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 CONTENTS

CONTRIBUTORS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii
PREFACE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix

Clinical Studies of Camptothecin and Derivatives

OTTO SOEPENBERG, ALEX SPARREBOOM, AND JAAP VERWEIJ
I. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
II. Mechanisms of Action . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
III. Mutagenicity and Resistance Mechanisms . . . . . . . . . . . . . . . . . 7
IV. Irinotecan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
V. Topotecan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
VI. Considerations of Route of Administration . . . . . . . . . . . . . . . . 27
VII. Investigational Derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . 29
VIII. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

Tremorgenic and Nontremorgenic 2,3-Fused

Indole Diterpenoids
HEATHER SINGS AND SHEO SINGH
I. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
II. Isolation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
III. Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125
IV. Biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144
V. Structure and Tremorgenicity . . . . . . . . . . . . . . . . . . . . . . . . 155
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158

The Daphniphyllum Alkaloids

JUN’ICHI KOBAYASHI AND HIROSHI MORITA
I. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165
II. Structures of Representative Alkaloids . . . . . . . . . . . . . . . . . . . 170
III. Biosynthesis and Biogenesis . . . . . . . . . . . . . . . . . . . . . . . . . 184
IV. Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193
V. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203

v
vi CONTENTS

The Manzamine Alkaloids

JIN-FENG HU, MARK T. HAMANN, RUSSELL HILL,
AND MICHELLE KELLY

I. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207
II. Isolation and Structure Elucidation from Marine Sponges . . . . . . . . 214
III. Biogenesis and Biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . 223
IV. Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 264
V. Pharmacology. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 270
VI. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 278

Sesquiterpene Pyridine Alkaloids

LUCIANO M. LIÃO
I. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287
II. Evoninate Sesquiterpene Pyridine Alkaloids and Derivatives . . . . . . . 289
III. Wilfordate Sesquiterpene Pyridine Alkaloids and Derivatives . . . . . . 307
IV. Edulinate Sesquiterpene Pyridine Alkaloids . . . . . . . . . . . . . . . . 315
V. Cassinate Sesquiterpene Pyridine Alkaloids . . . . . . . . . . . . . . . . 317
VI. Lower Molecular Weight Sesquiterpene Pyridine Alkaloids. . . . . . . . 317
VII. Non-Celastraceous Sesquiterpene Pyridine Alkaloids . . . . . . . . . . . 318
VIII. Biosynthesis and Biogenesis . . . . . . . . . . . . . . . . . . . . . . . . . 322
IX. Biological Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 330
X. Taxonomic Considerations. . . . . . . . . . . . . . . . . . . . . . . . . . 334
XI. Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 334
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 339

Chemical and Biological Aspects of Melanin

D.P. CHAKRABORTY AND SHYAMALI ROY
I. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 345
II. Biogenesis of Melanin . . . . . . . . . . . . . . . . . . . . . . . . . . . . 347
III. Natural Melanins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 354
IV. Investigations of Melanins by Physical Methods. . . . . . . . . . . . . . 373
V. Synthetic Melanins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 376
VI. Physicochemical Properties and Biological Functions of Melanins . . . . 379
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 384

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 393
 CONTRIBUTORS

Numbers in parentheses indicate the pages on which the authors’ contributions begin.

D.P. CHAKRABORTY (345), Institute of Natural Products, Calcutta 700 036, India

MARK T. HAMANN (207), Department of Pharmacognosy and National Center for

the Development of Natural Products (NCNPR), School Pharmacy, The
University of Mississippi, University, Mississippi, USA

RUSSELL HILL (207), The Center of Marine Biotechnology (COMB), University of

Maryland Biotechnology Institute, Columbus Center, Baltimore, MD, USA

JIN-FENG HU (207), Department of Pharmacognosy and National Center for the

Development of Natural Products (NCNPR), School Pharmacy, The University of
Mississippi, University, Mississippi, USA

MICHELLE KELLY (207), National Center for Aquatic Biodiversity and Biosecurity,
National Institute of Water and Atmospheric Research (NIWA) Ltd., Newmarket,
Auckland, New Zealand

JUN’ICHI KOBAYASHI (165), Graduate School of Pharmaceutical Sciences,

Hokkaido University, Sapporo 060, Japan

LUCIANO M. LIÃO (287), Instituto de Quimica, Universidade Federal de Goiás,

Goiânia, GO, 74001-970, Brazil

HIROSHI MORITA (165), Graduate School of Pharmaceutical Sciences, Hokkaido

University, Sapporo 060, Japan

SHYAMALI ROY (345), Institute of Natural Products, Calcutta 700 036, India

SHEO SINGH (51), Merck Research Laboratories, Rahway, NJ 07065, USA

HEATHER SINGS (51), Merck Research Laboratories, Rahway, NJ 07065, USA

OTTO SOEPENBERG (1), Department of Medical Oncology, Erasmus MC – Daniel

den Hoed Cancer Center, Rotterdam, The Netherlands

vii
viii CONTRIBUTORS

ALEX SPARREBOOM (1), Department of Medical Oncology, Erasmus MC – Daniel

den Hoed Cancer Center, Rotterdam, The Netherlands

JAAP VERWEIJ (1), Department of Medical Oncology, Erasmus MC – Daniel den

Hoed Cancer Center, Rotterdam, The Netherlands
 PREFACE

This volume marks the 60th in this series, which began publication in 1950
under the editorship of the late Professor R.H.F. Manske. Through the
successive editorships of Professor Russell Rodrigo and Dr. Arnold Brossi, the
series evolved in scope in an effort to try to keep up with the burgeoning field of
‘‘alkaloids.’’ As Editor, I have tried to continue this evolutionary process,
blending together diverse chapters on the discovery, biological evaluation, and
clinical applications of alkaloids. In this volume, the six chapters reflect areas
of alkaloid research which are new to the series and others which require
updating because of the recent progress.

In Chapter 1, Soepenberg, Sparreboom, and Verweij present a detailed

discussion of the clinical aspects of camptothecin, one of the most important
alkaloids to be studied in the past 40 years. In Chapter 2, Sings and Singh
describe the various 2,3-fused indole diterpenoid derivatives and their powerful
biological effects. A much needed update on the Daphniphyllum alkaloids is
offered by Kobayashi and Morita in Chapter 3.

A significant group of marine alkaloids, the manzamines, which have

attracted a lot of interest in the past few years, is reviewed by Hamann and
colleagues in Chapter 4, and Lião summarizes in Chapter 5 the advances that
have been made in the sesquiterpene alkaloids, a group not reviewed previously
in the series. Finally, following the untimely death of Professor D.P.
Chakraborty, Dr. Shyamali Roy agreed to complete the work of updating the
field of melanin chemistry and biology.

Geoffrey A. Cordell
University of Illinois at Chicago

ix
 —CHAPTER 1—

CLINICAL STUDIES OF CAMPTOTHECIN

AND DERIVATIVES

OTTO SOEPENBERG, ALEX SPARREBOOM, AND JAAP VERWEIJ

Department of Medical Oncology, Erasmus MC – Daniel den Hoed Cancer

Center, Rotterdam, The Netherlands

I. Introduction
II. Mechanisms of Action
III. Mutagenicity and Resistance Mechanisms
IV. Irinotecan
V. Topotecan
VI. Considerations of Route of Administration
VII. Investigational Derivatives
VIII. Conclusions
References

I. Introduction

About a half century ago, thousands of natural products were chemically

screened in a program initiated by the National Cancer Institute to search for
steroidal sapogenins which would be cortisone precursors. This program led to the
discovery of the plant alkaloids, the camptothecins (CPTs) – isolated from the stem
wood of the Chinese tree Xi Shu or Camptotheca acuminata (Decaisne, Nyssaceae)
(1–5). Initial in vitro and in vivo studies using the chloroform extract of the aqueous
ethanolic residue of C. acuminata suggested widespread antitumor activity (2,3).
Camptothecins are related to the monoterpenoid indole alkaloids, and the parent
alkaloid was shown to be a high-melting substance [molecular weight (MW)
348.111] comprising a unique and highly unsaturated pentacyclic-ring structure
(1,3). The structures of CPT and selected analogs are shown in Fig. 1.

The six-membered quinoline B-ring and the five-membered C-ring are

formed by a ring expansion and contraction sequence of reactions. The E-ring
presents a -hydroxylactone system which can undergo a pH-dependent reversible

THE ALKALOIDS, Vol. 60 Copyright ß 2003 Elsevier (USA)

ISSN: 0099-9598 1 All rights reserved
DOI: 10.1016/S0099-9598(03)60001-5
2 SOEPENBERG ET AL.

