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Synthesis of carboplatin and studies on its biotransformation products

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Synthesis of carboplatin and studies on its biotransformation products

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SYNTHESIS OF CARBOPLATIN AND STUDIES ON ITS
BIOTRANSFORMATION PRODUCTS
by
Darshana Palekar
A Thesis Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
Master of Science
(Pharmaceutical Sciences)
August 1996
Copyright 1996 Darshana Palekar
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UNIVERSITY O F SO U TH ER N CALIFORNIA
TH E GRADUATE SCHOOL
UNIVERSITY PARK
LOS ANGELES. CALIFORNIA 9 0 0 0 7
This thesis, written by
Darshana Palekar
under the direction of hJzC . Thesis Committee,
and approved by all its members, has been pre
sented to and accepted by the Dean of The
Graduate School, in partial fulfillment of the
requirements for the degree of
Master o f Science
Dtum
Tint, J u ly 31, 1996
THESIS CO,
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ACKNOWLEDGEMENTS
I would like to take this opportunity to express my sincere thanks and
appreciation to my advisor Dr. Walter Wolf for his thoughtful discussion, scientific
advice and prudent judgement during the course of this work. I am thankful to the
Department of Pharmaceutical Sciences for giving me a chance to undertake this
research. I am also grateful to Dr. Eric Lien and Dr. Manbir Singh for serving on my
committee.
I am thankful to Mr. Shijun Ren for his thoughtful discussion on the synthesis
of carboplatin. I am grateful to my friends and colleagues for their constant support
during the course of this work. I am also grateful for the constant encouragement and
moral support of my husband, parents and brother. Finally I am thankful to the
Almighty for His kind blessings.
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This thesis is dedicated to my parents and my dear husband, Vishwesh, whose
constant encouragement and support made this experience rewarding and enjoyable.
iii
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TABLE OF CONTENTS
Eagg-ftoi
ACKNOWLEDGEMENTS....................................................................................... ii
LIST OF TABLES..................................................................................................... v
LIST OF FIGURES................................................................................................... vi
ABSTRACT.............................................................................................................. viii
INTRODUCTION...................................................................................................... 1
Cancer and Chemotherapy................................................................................ 1
Platinum Antitumor Agents.............................................................................. 3
Chemistry................................................................................................. 3
Pharmacology........................................................................................... 7
Pharmacokinetics..................................................................................... 9
Biotransformation Products................................................................. 14
Mechanism of Action............................................................................ 17
Radiolabeling of Carboplatin.................................................................. 23
PURPOSE OF STUDY............................................................................................. 25
SPECIFIC AIMS....................................................................................................... 26
MATERIALS AND METHODS............................................................................... 27
Study of Reaction Kinetics............................................................................... 27
Microscale Synthesis of Radiolabeled of [1 9 5 m Pt]-Carboplatin........................ 29
Study of the Interaction of Carboplatin and Cisplatin with Guanosine 37
RESULTS AND DISCUSSION................................................................................ 39
Study of Reaction Kinetics............................................................................... 39
Microscale Synthesis of Radiolabeled of [I9 5 m Pt]-Carboplatin........................ 45
Study of the Interaction of Carboplatin and Cisplatin with Guanosine 53
Carboplatin Stability in Aqueous Medium............................................. 54
Carboplatin Stability in Isotonic Saline (0.9%)...................................... 54
Interaction of Cisplatin with Guanosine in Isotonic Saline (0.9%)........ 58
Interaction of Carboplatin with Guanosine in Isotonic Saline (0.9%).. 64
Interaction of Carboplatin with Guanosine in Aqueous Medium 69
Proposed Model for the Activation of Carboplatin................................ 70
iv
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Page No.
Calculation of Log P for Carboplatin.................................................... 73
SUMMARY............................................................................................................ 74
BIBLIOGRAPHY................................................................................................... 76
APPENDIX............................................................................................................. 82
V
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LIST OF TABLES
Table No.
1. Retention Times for Various Compounds on the Phenyl Column
used in HPLC Assay
2. % Yields for the Conversion of cis-Pt(NH3 )2 l2 to Carboplatin
Measured by HPLC as a Function of time
3. % Radiochemical Yields and Losses During the Synthesis of l9 5 m Pt-
carboplatin
4. Retention Time for Various Compounds on a Phenyl Column
5. Relative Amounts of Various Products Obtained During the
Interaction of Cisplatin with Guanosine in Isotonic Saline (0.9%) at
37°C
6. Relative Amounts of Various Products Obtained During the
Interaction of Carboplatin with Guanosine in Isotonic Saline (0.9%)
at 37°C
7. Log P Values of Some Platinum Complexes
Page No.
40
44
46
53
62
68
73
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LIST OF FIGURES
Figure Page
No. No.
1. Chemical Structures of Carboplatin and Cisplatin 4
2. Substitution Reactions of Carboplatin with Various Nucleophiles 6
3. Subcellular Distribution (% Injected Dose/gm) of 1 9 5 m Pt in Various
Components of the Tumor Following an IV Bolus Dose of 15mg/kg of
[1 9 5 m Pt]-carboplatin (Dowell, 1995) 13
4. Bifunctional Adducts of Platinum Analogs with DNA 18
5. Structure of the Bisubstituted Product Formed During the Interaction of
Carboplatin with Guanosine Involving the N7 Position of Guanosine 19
6. Reaction Steps Involved in Method (i) and (ii) for Carboplatin Synthesis
28
7. Simplified Schematic of the Semi-automated Setup for the Radioactive
Synthesis of [l9 S m Pt]-carboplatin 31
8. Outline of the Critical Steps Involved in the Synthesis of [1 9 5 m Pt]-
carboplatin 32
9. Typical HPLC Analysis Profile for the Reaction Mixture of Method (i) 41
10. % Yield at Various Time Points for the Methods (i) and (ii) 43
11. Thin Layer Chromatographic (TLC) Analysis of [I9 5 m Pt]-carboplatin on
Silica Gel Plates 47
12. UV Spectrum of [,9 S m Pt]Na2 PtCl6 in 1M HC1 50
13. HPLC Profile of Carboplatin in Isotonic Saline (0.9%) at 37°C 56
14. Formation of Cisplatin from Carboplatin in Isotonic Saline (0.9%) at
37°C 57
vii
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Figure Page
No. No.
15. Possible Pathways for the Interaction of Cisplatin with 2 eq. Guanosine 58
16. HPLC Profile for the Interaction of Cisplatin with 2 eq. Guanosine in
Isotonic Saline(0.9%) at 37°C 60
17. Relative Amounts of the Interaction Products of Cisplatin with 2 eq.
Guanosine in Isotonic Saline (0.9%) at 37°C 61
18. Possible Pathways for the Interaction of Carboplatin with 2 eq.
Guanosine 65
19. HPLC Profile for the Interaction of Carboplatin with 2 eq. Guanosine in
Isotonic Saline (0.9%) at 37°C 66
20. Relative Amounts of the Interaction Prodcuts of Carboplatin with 2 eq.
Guanosine in Isotonic Saline (0.9%) at 37°C 67
21. Proposed Model for the Activation of Carboplatin 72
viii
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ABSTRACT
Carboplatin is one of the most promising second generation analog of the
antitumor agent cisplatin. The use of radiolabeled [1 9 S m Pt]-carbopIatin for non-invasive
studies has generated considerable interest to help understand the localization of
carboplatin at the tumor site. The use of [l9 5 m Pt]-carbopIatin for these studies requires
a suitable quality controlled synthesis procedure for the manufacture of the
radiopharmaceutical in high yields under reproducible conditions. The reaction kinetics
of two syntheses routes, namely (i) reaction of cis-diammine diaquo platinum (II)
sulfate and the barium salt of 1,1-cyclobutane dicarboxylic acid and (ii) reaction of cis-
diammine diaquo platinum (II) nitrate and 1,1-cyclobutane dicarboxylic acid were
compared to optimize the synthesis of [I9 5 m Pt]-carbopIatin. Studies on the reaction
kinetics of these two methods of synthesis revealed method (i) to be considerably faster
with chemical yields in excess of 72% within the first five hours in comparison to
method (ii), thus making method (i) highly suitable for [l9 5 m Pt]-carboplatin synthesis.
This improved method was then adapted for the microscale synthesis of [1 9 5 m Pt]-
carboplatin to obtain high yields (greater than 60%) of the radiolabeled compound. The
increased yields are a direct consequence of the reduction of the chemical losses (from
30% to 16%), associated with the conversion of cis-Pt(NH3)2l2 to carboplatin. This
improved semi-automated method can now be used routinely for the microscale
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synthesis of [1 9 5 m Pt]-carbopIatin, under reproducible conditions with minimal radiation
exposure to personnel.
The antitumor action of carboplatin is believed to be related to its ability to
form bifunctional lesions between various DNA bases, with a greater preference for the
N7 position of the guanine base on DNA. However, the species responsible for
interacting with DNA is hitherto unknown. In as much as conversion to cisplatin is a
possibility, stability of carboplatin in isotonic saline and its interaction with guanosine
were studied to help gain a better understanding of its mechanism of action. Studies on
the biotransformation products of carboplatin in the presence of isotonic saline revealed
the presence of cisplatin as the only product as determined by high performance liquid
chromatography (HPLC). HPLC analysis of the interaction of carboplatin and cisplatin
with guanosine in isotonic saline at 37°C revealed that both carboplatin and cisplatin
form the same adducts with guanosine namely, the mono (Cl-Pt-G) and bisubstituted
(G-Pt-G) complexes. However, the interaction of carboplatin with guanosine was very
slow as compared to cisplatin and in the presence of high [Cl' ] was found to be
mediated via cisplatin. Based on these results, it is proposed that the activation of
carboplatin in vivo proceeds primarily by the conversion of carboplatin to cisplatin
which on diffusion into the cell form the more reactive aquo species that interacts with
DNA.
X
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INTRODUCTION
Cancer and Chemotherapy
Cancer encompasses a large group of diseases characterized by uncontrolled
growth and spread of abnormal cells. These cells may grow into masses of tissue
called tumors. Some tumors are benign, while others are malignant (cancerous). In
the beginning, cancer cells usually remain at the original site and the cancer is said to
be localized. Later cancer cells can metastasize, either by direct extension of growth
or by cells becoming detached and carried through the lymph or blood systems to
other parts of the body. Metastasis may be regional - confined to one region of the
body - when cells are trapped by lymph nodes. If left untreated, however, the cancer is
likely to spread throughout the body. This condition is known as advanced cancer and
usually results in death.
It is estimated by the American Cancer Society that about 547,000 Americans
now living, will die of cancer averaging about 1500 people per day. In 1995 alone,
eight million people living in the US were diagnosed as having cancer, with an
estimated one million new cases (Cancer Facts and Figures, 1995).
