 160, Beckman Associater). The output was connected
to both a 10-mV potentiometric integrator (Model 3390 A, Hewlett Packard) and
recorder (Model 585, Linear Instruments Co.)
(2) UV spectrophotometer:
UV/VIS spectrophotometer (Model Lambda 3A,
Perkin-Elmer) was used to measure the creatinine concentration in serum and urine
samples. Disposable semi-micro cuvettes (VWR Scientific Inc.) were used to hold
the test solution.
(3) Materials used for surgery: Silastic (Dow Corning Corporation), PE 50 (Clay
Adams Inc.) and 22 gauge needles (Becton Dickinson and Company) were used for
making catheters.
Metabolic cages (Nalge Company), infusion pumps (syringe
infusion pump 22, Harvard Apparatus), 10 ml glass syringes (Propper & Sons. Inc.)
and 1 ml sterile plastic syringes (Becton Dickinson and company) were used in the
study.
(4) Centrifuge: A Model TJ-6 centrifuge (Beckman) was used for separating serum.
3.2 Pharmacokinetic Studies
3.2.1 Catheter
The catheter is made from PE 50, silastic and a metal portion. The metal
portion is simply a segment (3-4 mm) of 22 a gauge needle. The needle is placed
1/3 - 1/2 of the way into the PE 50, and the silastic is forced over the PE 50, then
both pieces of tubing are tied to the metal piece. This particular cannula is bent to
42
accommodate the jugular vein. The cannulas are bent by wrapping around glass
tubing and immersing into hot water.
3.2.2 Animal Surgery
Male Sprague-Dawley rats (220 g - 320 g) supplied by Harlan Sprague Dawley
Inc. (Indianapolis, IN ) were used in the study. The rats were allowed to acclimate
to the facility for one week prior to the experiment. Forty-eight hours before the
experiment, two cannulas were implanted (both right and left jugular vein), one
catheter for long term infusion and another catheter for sampling during the infusion
(a preliminary study has shown that AmB is not adsorbed onto the catheter). The
surgical procedures were modifications of Weeks (Weeks et al., 1964). Briefly, rats
were placed under light ether anesthesia and the dorsal-neck-shoulder blade area
shaved for exteriorizing the catheters. The right and left ventral neck were shaved
for jugular vein cannulation. One small incision was made in the skin between the
shoulder blades and other incisions were made over the area on the ventral neck
(either right or left) where the jugular vein can be seen to pulsate below the skin
surface.
The jugular vein was isolated and ligated.
A probe was pushed
subcutaneously from the dorsal neck opening through the ventral neck opening and
the PE 50 end of the cannula was pulled through the probe to the ventral side. The
cannula was inserted into the jugular vein and tied. The cannula was fixed to the
neck muscle by a suture. Both incisions were sutured. About 2-3 cm of the cannula
43
filled with saline protuded from the shoulder blade area and the cannula was plugged
with a pin.
3.2.3 Pharmacokinetic Studies
According to the preliminary study, it appears that a minimum of 40 hours
(four half-lives) is required to reach 94% of the steady-state plasma concentration.
Therefore, a two-step infusion method proposed by Wagner (Wagner et al., 1974)
was used in the study. Simulations were performed by substituting the measured
pharmacokinetic parameters of AmB ( Chow et al. 1992) into the equations derived
by Wagner to estimate the plasma concentrations at different times as well as the
times required to reach the steady-state condition based on the infusion rates chosen.
Simulation results suggest that by employing this two-step infusion method we can
achieve the desired steady state concentration within eight hours. Based on the
simulation, a protocol using a forty min fast infusion rate followed by a 520 min slow
infusion rate was chosen. The infusion rate used for the fast infusion was 0.025
ml/min and the infusion rate used for the slow infusion was 0.01 ml/min.
Male Spraque-Dawley rats were used as the animal model. Twelve rats were
randomly selected and divided into three groups to receive different doses (low,
medium and high dose). The long term intravenous infusion was given through a left
jugular vein catheter which was surgically implanted as described previously. During
the infusion, rats were housed separately in metabolic cages and had access to water
44
and food. Blood samples were collected at appropriate times via a right jugular vein
catheter (implanted two days prior to the experiment) during infusion and after
infusion. Urine samples were collected at different time intervals during and after
drug administration.
Rats were weighed before drug infusion. Intravenous infusion solutions were
prepared immediately before the study.
Infusion syringes were covered with
aluminum foil to protect from light. At the end of the fast infusion, (40 mins, 0.025
ml/min infusion rate), the infusion line (with one end connected to the implanted
catheter and another end connected to the infusion syringe) was switched to another
infusion syringe which contained the slow infusion solution. The second infusion rate
was 0.01 ml/min during an infusion time of 520 mins.
3.3 Sampling
The blood samples were collected prior to drug administration and at
appropriate times up to 90 hours after the end of the infusion. Blood samples (0.35
ml) were obtained at the desired times from the implanted cannula. One tenth ml
of blood (which was subsequently re-injected and followed by 0.4 ml of saline) was
withdrawn prior to sampling in order to avoid an artifact due to sample
contamination by saline or blood trapped in the cannula. Time points for taking
blood samples were 0, 40, 370, 460, 560, 840, 1420, 1930, 2300, 3035, 3640, 4486,
5115, and 6165 min. Once blood samples were collected, they were centrifuged at
45
3000 rpm for 3 mins. Serum was collected and placed into a brown glass vial and
stored at -20°C until analysis.
During and after AmB infusion, urine cups were covered with aluminum foil
and put in an ice bath to protect the sample from light to avoid degradation. The
urine samples were collected at different time intervals up to 130 hours after the end
of infusion. The volume of urine was measured and the samples were stored at 20°C before analysis.
3.4 Preparation of Intravenous Solution
I.V. infusion solutions were prepared immediately before the experiment.
AmB, in the original Fungizone bottle (containing 50 mg AmB), was diluted with 10
ml sterile water to form a 5 mg /ml suspension.
Five percent sterile dextrose
injection was used for further dilution to the desired concentration. The resulting
infusion solution is clear without presence of precipitation. The concentrations of the
infusion solutions used for the fast infusion in the three different dose groups were
199.2, 796.4, and 1990.8 g/ml.
The concentrations of the three slow infusion
solutions were 21, 80, and 200 g/ml.
The infusion solutions were protected from
light by aluminum foil.
Intravenous solution stability (in 5% dextrose ) was examined by Kintzel
(Kintzel, 1991). The intravenous solutions at concentrations of 920,1200,1400 ug/ml
were stable at room temperature for up to 36 hours. No precipitation, turbidity, gas
46
formation or color change was observed.
3.5 Sample Preparation
Serum samples were treated with methanol before analysis by HPLC. Three
hundred 1 methanol (containing a certain amount of internal standard) was added
to 100 1 serum sample or standard serum sample. The mixture was vortexed and
centrifuged at 3000 rpm for 10 mins. The supernatant was concentrated under
nitrogen at room temperature, then reconstituted with 150 1 mobile phase and
injected onto the HPLC system.
Urine samples were diluted with 1 to 4 volumes of mobile phase according to
the AmB concentration. The diluted urine samples were vortexed for 1 min and
centrifuged at 3000 rpm for 3 mins. One hundred 1 supernatant of the diluted urine
sample was added to 100 1 mobile phase containing a certain amount of internal
standard. The mixture was vortexed and injected onto the HPLC column.