Figure 1. Lactone–carboxylate interconversion and chemical structures of six- and

seven-membered E-ring camptothecin analogs.

hydrolysis. Camptothecin forms the sodium salt of a hydroxy acid after adding
alkali and is relactonized on acidification. Both the E-ring and the D-ring, which
has a conjugated pyridone moiety, are essential structural features for the
antitumor activity of CPT (3).
 CLINICAL STUDIES OF CAMPTOTHECIN AND DERIVATIVES 3

Triggered by the preclinical antitumor activity, early Phase I clinical trials

were performed in the early 1970s using CPT as a water-soluble sodium salt
formulation, because of its ease of use as an intravenous (i.v.) formulation (6).
Although in the first Phase I study five, short-lasting partial remissions were
reported in 18 patients with primarily gastrointestinal cancers, these results could
not be confirmed in subsequent Phase I and II studies in the USA (7–9). In contrast,
a large program in the People’s Republic of China involving up to 1000 patients
treated with the sodium salt formulation of CPT reported favorable results, but for
a variety of reasons these had to be interpreted with caution (3). However,
unpredictable severe adverse events, i.e., life-threatening diarrhea, considerable
hemorrhagic cystitis, and myelosuppression, likely related to the poor aqueous
solubility of the parent compound, led to the suspension of the further development
of this antineoplastic agent during the next two decades. The recognition that the
nuclear enzyme DNA topoisomerase I was the prime target in the mechanism of
action of the CPTs, and the possibility to develop numerous semisynthetic CPT
analogs with improved aqueous solubility, and, in consequence, a more predictable
toxicity profile, resulted in renewed interest in these cytotoxic agents.

Currently, two CPT derivatives, irinotecan and topotecan, are registered for
use in oncologic practice. Irinotecan is registered for use in metastatic colorectal
cancer. Topotecan is approved for the treatment of patients with cisplatin-
refractory ovarian cancer and for small-cell lung cancer (SCLC) after the failure of
first-line chemotherapy. During the last 10 years, knowledge of the pharmaco-
kinetic and pharmacodynamic properties of the semisynthetic CPT analogs has
significantly increased. The next challenge in their development will be to maximize
efficacy and to minimize side effects in the treatment by biomodulation of
the pharmacokinetic and pharmacodynamic properties. For instance, the
pharmacological profile of irinotecan is extremely complex and the various
processes involved in drug elimination, either through metabolic breakdown or
excretion, likely impact substantially on interindividual variability in drug
handling. Strategies to individualize irinotecan administration schedules based on
patient profiles in enzyme and protein expression or by co-administration of
specific agents modulating side effects are under investigation (10). Once the results
of pharmacogenetics can be more crystallized, this may ultimately lead to more
selective or ‘‘tailor-made’’ dose scheduling for patients to adjust the ‘‘fine-tuning’’
administration of these agents.

In this review, we will focus on the clinically important aspects of the CPT
alkaloids with an emphasis on their mechanisms of action and resistance, their
pharmacokinetic and pharmacodynamic behavior, and their routes of administra-
tion.

II. Mechanisms of Action

The cytotoxic CPTs belong to the class of topoisomerase I inhibitors. DNA

topoisomerases are essential enzymes found in all nucleated cells. These enzymes
4 SOEPENBERG ET AL.

are involved in the regulation of DNA topology and are necessary for the
preservation of the integrity of the genetic material during DNA metabolism,
e.g., RNA transcription, DNA replication, recombination, chromatin remodeling,
chromatin condensation, and repair during cell division (11,12). Based on
their different reaction mechanism and cellular function, there are two types of
DNA topoisomerases (type I and type II). Their characteristics are summarized
in Table I.

Human topoisomerase I is a monomeric
91-kDa polypeptide of 765

amino acids encoded by an active-copy gene located on chromosome 20q12–13.2
and two pseudogenes on chromosomes 1q23–24 and 22q11.2–13.1 (13–15). The
coding sequence of the gene is split into 21 exons spread over at least 85 kilobase
pairs of human genomic DNA (16). The human topoisomerase I gene promoter is
influenced by positively and negatively acting transcription factors, which are
described in more detail elsewhere (16,17).

This protein is comprised of four major domains: a highly charged NH2-

terminal domain (MW 24 kDa), a conserved core domain (MW 56 kDa), a

TABLE I.
Differentiation of Human DNA Topoisomerases Type I and II.

Type I topoisomerase
– Monomeric protein, molecular weight
91 kDa
– Single-copy gene located on chromosome 20q12–13.2
– Transiently breaks one strand of duplex DNA and forms a 30 -phosphotyrosine
covalent intermediate
– Single-step changes in the linking number of circular DNAs
– Its expression is continuous during the cell cycle and in quiescent cells
– Mainly involved in relaxation of supercoiled DNA during RNA transcription
– ATP independent
Type II topoisomerase
– Homodimeric protein, molecular weight 170 kDa (isoenzyme II ) and 180 kDa
(isoenzyme II )
– Single-copy gene located on chromosome 17q21–22 (isoenzyme II ) and
chromosome 3p24 (isoenzyme II )
– Breaks both strands of duplex DNA and forms a pair of 50 -phosphotyrosine
covalent intermediates
– Generates a gate through which another region of DNA can be passed
– Double-step changes in the linking number of circular DNAs
– Its expression increases during S-phase of the cell cycle (especially isoenzyme II )
and almost absent in quiescent cells (primarily expression of isoenzyme II )
– Involved in DNA replication, recombination, RNA transcription, and repair
– ATP dependent
 CLINICAL STUDIES OF CAMPTOTHECIN AND DERIVATIVES 5

positively charged linker domain (MW 7 kDa), and a highly conserved COOH-
terminal domain (MW 6 kDa) containing the active-site thyrosine, i.e., the
nucleophilic Tyr723 amino acid residue (15,18). Human topoisomerase promotes the
relaxation of both positively and negatively torsionally strained (supercoiled)
duplex DNA with equal efficiency so that transcription and replication can proceed
(15). Hereby, a transesterification reaction takes place by which topoisomerase
I cleaves one strand of the double-helix structure of DNA using the C4-oxygen
atom of the active-site tyrosine (Tyr723) residue and constitutes a transient, covalent
phosphotyrosyl intermediate with the 30 end of the nicked DNA strand, the
so-called cleavable complex. This way it changes the linking number in single steps
(15,19). Later, the energy of this covalent attachment is recycled for the reverse
transesterification reaction that reseals the DNA strands (religation) and liberates
the enzyme. Consequently, for these intertwining processes neither energy cofactors
nor metal cations are required by human topoisomerase I (20).

Recent studies have revealed more detailed insights in the three-dimensional

structure of human topoisomerase I and its interactive function with the DNA
molecule (21,22). In Table II, the principal structural characteristics of human
topoisomerase I are summarized.

The mechanism of DNA relaxation after formation of the covalent complex

and before religation is still not completely elucidated (21). So far, two mechanisms
are supposed, viz. the strand-passage model and the free-rotation model. Both
models represent the two extremes of a continuum in the conceptual framework
for how topoisomerase I might effect changes in linking number (21,22). In the
strand-passage (or enzyme-bridging) model, it is hypothesized that the intact DNA
strand is passed through an enzyme-bridged gate, which is made by the covalent
linkage of the 30 end and by noncovalent binding to the 50 end of the broken strand.
On the other hand, in the free-rotation model, relaxation of torsonially strained
duplex DNA is possible due to releasing of the 50 end of the broken strand from the
active site and as a result is allowed to rotate freely about the complementary
unbroken strand (15,22). X-Ray crystallographic studies with complexes of human
topoisomerase I and DNA lead to the proposal that the relaxation of supercoiled
DNA elapsed by a controlled rotation mechanism (21,22).

In the controlled rotation model, the DNA structure is allowed to rotate

completely free at 30 intervals downstream of the cleavage site round the intact
DNA strand modulated by the interaction of the nose-cone helices of subdomains I
and II in a positively charged cavity formed by the cap of the enzyme and the linker
domain (15). In vitro studies with reconstituted human topoisomerase I have
revealed the more precise function of the linker region. During the normal
relaxation of supercoiled DNA it acts to slow the religation (18). From these
studies, it is also hypothesized that the inhibition of DNA relaxation caused by
CPT – by stabilizing the cleavable complex – depends on a direct effect of the
cytotoxic agent on DNA rotation which is mediated by electrostatic interactions
between the linker domain and DNA (18).
6 SOEPENBERG ET AL.