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The cure for cancer aims at early detection, followed by treatment. Along with
surgery and radiation therapy, chemotherapy plays an important role in cancer
treatment. Chemotherapeutic agents are believed to destroy cells according to first
order kinetics, implying that the same proportion of cells is killed for each dose of the
agent (Malpas, 1991). There are currently 42 FDA approved antineoplastic drugs,
which can be used alone or in combination with other agents, to treat a wide variety of
neoplasms. While a majority of these agents act by inhibiting synthesis of nuclear
materials, such as DNA or RNA, others block key enzymes involved in the synthesis
of various DNA bases. Based on their mechanisms of action, these agents are broadly
grouped into alkylating agents, antimetabolites, plant alkaloids, antibiotics, enzymes,
hormones and hormone inhibitors. The effectiveness of chemotherapy to a large
extent depends on the type of cancer, its sensitivity to the selected agent and the
relative amount of drug reaching the target site. In addition, knowledge of
pharmacokinetics and the biotransformation products of the drug in vivo are essential
for the success of the chemotherapeutic regimen.
2
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Platinum Anti-tumor Agents
Complexes containing platinum as the central atom have acquired significant
importance because of their activity as anticancer agents. Platinum antitumor
complexes were first discovered by Rosenberg (Rosenberg et al., 1969) in 1969, when
he observed inhibition of the growth of E. coli cells in a medium containing platinum
electrodes. The first platinum compound to be used clinically for its antitumor activity
was cisplatin (Figure 1). Although, cisplatin was effective against a wide range of
tumors, it exhibited significant nephrotoxicity. Since then about 200 different analogs
of cisplatin have been synthesized, in an effort to reduce the side effects associated
with the use of the parent drug. Of these, carboplatin is the only analog currently in
clinical use, with an antitumor spectrum similar to cisplatin while exhibiting a lower
toxicity profile.
Chemistry
Carboplatin [(l,l-cyclobutanedicarboxylato)diammine platinum (II)] (Figure
1) is a coordination complex, containing a central platinum atom bound to two
ammonia ligands and a bidentate cyclobutane dicarboxylate group (instead of the two
chloro groups of cisplatin). Of the two sets of ligands, the ammonia ligands are inert
3
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substitution, unless the nitrogen-platinum bonds are activated by sulfur coordinated to
platinum at the tram position. (Jennerwein et al., 1995). In contrast, the bidentate
cyclobutane dicarboxylate ligand (CBDCA) (see Figure 1) is relatively labile and can
undergo nucleophilic substitution with various nucleophiles present in biological
fluids.
Although the bidentate CBDCA can undergo nucleophilic substitution, it
displays relatively lower reactivity compared to the Cl' ligands in cisplatin. Borch
(1987) has shown that the rate of hydrolysis of carboplatin in water is two orders of
magnitude slower as compared to the hydrolysis of cisplatin. Thus, the replacement of
the chloro ligands of cisplatin by the six membered CBDCA, as in carboplatin,
enhances its chemical stability and results in reduced nephrotoxicity and ototoxicity
characteristics. The lower reactivity of carboplatin with the nucleophilic sites on DNA
0
I I
0
Carboplatin
Cisplatin
Figure 1: Chemical Structures of Carboplatin and Cisplatin
4
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may also account for the higher dose needed to obtain an antitumor effect similar to
cisplatin.
In comparison to the substitution reactions of cisplatin with various
nucleophiles, limited data is available on the interaction of carboplatin with
nucleophiles commonly present in biological fluids. Carboplatin has been shown to be
relatively stable in aqueous solutions, although Harland et al. (1984) believe that the
CBDCA ligand is replaced with one or more water molecules to produce a reactive
aquated platinum species. There is also evidence that carboplatin may undergo
nucleophilic reactions with chloride ions resulting in conversion to cisplatin (Cheung
et al., 1987). These various substitution reactions are shown in Figure 2. Other
nucleophiles present in blood including thiol and thiocyanate groups present in amino
acids and proteins could also react with carboplatin by nucleophilic substitution. The
reaction of carboplatin with these various species will depend upon the concentration
of these various nucleophiles and their individual reaction rates. These reactions are
important as the resultant platinated species would be expected to differ in their
pharmacokinetic behavior and their reactivity with biomolecules in vivo.
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o
i!
h 3N \ / O —
/ p\
H3N / o
Carboplatin
H 3N X / C l
cr
h 2 o
R-SH
h 3n
Pt
/ \
Cl
(reactive)
H3N \ / B 2 °
h3n
Pt
/ \
H jO
Amino Acids (AA)
Proteins
(reactive)
R - S - P t - S - R
(inactive)
\
/
Pt AA
(Inactive)
\
I Pt Proteins
(Inactive)
Figure 2: Substitution Reactions of Carboplatin with Various Nucleophiles
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Pharmacology
Carboplatin has an antitumor spectrum similar to cisplatin. It appears to be
active in the treatment of ovarian cancer and germ cell tumors and moderately
effective in bladder cancer, squamous cell carcinoma of the head, neck and small cell
lung cancer (Van der Vijgh, 1991). Its activity is uncertain in the case of cervical
cancer, acute leukemia, soft tissue sarcoma and breast cancer (Muggia, 1989).
Carboplatin is administered either as a single bolus iv injection at four weeks
interval (Motzer et al., 1987) or 30-60 min. infusion repeated every four weeks
(Cantwell et al., 1986). Intraperitoneal administration has also been used in tumors
restricted to the peritoneal cavity. In the USA, it is recommended that carboplatin be
administered at an initial dose of 360 mg/m (Wagstaff et al., 1989). Subsequent
doses should be adjusted according to the nadir of the WBC and platelet counts.
Doses should be reduced in patients with renal impairment or at risk of
myelosuppression. When administered orally, it shows poor bioavailability and severe
gastrointestinal toxicity (nausea and vomiting).
Whilst the therapeutic profile of carboplatin appears to be quite similar to
cisplatin, the toxicity profile of the two drugs are significantly different. Near its
maximum tolerated dose (MTD) of 400 mg/m2 , carboplatin is less nephrotoxic,
7
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neurotoxic, ototoxic and emetogenic as compared to cisplatin as it approaches its own
MTD of 100 mg/m2 (Canetta et al., 1988). At a dose of 400 mg/m2 of carboplatin
myelosuppression, predominantly thrombocytopenia is often dose-limiting.
Leucopenia and anemia also occur with relatively high frequency, but are usually less
severe. Carboplatin induced thrombocytopenia and leucopenia appear to be dose
related in severity and are reversible following cessation of carboplatin therapy (Bacha
et al., 1986; Rozencweig et al., 1983).
In the course of phase I and II clinical trials, occasional cases with reversible
increase in serum creatinine or decrease in glomerular filtration rates have been
reported, but in most trials significant nephrotoxicity did not occur. At carboplatin
doses of upto 450 mg/m , Tait et al. (1988) observed no deterioration of renal function
in patients with renal cancer. However, at doses exceeding 1000 mg/m2 , reversible
renal dysfunction and hepatotoxicity as well as ototoxicity become apparent (Shea et
al., 1989). Nausea and vomiting occurs in most patients receiving over 120 mg/m2 of
carboplatin, but seldom persists over 24 hours and is easily managed with standard
antiemetics. Some of the other side effects associated with carboplatin use include
abnormal liver function tests, skin rashes, hypersensitivity, stomatitis, murositis, and a
flu-like syndrome (Wagstaff et al., 1989).
8
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Pharmacokinetics
The blood pharmacokinetics of carboplatin in humans has been studied
following a variety of schedules and over a range of doses (20-500 mg/m2 ). Following
iv administration of carboplatin, the plasma clearance of ultrafilterable platinum
follows a bi-exponential curve, with a distribution half life in plasma of 23 minutes
and an initial drug elimination half-life of 120 min. (Elferink et al., 1987). The
terminal half-life of platinum in blood is 5.8 days, representing platinum that has been
irreversibly bound to plasma proteins.
Carboplatin once introduced into the living system can undergo reaction with
various nucleophiles such as amino acids, peptides and proteins commonly found in
blood. The blood pharmacokinetics of carboplatin, therefore includes not only the
parent compound but also ultrafilterable and total platinum. Ultrafiterable platinum
represents free carboplatin and non-protein bound platinum species, while total
platinum represents the sum of protein bound and non-protein bound platinum.
Elferink et al. (1987) have shown that the plasma concentration-time curves for
carboplatin, ultrafilterable platinum and total platinum in plasma are quite similar over
the first 6 hrs., but beyond that, the concentration-time curve for total platinum is
always higher than that for carboplatin and ultrafilterable platinum. This implies that
protein binding occurs slowly and only to a small extent, as would be expected based
9
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on the chemical reactivity of carboplatin. The binding of platinum to plasma proteins
is essentially irreversible, and this platinum is therefore not available to exert any
pharmacological activity (Cole et al., 1980). The elimination of platinum bound to
these molecules is believed to be through protein turnover (Vemorken et al., 1978).
Thus, in comparison to cisplatin, carboplatin has significantly lower reactivity
both in vivo and in vitro, presumably as a result of the bidentate CBDCA ligand which
holds the platinum in a stable six-membered ring. This influences its biodistribution
and pharmacokinetic properties in-vivo. Van der Vijgh et al. (1986) found that
carboplatin reacts with plasma proteins in vitro with a half-life (tI/2 ) of approx. 48.6
hours, in comparison to 1.7 hours for cisplatin. Similarly, in vivo, the plasma free
platinum half-life after iv administration of carboplatin was reported to be 23 min. as
compared to 6 min. for cisplatin (Elferink et al., 1987; Vermorken et al., 1984).
Studies on the tissue distribution of carboplatin have been performed in
humans (Newell et al., 1987) and mice (Siddik et al., 1988). Following an iv bolus
dose of 80 mg/kg in mice, significant accumulation of platinum was observed in
kidneys, liver, ileum, skin, ovaries, uterus and bile at the end of 4 hours (Siddik et al.,
1988). Studies on the tissue distribution of carboplatin in patients treated with high
doses of carboplatin (1600 mg/m ) showed highest concentration in the liver, kidney,
10
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skin and tumor at the end of 14 days (Newell et al., 1987). Interestingly, platinum
levels were found to be greater in the tumor than in the corresponding normal tissue.
Siddik et al. (1986) have studied the subcellular distribution of carboplatin in
the kidneys of mice and rats. They observed that three days after carboplatin
administration, the highest amount of platinum (65-77 %) was localized in the cytosol,
15-16 % in the mitochondrial fraction and 2-4% in the microsomal fraction. Platinum
concentration in the nuclear fraction was greater in the mouse (16 %) than in the rat (4
%).
Dowell (1995) studied the subcellular distribution of radiolabeled l9 5 m Pt-
carboplatin in tumored Sprague Dawley rats bearing the walker 256 carcinosarcoma.