3.6 AmB Assay by HPLC
AmB concentrations were determined using the modification of the HPLC
method described in the literature (Granich et al., 1986). HPLC methods have
proven to offer a faster, more accurate and more reproducible alternative to bioassay
for pharmacokinetic study. A reversed phase Nova-Pak C18 (Waters) column was
employed in the assay. Analysis was performed with a Beckman Model 110B liquid
47
chromatograph system with a fixed wavelength detector. The mobile phase consisted
of Na2HP04-KH2P04 buffer (4 mM, pH 7.0) : acetonitrile = 69 : 31 (v/v). The
mobile phase was filtered though a 0.45 m Nylon 66 filter membrane and degassed
by sonication under vacuum. A flow rate of 1.2 ml/min was used. The sample
injection size was 100 1. The ultraviolet spectrum of AmB in DMSO/methanol is
shown in Figure 4 indicating maximum absorption at 405 nm and 382 nm. Since the
maximum absorption of the internal standard is about 405 nm, the chosen wavelength
of 405 nm was near the maximum for both compounds. AmB and internal standard
had retention times of 3.5 min and 5.5 min, respectively.
3.7 Calculations
Standard curves for the assays were constructed and least-squares linear
regression analysis was performed. Standard curves were linear from 0.015 g/ml
to 1.75 g/ml.
An example of a standard curve is shown in Figure 5. The lowest
correlation coefficient among the standard curves for any assay was 0.97. The intraday and inter-day reproducibilities of the method were examined by HPLC injection
during one day and on each of three different days, respectively. The intra-day
coefficients of variation of the assay in three different days ranged from 1.89 % to
9.09 % at 0.05 ug/ml and ranged from 1.96 % to 4.12 % at 0.5 ug/ml. For the interday assay, the coefficient of variation was 8.33 % at 0.05 ug/ml and 3.33 % at 0.5
ug/ml. Unknown concentrations of AmB were calculated by comparing the observed
48
1
1
1
•
1
1
1
'
406
t
1
38?
1
AMPHOTERICIN B
(DMSO/CH3OH)
LU
U
z
<
CD
cc
3f»3
o
on
1
CD
<
>
Z3
345
t
250
>
300
1
350
1
1
400
V
«
1
450
.
1
500
WAVELENGTH (nmj
Figure 4. Ultraviolet absorption spectrum of
amphotericin
B
in
DMSO/CH,OH
solution
(concentration 5.45 /ig/ml).
:.477X —0.0247
= 0.999
3-
0.000
0.500
1.000
1.500
2.000
AmB serum concentration
Figure 5.
Plot of standard curve of
amphotericin B in serum.
Peak area ratio
versus AmB serum concentration.
50
peak height ratio of AmB to internal standard as a function of AmB concentration
on the standard curves.
3.8 Creatinine
3.8.1 Creatinine Assay
Creatinine clearance is a useful index for evaluating glomerular filtration rate
and for assessing renal function.
Low creatinine clearances occur in patients with
nephritis and renal obstruction.
It is necessary, therefore, to develop methods to
measure creatinine concentration. The most commonly used method for measuring
creatinine concentration is the Jaffe alkaline picrate method described by Jaffe 100
years ago.
However, the method is subject to interference from endogenous
constituents (glucose, acetoacetic acid, acetone, etc.) of plasma and urine, and it is
influenced by analytical condition. The assay always gives an overestimated value of
the creatinine concentration.
Numerous modifications have been made to the Jaffe method in order to
improve the specificity of the assay. The method used in this experiment was
developed by Slot (Slot et al., 1965).
The basic principle is that under acid
conditions the creatinine-picrate color fades faster than the interfering chromogens.
The diagnostic creatinine kit is based on this principle and it is widely used in
hospital and clinical laboratories due to its simplicity, although it is a non-specific
method.
51
The procedure for this method is as follows: 0.1 ml water, 0.1 ml of standard
creatinine solution (concentration of 0.3 mg/dl of creatinine) and 0.1 ml test serum
or diluted urine sample were each separately added to three different cuvettes. Onetenth ml alkaline picrate solution was added to all three cuvettes, mixed well and
maintained at room temperature for 8 mins. The initial absorbance of the standard
and test (versus blank as reference) was determined at 500 nm. Then, 0.1 ml acid
reagent was added to the cuvette, mixed immediately and thoroughly. After reacting
for exactly 5 min, the final absorbance was determined. Creatinine concentration was
obtained by using the following equation:
initial A. - final A,
me
mQ
Creatinine P^] =
x 3^
dl
initial As - final As
dl
(1)
where initial At and finial At are initial and final absorbances of the test sample and
initial \ and final As are initial and final absorbances of the standard creatinine
sample.
3.8.2 Creatinine Clearance
Creatinine concentrations in serum and urine samples were measured before
and after drug administration in order to compare differences in creatinine clearance
(CLcr) values.
Creatinine clearance can be calculated by the ratio of creatinine excretion rate
in urine determined over selected time intervals and the serum concentration. The
52
equation is as follows:
CLcr =
bXuJAt
»
~C~
(2)
where Ccr is the creatinine concentration in serum and A Xucr/A t is the excretion rate
of creatinine in urine over the time interval T. Excretion rate is given by:
AXuJAt =
Cur'cr V°lur
T
(3)
where Cur cr is the creatinine concentration in urine and Volur is the volume of urine
over the time interval T.
3.9 Data Analysis
The pharmacokinetic data were analyzed by both the model-independent and
model-dependent procedures.
3.9.1 Model-Independent Method
In the model-independent procedure, AmB serum concentrations were plotted
versus time on semi-logarithmic graph papers. Data in the post-distribution phase
of AmB concentration versus time profile were analyzed by log linear regression to
obtain the slope which presents the terminal disposition rate constant, 6. The
disposition half-life, TH, was calculated as follows:
53
where fi is the terminal phase rate constant.
T1/2 was also estimated based on urine data. The relationship between In
excretion rate (In A Xu/A t) versus time at the midpoint of the urine collection interval
should yield a straight line with a slope of fi, as illustrated in Figure 6. The fi value
can be obtained by using linear regression analysis.
Harmonic mean half-lives (Hn/2) and variance were estimated by using a
jacknife technique (Lam et al., 1985) in order to present the population mean halflives. A harmonic mean half-life can be determined from the following equation:
Ht, = n
n
' <— + —+••• + —)
*1/2, 1
*1/2, 2
*1/2, n
(5)
where n is the number of subjects.
The standard deviation of the harmonic mean half-life is given by:
E (//,.
\ (ti-1) i=l
SD =
(6)
- Hf
where Hj and H are given by:
"> - ~—i
+
*1/2,1
*1/2,2
+ ... +
r1—i
+
*1/2,1-1
1 "
H = - Y,HI
n i-1
*l/2,i+l
+ ... +
r
<7>
*l/2^i
(8)
InAXu/At
Tmid
Figure 6. A plot of the relationship between
in urine excretion rate (In AXu/At) and time
at the midpoint of urine collection. The slope
is -p.
55
The AmB area under the serum concentration versus time curve (AUC) was
calculated using the trapezoidal rule. The equation is as follows:
auc; = icA + Iekc, • cwx<,., - <,)] • £
2
2<=i
(»>
p
where Cj is the concentration at time t;. AUCn°° can be calculated by the term, Cn/B,
in equation 9, where Cn is the last measured concentration.
The first moment of the AmB concentration-time profile is the total area
under the curve (AUMC) resulting from a plot of the product of AmB concentration
and time versus time. AUMC is given by:
aumc; = lc,tf * Iekca +
2
2j=i
- oi+ ,Jr- *
p
(10)
pz
By using serum data together with urine data, other pharmacokinetic
parameters can be calculated. The systemic clearance CLS was obtained using the
following equation:
CL = Y,dose
S
AUCQ
(11)
where S dose is the total dose infused (including the fast infusion dose and the slow
infusion dose), AUC0°° is the area under the concentration-time curve from time 0
to infinity.
Also, CL was calculated from:
56
a, =
where Kq is the slow infusion rate,
K0
/
ss
(12)
is the AmB serum concentration at steady-
state condition.