TABLE II.
Structural Characteristics of Human DNA Topoisomerase Type I.

Two domains with essential functions for catalytic activity and relaxation function
I. Core domain (MW 56 kDa)
– Amino acid residues 215–635 (Ile215–Arg635)
– Subdomain I
– Amino acid residues 215–232 and 320–433
– Two helices and nine strands
– Subdomain II
– Amino acid residues 233–319
– Five helices and two strands
– Subdomain III
– Amino acid residues 434–635
– Ten helices and five strands
– Contains all active-site residues except the Tyr723
– Extends from the top half of the molecule downward through two long
helices that functions like a hinge that opens and closes the enzyme around
the DNA
j Subdomains I and II are folded tightly together and form the ‘‘cap’’ (top-lobe)
region of the enzyme
j Subdomains I and II have two long ‘‘nose-cone’’ helices ( 5 and 6) that make
an
90 angle with each other and come together in a ‘‘V’’-figure at a point 25 Å
away from the body of the molecule, and enclose a triangular-shaped empty
space between them
j Subdomain III forms the bottom lobe of the enzyme
j Subdomains I and III interact via two short ‘‘lips’’ opposite from the long-hinge
helices
II. COOH-terminal domain (MW 6 kDa)
Amino acid residues 713–765 (Gln713–Phe765)
Contains the active-site (catalytic) thyrosine Tyr723
Two domains without essential functions for catalytic activity and
relaxation function
III. NH2-terminal domain (MW 24 kDa)
– Amino acid residues 1–214 (Met1–Gly214)
– Highly charged, very few hydrophobic amino acids, is largely disordered, and
contains several nuclear-targeting signals
– Involved in nucleolar localization through interactions with nucleolin
IV. Linker domain (MW 7 kDa)
– Amino acid residues 636–712 (Pro636–Lys712)
– Coiled-coil structure that is positively charged
– Reaches 50 Å away from the body of the enzyme
 CLINICAL STUDIES OF CAMPTOTHECIN AND DERIVATIVES 7

When the attachment of CPT is modeled in the three-dimensional structure

of human topoisomerase I, it is believed that the DNA duplex is extended in such
a way that carbon positions 7 and 9 of the CPT structure are facing out into
open space. These carbon positions are very accessible to chemical modifications.
Consequently, this gives the opportunity to expand the potency of modified
CPT analogs by auspicious interactions of these derivatives with distant proteins or
DNA atoms at the binding site (22).

Stabilization of the cleavable complex by CPTs is not sufficient in itself for

the induction of cell death because the complex can reverse spontaneously in a
short time. The lethal effects of these drugs are caused by the interaction between a
moving replication fork (or transcription process) and the drug-stabilized cleavable
complex, resulting in irreversible arrest of DNA replication and the formation of
a double-strand break located at the fork. This so-called fork-collision model
leads to the arrest of the cell cycle in the S/G2-phase, and finally to apoptosis (23).
As cells in the S-phase division are up to 1000-fold more sensitive to topoisomerase
I inhibitors than cells in G1- or G2/M-phases after exposure, the cytotoxicity of
these agents is considered S-phase specific (24–27).

The time of persistence of the drug-stabilized cleavable complex depends

on the production and/or repair of replication or transcription lesions. In
general, transcription lesions require longer persistence, i.e., greater stability of the
cleavable complex, than do replication lesions (28). Unfortunately, the fraction of
replicating cells in tumor tissues is underrepresented. This means that cell kill
results predominantly from the transcription lesion mechanism. For this reason,
the potency of different CPT analogs to stabilize the cleavable complex is of
paramount importance for efficacious therapeutic use (28). Of relevance for all
topoisomerase I inhibitors are the data from in vitro experiments which revealed
that the cytotoxicity increases with the duration of exposure. Short-time exposures
to high concentrations are less effective than long-term exposures to low
concentrations (29,30).

III. Mutagenicity and Resistance Mechanisms

Owing to their mechanism of action, topoisomerase I inhibitors are a

double-edged sword, because besides their cytotoxic properties, these agents are
potential mutagens, although their mutagenicity and oncogenicity still remain to be
elucidated (31–33). This is stressed by the fact that mutations may lead to drug
resistance, limiting further treatment, or to the development of secondary
malignancies. If mutations arise in germ cells, this could eventually be transmitted
to subsequent generations. Furthermore, the tendency to administer topoisomerase
I inhibitors in protracted schedules or prolonged exposure regimens could
hypothetically lead to an increased risk of mutagenicity (34,35).

The stabilization of the cleavable complexes by topoisomerase I inhibi-

tors disrupts the DNA integrity and interferes with the normal processes of
8 SOEPENBERG ET AL.

DNA topology, including replication, transcription, DNA repair, chromosome

condensation, and chromosome separation (31). The formation of these drug-
induced cleavable complexes is essential, but is not sufficient in itself to cause
cytotoxicity. This implies that cells need to undergo DNA synthesis to yield
maximum toxicity (31,36–38). Experimental studies showed that in the presence of
topoisomerase II inhibitors chromosomal aberrations arise during replication
which seems a more important cause of cytotoxicity (31,36,39). Furthermore, it was
postulated that recombination processes must be initiated to bypass the replication
block created by topoisomerase II inhibitor-stabilized complexes, and that some
of these events cause aberrant, illegitimate, or nonhomologous recombination,
which may lead to cytotoxicity and/or mutations. Nonhomologous recombination
that causes deletions of an essential gene, partially or completely, resulted in the
loss of gene products and finally to cell death. In contrast, aberrant recombination
or rearrangement causing deletion of a suppressor gene, or activation of a
proto-oncogene, stimulated cell growth with the potential to induce secondary
cancers (31).

Similar data exist for topoisomerase II inhibitors (31), one use of which has
been related to the occurrence of acute myeloid leukemia (40–51). These secondary
leukemias are characterized by a short induction period and present as
myelomonocytic or monocytic leukemia, rather than myelodysplasia (52). The
rearrangements are usually distinguished by balanced chromosomal translocations
involving either the MLL (ALL-1, HRX) gene at 11q23 or the AML1 gene
21q22 (53,54).

In principle, topoisomerase I inhibitors can produce similar molecular

alterations as those caused by topoisomerase II inhibitors. Consequently, they may
have similar clinical consequences (31). Direct comparative in vitro studies have
shown that, on a molar basis, the topoisomerase I inhibitors were more mutagenic
than the topoisomerase II-inhibitor etoposide (31). Topotecan was found to be less
mutagenic than the parent compound CPT.

So far, the clinical use of topoisomerase I inhibitors has not been linked to
secondary malignancies. However, the relative survival time of patients treated with
irinotecan or topotecan as compared with those treated with epipodophyllotoxins
(e.g., testicular cancer or hematological malignancies) possibly results in a less
clinically apparent mutagenic risk of topoisomerase I inhibitors.

As with decreased levels of topoisomerase I, various point mutations of

topoisomerase I in different CPT-resistant cell lines have been associated with CPT
resistance (55–63). The different point mutations in human topoisomerase I in
several CPT-resistant cell lines are summarized in Table III. These point mutations
cause alterations to the topoisomerase I enzyme, resulting in decreased
topoisomerase I catalytic activity or impaired binding of CPT to topoisomerase I
(55,64). In some models, single amino acid changes resulted in partial resistance,
while double mutation induced a synergistic resistance (55).
 CLINICAL STUDIES OF CAMPTOTHECIN AND DERIVATIVES 9

TABLE III.
Different Point Mutations of Human Topoisomerase I.

CPT-resistant cell line Point mutation Reference

CPTR-2000 cell line Gly717 to Val, and Thr729 to Ile (55)

CPT-K5 cell line Asp533 to Gly*, and Asp583 to Gly (58)
Asp583 to Gly
PC-7/CPT cell line Thr729 to Ala (59)
By in vitro mutagenesis Gly363 to Cys (60)
Chinese hamster DC3F/C-10 Gly505 to Ser (62)
U-937/CR cell line Phe361 to Ser (63)

*Only Asp533 to Gly is responsible for resistance.