The animals were administered an iv bolus dose of carboplatin (15 mg/kg). At various
time points, the animals were sacrificed and the tumor tissue excised. The tumor
tissue was suitably processed and separated into four fractions: (i) free drug + low
molecular weight proteins (LMW) (ii) high molecular weight proteins (HMW) (iii)
chromosomal proteins (CP) (iv) DNA. The individual fractions were assayed for
l9 5 m Pt using a gamma counter. Figure 3 shows the subcellular distribution of l9 5 m Pt as
% Injected Dose (ID)/ gm of tumor tissue at various time points. The total amount of
l9 5 m Pt in the tumor was relatively low (< 2%). At initial time points (5 and 15 min.),
the highest amount of 1 9 5 m Pt was present in the cytosolic fractions (free + LMW and
11
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HMW proteins). In the nuclear fractions, a greater amount of l9 5 m Pt was bound to the
chromosomal proteins as compared to DNA. Between 60 and 240 minutes (see Figure
3), the measured i9 5 m Pt concentration in the nuclear fraction increased relative to the
cytosolic fraction. This suggests that chromosomal proteins may influence the
cytotoxicity of carboplatin, although its exact role is not clear at the present time.
Carboplatin is predominantly excreted via the kidneys. Most platinum was
excreted during the first 6 hours, with about 77% of the administered dose present in
the urine at 24 hours in patients without severely impaired renal function (GFR > 60
ml/min.). At least 50% of the excreted platinum was found to be in the form of intact
carboplatin. Egorin et al. (1984) and Harland et al. (1984) have shown that total body
or renal clearances of plasma free platinum following carboplatin administration
correlate with pre-treatment glomerular filtration rates (GFR) and that the slope of the
regression line does not differ significantly from unity. Thus glomerular filtration is
implicated as the sole mechanism of renal elimination of carboplatin. This is in sharp
contrast to the elimination of cisplatin where tubular secretion of cisplatin or its
metabolite along with glomerular filtration is believed to be involved in the renal
elimination of cisplatin. Since tubular secretion leads to the accumulation of cisplatin
or its metabolite in the renal tubules, this is also believed to be responsible for the
higher nephrotoxicity of cisplatin (Jacobs, 1986).
12
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Figure
0.35
Free + Low Mol. Wt. Proteins
High Mol. Wt. Proteins
Chromosomal Proteins
0.30
0.25
DNA
§>
0.20
< D
c / 1
O
Q
T3
■ < — >
o 0.15
< D
^ 0.10
0.05
-f .-------
0.00
y ----
---▼
50 0 100 150 250 200
Time (min)
3: Subcellular Distribution (% Injected Dose/gm) of I9 5 m Pt in Various
Components of Tumor Following an IV Bolus of 15 mg/kg [I9 5 m Pt]-
carboplatin (Dowell, 1995).
13
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Biotransformation Products
It is believed that the biotransformation of platinum complexes involves simple
ligand exchange reactions rather than enzyme mediated processes. In biological
systems, the extent to which these reactions occur, depends primarily on the affinity
and availability of the substituting ligands. The order of affinity of Pt(II) for
commonly occurring compounds in biologic fluids is: sulfur > nitrogen > chloride >
water, while the order in terms of the concentration is the reverse.
The aquation chemistry of cisplatin has been thoroughly investigated.
Cisplatin undergoes rapid hydrolysis in water to produce a series of aquo species,
formed by the displacement of the chloride ligands by water molecules. Of these, the
diaquo species has been shown to be approximately 1000 times more reactive than
cisplatin and therefore is believed to be more toxic (Daley-Yates et al., 1982). The
extent of hydrolysis depends upon the chloride ion concentration and pH of the
solution. It is believed that at the high chloride ion concentration found in plasma
(103 mM), cisplatin is present predominantly in the neutral form. It is proposed that
the drug enters the cells by passive diffusion in this neutral form. However, after
diffusing into the cells, where the chloride ion concentration is lower (4 mM), it
undergoes hydrolysis to produce the more reactive aquated species which are then
believed to react with DNA, to bring about its cytotoxic action.
14
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While there has been great emphasis on the study of the aquation chemistry of
cisplatin to help explain its antitumor activity and toxicity profile, the presence of
ligands other than water should not be neglected. Plasma contains a wide variety of
substances ranging from amino acids, peptides and proteins to urea, fatty acids and
lipids, many of which contain sulfur and nitrogen moieties capable of reacting with
platinum compounds. Daley-Yates et al. (1983) studied the biotransformation of
cisplatin in vivo using high performance liquid chromatography (HPLC) and reported
eight different biotransformation products of cisplatin in protein free plasma of rats 2
hours after dosing with cisplatin (15 mg/kg ip). Three of these have been identified as
mono and bisubstituted complexes of cisplatin with methionine and cis-diammine-
diaquo-platinum (II), its hydrolysis product. The methionine substitution complexes
have been shown to be devoid of any antitumor and nephrotoxic properties while the
hydrolysis product was shown to contribute to the nephrotoxicity associated with
cisplatin use (Daley-Yates, 1986).
On the other hand, carboplatin by virtue of its CBDCA ligand, is relatively
inert. Studies on the hydrolysis of carboplatin in water show that it is relatively stable
with <1% decomposing over a period of 43 hours (Tinker et al., 1987). However, in
the presence of isotonic saline (0.9%) at 37°C at the end of 24 hours, about 10% of
carboplatin gets converted to a material that behaves chromatographically identical to
15
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
cisplatin (Daley-Yates, 1986). The possibility of a similar conversion of carboplatin to
cisplatin in vivo, by the displacement of the cyclobutane dicarboxylate ligand has been
alluded to by several workers. (Cleare et al., 1980). Tinker et al. (1987) have also
detected the presence of cisplatin in protein free plasma of rats, as early as 30 min.
after dosing with carboplatin (15 mg/kg iv). Due to the lower reactivity of carboplatin
in comparison to cisplatin, it is proposed that subsequent biotransformation products
may be formed via cisplatin. In fact, several workers have alluded to cisplatin as the
active agent responsible for the antitumor activity of carboplatin (Daley-Yates, 1986).
The presence of high chloride ions in human plasma (in vivo) may favor the
conversion of carboplatin to cisplatin and its subsequent biotransformation.
16
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Mechanism of Action
It is widely believed that the antineoplastic activity of platinum compounds is
primarily related to its ability to bind and inhibit new DNA synthesis (Pascoe et al.,
1974; Harder et al., 1976), subsequently leading to cell death. Interaction of platinum
analogs with DNA has been shown to involve the formation of bifunctional crosslinks
between various DNA bases, with a greater preference for the N-7 position of the
guanine base on DNA. These crosslinks can either be between adjacent bases on a
single strand to form an intrastrand crosslink or between bases on adjacent strands to
form interstrand crosslinks (see Figure 4 and 5). Several in vitro and in vivo studies
have been performed to identify the nature of the adducts formed between cisplatin
and DNA. These studies show that the intrastrand N7,N7 d(GpG) and d(ApG)
chelates of the cis-Pt(NH3 )2 2 + moiety are the major platinum-DNA adducts. The
interstrand crosslinks represent less than 1% of the total DNA platination. Kinetic
studies further reveal that bifunctional binding to two guanine residues occurs more
rapidly than binding to guanine and adenine (Fichtinger-Schepman et al., 1985;
Eastman A., 1983; Eastman A., 1986).
17
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d(GpG) Adduct
H3N NH,
\ /
Pt
/ \
5 ' — G — G — 3'
• •
3 ' — c — C — 5'
A
d(GpXpG) Adduct
H3N n h3
/ \
5'— G - X - G — 3'
• • •
3 '— C — X — C — 5'
C
d(ApG) Adduct
H3N I nh3
\ /
Pt
/ \
5 '— A — G — 3'
• •
3 ' — T — C — 5'
B
Interstrand Crosslink
H,N NH,
Pt
- G 7- \ c -
— C — G —
Figure 4: Bifiinctional Adducts of Platinum Analogs with DNA. Lesions indicated in
panels A, B and C represent different intrastrand adducts which together
account for greater than 90% of the total platinum binding to DNA. The
lesion indicated in panel D is the interstrand crosslink which accounts for
less than 5% of total platinum binding to DNA. ( Adapted from Reed et al..
1996)
1 8
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Sugar
(Guanosine)
Figure 5: Structure of the Bisubstituted Product Formed During the Interaction of
Carboplatin with Guanosine Involving the N7 Position of Guanosine
19
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Recent studies on the interaction of carboplatin with DNA in vitro reveals that
carboplatin forms similar bifunctional adducts as cisplatin, with the intrastrand
crosslink cis-Pt(NH3)2 d(pGpG)(Pt-GG) between adjacent guanine residues as the
major product (Blommaert et al., 1995). In addition, small amounts of intrastrand
crosslinks (Pt-AG), between adjacent adenine and guanine residues and (G-Pt-G)
between two guanine residues separated by another base on the same strand were
observed. However, the formation of carboplatin-DNA adducts was very slow as
compared to cisplatin and almost 230-fold more carboplatin than cisplatin (molar
dose) was required to obtain equal levels of platination after 4 hours of incubation
(Blommaert et al., 1995).
Several investigators have tried to correlate the cytotoxicity of platinum
complexes to DNA interstrand and DNA intrastrand crosslinks. These studies have
yielded conflicting results. Some researchers believe the minor interstrand lesion to be
the most cytotoxic lesion (Pascoe, J. M., 1974; Roberts et al., 1987; Zwelling et al.,
1979), while others argue that the DNA intrastrand crosslinks are responsible for the
cytotoxicity of platinum compounds (Brouwer et al., 1981; Bruhn et al., 1990;
Strandberg et al., 1982). Thus, at the present time the DNA-platinum adduct
responsible for the cytotoxicity of platinum antitumor complexes is not known.
20
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A number of studies have been carried out on the reaction pathways involved
in the interaction of carboplatin with DNA (Knox et al., 1986; Itoh et al., 1991; Frey et
al., 1993). Knox et al. (1986) believe that aquation to be the rate limiting step in the
reaction of carboplatin with DNA. However, Itoh et al. (1991) and Frey et al. (1993)
have shown a greater probability for carboplatin reacting directly with guanosine
(Guo), to form the diadduct via a nucleophilic substitution, rather than through the
aquated intermediate. In vitro and in vivo studies have also shown that carboplatin, in
the presence of Cl' ions, is converted to cisplatin, leading to the belief that carboplatin
may be a prodrug for cisplatin (Daley-Yates, 1986; Tinker et al., 1987). It is likely
that one or more of these pathways would be involved in the intracellular reaction of
carboplatin with Guo.
Because of the slow reactions involved in the binding of platinum analogs with
DNA, other intracellular nucleophiles may compete with DNA for reaction. One such
reaction which may compete with DNA binding is platinum binding to glutathione
(GSH), a tripeptide thiol which accounts for the majority of intracellular non-protein
sulfhydryl contents of most cells. The reaction of cisplatin with GSH is believed to be
primarily mediated through the aquated species. While tumors resistant to cisplatin
have shown elevated levels of GSH, its role in tumor resistance to cisplatin remains
controversial. Carboplatin is believed to react with GSH very slowly with a half-life
21
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of approximately 40 hrs. at 5 mM GSH concentration (similar to the intracellular
concentration of GSH) (Wagstaff et al., 1989).