Renal clearance, CLr, was estimated by the following relationship.
CL, =
Xu',j
'•!AUCtJ
(13)
where Xutl12 is the amount of AmB excreted into urine during the time interval tl
to t2, AUCtlt2 is the area under the curve during the corresponding time intervals.
Renal clearance was also obtained by employing urinary excretion rate versus
plasma concentration at the midpoint of the collection interval. This relationship can
be described by:
CLr =
AX" 1 Af
(14)
^'mid
CLr can be obtained from the slow of the above relationship by performing a linear
regression analysis of plots of A Xu/A t versus Cmid.
Renal clearance at steady-state condition was the ratio of urinary excretion
rate at the steady-state condition and the serum steady-state concentration.
The apparent volume of distribution, usually termed Varca, is given by:
57
r,
E dose
area
=
^
AUCq
•
p
(15)
where 2 dose is the total dose infused.
The volume at the steady-state, Vss, is given by ( Perrier et al., 1982):
v
"
=
Dose
uose
AUC
AUMC-T.AUC.
T-AUC,
1
1 _ —2
-]-AT AUC.
^
2AUC1
2AUC
AUC
(16)
where Tj is the duration of the first infusion, T2 is the duration of the second
infusion, A T is the time interval between these two infusions, AUCj and AUC2 are
the areas under the AmB serum concentration versus time profiles for infusion one
and infusion two, respectively.
Fexc, the fraction of the AmB dose excreted into urine is determined
experimentally and can be calculated by:
Clr
F =
"
CLS
(17)
3.9.2 Model-Dependent Method
The AmB plasma concentration - time curve can be described by a
multicompartment model according to literature reports and on the basis of data
obtained from this laboratory. AmB data is best described by a two compartment
model based on our previous experiments.
58
In this two compartment model, AmB is assumed to distribute instantaneously
into the central compartment which represents tissues that are highly perfused by
blood. The drug is simultaneously but more slowly distributed into a peripheral or
tissue compartment which is poorly perfused by blood. The situation is illustrated
in Fig. 7.
Model-dependent analysis was conducted by using the computer program
ADAPT II (D'Argenio et al., 1992).
We have fit the data using the following
sequence: first, setting up the model file with the differential equations describing
the two compartment model; second, entering the experimental data; and, last, using
weighted least squares regression to estimate the pharmacokinetic parameters. Initial
estimated values are obtained from the analysis of preliminary results of experiments
conducted in this laboratory. Micro-rate constants K12, K21, K10 and volume Vc were
estimated by ADAPT n.
3.9.3 Statistical Analysis
CLS, CLr, V, Fex, Tlj2 and other pharmacokinetic parameters as a function of
dose were examined by linear regression analysis. The slope of the regression line
was tested for statistical significance. One-way analysis of variance was used to
examine the variance among three groups.
Creatinine clearances (before and after drug administration) were examined
by a paired Students t test.
59
A p value < 0.05 was considered to be significant.
All statistical analyses were performed with use of the MINITAB statistical
package (Minitab Inc, state college, PA).
XI
Central
Comoartment
21
12
Denpherai
X2
Comoartment
10
Figure 7.
Pharmacokinetic model for AmB
distribution and elimination. X, is the amount
of drug in the central compartment. X2 is the
amount of drug in the peripheral compartment.
K12 and K21 are the rate constants between the
two compartments. K10 is the elimination rate
constant from the central compartment.
61
CHAPTER 4
RESULTS
4.1 Analytical Method
AmB concentrations in serum and urine samples were determined by using
the HPLC assay described earlier. Briefly, the mobile phase (4mM NajHPO,, KH2P04 buffer, PH=7.0, and acetonitrile, 69:31 v/v) was pumped at a flow rate 1.2
ml/min through a C18 Nova-Pak column. The column effluent is monitored at 405
nm using a UV absorbance detector.
nitronaphthalene)
were
separated
AmB and internal standard (l-amino-4from
other
interfering
compounds.
Chromatograms of blank serum and spiked AmB serum sample are shown in Figure
8A and 8B. In the chromatogram, AmB and internal standard were completely
separated, with retention times of 3.59 min and 4.96 min, respectively.
The urine sample was diluted 1 to 4 times according to the AmB
concentration. The mobile phase was used to dilute the urine due to low solubility
62
of AmB in water. In addition, the mobile phase can improve the resolution of the
peak.
Standard curves for AmB were established by linear least-square regression
of the area ratio of amphotericin B/internal standard versus the AmB concentration
(r2 > 0.99). The standard curve was shown in Figure 5. Unknown concentrations
of AmB in the authentic samples were determined by comparison with the AmB
standard curve. The linear range of the standard curve was from 0.015 ug/ml to 1.75
ug/ml.
The within-day precision was assessed by analyzing triple samples for each
concentration in one day for three days, while the between-day variation was
examined by analyzing the results of three days. Two different AmB concentrations,
0.05 ug/ml and 0.5 ug/ml, were examined. The results are shown in Table 5. The
within-day % CV ranged from 1.89% to 9.09% for two different concentrations. The
between-day % CV was 9.12% and 3.33% for the concentrations of 0.05 ug/ml and
0.5 ug/ml, respectively.
The detection limit of the method for AmB in biological fluids was 15 ng/ml
at a sensitivity setting of 0.008 a.u.f..
4.2 Stability of AmB in Urine
According to the literature (Shadomy et al., 1973), light, temperature and pH
of the medium are thought to affect the stability of AmB. Wiest et al.(1991) found
(A)
(B)
r©
Figure 8. (A) Blank serum. (B) Spiked serum
sample with AmB concentration of 0.5 jug/ml.
3.59 min and 4.96 min are retention times of
AmB and internal standard, respectively.
Table 5.
Day
Intra-day Reproducibility of the AmB Assay in
Spiked Serum Samples (n=3)
Spiked conc.
(Mg/ml)
Observed conc.
(Mg/ml)
SD
CV%
1
0.050
0.500
0.048
0.510
0.003
0.010
6.25
1.96
2
0.050
0.500
0.053
0.510
0.001
0.021
1.89
4.12
3
0.050
0.500
0.044
0.530
0.004
0.011
9.09
2.08
Table 6.
Inter-day Reproducibility of the AmB Assay in
Spiked Serum Samples (n=9)
Spiked con.
(fxg/ml)
0.050
0.500
Observed con.
(jug/ml)
0.048
0.515
SD
CV%
0.004
0.017
9.12
3.33
65
a peak for a degradation product of AmB in the chromatogram when AmB was
heated, and in contact with acid or base. In this study, the stability of AmB in rat
urine was examined. During 24 hours, the initial concentration declined by up to
43% at room temperature, without protection from the light. However, no peak was
found for a degradation product. Therefore, precautions were taken in handing urine
samples to minimize the degradation. Aluminum foil was used to cover the urine
cup to protect from light and an ice bath was used to keep urine at a low
temperature during urine collection. With such precautions, AmB concentrations in
urine remained above 95% of the initial concentration at all times up to 24 hours
(see Table 6). The results suggest that amphotericin B in urine was stable under
these condition.
4.3 Pharmacokinetic Data
Male Sprague-Dawley rats were randomly divided into three different dose
groups. The experiment was designed to administer the drug with a two-step infusion
procedure to rapidly achieve a steady-state condition. The high dose group was given
a total dose of 4210.0 ug/kg, including 3069.8 ug/kg for the fast infusion rate and
1900 ug/kg for the slow infusion rate. The medium dose group was given a 1323.1
ug/kg total dose, with 837.8 ug/kg for the fast infusion rate and 331.5 ug/kg for the
slow infusion rate. In the low dose group, a total dose of 380.3 ug/kg was given, with
284.1 ug/kg and 96.3 ug/kg from the fast and slow infusions, respectively.
Table 7.