In a small clinical study involving eight non-small cell lung cancer (NSCLC)
patients treated with irinotecan, two-point mutations were identified that were
located near a site in topoisomerase I that was previously identified as a position
of a mutation in the CPT-resistant human lung cancer cell line PC7/CPT (64).
Although this is the first prospective clinical study which demonstrates that point
mutations in topoisomerase I occur after chemotherapy with irinotecan, further
clinical studies will be needed to verify if the occurrence of topoisomerase I gene
mutations relates to the occurrence of clinical resistance to topoisomerase I
inhibitors (64).

IV. Irinotecan

A. CLINICAL PHARMACOLOGY

Irinotecan (CPT-11; 7-ethyl-10-{4-[1-piperidino]-1-piperidino}-carbonyl-

oxycamptothecin) is a semisynthetic, water-soluble prodrug that requires hydrolysis
or de-esterification by carboxylesterases to form its active-metabolite SN-38
(7-ethyl-10-hydroxycamptothecin), which is 100–1000-fold more cytotoxic in vitro
than the parent compound (65). Irinotecan contains a dibasic, bispiperidine
substituent, linked through a carbonyl group to the hydroxyl at C-10, crucial for
water solubility.

Irinotecan pharmacology is extremely complex and still the subject of

research (65). It is known to be dependent on various enzyme and protein systems
for metabolic transformation and active transport, all regulating intestinal
absorption and hepatobiliary secretion mechanisms. Both irinotecan and SN-38
exist in an active lactone form and an inactive carboxylate form, through
an equilibrium that depends on the pH and the presence of binding proteins (65).
SN-38 is detoxified in the liver by the polymorphic enzyme uridine-diphosphate
glucuronosyltransferase 1A1 (UGT1A1) to SN-38 glucuronide (SN-38G) (66). This
isoenzyme is also responsible for bilirubin glucuronidation (67). Because of this,
10 SOEPENBERG ET AL.

patients with hereditary UGT enzyme polymorphism – e.g., familial hyperbili-

rubinemia conditions (Crigler–Najjar syndrome types I and II or Gilbert’s
disease) – are at increased risk for irinotecan-induced diarrhea due to decreased
glucuronidation of SN-38 (68).

Irinotecan showed a linear pharmacokinetic behavior (69–73). Both the

lactone form and the carboxylate form of irinotecan and SN-38 are detectable in
plasma shortly after i.v. administration (74). The AUC of SN-38 is only
approximately 4% of the AUC of irinotecan, which means that only a small
fraction of the dose is converted to the active form. After administration of
irinotecan as a short-time i.v. infusion over 30–90 min, there is a three-exponential
decline of the plasma concentration of the drug (75). The biological half-life of the
lactone form of SN-38 is 11.5 h. This is much longer than for other CPTs, for
instance topotecan (70,74). The clearance of irinotecan lactone (53.5 l/h/m2) is
approximately two times larger than that of topotecan. At steady state, the mean
volume of distribution of the total drug is 142 l/m2, representing approximately
four times the total body weight (74). When irinotecan is administered as a
protracted i.v. infusion over 7–21 days, the plasma levels of irinotecan, SN-38, and
SN-38G are comparable on days 7, 14, or 21 during the infusion. The AUC ratio of
SN-38 to irinotecan was 16% and was constant over the tested dose range (76).
This metabolic ratio was higher compared with the ratios found after short-time
infusion, namely 3–9%. This partly explains the relatively low maximum-tolerated
doses (MTDs) achieved with the continuous infusion regimens, which are thus due
to the greater conversion of irinotecan to the cytotoxic-metabolite SN-38.

Recently, Xie et al. reported a population analysis in 70 cancer patients on

the clinical pharmacokinetics of irinotecan and four of its metabolites (77). These
authors found that the interconversion between the lactone and carboxylate forms
of irinotecan was relatively rapid, with an equilibration half-life of 14 min in the
central compartment and hydrolysis occurring at a rate five times faster than
lactonization. Also, the same interconversion took place in the peripheral
compartments. The irinotecan lactone form had extensive tissue distribution (Vss,
445 l) in comparison with the carboxylate form (Vss, 78 l, excluding peripherally
formed irinotecan carboxylate) (77). The clearance of the lactone form (74.3 l/h)
was higher than the carboxylate form (12.3 l/h). These models revealed that there
was a preference of SN-38 and NPC to be formed out of the lactone form of
irinotecan, whereas APC could be modeled best by presuming formation from
irinotecan carboxylate (77). The interconversion between SN-38 lactone and
carboxylate was slower than that of irinotecan. SN-38 lactone is more tightly
bound to serum albumin than the carboxylate form, and for this reason the
dynamic equilibrium is preferential toward the lactone form. Although the
clearances for SN-38 lactone and carboxylate were similar, the lactone form had
more extensive tissue distribution (77).

Diarrhea originates from intestinal epithelium cell damaged by SN-38.

This reversible reaction can be modulated by changing the intestinal flora using
 CLINICAL STUDIES OF CAMPTOTHECIN AND DERIVATIVES 11

Figure 2. Summary of irinotecan-elimination (CPT-11) routes, showing esterase- (hCE1

and hCE2) mediated conversion to the active-metabolite SN-38, its further conjugation
into the glucuronic-acid derivative SN-38G by UGT1A1, the reversible deconjugation
by bacterial beta-glucuronidase (Beta-Glu), CYP3A-mediated conversion to the
oxidized derivatives APC and NPC, P-glycoprotein- (ABCB1) mediated cellular efflux
of irinotecan, and MDR1- (ABCC1), cMOAT- (ABCC2), and BCRP- (ABCG2)
mediated transport of SN-38.

antibiotics, e.g., neomycin or other beta-glucuronidase inhibitors, such as

cyclosporin A, which inhibits the cMOAT-mediated biliary excretion of
SN-38 (78,79). Furthermore, cytochrome P-450 3A4 (CYP3A4) metabolizes
irinotecan into several oxidized derivatives, including APC (7-ethyl-10-[4-N-5-
aminopentanoic acid)-1-piperidino]-carbonyloxycamptothecin) and NPC (7-ethyl-
10-[4-N-(-1-piperidino)-1-amino]-carbonyloxycamptothecin) (71,80–83). The
various genes with a putative role in irinotecan disposition are shown in Fig. 2.
Both metabolites are also poor inhibitors of topoisomerase I and only the minor-
metabolite NPC can be further converted to SN-38 (74,83,84). Because CYP3A4
is involved in the biotransformation of many other drugs, there is a potential
risk of clinically relevant drug interactions. A Phase I study of irinotecan in
malignant glioma patients revealed that the clearance of irinotecan was significantly
faster in patients who required cotreatment with anticonvulsants and glucocorti-
coids – inducers of the CYP3A4 enzyme – as compared with patients who did not
12 SOEPENBERG ET AL.

receive such concomitant treatment (85). A recent investigation indicates that a

significant interaction occurs between irinotecan and the herbal product
St. John’s wort because of CYP3A4 induction, resulting in 42% decreased
circulating concentrations of SN-38 (86). In contrast, inhibition of CYP3A4
activity, for example by ketoconazole, leads to substantially increased exposure
(
two-fold) to SN-38 (87). It has also been shown that the sequence of treatment
with irinotecan and infusional 5-FU affects the tolerability of this combination,
with the SN-38 area under the concentration versus time curve (AUC) being
40% lower (P<0.05) when irinotecan preceded 5-FU. This suggests that well-
defined 5-FU/irinotecan regimens are needed because the administration sequence
or the interval between the agents might affect treatment tolerance and perhaps
also activity (88).

Elimination of irinotecan occurs primarily in the feces via hepatobiliary and

intestinal secretion and secondarily in the urine (89). Approximately, 50% of the
total administered dose of irinotecan is recovered in urine (28%) and feces (25%) as
unchanged parent compound or its inactive metabolites (81). The hepatobiliary
elimination route is dependent on the presence of drug-transporting proteins,
notably P-glycoprotein and canalicular multispecific organic anion transporter
(cMOAT), present on the bile canalicular membrane (10). Because of the close
connection of irinotecan with liver biotransformation and elimination, the drug
should not be administered in case of increased bilirubin concentrations or elevated
transaminases (68,90).