In addition, both cisplatin and carboplatin also bind to nuclear proteins,
cytosolic proteins and mitochondria. However, the significance of the interaction of
platinum analogs with selected target sites other than DNA is not clear at the present
time. Pil et al. (1992) have identified high mobility group protein (HMG1), a nuclear
protein, which binds specifically to DNA containing cisplatin induced d(GpG) or
d(ApG) intrastrand crosslinks. While the exact role of HMG1 proteins in the
cytotoxicity of cisplatin is not known, it is proposed that binding of HMG1 to
cisplatin modified DNA may prevent recognition of the damaged nucleic acid by the
cellular repair mechanism, thereby inhibiting replication and eventually leading to cell
death. Alternatively, since the HMG1 proteins are believed to be involved in gene
transcription, the binding of HMGl to cisplatin-DNA adducts may prevent its
participation in gene transcription leading to internal cellular disarray and cell death
(Pil et al., 1992).
22
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Radiolabeling of Carboplatin
Several studies are currently being designed to define non-invasively, the
localization of carboplatin at the tumor site and subsequently apply pharmacokinetics
to the optimization of drug therapy. Detailed studies of this type are greatly
facilitated, when carboplatin is radiolabeled intrinsically with a radionuclide of
platinum. Of these, l9 S m Pt (t1 /2 = 4.02 days) is an ideal radiolabel for several reasons
(Wolf et al., 1977). Firstly, it emits penetrating gamma radiations (66-77 keV and 99
keV) which provide a high sensitivity of detection and permits measurement of
platinum at low concentrations in whole animals, tissues and in vitro biochemical
samples. Another advantage of 1 9 5 m Pt is that it decays by isomeric transition directly
to the stable isotope 1 9 5 Pt without producing any daughter radioactivities. l9 S m Pt also
has attractive properties for autoradiographic applications, since it emits three auger
electrons per disintegration.
The use of radiolabeled compounds for non-invasive studies requires a suitable
quality controlled synthesis procedure for the manufacture of the radiopharmaceutical
under reproducible conditions. The short half life of I9 5 m Pt (t1 /2 = 4.02 days) makes it
imperative that any synthesis procedure developed be rapid enough, with high yields,
so that the material synthesized can be used for non-invasive measurements. In
23
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addition, radiation exposure of personnel needs to be minimized. Since the original
work of Dhara et al. (1970) for preparing cis-[Pt(NH3 )2Cl2] (cisplatin), several
variations of the synthesis scheme have been adapted to synthesize Pt analogues such
as carboplatin. Baer et al. (1985) reacted Na2 PtI4 in solution with AgN03 to form the
diaquo species, which was then subsequently reacted with 1,1-cyclobutane
dicarboxylic acid (CBDCA) to give carboplatin labeled with l9IPt. Hill et al. (1990)
substituted Ag2S04 for AgN03 and used the barium salt of CBDCA for rapid
precipitation of BaS04 to increase the rate of non-radioactive carboplatin formation.
While these procedures produced carboplatin with high yields, no quantification of the
reaction kinetics of these methods has been reported. As a result, the applicability of
these methods for the rapid synthesis of radiolabeled l9 5 m Pt-carboplatin remains
limited.
24
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PURPOSE OF STUDY
Previous work performed in our laboratory by Anand et al. (1992) has led to
the development of a semi-automated setup and procedure for the synthesis of
[i95 m Pt]-cisplatin. The objective of this work was to develop a suitable procedure for
the routine microscale synthesis of radiolabeled [l9 5 m Pt]-carboplatin using this semi-
automated system. The synthesis was optimized by studying the reaction kinetics for
two procedures for the synthesis of carboplatin, reported by Baer et al. (1985) and Hill
et al. (1990) using HPLC.
The antitumor activity of carboplatin is believed to be related to its ability to
form bifunctional crosslinks with DNA. However the nature of the species reacting
with DNA to form the adducts is unknown at the present time. Studies to help identify
the active species has led to the hypothesis that the high CT ion concentration in
plasma may favor the conversion of carboplatin to the more reactive cisplatin species
and the antitumor action of carboplatin may be mediated via cisplatin. The
biotransformation products of carboplatin in the presence of high CT ion concentration
and the reaction of carboplatin and cisplatin with 2 eq. guanosine in the presence of
water and isotonic saline (0.9%) at 37°C was studied to identify the possible
extracellular and intracellular reaction pathways involved in the interaction of
carboplatin with DNA and its antitumor action.
25
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SPECIFIC AIMS
The specific aims of this study were as follows,
• To study the reaction kinetics for the synthesis of carboplatin by two methods
namely,
(i) reaction of m-diammine diaquo platinum (II) sulfate and the barium salt of
cyclobutane dicarboxylic acid, and
(ii) reaction of c/s-diammine diaquo platinum (II) nitrate and 1,1-cyclobutane
dicarboxylic acid.
• To develop a suitable method for the microscale synthesis of radiolabeled l9 5 m pt-
carboplatin with high yields under reproducible conditions.
• To study the stability and identify the biotransformation products of carboplatin
and its interaction with guanosine.
2 6
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MATERIALS AND METHODS
(A) Study o f Reaction Kinetics - A comparison of the reaction kinetics for
the reaction between (i) cfr-diammine diaquo platinum (II) sulfate and barium
salt of 1,1-cyclobutane dicarboxylic acid and (ii) m-diammine diaquo platinum
(II) nitrate and 1,1-cyclobutane dicarboxylic acid
Reagents and Materials
Carboplatin was obtained from Sigma Chemicals. Uridine, silver sulfate,
barium hydroxide and 1,1-cyclobutane dicarboxylic acid were obtained from Aldrich
Chemicals. All other chemicals were of analytical or reagent grade.
Figure 6 shows the individual steps and reagents used in synthesis methods (i)
and (ii). For method (i), the cfr-diammine diaquo platinum (II) sulfate was prepared
by reacting 0.052 mM cis-Pt(NH3 )2 l2 with slightly less than the stoichiometric amount
of Ag2S04 (0.98 eq.) for 4 hrs., as described by Hill et al. (1990). The barium salt of
1,1-cyclobutane dicarboxylic acid (BaCBDCA) was prepared in situ by reacting 0.057
mM (1.1 eq.) 1,1-cyclobutane dicarboxylic acid (CBDCA) with Ba(0H)2.8H20 (0.98
eq.). The cis-diammine diaquo platinum (II) sulfate and the barium salt of 1,1-
cyclobutane dicarboxylic acid, prepared as described above, were subsequently mixed
at room temperature. The precipitate of BaS04, which forms immediately was
27
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removed by centrifugation and the supernatant was used as the reaction mixture.
Samples of the reaction mixture were withdrawn at regular intervals (up to 46 hours),
suitably diluted and analyzed by automatic injection into an HPLC system.
For method (ii) (see Figure 6), the cis-diammine diaquo platinum(II) nitrate
was prepared by reacting 0.052mM cis-Pt(NH3)2l2 with 2 eq. of AgN03 for 30 min. as
described by Baer et al. (1985). Two variations of method (ii) were carried out by
reacting cis-diammine diaquo platinum(II) nitrate with 0.182 mM (3.5 eq.) and 0.052
mM (1 eq.) of 1,1-cyclobutane dicarboxylic acid (CBDCA) respectively. In each of
these syntheses, the reactants were mixed and allowed to react at room temperature.
Samples were withdrawn at regular intervals (upto 46 hrs.), suitably diluted and
analyzed using HPLC. These are referred to as method (ii) with 3.5 eq. CBDCA and 1
eq. CBDCA respectively.
M e th .Q d _ G )
cis-Pt(NH3 )2 l2 + Ag2 S04 ----------- ► cis-[Pt(NH3)2(H20 )2]2S0 4 + 2AgI |
cis-[Pt(NH3)2(H20 )2]2S0 4 + BaCBDCA----------► carboplatin + BaS04 j
MethoiUifl
cis-Pt(NH3 )2 I2 + 2AgN03 ----------- > cis-[Pt(NH3 )2 (H2 0)2](N03 )2 + 2AgI |
cis-[Pt(NH3 )2 (H20)2 ](N03 )2 + CBDCA------------► carboplatin
Figure 6: Reaction Steps in Method (i) and (ii) for Carboplatin Synthesis
28
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HPLC Assay
The kinetics of carboplatin formation by methods (i) and (ii) was monitored by
an HPLC system. The HPLC system consisted of a Perkin Elmer Series 410LC
solvent delivery pump, an ISS-100 autosampler and variable wavelength LC90 UV
spectrophotometric detector. The analytical column used was a reversed phase phenyl
Microsorb-MV™ column, 250 mm x 4.6 mm, 5pm particle size and 100°A pore size.
The mobile phase was a 20 mM ammonium phosphate buffer (adjusted to pH = 4,
using IN HC1) with a flow rate of 1.5 ml/minute. Uridine solution (100 pg/ml) was
used as an internal standard. The effluent was monitored using an UV detector at a
wavelength of 230 nm. Data collection and quantitation was performed using a LCI-
100 laboratory computing integrator. The ratio of peak height of carboplatin to the
peak height of uridine (internal standard) was used for quantitation purposes.
(B) Microscale Synthesis of Radiolabeled [1 9 S m Pt]-CarbopIatin
Production o fI9 5 m Pt
Thermal neutron irradiation of the enriched metallic platinum target, with
>95% as I9 4 Pt, for the reaction 1 9 4 Pt (n,y) 1 9 5 m Pt was carried out at the University of
Missouri Research Reactor (MURR). Typical irradiation times were 12-14 days at a
29
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
flux of 3x101 4 n/cm2 to yield 1 9 S m Pt with a specific activity of upto 22.2 Mbq (0.6
mCi/mg) Pt at the end-of-bombardment.
Microscale Synthesis o f [i9 5 m PtJ-Carboplatin
The synthesis was carried out in the semi-automated system previously
developed in our laboratory for the synthesis of radiolabeled [l9 5 m Pt]-cisplatin and
later adapted for the synthesis of [1 9 5 m Pt]-carboplatin. A simplified schematic of the
experimental system is shown in Figure 7. Some of the key features of this semi
automated system, include a conical bottom reaction vessel, fitted with a custom
designed, exchangeable six-inlet/outlet heads for addition/removal of various reagents.
In order to minimize handling of the radioactive material, transfers of all reagents into
and out of the reaction vessel were directed by 2-and 3-way low dead volume solenoid
valves, remotely operated by means of a control panel placed outside the radiation
area. The reaction vessel was placed in an aluminum block, suitably equipped with
heating and cooling elements. The temperature of the reaction vessel was closely
monitored by use of a miniature microprocessor temperature controller equipped with
a sensory probe inserted in the aluminum block. The system was maintained behind a
lead shield to keep radiation exposure to personnel to a minimum.