Stability of AmB in Urine with Protection from
Light and Kept at Low Temperature in Glass
Tubes (n=2).
Time (hour)
0
4
16
24
% Initial Concentration Remaining
102.8
102.1
103.1
96.0
Stability of AmB in Urine in Plastic Tubes
with the Same Precautions as Above (n=l)
Time (hour)
0
4
16
24
% Initial Concentration Rmaining
112.3
102.8
101.9
109.5
67
The serum concentration versus time profiles for the three different dose
groups are shown in Figures 9-11. The individual plasma and urine data for all
animals are summarized in Appendix A and B. AmB concentrations in blood can
reach steady-state conditions in about 300 mins after the initiation of the slow
infusion. The mean AmB steady-state concentrations for the low, medium and high
dose groups were 0.066 ± 0.017 ug/ml, 0.143 ± 0.023 ug/ml and 0.551 ± 0.12 ug/ml,
respectively. Linear regression analysis of the mean AmB serum concentration at
steady-state as a function of dose indicates that the mean steady-state concentration
increased linearly when the dose increased (Figure 12).
The terminal disposition rate constant, 6, was calculated by linear regression
of the last several points in the serum concentration - time profile. Linear regression
of the rate constants as a function of dose indicates that the parameter is doseindependent. One-way analysis of variance indicates that there were no significant
differences in rate constant among the three groups. The half-life can be estimated
from the rate constant obtained from the plasma data or from linear regression of
urinary excretion rate versus the midpoint of the urine collection interval (Table 8).
The half-lives (± SD) were 917 ± 252.6 min, 1043.3 ± 139.0 min and 909.5 ± 443.7
min in the high, medium and low dose groups, respectively. Linear regression of
half-life versus dose indicates that half-life was dose-independent, as illustrated in
Figure 13. One-way analysis of variance ( ANOVA) indicates that there were no
significant differences in half-life among those three groups.
68
The harmonic mean half-life is more meaningful than the arithmetic mean
half-life because the harmonic mean half-life represents the population mean of the
half-life under investigation. The harmonic mean half-life can be obtained based on
the method described in Chapter Three. The harmonic mean half-lives (± SD) were
866.24 ± 239.25 min, 1030.48 ± 124.69 min and 767.78 ± 1303.68 min for the high,
medium, and low dose groups, respectively.
The volume of distribution (Varea) in the high, medium and low dose groups
were 5.29 ± 1.50 L/kg, 5.89 ± 1.34 L/kg and 4.43 ± 1.65 L/kg, respectively, and
these results are shown in Table 8. Linear regression of volume of distribution
versus dose indicates that volume of distribution is dose-independent (Figure 14).
One-way analysis of variance indicates that there were no significant differences in
this volume among those three groups.
The systemic clearances were determined to be 4.03 ± 0.67 ml/min*kg, 3.92
± 0.81 ml/min»kg and 3.52 ± 0.72 ml/min* kg for the high, medium and low dose
groups, respectively. Systemic clearance values calculated from the steady-state
condition ( i.e., K^/C^ ) were very similar to those determined from area analysis
(Table 8).
Linear regression of systemic clearance versus dose indicates that
systemic clearance is dose-independent, as illustrated in Figure 15.
ANOVA
indicated there were no significant differences in clearance among the three groups.
However, renal clearances were 0.37 ± 0.04 ml/min*kg, 0.86 ± 0.24 ml/mimkg and
0.76 ± 0.33 ml/min*kg for the high, medium and low dose groups, respectively. The
69
renal clearances calculated from the steady-state condition were very similar to those
above cited (Table 8). The linear regression of renal clearance versus dose indicates
that renal clearance decreases when the total infused dose increases, suggesting dosedependent characteristics (Figure 16). One-way analysis of variance suggests that
renal clearance of the high dose group was significantly different from both the low
and medium dose groups.
However, there is no significant difference in renal
clearance between the medium and low dose groups, suggesting that the alteration
of renal clearance is not proportional to the total infused dose.
Figures 17 and 18 illustrate the profiles of renal clearance ( estimated from
the rate method ) versus serum concentration at corresponding time and the renal
clearance versus the serum concentration at the steady-state condition. Both graphs
indicate that renal clearance is inversely related to AmB serum concentration. In the
graph of renal clearance versus time in the high dose group (Figure 19), it seems that
renal clearance increased over time. However, with the high variation, it is difficult
to conclude that renal clearance increased with time. There were no alterations in
renal clearance as a function of time in the medium and low dose groups (Figure 19).
Urinary excretion accounted for 9.4 ± 1.2%, 22.0 ± 5.0% and 23.1 ± 13.0%
of total elimination in the high, medium, and low dose groups, respectively. The
linear regression of the fraction of the dose excreted in the urine versus dose
suggested that this fraction decreased when the infused dose increased, which is in
agreement with the change in renal clearance (Figure 20).
70
Renal clearance only accounted for 10-20% of total body clearance. The
mode of non-renal AmB elimination remains to be determined.
Serum and urine creatinine concentrations were determined using the picric
acid assay. Creatinine clearance was estimated using the method described in
Chapter Three and these results are shown in Table 8. Comparison of creatinine
clearance before and after AmB treatment in the three different dose groups is
shown in Figure 21. High AmB dose treatment resulted in significantly decreased
creatinine clearance (Figure 22), which suggests there is a change in glomerular
filtration rate. This finding agrees with the alteration in AmB renal clearance.
There were no significant decreases in creatinine clearances after AmB treatment in
the medium and low dose groups. The relationship between renal clearance and
creatinine clearance is shown in Figure 23. Both renal clearance and creatinine
clearance were found to decrease in the high dose group.
Model-dependent methods were also used to analyze the data. Serum data
were analyzed by the ADAPT n computer program developed by D'Argenio et al.
(1992) for nonlinear regression. Both two compartment and three compartment
models were designed and used to fit the experimental data. Based on the results
of the F test ( F value can be calculated based on weighted sum of squares and
degree of freedom) ( Boxenhaum et al., 1974), the two compartment model was
chosen as the best model to describe the pharmacokinetic characteristics of AmB.
Micro constant, K12, K21, K10, and central volume of distribution, Vc, were estimated
71
by running the ADAPT II program. The non-linear regression fitting curves are
provided in Appendix C.
The computer iteratively adjusted the values of all the parameters of the
model to minimize the differences between the theoretical curves describing the
serum concentration and observed data points.
Parameters estimated from the
computer program depend on the initial estimates given. Sometimes, giving different
initial estimates results in different computer-estimated parameters. However, the
initial estimates used in the fitting were obtained from the results of previous work
done in this laboratory ( Cai, Masters Thesis, 1991). This initial estimation problem
is responsible for the high variation of parameter valves among the individual
animals.
72
Table 8.
Dose
Mg/kq
Summary of Pharmacokinetic Parameters of AmB Based
Upon Model-Independent Analysis of the Data.