The cytotoxic activity of SN-38 is performed by producing intermediate

forms of drug-stabilized covalent DNA/topoisomerase I complexes, also referred
earlier as the cleavable complexes, as outlined previously. SN-38 exerts this
cytotoxic function by trapping cleavable complexes instead of inhibiting
topoisomerase I enzyme function. Antitumor activity of CPT analogs can be
predicted in vitro based upon gene-copy number, mRNA content, and protein
expression of topoisomerase I (90). The fact that in human tumor samples from
colon cancer, there was increased topoisomerase I expression and activity as
compared to normal mucosa, could partly explain the activity of irinotecan in this
disease (90–93).

B. DOSE-FINDING TRIALS

Phase I studies were performed first in Japan, later in the USA and Europe.
The recommended regimen of irinotecan in the USA is 125 mg/m2 administered
as a 90-min i.v. infusion once weekly for 4 or 6 weeks (70). In Europe, the approved
administration schedule of irinotecan is 350 mg/m2 given as an i.v. infusion
over 60–90 min once every 3 weeks (94), while a recent reevaluation indicated a
maximum-tolerable dose of 320 mg/m2, or 290 mg/m2 in patients with prior
abdominal/pelvic radiation therapy (95). Finally in Japan, the administration
schedule of irinotecan was developed as 100 mg/m2 every week or 150 mg/m2
every other week (96). Remarkably, the dose intensity (DI) of all applied dosage
 CLINICAL STUDIES OF CAMPTOTHECIN AND DERIVATIVES 13

regimens of irinotecan is approximately 100 mg/m2/week, which suggested a

schedule independency. This phenomenon can be explained by the long
half-life of SN-38, which is achieved after a single dosage of irinotecan.
Although the half-life of SN-38 does not fully support this approach, in an effort
to further explore the possible therapeutic advantage of prolonged exposure
to CPTs, protracted or repeated dosing regimens of irinotecan have been studied.
Irinotecan was administered as a short infusion repeated on 5 consecutive days,
as a 4-day i.v. continuous infusion weekly for 2 weeks every 3 weeks, and as a
14-day continuous i.v. infusion every 3 weeks (97). Since protracted i.v.
infusion is inconvenient for patients, an oral formulation of irinotecan is also
under study (71).

In the transatlantic Phase I clinical trials, the maximum DI of irinotecan

was achieved with short-time i.v. infusion once every 2 or 3 weeks (mean DI
125 mg/m2/week; range, 80–167 mg/m2/week), whereas the lowest DI was achieved
with continuous i.v. infusion (DI 27 and 47 mg/m2/week). In the
weekly  4 schedule and the daily i.v. infusion over 3 or 5 consecutive days, the
mean calculated DI was 83 mg/m2/week (range, 73–100 mg/m2/week). Thus,
the maximum DI achieved with prolonged exposure schedules of irinotecan is
2–3 times lower than that achieved with short-infusion administration. But, as
indicated previously, irinotecan is more effectively converted to SN-38
during protracted i.v. infusion. The SN-38 to irinotecan AUC ratios ranged from
16% to 24% (97). After oral administration of irinotecan, the absolute
bioavailability of the drug based on lactone measurements is relatively low (74).
However, the SN-38 to irinotecan AUC ratio, expressed on a molar basis, is
three times greater after oral administration than after i.v. infusion. Possibly, the
first-pass conversion of irinotecan to SN-38 in the intestine and liver could explain
this observation (74).

The principal dose-limiting toxicity (DLT) for all schedules used

was delayed diarrhea, with or without neutropenia (70,94,96). The frequency of
severe diarrhea (grade 3 or 4) was reported as 35% in the Phase I studies.
The incidence of this toxicity can be reduced by more than 50% if an
intensive treatment with loperamide is used as described in the safety guidelines in
Table IV (98).

Neutropenia was dose related, generally of brief duration and noncumu-

lative, and occurred in 14–47% of patients treated once every 3 weeks and less
frequently using the weekly schedule (12–19%) (98–102). In approximately 3% of
patients, the neutropenia was associated with fever. In one Phase I study, where
irinotecan was given as a 96-h continuous infusion for 2 weeks every 3
weeks, thrombocytopenia was also dose limiting (103). Due to inhibition of
acetylcholinesterase activity by irinotecan within the first 24 h after dosing of the
drug, an acute cholinergic reaction can be observed. The symptomatology of this
syndrome, as well as the other nonhematological toxicities of irinotecan, is
summarized in Table V.
14 SOEPENBERG ET AL.

TABLE IV.
Safety Guidelines for the Use of Irinotecan.

d Proper patient selection according to known risk factors:

– For severe neutropenia: Performance status; serum bilirubin level
– For severe diarrhea: Performance status; prior abdominopelvic radiation therapy,
hyperleucocytosis
d Proper training of the treating medical oncologist and his staff, and extensive
information to the patient:
– Including patient’s information leaflet about management of toxicities
d Early use of high-dose loperamide as soon as the first loose stool occurs.
– Recommendation:
4 mg Loperamide for the first intake and then 2 mg every 2 h at least
12 h After the last loose stool for a maximum of 48 h
d Early use of oral broad-spectrum antibiotics (e.g., fluoroquinolone) in case of:
– Any grade 4a diarrhea
– Febrile diarrhea
– Diarrhea with concomitant grade 3a or 4 neutropenia
– Failure of a 48-h high-dose loperamide therapy
d Dose reduction in case of:
– Grade 4 neutropenia (even asymptomatic)
– Grade 3 neutropenia concomitant with fever and/or infection
– Severe diarrhea
d Contraindication for use in patients with:
– WHOb performance status >2
– Predictable poor compliance
– Baseline serum bilirubin >1.5  UNLc
d Treatment only with caution and close survey between cycles in patients with:
– WHO performance status ¼ 2
– Baseline serum bilirubin between 1 and 1.5  UNL
– Prior abdominopelvic radiation therapy
– Baseline hyperleucocytosis
a
Grade 3 and 4 according to NCI common toxicity criteria (version 2.0).
b
WHO, World Health Organization.
c
UNL, upper normal limit.

C. ANTITUMOR EFFICACY

Phase II studies consistently revealed response rates of 10–35% to single-

agent irinotecan in advanced or metastatic colorectal cancer (89,90,104,105),
independent of the applied schedules. There was no apparent difference between the
applied schedules with respect to the median remission duration and median
survival time, respectively 6–8 months and 8–13 months (90).
 CLINICAL STUDIES OF CAMPTOTHECIN AND DERIVATIVES 15

TABLE V.
Main Toxicities of Irinotecan.

Hematological
a a
d Grade 3 and 4 neutropenia
– Dose and schedule dependent
– Lowest frequencies in protracted dose regimens
– Reversible, noncumulative, and of short duration
– Febrile neutropenia occurred in about 3% of patients
Nonhematological
d Acute cholinergic-like syndrome ( 9% of patients)
– Caused by rapid and reversible inhibition of acetylcholinesterase by lactone form
of irinotecan and can be induced by co-administration of oxaliplatin
– Symptoms may occur shortly or within several hours after drug administration,
and are short lasting, never life threatening
– Symptoms: diarrhea, gastrointestinal cramps, nausea, vomiting, anorexia,
asthenia, diaphoresis, chills, malaise, dizziness, visual-accommodation distur-
bances, salivation, lacrimation, and asymptomatic bradycardia
– Responsive to and preventable by subcutaneous administration of 0.25–1.0 mg
atropine
d Delayed-onset diarrhea
– Could be severe and unpredictable at all dose levels with increased intensity and
frequency at higher dose levels ( 34% of patients grade 3 or 4 after use of
loperamide) and occurred later than 24 h after drug administration with peak
incidence at day 5 or 6
d Nausea and vomiting ( 86% of patients, but  19% grade  3)
– Manageable with 5-HT3 antagonists
d Other common toxicities
– Fatigue ( 17%), mucositis ( 12%), skin toxicity ( 5%), asthenia, alopecia,
and elevated liver transaminases
d Less common toxicities
– Pulmotoxicity (pneumonitis), cardiotoxicity (bradycardia), microscopic hema-
turia, cystitis, dysarthria, and immune thrombocytopenia
a
Grade 3 and 4 according to NCI common toxicity criteria (version 2.0).

In a randomized Phase III study, comparing treatment with irinotecan

given as a 300–350 mg/m2 i.v. infusion every 3 weeks to best-supportive care
in patients refractory to previous treatment with 5-FU based chemotherapy, the
one-year survival rate was significantly greater for the irinotecan-treated group
than for the control group, 36% and 14% (P<0.01), respectively (99). Another
randomized Phase III study, comparing treatment with irinotecan to three different
continuous i.v.-infusion schedules of 5-FU in patients with previously treated
advanced colorectal cancer, revealed a survival advantage for the irinotecan-treated
group in comparison to the 5-FU-treated group (100). The one-year survival
rates were 45% and 32%, respectively (P<0.05).
16 SOEPENBERG ET AL.