30
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
V A L V E C -3
I ^ ----
| | | U G K O U T
WASTE
A r PURGE
VALVE D-*
Z a
V f
RADIATION X
AREA ^
Ar PURGE LEAD SHIELD
VALVE
F-S
COMMON
AEMRANE)
FILTER
REACTION VESSEL
;
VALVE B-2
~ £ > < H
SAFETY VALVE '
Ar PURGE
COMMON
TO**.
c c w T w o u ja
SYRINGE
UQ U O (N O U T)
VALVE A-1
WORK AREA
CONTROL PANEL
2-WAY SWITCHES
J T R T T U T T S "
0 0 0 0 0 0
9 2Si?o 9, 9 ,„
3-WAY SWITCHES
Figure 7: Simplified Schematic o f the Semi-automated Setup for the Radioactive Synthesis o f
[1 9 5 m Pt]-Garboplatin. Valve A-1 is an Inlet valve (3-way) for introducing reagents. The liquid is then
pushed along the inert Teflon PFE tubing by means o f argon gas. This valve is situated outside the
radiation area. Valve B-2 constitutes a safety valve (2-way). Tim ed opening o f this valve allows the
reagents to reach the reaction vessel. Valve C-3 (2-way), when activated (with safety valve D-4 closed
and the argon purge valve E-l open) represents the outlet for M IBK (step 2). Valve D-4 is a safety
valve (2-way) that ensures pressure equalization thus avoiding unwanted transfers due to pressure
differential. Valve E-5 is a 3-way valve that serves 2 purposes: (a) when actuated, it acts as a vent and
serves as an additional safety measure; (b) with the vent closed (and the argon purge orifice open) a
positive pressure is generated that is utilized to remove MIBK (step 2) and also to force the contents out
o f the reaction vessel. Valve F-6 is a 3-way filtration valve. The positive pressure forces the reaction
mixture out through the membrane filter and the clean filtrate is returned to the reaction vessel. All
valves (2- and 3-way) are Teflon PTFE low dead volume solenoid valves (0 to low retention volumes
in the orifice). Inert Teflon TFE tubings (o.d. X i.d. = 1/8” X 0.095”) were used in the entire setup. The
control panel, located outside the radiation area has toggle switches to operate 2- and 3- way valves. A
miniature microprocessor temperature controller with digital readings together with the control panel
comprises a M aster Control Box. (Reproduced from Anand et al., 1995)
31
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Enriched Pt Metal
I
irradiation
1 9 5 m p g
Aqua regia
[1 9 5 m Pt]H2PtCl6
| 2M NaCl
[1 9 5 m Pt]Na2PtCl6
0.5MN2 H4^HC1
[1 9 5 m Pt]Na2PtCl4
4N N aI
[1 9 5 m Pt]Na2Ptl4
8M N H4OH
cis-[1 9 5 m Pt]Pt(NH3)2I2
Ag2 S 0 4
cis-[195m Pt][Pt(NH3)2(H20 )2]S0 4
Ba salt of 1,1-cyclobutane
dicarboxylic acid
f ?
[1 9 5 m Pt]-carboplatin
Figure 8: Outline of the Critical Steps Involved in the Synthesis of [1 9 5 m Pt]-Carboplatin
3 2
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
An outline of the critical steps involved in the microscale synthesis of
radiolabeled platinum is shown in Figure 8. Volumes of all reagents listed below are
based on an initial amount of 0.1 mmol Pt.
1 . The quartz vial, containing 1 9 5 m Pt powder was cut by means of an electrically
operated tungsten carbide scorer and the powder poured remotely into the reaction
vessel shown in Figure 7. Approximately, 6.67 ml of freshly prepared aqua regia
(HC1:HN03 - 3:1) was added and the mixture heated with constant stirring, until all
the Pt powder had dissolved. A low flow of nitrogen was maintained through the
reaction vessel (0.5-1 p.s.i.), to aid the rapid dissolution of Pt, but prevent any
excess evaporation of aqua regia. Once all the Pt powder had dissolved to form
H2 PtCl6 solution, the flow of nitrogen through the reaction vessel was increased to
enhance the evaporation of aqua regia to almost dryness. At this stage, utmost care
was taken to prevent overheating and any thermal degradation of H2 PtCI6.
2. For the extraction of 1 9 9 Au, methyl isobutyl ketone (MIBK), prequilibrated with
2 M HC1 was added to the reaction mixture and thorough mixing of the organic
(MIBK) and aqueous (H2 PtCl6 ) phases was achieved by rapid bubbling of nitrogen
gas. After allowing the two phases to separate, the upper MIBK layer was
removed by creating a positive pressure (~5 p.s.i.) within the reaction vessel and
33
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
timed operation of the appropriate solenoid valves. The MIBK addition/extraction
TO O
was repeated once more to ensure complete removal of Au.
3. The hexachloroplatinic acid (H2 PtCl6 ) was converted to its sodium salt (Na2PtCl6 )
by the addition of 333pi of 2 M NaCl. The resulting solution was evaporated to
yield a yellow crystalline residue of Na2 PtCl6. This was followed by the complete
removal of HN03 by 3-4 additions-evaporations of 3.33 ml of 12 M HC1. The
resulting residue was dissolved in 10 ml of 1M HC1. 50pl of this solution was
withdrawn for spectrophotometric analysis of Na2 PtCl6. For the assay, the 50 pi of
solution withdrawn was further diluted with 15 ml of 1M HC1 and the absorbance
read at a wavelength of 262 nm was used for calculating the amount of Na2 PtCl6.
The volumes of all subsequent reagents were adjusted on a stoichiometric basis
with the amount of Na2 PtCl6 measured at this step. The solution containing
Na2 PtCl6 was then evaporated to dryness and traces of HC1 were removed by
addition of water and reevaporation to dryness. The final residue was dissolved in
approximately 0.5 ml water and cooled to 0°-4°C.
4. Further, the hexachloroplatinate was stoichiometrically reduced to
tetrachloroplatinate, under a blanket of gaseous nitrogen, by the addition of 105 pi
of 0.5 M N2 H4-2HC1. The addition of hydrazine resulted in a vigorous evolution
of N2. After the evolution of N2 had subsided, the solution was evaporated to
34
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
dryness to remove any excess HCL To ensure complete removal of HC1, the
additions-evaporations of water was repeated 1-2 times, while maintaining the
reaction mixture under nitrogen. Na2 PtCl4 was then converted to the dark brown
complex of Na2 PtI4 by the addition of 150 pi of 4 M Nal (1.5 x stoichiometric
amount) and allowing the reaction to proceed for 20 min. at room temperature.
5. The Na2 PtI4 was further converted to the yellow precipitate of c/j-Pt(NH3 )2 I2 by
the addition of 69 pi of 8 M NH4 OH (1.1 x stoichiometric amount). The reaction
vessel was tightly sealed to minimize NH3 losses and the mixture was heated to
40°C to accelerate the completion of the reaction. To compensate for loss of NH3
through volatilization, small amounts (~ 5 pi) of NH4 OH were added until the
supernatant was pale yellow in color. At this stage, the supernatant containing
excess iodides and variable amounts of free HC1 was removed by centrifugation.
The precipitate was washed 1-2 times with 0.001 M Nal to remove any excess Nal.
6. The further transformation of the solid c/s-1 9 5 r n Pt(NH3 )2 I2 to [I9 5 m Pt]-carboplatin
*
was carried out via methods (i) and (ii) respectively, as shown earlier in Figure 6.
Method (i) as described earlier involved the reaction of c«-l9 5 m Pt(NH3 )2 I2 with
0.098 mM Ag2S04 in 1-2 ml of water for 4 hours to yield the Agl precipitate and
a clear supernatant containing the diaquo species, namely cis-
[Pt(NH3 )2 (H2 0 )2 ]S04. The barium salt of 1,1-cyclobutane dicarboxylic acid
35
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
(BaCBDCA) was prepared in situ, by reacting 0.098 mM Ba(OH)2.8H2 0 with 0.11
mM CBDCA. The diaquo species was subsequently reacted with BaCBDCA, to
yield radiolabeled [I9 5 m Pt]-carboplatin. Similarly, method (ii) involved the
reaction of c/s-I9 S m Pt(NH3 )2I2 with 550 pi of 0.4M AgN03 (10% excess) at 60°C
for 30 min. to yield c/s-[Pt(NH3 )2(H2 0 )J(N 0 3 )2 and the Agl precipitate. The Agl
precipitate was removed by centrifugation and the supernatant containing cis-
[Pt(NH3 )2 (H20)2 ](N03 )2 was reacted with 350 pi of 1M CBDCA (3.5 eq.) for 48
hours at room temperature to produce [l9 S m Pt]-carboplatin.
Purity and Quality Control o f [I9 5 m Pt]-Carboplatin
The [l9 5 m Pt]-carboplatin synthesized was analyzed to determine its chemical,
radiochemical and radionuclidic purity. Thin layer chromatography (TLC) was used
to determine the chemical and radiochemical purity of [I9 5 m Pt]-carboplatin. For TLC,
the support employed was silica gel plates (Eastman Kodak) and the eluent was a
mixture of isopropanol and water in ratio of 7:3. Approximately 4-6 pg of the sample
was spotted and the purity of the radiolabeled carboplatin was determined
quantitatively, by counting the activity of both pure and impure spots using an
automated gamma counter. The Rf value (distance moved on the TLC) was compared
to that of pure, non-radioactive carboplatin to determine the purity of the sample.
36
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Radionuclidic purity was determined by obtaining a y-spectra for [1 9 5 m Pt]-
carboplatin synthesized and then comparing it with the reference spectra for 1 9 5 m Pt.
The spectra was obtained using a well counter equipped with a Nal detector and a
133
multichannel analyzer. The instrument was calibrated using a pure Ba source.
(C) Study of the Interaction of Carboplatin and Cisplatin with Guanosine (Guo)
Reagents
Carboplatin was obtained from Sigma Chemicals, while cisplatin was obtained
from Alfa Products. Guanosine and uridine were obtained from Aldrich Chemicals.
All other chemicals were of analytical reagent grade or HPLC grade.
Procedure
The stability of carboplatin in isotonic saline was monitored by incubating,
carboplatin(3 mg, 8.1 (imol) in 6 ml of 0.9% saline at 37°C. At appropriate intervals
samples were withdrawn and analyzed by HPLC.
The interaction of cisplatin with guanosine was studied by incubating cisplatin
(3 mg, 10 pmol) with a solution of guanosine (5.68 mg, 20 gmol) in 6 ml 0.9% saline
at 37°C. Samples were withdrawn at suitable intervals and analyzed by HPLC. The
interaction of carboplatin with guanosine was similarly monitored by incubating
37
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
carboplatin (3 mg, 8.1 pmol) with a solution of guanosine ( 4.58 mg, 16.2 pmol) in 6
ml 0.9% saline at 37°C.
Measurements
The HPLC analyses were performed on a Perkin Elmer Series 410LC
chromatograph equipped with an ISS-100 autosampler and variable wavelength LC90
UV spectrophotometric detector. A reversed phase phenyl Microsorb-MV column,
250 mm x 4.6 mm, 5 pm particle size and 100°A pore size was used. The mobile
phase was a 20 mM ammonium phosphate buffer (pH = 4) with a flow rate of 1.5
ml/minute. Uridine solution (100 pg/ml) was used as an internal standard. The
effluent was monitored using a UV detector at 230 nm. Data collection and
quantitation was performed using a LCI-100 laboratory computing integrator. The
ratio of the peak height of the drug to the peak height of the internal standard was used
for quantitation.