AUC
'rain/ml
0
min"
T1/2a
min
T1/2b
mm
Varea
L/kq
Vss
L/kq
3782.6
4145.9
4989.5
3921.9
AVG
SD
810.7
1161.1
1489.6
861.7
0.00065
0.00094
0.00058
0.00103
0.00080
0.00022
1066.2
737.2
1194.8
672.8
917.8
252.6
845.1
1019.1
805.8
1004.3
918.4
109.1
7.17
3.80
5.78
4.42
5.29
1.50
2.94
2.16
4.01
2.12
2.81
0.89
1220.0
1500.0
1672.2
900.2
AVG
SD
300.6
300.0
492.2
280.1
0.00056
0.00074
0.00066
0.00073
0.00067
0.00008
1237.5
936.5
1050.0
949.3
1043.3
139.0
949.3
1260.0
924.0
911.8
1011.3
166.6
7.25
6.76
5.14
4.40
5.89
1.34
2.75
2.92
2.53
1.33
2.38
0.72
323.7
351.8
439.5
395.3
391.2
AVG
SD
106.6
117.8
99.6
114.4
116.8
0.00045
0.00057
0.00108
0.00120
0.00117
0.00089
0.00036
1540.0
1215.8
641.7
557.5
592.3
909.5
443.7
529.0
1050.0
558.9
962.5
679.4
756.0
237.3
6.76
4.82
4.40
2.88
2.86
4.43
1.65
1.78
1.40
1.44
1.45
1.07
1.43
0.25
73
Table 8 (continued)
Summary of pharmacokinetic parameters
Clearance, ml/min kg
CLsd
CLre
CLpf
4.66
3.57
3.35
4.55
4.03
0.67
3.69
4.70
2.70
4.18
3.82
0.85
0.42
0.38
0.33
0.36
0.37*
0.04
0.41
0.39
0.37
0.40
0.39*
0.02
0.37
0.28
0.33
0.30
0.32*
0.04
4.24
3.19
3.02
4.19
3.66
0.64
0.090
0.106
0.099
0.079
0.094*
0.012
0.078
0.077
0.098
0.067
0.080*
0.013
4.06
5.00
3.39
3.21
3.92
0.81
3.94
5.03
3.17
6.56
4.68
1.47
0.77
1.15
0.59
0.92
0.86
0.24
0.72
1.12
0.62
0.82
0.82
0.22
0.45
0.76
0.49
0.58
0.57
0.14
3.29
3.85
2.80
2.29
3.06
0.67
0.190
0.230
0.174
0.287
0.220
0.050
0.111
0.084
0.143
0.182
0.130
0.042
3.04
2.99
4.75
3.46
3.35
3.52
0.72
3.55
4.98
2.75
2.44
2.34
3.21
1.10
0.76
1.34
0.59
0.53
0.60
0.76
0.33
0.63
1.14
0.80
0.62
0.74
0.79
0.21
0.60
0.95
0.44
0.56
0.58
0.62
0.19
2.28
1.65
4.16
2.93
2.75
2.75
0.93
0.250
0.448
0.124
0.153
0.179
0.231
0.130
0.197
0.317
0.100
0.162
0.172
0.190
0.080
CLSC
CLr8
cC
Fexc '
i
*Fexc
Plasma data
Urine data
From dose/AUC
From K q /CSS
From excretion rate vs plasma concentration
From excretion rate vs plasma concentration at steady-state
From amount excreted vs AUC
Non-renal clearance from, Clsc - Clre
Fraction of dose excreted unchanged in urine (Clre/Clsc)
Fraction of dose excreted unchanged in urine determined
experimentally
Significant difference
74
Table 9
Sununary of Creatinine Clearance Before and After Dosing
Dose
Before dosing
ug/kg
csea
AXucr/Atb
323.7
351.8
439.5
395.3
391.2
AVG
SD
1.84
1.90
1.84
1.30
1.50
1.68
0.26
8.84
9.82
9.60
10.81
8.92
9.60
0.80
1220.0
1500.0
1672.2
900.2
AVG
SD
2.90
1.43
1.89
2.00
1.82
0.84
3782.6
4145.9
4989.5
3921.9
AVG
SD
1.10
1.21
2.21
1.86
1.60
0.53
after dosinq
CLcrC
cse
AXucr/At
CLcr
17.27
17.95
17.28
27.53
19.70
19.95
4.35
1.81
2.10
2.89
2.13
1.22
2.03
0.60
8.23
9.95
8.78
10.34
9.46
9.35
0.86
16.36
16.45
10.06
16.07
25.68
16.92
5.59
7.34
8.93
11.86
6.88
8.75
2.25
11.30
25.39
19.07
11.62
16.85
6.73
2.02
2.54
2.06
2.19
2.20
0.24
7.66
8.14
10.33
8.08
8.55
1.20
16.93
13.03
15.24
12.46
14.42
2.06
6.04
9.62
9.46
8.73
8.46
1.66
19.89
28.81
14.86
16.18
19.94
6.29
3.10
2.50
3.03
2.98
2.90
0.27
7.78
7.50
6.90
7.67
7.46
0.39
9.09
10.87
7.91
8.88
8.99
0.89
®: Cse is the concentration of creatinine in serum, unit is ng/ml.
: excretion rate of creatinine in urine, jig/min.
creatinine clearance, ml/min«kg.
Table 10. Summary of Pharmacokinetic Parameters of AnB
Obtained by Model-Dependent Fitting Method.
V=
CLS
Dose
*12
K21
*10
Aig/kg
1/min
1/min
1/min
L/kg
3782.6
4145.9
4989.5
3921.9
AVG
SD
0.0025
0.0090
0.0024
0.0041
0.0045
±0.0031
0.0012
0.0020
0.0010
0.0026
0.0017
±0.0008
0.0027
0.0068
0.0040
0.0027
0.0040
±0.0019
1.865
0.712
0.979
1.869
1.356
±0.600
5.01
4.82
3.90
4.99
4.68
±0.53
1220.0
1500.0
1672.2
900.2
AVG
SD
0.0035
0.0226
0.0045
0.1984
0.0572
±0.0945
0.0009
0.0078
0.0013
0.0140
0.0060
±0.0062
0.0019
0.0023
0.0041
0.0059
0.0036
±0.0018
1.981
2.260
0.958
0.491
1.423
±0.836
3.79
5.11
3.93
2.92
3.94
±0.90
323.7
351.8
439.5
395.3
391.2
AVG
SD
0.0025
0.1582
0.0043
0.0219
0.0014
0.0377
±0.0679
0.0017
0.0192
0.0060
0.0336
0.0027
0.0132
±0.0148
0.0012
0.0030
0.0020
0.0023
0.0021
0.0021
±0.0007
2.320
0.691
2.439
1.626
1.689
1.753
±0.696
2.86
2.10
4.81
3.82
3.51
3.42
±1.02
ml/min kg
1.000
Dose= 380.3 ug/kg
0.100-r
11 i
0.010--
0.001
0
500
1000
1500
2000
2500
Time (min)
Figure
9.
Plot
of
mean
AmB
serum
concentration versus time in the low dose
group. Average infused dose was 380.3 jug/kg.
Each point is the mean of five rats.
The
cross-hatched vertical bars represent the
standard deviation.
The solid line connects
the points.
77
Dose= 1323.1 ug/kg
1.000--
0.100c
<L>
<J
o
o
0.010 +
0.005
1000
2000
3000
Time (min)
Figure
10.
Plot
of
mean
AmB
serum
concentration versus time in the medium dose
group. Average infused dose was 1323.1 /ig/kg.
Each point is the mean of four rats. The
cross-hatched vertical bars represent the
standard deviation.
The solid line connects
the points.
78
10.000
Dose =4210.0 ug/kg|
cn
3
c
o
1.000-
0.100c
(D
O
c
o
CJ
0.01040.005
1000
2000
3000
4000
Time (min)
Figure
11.
Plot
of
mean
AmB
serum
concentration versus time in the high dose
group. Average infused dose was 4210.0 /ng/kg.
Each point is the mean of four rats. The
cross-hatched vertical bars represent the
standard deviation. The solid line connects
the points.
0
1
2
3
4
Thousands
Dose (ugfcg)
Figure
12.
Plot
of
mean
AmB
serum
concentration at steady-state as a function of
dose. Each point is the mean of four rats.
The
solid
line
represents
the
linear
regression analysis of the data forced through
the origin ( Y = 0.000129 X; r=0.99 ).
5
80
!500
o high dose
• medium dose
A low dose
2000
1500--. A
A
0
0
O
•
• •
1000-
500-
•
O
o°
£
«
'
\
1000
2000
3000
1.