TABLE VI.
Phase II Studies of Single-Agent Irinotecan.

Tumor type Dose level and schedule Overall response rate (%)

SCLCa 100 mg/m2/wkc 47

NSCLCb 100 mg/m2/wk 32
Gastric cancer 100–150 mg/m2/1–2 wk 23
Pancreatic cancer 100–150 mg/m2/1–2 wk 10
350 mg/m2/3 wk 9
Breast cancer 100–150 mg/m2/1–2 wk 14–20
200 mg/m2/3–4 wk
350 mg/m2/3 wk 8
Cervical cancer 350 mg/m2/3 wk 15–23
125 mg/m2/wk  4 qd.6 wk 0–23
Ovarian cancer 100–150 mg/m2/1–2 wk 24
Head and neck cancer 75–125 mg/m2/wk  4 q.6 wk 21
Leukemia/lymphoma 40 mg/m2/de  5 q.3–4 wk 17–24
40 mg/m2/d  3 q.wk 38
a
SCLC, small-cell lung cancer.
b
NSCLC, non-small cell lung cancer.
c
wk, week(s).
d
q, every.
e
d, day(s).

Apart from colorectal cancer antitumor activity, single-agent irinotecan

was also active in Phase II studies in several other solid tumors summarized in
Table VI (65,74,106,107).

Based on the activity data in colorectal cancer derived from Phase I/II
studies on the combination of irinotecan with 5-FU/leucovorin, two randomized
Phase III studies were performed comparing this combination to single-agent
5-FU/leucovorin in the first-line treatment of metastatic colorectal cancer
(108,109). Saltz et al. randomized 683 patients to receive either a weekly  4
regimen of a 90-min i.v. infusion of irinotecan at a dose of 125 mg/m2 and
leucovorin at a dose of 20 mg/m2 as a 15-min i.v. infusion, followed by 5-FU i.v.
bolus at a dose of 500 mg/m2 (arm A, n ¼ 231), or conventional low-dose 5-FU/
leucovorin (arm B, n ¼ 226), or irinotecan at a dose of 125 mg/m2 for 4 consecutive
weeks every 6 weeks (arm C, n ¼ 226) (108). An intention-to-treat analysis showed
that the combination of irinotecan and 5-FU/leucovorin (arm A) yielded a
significantly higher remission rate (P<0.001), significantly longer progression-free
survival (P ¼ 0.004), and significantly longer median survival (P ¼ 0.04) in
comparison to single-agent 5-FU/leucovorin (arm B). There was no difference
between single-agent irinotecan (arm C) and 5-FU/leucovorin (arm B) in terms of
overall response rate, median time to disease progression, and median overall
survival time (108).
 CLINICAL STUDIES OF CAMPTOTHECIN AND DERIVATIVES 17

Severe diarrhea (grade 3 or 4) (arm A 23%; arm B 13%; and arm C 31%)
and neutropenia (grade 4) (arm A 42%; arm B 24%; and arm C 12%) were the
most prominent toxicities in this study, but these side effects did not preclude
the administration of approximately 75% of the prescribed doses of irinotecan
and 5-FU (108). The reverse side of the study was the bias of unreported toxic
deaths in the combination treatment arm (arm A).

Douillard et al. randomized 385 patients to (arm A, n ¼ 169) the

combination of irinotecan and an infusional schedule of 5-FU. The regimens
were: once weekly, irinotecan 80 mg/m2 with 5-FU 2300 mg/m2 by 24 h infusion,
and leucovorin 500 mg/m2 (arm A1); or every 2 weeks, irinotecan 180 mg/m2
on day 1 with 5-FU 400 mg/m2 bolus and 600 mg/m2 by 22 h infusion, and
leucovorin 200 mg/m2 on days 1 and 2 (arm A2) (109). For the control arm (arm B,
n ¼ 169), the regimens were: once weekly, 5-FU 2600 mg/m2 by 24 h infusion
and leucovorin 500 mg/m2 (arm B1); or every 2 weeks, 5-FU and leucovorin at the
same doses and administration as in arm A2 (109). Although there was a good
balance between both treatment arms for known risk factors, the number of
primary rectal cancers in arm A was slightly higher. Compared with the study by
Saltz et al., in this study proportionally more patients had received prior adjuvant
5-FU-based chemotherapy 10% versus 25%, respectively (90). An objective
response rate of 41% was reported in treatment arm A (combination irinotecan
with 5-FU and leucovorin) compared with 23% in the schedule of 5-FU and
leucovorin alone (arm B) (P<0.001) (109). Also, the median time to disease
progression (P<0.001) and median survival time (P<0.028) were statistically
significant in favor of the combination treatment arm (arm A) compared to the
control arm (arm B).

The overall response rate of treatment arm A1 was 51% and for arm
A2 38%. For the weekly single-agent 5-FU and leucovorin (arm B1) and biweekly
5-FU/leucovorin (arm B2), the overall response rates were 29% and 21%,
respectively (109). More severe neutropenia (29% vs. 21%, P<0.01) and severe
diarrhea (24% vs. 11%) were seen in the combination arm (arm A) compared with
the control arm (arm B). The neutropenia was not significantly associated with
fever or infection.

In a multiregression Cox model analysis, the influence of baseline

characteristics of patients on the efficacy of the cytotoxic therapy in terms
of time to progression and survival were determined. This analysis revealed
that excellent performance status (WHO 0 vs. 1 or more), extension of
organ involvement (1 vs. 2 or more sites), and normal values for lactate
dehydrogenase (LDH), bilirubin, and leucocytes were the major prog-
nostic factors for longer overall survival (90). A significant prolongation
of time to progression adjusted for normal values of LDH and disease extension
(only one organ site) (P ¼ 0.0001) was established for the combination irinotecan
and 5FU/leucovorin compared to single-agent 5-FU/leucovorin, in both
studies (90).
18 SOEPENBERG ET AL.

Combined analysis of both studies confirmed that the addition of

irinotecan to 5-FU/leucovorin significantly increases response rate, median time
to disease progression, and median time to survival, especially for patients with
excellent prognostic characteristics will have a survival benefit of this combination
therapy (90). The abovementioned treatment schedules of both studies are
approved by the FDA as first-line chemotherapy for patients with metastatic
colorectal cancer (90). Yet, randomized trials to evaluate the merits of this
combination in the adjuvant setting are ongoing (74). Furthermore, clinical trials
evaluating the antitumor efficacy of irinotecan in combination with an oral
fluoropyrimidine (capecitabine), which may replace infusional 5-FU, are currently
in progress (74).

D. OTHER COMBINATION CHEMOTHERAPY TRIALS

The combination of irinotecan and raltitrexed was evaluated in two Phase I

studies, whereby asthenia was found as the DLT in both studies. The recommended
doses were irinotecan 350 mg/m2 and raltitrexed 3 mg/m2 once every 3 weeks
(110,111).

In vitro and in vivo studies have revealed synergism or additivity between

irinotecan and cisplatin in numerous tumor cell lines and human tumor xenografts
(112–120). The mechanism of interaction between both agents involves a delay
by the topoisomerase I inhibitor in the reversal of cisplatin-induced DNA
interstrand cross-links (ISCs) without modifying the formation of ISCs (120,121).
No alteration in cleavable complexes formation or reversion was observed.

All Phase I studies on the combination of irinotecan and cisplatin,

except for one, focused on fractioned dose schedules for both agents (120,122–128).
Neutropenia (grade 4 in 35% of the patients) and diarrhea were the dose-limiting
toxicities. In only 4% of all patients neutropenia was complicated by fever.
A 33% and 65% increase in DI of irinotecan could be achieved by adding G-CSF
to schedules with neutropenia as the DLT (126,127). Only one Phase I study
involved the 3-weekly administration and studied the relevance of sequence of
drug administration (128). Patients were randomized to receive irinotecan,
immediately followed by cisplatin in the first course, and the reversed sequence in
the second course or vice versa. Significant differences in toxicity between the
treatment schedules were not observed. Neither could a pharmacokinetic
interaction be discerned. In addition, irinotecan had no influence on the platinum
DNA-adduct formation in peripheral leukocytes in either sequence (128).
Apparently there is no administration sequence that should clearly be favored
for this particular topoisomerase I inhibitor.