38
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RESULTS AND DISCUSSION
(A) Study of Reaction Kinetics - A comparison of the reaction kinetics for the
reaction between (i) cu-diammine diaquo platinum (II) sulfate and barium salt of
1,1-cyclobutane dicarboxylic acid and (ii) m-diammine diaquo platinum (II)
nitrate and 1,1-cyclobutane dicarboxylic acid
Published assay methods for the detection of carboplatin, including atomic
absorption spectroscopy, atomic emission spectroscopy and X-ray fluorescence are not
specific to the intact drug (De Waal et al., 1990). Other methods designed for the
measurement of drug in the body and biological fluids were not suitable for the
purpose of this study (De Waal et al., 1990). Therefore, in order to quantitate the yield
of carboplatin from cis-diammine diaquo platinum(II) and 1,1-cyclobutane
dicarboxylic acid, it was essential to use an analysis method which would separate the
three compounds of interest. A new HPLC assay was therefore developed for the
study, using a reversed phase phenyl column and 20 mM ammonium phosphate (pH =
4) as the mobile phase. Uridine was used as an internal standard. Typical retention
times for the various species is shown in Table 1 .
39
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Table 1: Retention Time for Various Compounds on the Phenyl Column used
in HPLC Assay
Compound Retention Time (min.)
cis-diammine diaquo platinum (II) 2.0
Carboplatin 4.7
Uridine 6.0
1,1-cyclobutane dicarboxylic acid 8.6
A typical HPLC profile for the analysis of carboplatin with uridine as an
internal standard is shown in Figure 9. The standard curve was linear over a range of
0.5 pg/ml to 50 |ig/ml, with a mean slope of 0.18737 and y-intercept of 0.01879. The
correlation coefficient for the standard curve was 0.9996. The assay was validated for
between-run and within-run precision and accuracy by assaying six different
concentrations on five different days.
4 0
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b
t = 90 min.
Figure 9: Typical HPLC Analysis Profile for Reaction Mixture of Method (i)
a: diaquo species (rt = 2 min.), b: carboplatin (rt = 4.7 min.) and
c: uridine (rt = 6.0 min, internal standard).
41
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The comparative reaction kinetics for the formation of carboplatin by methods
(i) and (ii) is shown in Figure 10. Table 2 compares the % yields at various time
points for methods (i) and (ii), the latter with 3.5 eq. and 1 eq. of CBDCA. In the case
of method (i), the formation of carboplatin from cis-diammine diaquo platinum (II)
sulfate and barium salt of 1,1-cyclobutane dicarboxylic acid proceeds rapidly at initial
time points reaching maximum concentrations in about 4-5 hours. At the end of 5
hours, method (i) yields about 72% conversion which increases to about 80% at the
end of 46 hours. In contrast, the formation of carboplatin using cis-diammine diaquo
pIatinum(H) nitrate and 1 eq. of 1,1-cyclobutane dicarboxylic acid proceeds very
slowly yielding less than 10% conversion within the first five hours. At the end of 46
hours, method (ii) using 1 eq. of CBDCA yields approx. 30% carboplatin. The use of
excess of 1,1-cyclobutane dicarboxylic acid (3.5 eq.), results in a slight increase in
yield (about 60%) at the end of 46 hrs. Thus, method (i) results in high yield of
carboplatin while significantly reducing the reaction time.
4 2
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■ D iaquoSO , + I eq. B aC B D C A
O D iaquo(N O j)z + 3.5 eq. C B D C A
□ DiaquoCNO j)^ + 1 eq. C B D C A
80
60
0 5 10 15 20 25 30 35 40 45 50
T im e (hrs)
Figure 10: % Yield At Various Time Points for Methods (i) and (ii)
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Table 2: % Yields for the Conversion of cis-Pt(NH3 )2 l2 to
Carboplatin Measured by HPLC as a Function of Time
Time (hrs.) % Yield
Method I Method II
(using 3.5 eq.
CBDCA)
Method II
(using I eq.
CBDCA)
0.5 31.7 0.3 0.2
1 49.6 1.0 0.7
2 66.5 3.0 2.0
4 71.5 9.4 5.3
10 75.0 20.4 10.0
24 77.9 46.7 22.8
40 78.9 59.6 27.9
46 80.6 62.9 29.2
The short reaction times and high yields obtained using method (i) can be
attributed to the conversion of 1,1-cyclobutane dicarboxylic acid to the carboxylate
anion due to the precipitation of the BaS04, making it a better attacking group as
compared to the free acid, as would be the case for method (ii). In addition, the
removal of the barium sulfate precipitate by centrifugation essentially make the
reaction irreversible. This facilitates the rapid progression of the forward reaction,
resulting in higher yields of carboplatin.
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(B) Microscale Synthesis of Radiolabeled [1 9 S m Pt]-Carboplatin
The radiochemical yields obtained for four [l9 5 m Pt]-carboplatin synthesis is
shown in Table 3. Major losses associated with the conversion of cis-
[i95m ptjpt(NH3 ) ^ tQ [I9 5 m pt].carboplatin were reduced from about 30% to about 16%
(see Table 3), by use of Ag2S04 and the barium salt of 1,1-cyclobutane dicarboxylic
acid. This resulted in an overall increase in yield of [1 9 5 m Pt]-carboplatin from 50% to
64%. The use of Ag2 S04 and BaCBDCA also resulted in a significant reduction
(about 20 hours) in the reaction time and therefore higher specific activity of the final
product.
Thin layer chromatography (TLC) analyses at the end of 46 hours on [l9 5 m Pt]-
carboplatin synthesized, confirm that the samples were of high chemical purity
(>95%). Thin layer chromatographic analyses on silica gel plates, shown in Figure 11,
revealed a single spot (Rf = 0.7), indicating the absence of any other radiolabeled
platinum complexes. The Rf value obtained for the radiolabeled compound compared
well with that obtained with unlabeled, pure carboplatin.
4 5
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Table 3: % Radiochemical Yields and Losses During the Synthesis of 1 9 S m Pt-
carboplatin
Material Method (i) Method (ii)
(using 3.5 eq.
CBDCA)
Synthesis No.
1 2
Synthesis No.
1
Yields (%) at end o f 46 hours
cis-[l9 5 m Pt]Pt(NH3 )2 I2* ’
[9 S m Pt]-carboplatin
81.3 79.3
63.3 62.7
81.0
50.5
Losses (%)
Conversion of [I9 5 m Pt]Na2 PtCl6 to cis-
[I9 5 m Pt]Pt(NH3 )2I2
Conversion of cis-[1 9 5 m Pt]Pt(NH3 )2 I2 to
[l9 5 m Pt]-carboplatin
18.7 20.7
18.0 16.6
19.0
30.5
* - % yields and losses reported relative to the activity o f [l,;> in Pt]-Na2 PtCl6 defined as 100%.
** - Values obtained by subtracting the amount o f radioactivity o f the supernatant and
washings from the initial activity o f [l9 5nlPt]-Na2PtCl6 (100%).
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
900000
800000 -
700000 -
u
3
C
ii 500000 -
600000 -
400000 -
| 300000 -
u
200000 -
100000 -
2 3 4 5 6 7 8 9 10
Distance Moved by Solvent Front
Figure 11: Thin Layer Chromatographic (TLC) Analysis of [1 9 5 m Pt]-CarbopIatin on
Silica Gel Plates
47
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Analyses on the radionuclidic purity indicates the presence of [Pt-I95m] as the
only radioactive isotope present, as identified by gamma rays of 66-77 keV, 99 keV
and 129 keV.
The present work is an extension of the semi-automated method for the
synthesis of cisplatin, previously described by Anand et al. (1992), to the synthesis of
radiolabeled carboplatin with suitable modifications. No major changes were made to
the procedure used for the synthesis of [l9 5 m Pt]-cisplatin up to the formation of cis-
[i9smpt]pt(NH3 )2 i2. The results of the reaction kinetics revealed that the method
utilizing Ag2S04 and barium salt of 1,1-cyclobutane dicarboxylic acid resulted in
higher yields at faster rates and was therefore selected as the method of choice for the
synthesis of radiolabeled carboplatin. A similar approach, using cis-[I9 5 m Pt]Pt(NH3 )2 l2
and Ag2 CBDCA to hasten the formation of [1 9 5 m Pt]-carboplatin has been reported by
Jackson et al. (1991) with yields upto 43%. The present work represents a further
refinement over the previous methods and is further characterized by a remotely
operated semi automated system that appears to be suited for the routine microscale
production of radiolabeled carboplatin. Na2 PtCl6 was employed in preference to
K2 PtCl6 and H2 PtCl6, since it is more water soluble than the former and has better
thermal stability as compared to the latter. This permits use of concentrated solutions
with rapid reduction to Na2 PtCl4 without Pt° metal formation. Furthermore, more
48
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reliable spectrophotometric results are obtained with the use of Na2 PtCl6 as starting
material.
The complete removal of residual HN03, in step 3 of the synthesis scheme
described above, was essential because of possible interference of HN03 , both in the
accurate determination of Pt(TV) in Na2 PtCl6 and in the reduction step (step 4), where
it would slow the rate of the forward reaction (Hoeschele et al., 1982). This was
achieved by monitoring the ratio of the absorbance at 261 nm to that at 231 nm as
shown in figure 12. Typical values of greater than 7.2 were obtained and served as a
measure of the [HN03 ] and also as an index of the reproducibility of Na2PtCl6
solutions (Hoeschele etal., 1982).
49
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0.370
s
A
<
0.000
nm 200 280 320
Wavelength
Figure 12: UV Spectrum of [I9 5 m Pt]Na2PtCl$ in
400
MHCI
50
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Several workers (Anand et al., 1992; Hoeschele et al., 1982) have shown that
precise stoichiometric amounts of the reducing agent and subsequent reagents are
essential for obtaining high yields of the final product. This meant that an accurate
determination of the amount of Na2 PtCl6 is crucial to the success of the microscale
synthesis. This was achieved by determining the amount of Na2 PtCl6
spectrophotometrically as detailed in step 3. This may be due to the fact, that the
reduction potential for the reduction of PtCl6 2 ' to PtCl4 2 ' is 0.77V, while that for the
reduction of PtCl4 2 ' to Pt° is 0.75V. Thus, a very small difference of 0.02V exists as a
barrier between the reduction of platinum(IV) to platinum(II) and not to platinum(O).
This could possibly help explain the extreme sensitivity of the reduction step 4, to the
amount of reducing agent used. An excess of N2 H4-2HC1 results in the formation of
hydrazine-Pt (II and IV) complexes, while a deficiency of N2 H4-2HC1 leads to
incomplete reduction and hence to the presence of unreduced Pt(TV). On subsequent
addition of Nal and NH4OH, solutions containing excess hydrazine-2HCl will be
reduced to Pt°, while solutions containing less than the stoichiometric amount of
N2 H4*2HCl leads to the formation of brown-black colored cis-Pt(NH3 )2 I2,
contaminated with dark colored Pt(TV) compounds of unknown composition. The
reduction step has also been shown to be pH (Wolf et al., 1977a) and temperature
(Manaka, 1980) sensitive.