4000
»
5000
6000
Dose( / u,g/kg)
Figure 13.
Plot
function of dose.
of
AmB
half-life
as
a
81
O high dose
• medium dose
A low dose
A
O
•
O
O
A
A
O
••
•
A
0
-
"
"
i
i
i
1000
2000
3000
4000
5000
6000
Dose (ju,g/kg)
Figure 14. Plot of volume of distribution of
AmB as a function of dose.
82
O High dose
• Medium dose
A Low dose
8 -
A
4-1-
0
qd
•
H
1000
O
1
2000
DOSE
1
3000
1
4000
O
h
5000
6000
G^g/kg)
Figure 15. Plot of AmB systemic clearance as
a function of dose.
83
2.000
O high dose
• medium dose
A low dose
.500c
E
E
1.000-
o
0.500-
0.000
0
1000
2000
3000
4000
5000
6000
Dose (Aig/kg)
Figure 16. Plot of AmB renal clearance as a
function of dose. Renal clearance determined
from excretion and plasma concentration. The
solid line is a linear regression fit of the
data ( Y=-0.00011X+0.883; r=0.40; p>0.05 ).
84
2.000
cn
c
O high dose
• medium dose
A low dose
1.500O
E
1.000 +
A
*
•
t:
o
°^ o
°' 500 '^c
0.000
0.000
o
o
0.500
1.000
AmB serum concentration (ug/ml)
Figure 17.
Plot of AmB renal clearance
versus
AmB
serum
concentration.
Renal
clearance determined from excretion rate and
plasma concentration.
85
1.500
O high dose
• medium dose
A low dose
A
•
J? 1.000
c
E
A®
A •
AA
•
_i 0.500
o
<8
0.000
0.000
o
I
1
U
0.200
0.400
0.600
I
0.800
1.000
AmB serum concentration at steady-state
condition (ug/ml)
Figure 18. Plot of AmB renal clearance versus
AmB serum concentration at steady-state.
Renal clearance determined from excretion rate
and plasma concentration.
0.400 - •
0.200
0.000
1500
2000
0.400 --
0.200--
0.000 -j
0
1
1
1
1
500
1000
1500
2000
2500
0.400--
0.000 -I
0
1
1
1
1
500
1000
1500
2000
2500
Time (mln)
Figure 19. Plot of AmB renal clearance as a function of time
in the low (A), medium (B), and high (C) dose groups. Renal
clearance determined from excretion rate and plasma
concentration. Each point is the mean of four rats. The crosshatched vertical bars represent the standard deviation.
87
0.600
O high dose
• medium dose
A low dose
A
0.400
0.300 +
•
0.200
v
-
*
.
.
A
0.100
0.000
0
i
i
1
t
1
1000
2000
3000
4000
5000
6000
Dose (fj,g/kg)
Figure 20. Plot of the fraction of the dose of AmB excreted
into the urine as a function of dose. This fraction calculated
from, CLr/CLs. The solid line represents the linear regression
fit of the data (Y=-0.000037X + 0.254; r2=0.40; p>0.05).
Figure 21. Plot of the individual creatinine clearance before
and after AmB treatment in the low (A), medium (B) and high
(C) dose groups. The circles with vertical cross-hatched bars
represent the mean and standard deviation.
after dosing
before dosing
30-
20-
low dose
medium dose
high dose
* significant difference
Figure 22.
Bar graphs illustrating the mean
creatinine clearance values prior to and after
AmB treatment as a function of dose.
The
cross-hatched vertical bars represent the
standard deviations.
90
2.000
O high dose
• medium dose
A low dose
^ 1.500-cn
c
E
\
E
1.000-
L-
o
0.500-
0.000
0
10
20
CLcr (ml/min kg)
Figure 23.
Plot of the relationship between
the AmB
renal
clearance and
creatinine
clearance in three different groups.
The
solid line is a regression fit of the data
(Y=0.0575X-0.0564; r=0.375; p<0.05). Value in
parentheses not used in analysis.
30
91
2.000
O High dose
• Medium dose
A Low dose
1.500
O
_c
1.000
J,
u
d
6 A
O
•&
0.500
8
0.000
0.000
0.020
0.040
0.060
0.080
0.100
Urine Flow (ml/min)
Figure 24. Plot of the renal clearance versus
urine flow.
92
CHAPTER 5
DISCUSSION AND CONCLUSIONS
5.1 Discussion
Obtaining an accurate concentration measurement in biological samples is the
foundation for studying the pharmacokinetic characteristics of AmB. The HPLC
method that we modified offers a sensitive, precise, specific and reproducible
measurement of the plasma and urine concentrations of AmB.
The final
chromatographic conditions adopted represented a compromise between analysis
time, peak shape symmetry, and resolution from other interfering substances.
Our method for extraction of serum samples uses methanol as a
93
deproteinizing agent with a 1 : 3 volume ratio of sample : organic solvent.
Deproteinization is relatively complete and the supernatant may be quickly
evaporated in only a few minutes under a steam of nitrogen. Precautions were taken
during urine collection.
These precautions have proven to efficiently avoid
degradation of AmB during the urine collection (i.e., protection from light and
cooling on ice).
For nearly 30 years AmB has been the principal chemotherapy agent used to
treat systemic mycoses. However, the pharmacokinetic information about this drug
remains limited. The results of this study provide information on half-life, volume
of distribution, and clearance of AmB in an animal model. The linearity of the
disposition kinetics of AmB has also been determined in the study.
Based upon results from preliminary studies (Chow et al., 1992), it will take
at least 40 hours to reach 94% of AmB steady-state plasma concentration via a
constant rate infusion.
The traditional approach to attain and maintain an
immediately desired steady-state drug plasma concentration is achieved by
administering a simultaneous intravenous bolus and infusion. However, this regimen
may lead to potential toxicity to AmB whose kinetic behavior was described by a
multicompartment model.
In addition, an intravenous bolus of AmB causes
precipitation of the drug due to its low water solubility ( based on a preliminary
study). Therefore, a two-step infusion method proposed by Wagner (Wagner et al.,
1974) was used to rapidly achieve AmB steady-state plasma concentrations. An
94
initial simulation was performed by substituting the pharmacokinetic parameters into
equations defined by Wagner. Based on the computer simulation and a preliminary
study, steady-state conditions are achieved within eight hours. The AmB steady-state
plasma concentration is related to the dose infused (Figure 12). At the steady-state,
the systemic clearance estimated based on steady state concentration and infusion
rate should have minimal error if a real steady-state has been attained. Also, the
renal clearance under this condition should also be accurate.
Atkinson and Bennett (1978) reported the pharmacokinetic results in humans.
They found that the behavior of AmB is best described by a three compartment
model. The terminal half-life of 15 days is due to the slow return of AmB from the
slowly equilibrating peripheral compartment.
The volume of the peripheral
compartment accounts for an average 80% of the total distribution volume of 4
Liters/kg. The half-life reported by Atkinson was calculated by measuring AmB
concentrations over several days ( from the fortieth day in one patient and from the
sixth day in another patient) after cessation of therapy.
In our study, a two
compartment model was chosen to fit the plasma data based on the results of a
statistical test. The half-life of AmB is approximately 15 hours. Although there are
some variations in the half-lives among three groups, the linear regression indicates
that half-life is dose-independent. The central volume of distribution is about 1.2
L/kg and accounts for 23% of the total volume of distribution. The differences in
half-life between literature reports and our results are due to the different species
95
studied, although other factors such as analytical methods used to determine AmB
concentrations may have played a role.
In addition, Atkinson and Bennett (1978) reported that the rate of transport
from the peripheral tissues to the central compartment is relatively slow when
compared to the rate of transport from the central compartment to the peripheral
compartment. Similarly, we observed the same phenomena in our study as well as
in a preliminary study in this laboratory. The rate constant of the drug moving from
the peripheral compartment to the central compartment is one-fourth of the rate
constant from the central compartment to the peripheral compartment.