Phase II studies of this combination indicate high levels of activity in

various tumors, but none of these studies were randomized, so their interpretation
is difficult (74). A Phase III study using this combination as first-line chemotherapy
for patients with extensive disease of SCLC showed a significant improvement
 CLINICAL STUDIES OF CAMPTOTHECIN AND DERIVATIVES 19

in the one-year survival rate (60%) in comparison with conventional treatment

with etoposide and cisplatin (40%) (P ¼ 0.005) (129).

The combination of irinotecan and oxaliplatin was evaluated in Phase I

studies using a once every 2 weeks and once every 3 weeks schedule with
neutropenia, and a combination of diarrhea and neutropenia as dose-limiting side
effects in the different schedules, respectively (130–132). Remarkably, the
interaction of both drugs showed acute cholinergic toxicities, whose severity is
potentiated by oxaliplatin (133–135). The recommended dose of oxaliplatin is 85
mg/m2 for both schedules. The recommended dose of irinotecan is 175 mg/m2 for
the once every 2 weeks and is 200 mg/m2 for the once every 3 weeks. In a Phase II
study in patients with advanced colorectal cancer, this combination was compared
with raltitrexed as first-line treatment, which showed an acceptable toxicity profile
after dose reduction of irinotecan to 150 mg/m2 once every 3 weeks (136).

In Phase I and/or II studies, irinotecan is also combined with other

cytotoxic agents, including etoposide (137–142), carboplatin (143–145), docetaxel
(146,147), paclitaxel (148,149), mitomycin-C (150,151), and also the triplet
combination with carboplatin and docetaxel (152,153). This topic about
combination treatment is reviewed in more detail by De Jonge et al. (120). The
combination of irinotecan and etoposide seems to be hepatotoxic (154).

V. Topotecan

A. CLINICAL PHARMACOLOGY

Topotecan (TPT; 9-dimethylaminomethyl-10-hydroxycamptothecin) is

a semisynthetic, water-soluble CPT derivative, synthesized by modification of
10-hydroxycamptothecin (155). It undergoes CYP3A-catalyzed metabolism to
N-desmethyl topotecan. N-Desmethyl topotecan is a less-active metabolite of which
only low plasma levels were found, suggesting minimal CYP3A-related conversion.
Nonetheless, clinical trials have shown significantly altered clearance of topotecan
in patients on CYP3A-inducing anticonvulsants, similar to that observed with
irinotecan (65). Both topotecan and N-desmethyl topotecan exist in an active
lactone and inactive carboxylate form in a similar equilibrium (156). Only a very
small amount of total topotecan is present in the lactone form at equilibrium.
At physiological pH, most topotecan is in the inactive carboxylate form, whereas
in an acidic environment the ratio is opposite (157). These circumstances
have practical consequences because, on reconstitution in normal saline, a large
amount of topotecan is converted to the carboxylate form, whereas at lower pH of
5% dextrose for injection, the maximum extent of conversion is 10% with
equilibrium being achieved within half an hour (74,158). For this reason, a
new parenteral formulation has been produced that contains tartaric acid in the
infusion diluent to provide a sufficiently low pH to maintain the agent in the
essential lactone form (159).
20 SOEPENBERG ET AL.

Both the carboxylate form and the lactone form of topotecan may undergo
further metabolism into an UGT-mediated glucuronide product (i.e., topotecan-O-
glucuronide and N-desmethyl topotecan-O-glucuronide) (160). This is a reversible
transformation because beta-glucuronidase is able to reform topotecan and
N-desmethyl topotecan. Because topotecan is metabolized in the liver only to a
minor extent, it is not surprising that the pharmacokinetics in patients with
impaired liver function did not significantly differ from those in patients with
normal hepatic function (161). In contrast, patients with moderately impaired renal
function had significantly reduced plasma clearance (162). As a clinical practical
consequence, dose modifications are recommended for patients with impaired renal
function and are not required for patients with liver dysfunction (161,162).

Topotecan is most commonly administered as a 30-min i.v. infusion (74).

Within 5–10 min after the end of the infusion plasma concentrations of the
carboxylate form exceed those of the lactone form. Under the abovementioned
circumstances, the AUC ratio of the lactone to total drug ranges from 30% to 40%.
The same AUC ratio of both compounds in plasma is achieved when topotecan is
administered as a prolonged infusion once steady-state concentration is reached
(163–172). After i.v. infusion of topotecan, the plasma levels of the lactone form
and total drug descend in a biexponential way with similar terminal half-lives of
2.4–4.3 h. Compared with most other CPT derivatives, the biological half-life of
topotecan is short. Consequently, no drug accumulation will occur when five
daily doses of topotecan are administered by a 30-min i.v. infusion at an interval
of 24 h, which is the recommended dosing regimen for topotecan (74).

Over the range of tested doses of 0.4–22.5 mg/m2, topotecan showed linear
pharmacokinetic behavior for both the total drug and intact lactone species. This
pharmacokinetic behavior did not change significantly when topotecan was given
daily (74). The total body clearance of topotecan on the average is 27.4 l/h/m2 for
the lactone form and 13.4 l/h/m2 for the total drug (164–171). Owing to its
hydrophilic properties, topotecan showed a moderate, steady-state apparent
volume of distribution, being only approximately two times the body weight for the
lactone species and total drug. In comparison with other CPT analogs, the fraction
of topotecan bound to plasma proteins is much lower. Possibly, this property
accounts for the more efficacious penetration of topotecan in cerebrospinal fluid
(173–175) and pleural fluid and ascites (176) as compared to other CPT derivates.

Furthermore, the plasma pharmacokinetics of topotecan did not change

due to the presence of third spaces, i.e., ascites and/or pleural effusion (176). The
ratio between the exposure to topotecan in the third space and in the plasma
compartment was 55%. Consequently, topotecan can be safely administered to
patients with malignant ascites and pleural effusion, and it might contribute to local
tumor effects due to its substantial penetration in the third space (176).

A Phase I study on the intraperitoneal (i.p.) administration of topotecan

revealed that the MTD was 20 mg/m2 once every 3 weeks delivered by the i.p.
 CLINICAL STUDIES OF CAMPTOTHECIN AND DERIVATIVES 21

route, achieving cytotoxic plasma levels of topotecan, with acceptable toxicity and
avoiding myelotoxicity (177). Peritoneal total topotecan was cleared from the
peritoneal cavity at 0.4  0.3 L/h  m2 with a half-life of 2.7  1.7 h. The mean
peritoneal to plasma AUC ratio for total topotecan was 54  34% (177). These data
were in good agreement with those from a Phase I study using a topotecan 24-h
continuous i.p. infusion (178). The terminal half-life for peritoneal topotecan and
for plasma total topotecan was similar for both studies (177,178). These data
suggest that it would be possible to combine the i.p. administration of topotecan
with other active chemotherapeutic agents.

In a randomized trial on topotecan administered either orally or

intravenously, Herben et al. found that the urinary and fecal excretion of
unchanged drug were the major routes of elimination (179). The principal route
of excretion was via the kidneys, accounting for approximately 49% of the
intravenously administered dose and 20% of the oral dose (179). Furthermore,
approximately 18% and 33% of the i.v. and oral dose of topotecan, respectively,
was excreted unchanged in the feces. Elimination through other routes was
approximately 28% of the i.v. dose and 43% of the oral dose.

As mentioned earlier, in patients with impaired renal function the clearance

of total topotecan is reduced, i.e., a 33% decrease in patients with a creatinine
clearance (CrCl) ranging from 40 to 59 ml/min and a 75% decrease in patients with
CrCl between 20 and 39 ml/min, compared to patients with normal renal function
(i.e., CrCl  60 ml/min) (162). In patients with reduced renal clearance of
topotecan, a second plasma peak was seen after the end of infusion due to increased
bile excretion which, in turn, leads to enterohepatic recycling (162,179).
Nevertheless, this is likely not of clinical relevance.

The oral bioavailability of topotecan is influenced by different factors.