51
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The use of six mole equivalent Nal in step 4 enhances the formation and
stability of the PtCLt2 " (Hoeschele et al., 1982), while at the same time reducing the
formation of surface scum which occurs in solutions containing the stoichiometric
amount of Nal. The excess amount of Nal also reduces the chance of Ptl2 precipitation
(Baer et al., 1985).
One key feature of this work is the study of the reaction kinetics of the methods
described by Hill et al. (1990) and Baer et al. (1985), to optimize the reaction
conditions for the synthesis of radiolabeled carboplatin. These methods after suitable
modification were then successfully used for the microscale synthesis of [l9 5 m Pt]-
carboplatin using the semi automated setup. The use of cis-diammine diaquo platinum
(II) sulfate and barium salt of 1,1-cyclobutane dicarboxylic acid results in a significant
increase in the yield of the final product. By avoiding multiple transfers of reaction
mixture, high chemical and radiochemical yields of up to 64% were obtained
reproducibly on a routine basis. Moreover, since the process is semi-automated,
radiation exposure to personnel is minimized. This procedure can now be used for the
routine production of [l9 5 m Pt]-carboplatin in the laboratory for clinical studies.
52
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(C) Study of the Interaction of Carboplatin and Cisplatin with Guanosine
The ligand substitution reaction in square planar platinum (II) complexes such
as carboplatin with guanosine may either,
(i) proceed through the solvent assisted substitution pathway in which the first reaction
is a nucleophilic substitution of the CBDCA ligand with solvent ions such as H2 0 or
Cl' ions to form the aquated species or cisplatin respectively, followed by the reaction
of the resulting complex with guanosine.
(ii) involve the direct nucleophilic SN2 substitution of the bidentate leaving group, i.e.
CBDCA, with guanosine.
The interaction of carboplatin and cisplatin with guanosine were followed
using an HPLC system consisting of a phenyl column and 20 mM ammonium
phosphate as the mobile phase. The retention time for various species is shown in
Table 4.
Table 4: Retention Time for Various Compounds on a Phenyl Column
Compound Retention Time
(min.)
Cisplatin 2.24
Carboplatin 4.7
Uridine 6.0
Guanosine 14.13
G-Pt-G (biadduct) 19.89
Cl-Pt-G (monoadduct) 21.4
53
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Carboplatin Stability in Aqueous Medium
The aqueous solution of carboplatin at 37°C was monitored by HPLC over a
period of 48 hours with negligible change in carboplatin concentration. These results
are in support of previous findings (Daley-Yates, 1986; Tinker et al., 1987) that the
bidentate cyclobutane dicarboxylate ligand chelating to platinum renders carboplatin
stable to hydrolysis. This indicates that the solvent H2 0-assisted pathway for
carboplatin transformation can be neglected.
Carboplatin Stability in Isotonic Saline (0.9%)
The presence of C1‘ ions in isotonic saline (0.9%) results in the conversion of
carboplatin to cisplatin, as shown in Figure 13. In addition to the peaks for carboplatin
and uridine (internal standard), the HPLC chromatogram at 37°C shows a single peak
for cisplatin (rt = 2.24 min.) with a corresponding decrease in carboplatin
concentration. As shown in Figure 14, about 5% of carboplatin is converted to
cisplatin at the end of 24 hours with almost 33% conversion at the end of 4 days. A
pseudo-first order rate expression was fitted to the data shown in Figure 14 to obtain a
kobs = 3.4*1 O '3 hr’1 with a half life of 161.9 hours. These results are in agreement with
that reported by Tinker et al., (1987) and Cheung et al., (1987). Tinker et al., (1987)
54
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also observed a more rapid formation of cisplatin in vivo compared to in vitro studies,
suggesting the importance of the in-vivo transformation of carboplatin to cisplatin.
55
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
b
U l
Figure 13: HPLC Profile of Carboplatin in Isotonic Saline (0.9%) at 37°C
a: cisplatin (rt = 2.24 min.), b: carboplatin (rt = 4.7 min.), c: uridine
(rt = 6.0 min., internal standard.).
5 6
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
♦ Carboplatin
■ Cisplatin
120
100
c
3
O
a
E
o
o
40
|l- *
0 1000 2000 3000 4000 5000
Time (mins.)
Figure 14: Formation of Cisplatin from Carboplatin in Isotonic Saline (0.9%) at 37°C
5 7
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Interaction of Cisplatin with Guanosine in Isotonic Saline (0.9%)
The two chloride ligands of cisplatin are replaceable with H2 0 to give the
corresponding aqua species. Therefore, the interaction of cisplatin with guanosine may
proceed through pathways A and/or B as shown in Figure 15.
Pathway (A)
Guanosine
H3N \ / c i
p t
t / \
H^N Guanosine
Guanosine
h 3 n n / C l
H 3 N
P t
Pathway (B)
ci
H2 0
H3N x /G u a n o s in e
Pt
/ \
h3 n Guanosine
Guanosine
h 3n x / h 2o
Pt
h 3n H jO
Guanosine H3N \ / H , 0
pt
h 3n
/ \
Guanosine
Figure 15: Possible Pathways for the Interaction of Cisplatin with 2 eq. Guanosine
58
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It is likely that pathway B is negligible due to the high chloride concentration, in 0.9%
saline. Consequently, the interaction of guanosine with cisplatin will proceed via the
monosubstitution followed by the bisubstitution of chloride ion with guanosine, as
shown in Figure 15. The relative concentrations of the adducts in solution are a
function of the rate constants k[ and k2 . The HPLC profile for guanosine interaction
with cisplatin at 37°C, shown in Figure 16, shows two peaks - the monosubstituted
(Cl-Pt-G) complex eluting at rt = 21.4 min. and the bisubstituted (G-Pt-G) at rt = 19.89
min. The monoadduct concentration goes through a maximum, indicating that the k[
and k2 are comparable with kt slightly greater than k2. As shown in Figure 17 and
Table 5, the series reaction proceeds via the rapid formation of the monosubstituted
complex followed by the bisubstituted complex. Other studies by Itoh et al. (1991) on
the interaction of cisplatin with guanosine at 40°C have yielded similar results with
about 48% cisplatin reacting with guanosine at the end of 24 hrs.
59
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Figure 16: HPLC Profile for the Interaction of Cisplatin with 2 eq. Guanosine in
Isotonic Saline (0.9%) at 37°C. a: cisplatin (rt = 2.23 rain.), c: uridine (rt =
6.0 min., internal standard), d: guanosine (rt = 14.13 min.), e: G-Pt-G (rt =
19.89 min.), f: Cl-Pt-G (rt = 21.4 min.)
60
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
1.4
1.3
1.2
l.l
1.0
.2 09
t2 0.8
. f 0.7
0
1
j* 0.6
e u
0.5
0.4
0.3
0.2
0.1
0.0
0 20 40 60 80 100 120 140
Time (hours)
Figure 17: Relative Amounts of the Interaction Products of Cisplatin with 2eq.
Guanosine in Isotonic Saline (0.9%) at 37°C
■ Cisplatin
A Cl-Pt-G
T G-Pt-G
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Table 5: Relative Amounts o f Various Products Obtained during the Interaction of
Cisplatin with Guanosine in Isotonic Saline (0.9%) at 37°C
Time(h) Peak Height Ratio
Cisplatin
(rt = min.)
(G-Pt-G)
(rt = 19.89 min.)
(Cl-Pt-G)
(rt = 21.4 min.)
0.53 0.865 0.005 0.016
20.13 0.622 0.103 0.411
23.16 0.598 0.144 0.494
93.00 0.223 0.800 0.579
96.53 0.210 0.963 0.641
123.00 0.163 1.262 0.614
* - Peak height ratio relative to uridine used as internal standard.
62
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
The commonly accepted model for the mechanism of action of cisplatin
proposes that once the drug enter the cell, the low chloride ion concentration (4 mM)
inside the cell facilitates the conversion of cisplatin to the aquated species. These
aquated species which are more reactive than cisplatin readily platinate proteins,
amino acids and attacks DNA to form lesions believed to be responsible for the
cytotoxic action of cisplatin. The oft-cited 4 mM value for the intracellular C f ion
concentration is derived from the application of the Nemst equation to the passive
distribution of Cl' ions across plasma membrane in muscle or nerve cells (McCaig et
al., 1984). While this calculation seems to be valid in the case of muscle cells where
there are no membrane channels or exchangers for Cl' ions, Jennerwein et al. (1995)
argue that this value may not be valid in case of tumor cells. Jennerwein et al (1995)
in recent studies have actually measured the intracellular Cl' ion concentration inside
tumor cells and have shown it to be much higher (31-55 mM) than 4 mM as normally
perceived. They have further shown that reduction of the intracellular Cl' ion
concentration may have little or no effect on the cytotoxicity of cisplatin. This
suggests that aquation of cisplatin while probable, may not necessarily be an essential
step for the platination of DNA by cisplatin. Alternatively, cisplatin may be able to
directly react with guanine bases on DNA to produce the cytotoxic lesions.
63
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Interaction of Carboplatin with Guanosine in Isotonic Saline (0.9%)
The bidentate CBDCA ligand can be substituted by the Cl* ions present in
saline to give cisplatin or undergo direct attack by guanosine via a pathway shown in
Figure 18, similar to the pathway proposed earlier for cisplatin in Figure 15. Figure 19
shows the HPLC profile for the interaction of carboplatin with 2 eq. guanosine in 0.9%
saline at 37°C. In addition to carboplatin and uridine (internal standard), the
chromatogram shows three peaks - cisplatin eluting at rt = 2.24 min., and as before,
the monosubstituted (Cl-Pt-G) complex eluting at rt = 21.4 min. and the bisubstituted
(G-Pt-G) at rt = 19.89 min. Figure 20 and Table 6 shows the progress of the reaction
as a function of time. The carboplatin is first converted to cisplatin which rapidly
interacts with guanosine to give the monosubstituted adduct (Cl-Pt-G) followed by the
bisubstituted adduct (G-Pt-G). At the end of 48 hours the amount of mono substituted
complex (Cl-Pt-G) is higher than the amount of bisubstituted complex (G-Pt-G). The
concentration of the monoadduct reaches a maximum and at the end of 7 days the
bisubstituted complex (G-Pt-G) is more predominant indicating a series of reactions
similar to the interaction of cisplatin with guanosine, with one additional step, namely
the reaction of carboplatin to give cisplatin. The interaction of carboplatin with
guanosine, thus proceeds through the formation of cisplatin in the presence of high Cl"
ion concentration, rather than the direct attack of the guanosine on the CBDCA ligand.