This
information, along with the analysis provided by Atkinson and Bennett (1978),
supports the hypothesis of prolonged AmB storage in tissues. The large volume of
distribution observed coincides with this hypothesis.
Renal clearance was reported to account for the elimination of less than 10
% of the total dose (Bindschadler et al., 1969).
In our study, renal excretion
accounted for 10 -20 % of the total dose. This difference may be attributed to
species difference. In rhesus monkeys, high concentrations of AmB have been found
in bile following intravenous administration (Lawrence et al., 1980). In dogs, serum
AmB concentrations were about 19 % higher during periods of biliary obstruction
and biliary elimination was estimated to be 19 % of the dose (Craven, et al., 1979).
Therefore, biliary elimination and urinary elimination may account for a certain
fraction of the dose. The fate of the remaining 60 % - 70 % of the dose of AmB is
96
unknown. A possible reason for failure to find most of the administered dose might
be metabolic conversion of AmB. However, no metabolites of AmB have been
identified both in human and animals.
Another possible reason is due to the
limitation of assay methods, such that very low concentrations of AmB excreted over
a prolonged period may be undetectable. This undetectable drug in biological
samples is likely to be the portion of the missing dose. The hypothesis of prolonged
tissue storage of AmB agrees with this observation.
The renal clearance has been found to account for 5 % of creatinine
clearance, similar to the result reported by Aktinson. Systemic clearance of AmB
was about 3 ml/min*kg, and was found to be dose-independent over the dose range
studied. This is consistent with the preliminary results from this lab and results
reported by Chabot (1989).
However, renal clearance was found to decrease
significantly from 0.80 ml/min* kg to 0.4 ml/min* kg as dose increased. The renal
excretion of drugs usually involves three processes: glomerular filtration, renal
tubular secretion, and passive or active reabsorption from the renal tubular lumen.
Glomerular filtration is a passive process and may be assumed to be a function of
the free concentration of drug in plasma, if the glomeruli are intact. Renal tubular
secretion is an active and saturable process because it needs carriers and energy to
accomplish transport. Renal tubular reabsorption of most drugs involves passive
diffusion of unionized molecules from the renal tubular lumen. Therefore, the rate
of reabsorption is proportional to the concentration gradient of diffusible drug across
97
the renal tubular boundary, and may be affected by urine flow rate and by the urine
pH. Renal clearance from our study was found to be similar or even lower than
glomerular filtration ( product of unbound fraction of drug obtained from the
literature and GFR ) depending on the unbound fraction used to calculate
glomerular filtration. This comparison dose not provide a clear indication of the
mechanisms responsible for renal excretion of AmB.
The alteration of renal clearance can result from a number of underlying
mechanisms. First, one may consider alteration in the glomerular filtration process.
Glomerular filtration may be affected by both the integrity of the nephron and
plasma protein binding.
Impairment of renal function after high dose AmB
treatment is a possible explanation for the observed dose-dependent renal clearance.
Signs of renal impairment noted in our study, by measuring creatinine clearance,
tends to support the above hypothesis.
Alterations in plasma protein binding are
known to affect glomerular filtration and renal clearance. Renal clearance usually
increases when plasma protein binding decreases.
For example, progressive
saturation of protein binding at high drug concentration can increase the unbound
fraction and consequently, increase renal clearance.
However, the direction of
alteration in renal clearance does not agree with the observed results, suggesting a
minimal contribution from this process. A second consideration is the existence of
active renal tubular secretion. The dose-dependent renal clearance can also be
explained by the saturation of the active secretion process at high AmB
98
concentrations.
A plot of renal clearance versus plasma amphotericin B
concentration results in a profile characteristic of a saturable active secretion process
(Figure 20).
Finally, there is the possible existence of tubular passive reabsorption. There
are no known alterations in urine flow rate and urine pH as the AmB doses
increased (Figure 24). Thus, alterations in the reabsorption process are least likely
to be responsible for the dose-dependent renal clearance.
Because renal clearance is a small part (10-20 %) of systemic clearance, the
change in renal clearance will not dramatically affect total body clearance.
5.2 Conclusion
The most important conclusion of this research is the observation that
elimination half-life, volume of distribution, and systemic clearance of amphotericin
B are dose-independent in the rat. However, the dose-dependent renal clearance
may be due to either the nephrotoxicity associated with AmB or saturable active
secretion.
5.3 Suggestions for Further Work
Further work may emphasize (1) characterizing the distribution, metabolism
and elimination of amphotericin B (2) understanding factors affecting the disposition
kinetics of Amphotericin B (3) examining amphotericin B-associated nephrotoxicity
99
as a function of dose, dose rate and disease states.
100
APPENDIX A
Serum and urine AmB concentration versus time data after administration of
AmB.
Table A.l
AmB Concentration (jLtg/ml) vs Time
( Low Dose )
Dose la
Dose 2b
Dose 3°
Dose 4d
Dose 5e
40
0.091
0.077
0.128
0.139
0.165
370
0.049
0.041
0.070
0.069
0.095
460
0.055
0.046
0.049
0.102
0.072
560
0.049
0.045
0.069
0.095
0.081
840
0.029
0.049
0.039
0.051
0.040
1420
0.026
0.039
0.023
0.017
0.027
1930
0.017
0.026
0.012
0.014
0.011
Time
(min)
323.7
: 351.8
439.5
: 395.3
e: 391.2
Mg/kg
Mg/kg
/ig/kg
Mg/kg
Mg/kg
Table A.2
Time
(min)
Dose 1
1220.0 M9/kg
AmB Concentration ( n q / m l ) vs Time
( Medium Dose )
Dose 2