64
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Pathway (A)
Guanosine
H N . o— c
p /
h3n
/ \ O
Guanosine
Guanosine
H , N \ / O -
/ p\
h 3n x o -
Pathway (B)
h 3 n x / C l
h 3 n
Pt
/ \
Cl
H3N \ /G uanosine
h 3n
Pt
/ \
Guanosine
2 Guanosine
* The interaction o f cisplatin with guanosine may proceed via pathways similar to
those shown in Figure 15
Figure 18: Possible Pathways for the Interaction of Carboplatin with 2 eq. Guanosine
65
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
d
Figure 19: HPLC Profile for the Interaction of Carboplatin with 2 eq. Guanosine in
Isotonic Saline (0.9%) at 37°C. a: cisplatin (rt = 2.24 min.), b: carboplatin
(rt = 4.7 min.), c: uridine (rt = 6.0 min., internal standard), d: guanosine
(rt = 14.13 min.), e: G-Pt-G (rt = 19.89 min.), f: Cl-Pt-G (rt = 21.4 min.)
6 6
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
5.0
4.5
4.0
3.5
o
at
x:
o o
3.0
• Carboplatin
■ Cisplatin
A Cl-Pt-G
T G-Pt-G
•S' 2'5
X
1 2.0
a.
0.5
0.0
0 20 40 60 80 100 120 140 160 180 200 220 240
Time (hours)
Figure 20: Relative Amounts of the Interaction Products of Carboplatin with 2eq.
Guanosine in Isotonic Saline (0.9%) at 37°C
67
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Table 6: Relative Amounts of Various Products Obtained during the Interaction of
Carboplatin with Guanosine in Isotonic Saline (0.9%) at 37°C
Time (h) Peak Height Ratio’
Cisplatin
(rt = 2.24 min.)
Carboplatin
(rt = min.)
(G-Pt-G)
(rt= 19.89
min.)
(Cl-Pt-G)
(rt = 21.4 min.)
0.50 - 4.495 - -
21.50 0.0303 4.085 - 0.0702
24.00 0.0432 4.194 0.0211 0.0798
51.58 0.0874 4.030 0.1330 0.2443
171.33 0.1768 3.615 1.4084 0.6145
217.80 0.1752 3.507 1.8741 0.5722
* - Peak height ratio relative to uridine used as internal standard.
68
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Interaction of Carboplatin with Guanosine in Aqueous Medium
As discussed above, carboplatin is relatively stable in terms of the substitution
of the bidentate CBDCA ligand with H2 0 . Thus, unlike in the presence of Cl' ions,
the interaction of carboplatin with guanosine in aqueous medium proceeds directly via
the direct attack of guanosine as shown in Figure 18. The interaction of 2 eq.
guanosine with carboplatin at 37°C in an aqueous medium was monitored by HPLC.
The only detectable product was the diadduct (rt = 19.89 min.) with no peak being
observed for the product containing one guanosine (monoadduct). This interesting
difference suggests that in an aqueous medium, the binding of the second guanosine
molecule may be very rapid (k2 » k[) with the rate determining step involving the
reaction between carboplatin and the first guanosine molecule, proceeding according
to second order kinetics. Itoh et al. (1991)has reported similar results on the
interaction of carboplatin with guanosine in aqueous medium.
69
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Proposed Model for the Activation of Carboplatin
The above results indicate that in the presence of high Cl' ion concentration,
the predominant pathway for the interaction of carboplatin with guanosine involves
the conversion of carboplatin to cisplatin followed by guanosine attack to give
measurable quantities of the monoadduct (Cl-Pt-G) and then the diadduct (G-Pt-G). In
contrast, in the absence of C 1‘ ions, guanosine interacts with carboplatin directly, at a
much slower rate, with detectable quantities of only the biadduct (G-Pt-G) species.
Based on this study and the high extracellular Cl' ion concentration commonly
found in blood plasma, it is proposed that the conversion of carboplatin to cisplatin
may be a key step responsible for the extracellular activation of carboplatin in-vivo.
As shown in Figure 21, the cisplatin formed from carboplatin will then diffuse into the
cell. It has been previously reported that cisplatin uptake by the cell is almost 8-20
times higher than that of carboplatin (Dowell, 1995; Los et al., 1991). Morever,
Mauldin et al. (1986) have shown that the rate of uptake of platinum analogs
containing the bidentate ligand is significantly slower than that for compounds
containing Cl' ions, in support of the proposed rapid diffusion of cisplatin into the cell.
Once inside the cell, cisplatin may hydrolyze to form the reactive mono aquo and/or
diaquo species or directly react with its cellular target i. e. DNA. Thus, there is a high
possibility that the anti-tumor action of carboplatin may be mediated via cisplatin.
70
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Daley-Yates (1986) and Tinker et al. (1987) have proposed similar pathways for the
activation of carboplatin in vivo. This is important especially since the carboplatin
dose administered is 5-10 times that of cisplatin. In addition, carboplatin, as such, can
also diffuse into the cell, as shown in the model proposed in Figure 21. At
intracellular CT ion concentrations of 31-55 mM reported by Jennerwein et al. (1995)
in tumor cells, there may be a some conversion of carboplatin to cisplatin, with
subsequent conversion of cisplatin to the more reactive aquo species. The intracellular
reactions of carboplatin and the cisplatin formed, are thus likely to be influenced by
the Cl* ion concentration within the cell. Due to the high stability of carboplatin in
aqueous medium, as shown above, it is likely that formation of the aquo species
directly from carboplatin may be negligible. Other possible biotransformation
products such as platinum-amino acids and the platinum-protein complexes formed in
plasma are reported to be inactive and unable to diffuse into the cell (Chaney et al.,
1987). A similar model has been described for the activation of Pt(mal)(dach) which
contains a bidentate malonate ligand akin to the 1,1-cyclobutane dicarboxylic ligand of
carboplatin (Chaney et al., 1987). Further in-vivo and in-vitro studies in cell cultures
are needed to better understand the exact cellular mechanism of the anti-tumor action
of carboplatin. An understanding of the mechanism of cytotoxic action of carboplatin
may help provide a better rationale for the design of future platinum analogs with
improved therapeutic indices.
71
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o
I I
o---
H,N
O
proteins
Carboplatin Cisplatin
Proteins
AA
slow
rapid
EXTRACELLULAR
INTRACELLULAR
h3n \ / O — c.
o—
H,N
Cl-
(reactive)
a
(reactive) / \
h3n h 2o
/
h 3n \ / H ,0
Pt
/ \ (reactive)
h3n h , o
Figure 21: Proposed Model for the Activation of Carboplatin
7 2
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Calculation of Log P of Carboplatin
The partition coefficient of carboplatin is an important factor which will
govern its distribution in various body tissues. Table 7 shows the log P values of some
platinum complexes and their relative solubilities. The calculated log P of carboplatin
seems to be in good agreement with its water solubility.
Table 7: Log P Values of Some Platinum Complexes
Compound Cisplatin Carboplatin Diaquo Nitrate
Solubility in Water 1 mg/ ml 15 mg/ml freely soluble
Log P -2.21 -3.05 * -3.36
- Calculated as shown below.
L ° g ^Carboplatin ~ L o g P>Pt(NH3)2 + 71 -(00C)2CH(CH2)3
- ( Log P cisp latin - 2 7 1 ) + ( 27t _cooH + n C4H6 )
= -1.43 - 2(0.39) +2 (-1.26) + 1.68
= -2.21-0.84
= -3.05
73
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SUMMARY
Carboplatin is one of the most promising second generation analogs of the
antitumor agent cisplatin. The use of radiolabeled [l9 5 m Pt]-carboplatin for non-
invasive studies has generated considerable interest to help understand the localization
of carboplatin at the tumor site. The use of [l9 5 m Pt]-carboplatin for these studies
requires a suitable quality controlled synthesis procedure for the manufacture of the
radiopharmaceutical in high yields under reproducible conditions. Studies on the
reaction kinetics of two methods of carboplatin synthesis namely (i) reaction of cis-
diammine diaquo platinum (II) sulfate and the barium salt of 1,1-cyclobutane
dicarboxylic acid and (ii) reaction of cis-diammine diaquo platinum (II) nitrate and
1,1-cyclobutane dicarboxylic acid, revealed method (i) to be considerably faster with
chemical yields in excess of 72% within the first five hours in comparison to method
(ii) which resulted in 62% yield after a 46 hour reaction period, thus making method
(i) highly suitable for [I9 5 m Pt]-carboplatin synthesis. This improved method was then
adapted for the microscale synthesis of [1 9 5 m Pt]-carboplatin to obtain high yields
(greater than 60% overall product yield) of the radiolabeled compound. The increased
yields were found to be a direct consequence of the reduction of the chemical losses
(from 30% to 16%), associated with the conversion of cis-Pt(NH3 )2 I2 to carboplatin.
This improved semi-automated method can now be used routinely for the microscale
74
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synthesis of [l9 5 m Pt]-carboplatin, under reproducible conditions with minimal radiation
exposure to personnel.
The antitumor action of carboplatin is believed to be related to its ability to
form bifimctional lesions between various DNA bases, with a greater preference for
the N7 position of the guanine base on DNA. Stability of carboplatin in isotonic
saline and its interaction with guanosine were studied to help gain a better
understanding of its mechanism of action. Studies on the biotransformation products
of carboplatin in the presence of isotonic saline revealed the presence of cisplatin as
the only product as determined by high performance liquid chromatography (HPLC).
HPLC analysis of the interaction of carboplatin and cisplatin with guanosine in
isotoinc saline at 37°C revealed that both carboplatin and cisplatin form the same
adducts with guanosine namely, the mono (Cl-Pt-G) and bisubstituted (G-Pt-G)
complexes. However, the interaction of carboplatin with guanosine was very slow as
compared to cisplatin and in the presence of high Cl' ions was found to be mediated
via cisplatin. Based on these results, it is proposed that the activation of carboplatin in
vivo is likely to proceed primarily by the conversion of carboplatin to cisplatin which
on diffusion into the cell forms the more reactive aquo species that interacts with
DNA.
75
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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
APPENDIX
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P e a k Height Ratio
10
y = 0.18737 x - 0.0179
(r2 = 0.9996)
9
8
7
6
5
4
3
2
1
0
0 5 10 15 20 25 30 35 40 45 50
Concentration (pg/ml)
Figure 1: Standard Curve for Carboplatin using Uridine as Internal Standard.
Curve prepared using 475 pi of carboplatin solutions having
concentration 2, 5, 10, 20, 50 pg/ml + 25 pi of uridine solution (100
pg/ml)
83
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P e a k Height Ratio
1.0
0.9
y = 0.04999 x + 0.00089
(r2 = 0.9998)
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
0 2 10 12 14 16 18 20 4 6 8
Concentration (pg/ml)
Figure 2: Standard Curve for Cisplatin using Uridine as Internal Standard.
Curve prepared using 475 pi of cisplatin solutions having
concentration 1,2, 5, 10, 20 pg/ml + 50 pi of uridine solution (100
pg/ml)
8 4
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Creator Palekar, Darshana (author)

Core Title Synthesis of carboplatin and studies on its biotransformation products

Degree Master of Science

Degree Program Pharmaceutical Sciences

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag chemistry, pharmaceutical,health sciences, oncology,Health Sciences, Pharmacy,OAI-PMH Harvest

Language English

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