1500.0 Mg/kg
Dose 3
1672.2 jLtg/kg
Dose 4
900.2 (ig/kg
40
0.411
0.340
1.160
0.198
370
0.154
0.119
0.222
0.081
460
0.129
0.134
0.208
0.098
560
0.111
0.122
0.180
0.098
840
0.051
0.098
0.101
0.097
1420
0.071
0.072
0.066
0.075
1930
0.030
0.073
0.043
0.067
2300
0.051
0.038
0.047
0.059
3035
0.024
0.030
0.022
0.031
Table A.3
Time
(min)
Dose 1
3782.6 /ig/kg
AmB Concentration (|zg/ml) vs Time
(High Dose)
Dose 2
4145.9 /xg/kg
Dose 3
4989.5 /xg/kg
Dose 4
3921.9 Mg/kg
40
1.410
3.020
3.360
1.350
370
0.532
0.464
0.907
0.505
460
0.448
0.567
0.882
0.499
560
0.323
0.475
0.595
0.415
840
0.219
0.229
0.271
0.263
1420
0.105
0.141
0.184
0.153
1930
0.109
0.126
0.117
0.130
2300
0.072
0.121
0.088
0.122
3035
0.054
0.027
0.047
0.033
3640
0.032
0.033
0.065
0.026
104
Table A.4
DOSE
(ug/kg)
3782.6
Urine Amphotericin B Concentration versus Tims Data
TIME
(min)
0-363
363-547
547-798
798-1359
1359-2018
2018-2279
2279-3006
3006-3614
3614-4655
4655-5175
5175-6131
6131-8048
VOLUME
(ml)
EXC. RATE
(ug/min)
10
12.5
12
13
6
5
8
8
12
5
8
11
0.037
0.056
0.043
0.027
0.014
0.016
0.006
0.006
0.003
0.002
0.0008
0.0004
0-362
362-549
549-801
801-1363
1363-2023
2023-2281
2281-3008
3008-3616
3616-4655
4655-5177
5177-6133
6133-8053
8
8
12
16
8
4
10
8
12
2
10
13
0.038
0.054
0.047
0.022
0.018
0.018
0.011
0.009
0.004
0.003
0.002
0.0005
0-348
348-546
546-798
798-1360
1360-2020
2020-2277
2277-3013
3013-3610
3610-4650
4650-5169
5169-6127
6127-8046
7.5
8
16
19
12
5
10
6
14
8
12
22
0.043
0.097
0.085
0.038
0.031
0.028
0.012
0.012
0.008
0.008
0.004
0.002
4145.9
4989.5
3921.9
0-346
346-600
600-801
801-1364
1364-2020
2020-2279
2279-3002
3002-3608
3608-4651
4651-5166
5166-6126
6126-8042
7.5
12
5
9
7.5
3
11
5.5
8.5
1.5
5
10
0.042
0.059
0.030
0.014
0.017
0.013
0.009
0.005
0.002
0.001
0.001
0.0003
0-395
395-573
573-880
880-1448
1448-2273
2273-3069
3069-3690
3690-4529
4529-5731
8
4
4
6
8
10
8
10
12
0.021
0.023
0.011
0.006
0.006
0.003
0.002
0.001
0.0005
0-390
390-605
605-899
899-1447
1447-2272
2272-3067
3067-3689
3689-4530
4530-5730
10
9.5
9
10
13
12
9
12
20
0.017
0.036
0.026
0.011
0.011
0.007
0.005
0.001
0.001
0-406
406-598
598-812
812-1475
1475-2138
2138-2519
2519-3315
3315-3884
3884-4877
4877-6771
19
7
4
8
3.8
3
4.5
3
8.2
12
0.049
0.044
0.033
0.018
0.016
0.013
0.009
0.007
0.005
0.002
1220.0
1550.0
106
900.2
0-404
404-597
597-809
809-1475
1475-1890
1890-2275
2275-3067
3067-3635
3635-4630
4630-6525
10
3.5
3
13
6
4
10.5
4.5
7
26
0.026
0.024
0.017
0.014
0.010
0.008
0.006
0.004
0.001
0.001
0-407
407-591
591-814
814-1474
1474-1891
1891-2273
2273-3072
3072-3640
10
3
3.8
11.5
3.5
4.8
20
11
0.014
0.009
0.013
0.005
0.002
0.002
0.002
0.001
0-407
407-594
594-813
813-1475
1475-1890
1890-2273
2273-3070
3070-3635
11
4.5
3
9
3
4
7.5
5.2
0.016
0.015
0.013
0.011
0.006
0.004
0.001
0.0007
0-360
360-610
610-809
809-1471
1471-1986
1986-2276
6
4.7
2
6
3.5
2.5
0.009
0.015
0.007
0.003
0.0006
0.0004
0-363
363-612
612-810
810-1472
1472-1986
1986-2281
2281-3014
7
6.5
3.5
9
3.5
2
7.5
0.009
0.013
0.015
0.006
0.002
0.002
0.0004
351.8
107
391.2
0-365
365-613
613-811
811-1474
1474-1987
1987-2282
2282-3015
6
4
4
10
4
4
10
0.014
0.017
0.007
0.008
0.003
0.002
0.0005
108
APPENDIX B
Serum AmB concentration versus time plots in rats after administration of
- .52 5 /
0.100
•• •
c
o
CJ
CD
E
<
0 010
500
1500
1000
2000
2500
Dose=351 8 £ig/kg
0.100 --
••
0.010
500
1000
1500
2000
2500
Time (min)
O
VO
1 000
l)ose = 395 -5 /jg/kg
0 100
0.010 -
0 001
500
1000
2000
2500
1.000
Dose=391.2 ng/kg
0 100
0.010
I—
0.001
500
1000
Time (min
2000
2500
AmB Concentration
III
(/ig/ m l )
1 000
Uose= 1220 /zg/kg
~
0 100
—I.
0010
500
1000
1—
1500
2000
-I
2500
I
1
3000
3500
4000
1.000
Pose= 1500 0 //g/t-g
-
o
0 100
0.010
500
1000
1500
2000
lime (min)
1
1
1
2500
5000
3500
4000
AmB Concentrotion (/xg/ml)
£11
AmB Concentrotion (/xg/ml)
10 000
f>ose = 3782 6 /jg/kg
1.000
0.100 - -
—I
0.010 -
500
1000
1500
2000
f
3000
3500
4000
10.000
Dose-4145 9
itq/Uq
1.000 --
•••
0.100 --
0.010
500
1000
1500
2000
Time (niin)
3000
3500
4000
10.000
Dose=4989 5 ng/kg
1.000
•*
0.100 --
0.010
500
-I
(-
1000
1500
2000
2500
3000
3500
4000
10.000
Dose=3921.9
/aq/kg
1.000
0.100
0.010
500
1000
1500
2000
Time (min)
2500
3000
3500
4000
116
APPENDIX C
Serum AmB concentration versus time fitted curves generated by program
ADAPT n.
10.0-q
Dose=323.7 ug/kg
1.0-
3
C
o
•H
•P
J"
o. i -
•P
C
<D
o
c
8
o.oi-
ffl
e
<
1. O E - 3
11
|| | l I I I I I | I I I I I I I I I |I I I I I I I I I| I I I I I I I I I | I I I I I I I I I |
0.50k
1.00k
l.SOk
2.00k
2.SOk
Time (min)
10.0-q
3
l. o-d
C
o
•P
00
o.i-
Dose=351.8 ug/kg
•H
•p
c
a)
o
c
o
O
o.oi -
CQ
I
1•OE-3 "I I I I I I I I I | I I I I I 1 I I I | I I I I I I I I I | I I b • i i i i i | • i i (• i i J I |
0.50k
1.00k
1.50k
2.00k
Time (min)
Note: symbols represent the observed data,
line represents the fitted curve generated
by ADAPT II.
2.60k
AmB concentration (ug/ml)
AmB concentration (ug/ml)
O
O
H
""i
O
H
H
O
H
O
O
\
K)
cno
AmB concentration (ug/ml)
J
' ' ' "ill
i
• • i mil
|
I
l
i l i mil
c
8
120
10.0-^
Dose=1220.0 ug/kg
1.0-
0 .1-
0 . 01 -
. 0E-3
11111111111111 11 11|I IItII I I 11lll11 I I I 11I Il11 II I I11 I I I 11 I I I 11 II I I i I l I |
0.50k
1.00k
1.50k
2.00k
2.50k
3.00k
3.50k
Time (min)
10 0 —i
Dose=1500.0 ug/kg
1.0-
o. i-
1111111111
0 . 50k
1111111 n 111H1111111 u 111111 u 11} 111111111111111111 * I
1.00k
1.50k
2.00k
Tim© (min)
2.50k
3.00k
3.50k
10. 0 -a
Dose=1672.2 ug/kg
1 . 0-=
0.1-
1 'I
I I I I I I 1 I | I I I I I I I I I| I I I I I I I I I I I I
0.50k
1.00k
i M I |) M I 1 I I I I |
1.50k
3.00k
3.50k
Time (min)
10.0-q
Dose=900.2 ug/kg
1.0-
0 .1-
0 . 01 -
l.OE-3
0. 50k
1.00k
1. 50k
2.00k
Time (min)
2.50k
3.00k
3.50k
AmB concentration (ug/ml)
AmB concentration (ug/ml)
O
K
K
O
M
O
O
CO
AmB concentration (ug/ml)
AmB concentration (ug/ml)
K
O
O
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H
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3
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124
REFERENCE:
Andreoli, T.E., "On the Anatomy of Amphotericin B Cholesterol Pores in Lipid
Bilayer Membranes." Kidney Int., Vol.4, pp.337-345, 1973.
Atkinson A.J., J.E. Bennett, "Amphotericin B Pharmacokinetics in Humans".
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