Firstly, the relatively high pH in the intestines leads to conversion to the
carboxylate form, which is poorly absorbed by the intestinal walls (180). Secondly,
the bioavailability is reduced by protein-mediated drug transport of topotecan. The
breast cancer-resistance protein (BRCP), a protein of the adenosine triphosphate
(ATP)-binding cassette family of transmembrane transporters, is highly expressed
in the small intestine and mediates the apically directed drug transport of
topotecan (179,181). Thirdly, the bioavailability is partly influenced by the binding
of topotecan to food, proteins, and intestinal fluids, or by decomposition in the
gastrointestinal fluid. Furthermore, the metabolism to N-desmethyl topotecan was
demonstrated as a minor mechanism for elimination (179).

Population pharmacokinetic studies in patients treated with intravenously

administered topotecan revealed that patient characteristics (i.e., gender, height,
weight) and laboratory values (i.e., serum creatinine concentration) give
a moderate ability to predict the clearance of topotecan in an individual patient
(182). Although a significant correlation between the clearance of topotecan
and patient age was not established, pharmacokinetic results of a greater number
22 SOEPENBERG ET AL.

of patients are warranted in order to make more definite conclusions on this

specific issue.

B. DOSE-FINDING TRIALS

Numerous Phase I clinical trials with topotecan in different schedules of

drug administration have been performed (183). Based on the in vitro data on long-
term exposure and the fact that efficacy of the drug has been demonstrated to be
dependent on the schedules of administration, two schedules were selected for
Phase II studies. Firstly, there is a 30-min i.v. infusion daily for 5 consecutive days
every 3 weeks, at a dose of 1.5 mg/m2/day. In this schedule, the DLT is short
lasting, noncumulative myelosuppression (184–186). Nonhematological toxicities
are usually mild and reversible and include nausea, vomiting, fatigue, alopecia, and
sometimes diarrhea. Phase II studies with the drug administered in this schedule
revealed response rates ranging from 9.5 to 25% in pretreated patients with ovarian
cancer, and response rates of 10–39% in patients with SCLC (187–190). In
addition, a comparative randomized, multicenter trial in which patients with
recurrent ovarian cancer were treated, showed that topotecan was at least as
effective as paclitaxel in terms of response rate (20% vs. 13%), median duration of
response, and median time to progression (191–197). In other tumor types,
topotecan was much less active (198–217). A summary of the safety profiles and
clinically observed toxicities is provided in Tables VII and VIII, respectively
(218,219).

Secondly, various schedules focusing on the continuous infusion of

topotecan have been studied, including a 24-h infusion weekly, and every 3 weeks, a
72-h infusion administered weekly, every 14 days and every 21 days, a 120-h
infusion every 3–4 weeks, and 21-day low-dose continuous infusion every 4 weeks
(27). In addition to the DLT of leucocytopenia, the longest infusion schedules
also induce thrombocytopenia.

With the continuous i.v. administration for 21 days every 28 days, the
MTD was 0.53 mg/m2/day (220). The steady-state concentration of lactone
topotecan was only approximately 4 ng/ml. No consistent relationship between
drug level and hematological toxicity was found. Of interest, the Phase I study
showed several partial tumor responses in tumor types which were initially
chemotherapy resistant (220). In a Phase II study with this regimen in patients
with progressive and platinum-refractory ovarian cancer, the response rate was
37% (221).

Since 21-day continuous infusion is inconvenient, an oral formulation of

topotecan was also investigated (166). The oral bioavailability of topotecan is
approximately 30–35% with acceptable interpatient variation and independent
of the formulation used. In view of this, the oral route of administration may
provide a more convenient approach to clinical-prolonged exposure (166,222,223).
After oral administration, topotecan is rapidly absorbed with peak plasma
 CLINICAL STUDIES OF CAMPTOTHECIN AND DERIVATIVES 23

TABLE VII.
Safety Guidelines for the Use of Topotecan.

d Proper patient selection according to known risk factors:

– For severe neutropenia: Renal impairment, prior myelosuppressive chemotherapy
(both platinum analogs and alkylating agents), prior bone marrow transplantation,
prior wide-field radiotherapy
– For severe thrombocytopenia: Renal impairment, prior myelosuppressive che-
motherapy (especially carboplatin)
d Proper training of the treating medical oncologist and his staff, and extensive
information to the patient:
– Including a patient information leaflet about the management of toxicities
d Dose reduction in case of:
– Grade 4a neutropenia >2 weeks (with G-CSFb)
– Grade 3a neutropenia concomitant with fever and/or infection
– Complicated grade 3 or 4 thrombocytopenia
– Renal impairment; creatinine clearance <60 ml/min and extensive prior therapy;
creatinine clearance <40 ml/min and minimal prior therapy
– Treatment delay >2 weeks
– Elevated bilirubin in combination with poor performance status and
comorbidities
d Contraindication for use in patients with:
– WHOc performance status >2
– Predictable poor compliance
– Creatinine clearance <20 ml/min
– Baseline serum bilirubin >1.5  UNLd
d Treatment only with caution and close survey between cycles in patients with:
– WHO performance status ¼ 2
– Creatinine clearance <60 ml/min
– Prior myelosuppresive chemotherapy (carboplatin)
– Prior abdominopelvic radiation therapy
a
Grade 3 and 4 according to NCI common toxicity criteria (version 2.0).
b
G-CSF, granulocyte colony-stimulating factor.
c
WHO, World Health Organization.
d
UNL, upper normal limit.

concentrations reached at 0.6–0.78 h after intake (157). Lactone to carboxylate

ratios was comparable after oral and i.v. administration (166,223). No relationship
was found between bioavailability and age, gender, performance status, and
the presence of liver metastases. Currently, topotecan is supplied in gelatin
capsules and is administered at least 10 min before a meal, although combination
with high-fat meal only led to a small decrease in the rate of absorption, not in the
extent of absorption (224).
24 SOEPENBERG ET AL.

TABLE VIII.
Main Toxicities of Topotecan.

Hematological
a
d Grade 4 neutropenia (81% of patients)
– Dose related, reversible, noncumulative
– (Extensive) prior treatment dependent
– Median onset occurred on day 9
– Median duration of 6 days (in 12% of patients duration longer than 7 days)
– Febrile neutropenia or infections occurred in 26% of patients
d Grade 4 thrombocytopenia (26% of patients)
– Dose related, reversible, noncumulative
– (Extensive) prior treatment dependent
– Platelet transfusions necessary in 13% of patients
d Grade 4 anemia (40% of patients)
– Dose related, reversible, noncumulative
– Blood transfusions necessary in 56% of patients
Non-hematological
d Other common toxicities
– Alopecia, nausea, vomiting, fatigue, stomatitis, constipation, diarrhea, abdom-
inal pain, asthenia
a
Grade 4 according to NCI common toxicity criteria (version 2.0).

In order to mimic a prolonged exposure regimen and based on the relatively

short half-life of the drug (average 2.4 h), a twice daily oral administration
schedule for 21 days every 28 weeks was studied (222). The dose-limiting side effect
was diarrhea, at a dose of 0.6 mg/m2 twice daily. It occurred in 55% of patients
with a median day of onset on day 15 (range, 12–20) and resolved after a median of
8 days (range, 7–16). Administration of high-dose loperamide did not limit the
diarrhea (222). The hematological toxicity was mild and comprised mainly of
neutropenia (35%).

Because of this diarrhea occurring beyond day 15, and in view of the
emerging insights that topoisomerase I inhibition might be no longer optimal
after 14 days of continuous drug administration, a shorter schedule was
investigated (224,225). Patients were treated once daily or twice daily for 10 days
every 21 days (224). In the once-daily regimen, dose-limiting thrombocytopenia
and diarrhea was seen at a dose of 1.6 mg/m2/day. The DLTs were similar
occurring at a dose of 0.8 mg/m2 b.i.d. (157,224).

Because of the persistence of diarrhea as a side effect in the 10-day schedule,

finally a 5-day schedule was studied (226). The DLT was neutropenia similar
to the i.v. drug use, with a nadir between days 8 and 15 and median duration of
 CLINICAL STUDIES OF CAMPTOTHECIN AND DERIVATIVES 25

6 days (range, 2–12). Nonhematological toxicities were mild to moderate. Moderate

to severe diarrhea ( CTC grade 2) was observed in 21% of the patients and this
event was self-limiting. The recommended dose was 2.3 mg/m2/day. Assuming
an average body surface area in patients of 1.75 m2, the recommended dose of
2.3 mg/m2/day equals a fixed dose of 4 mg/day. Pharmacokinetics and toxicity
were studied at this fixed dose in order to ascertain whether dosing based on per
square milligram offered any advantage over flat dosing. Such an advantage was
not found (226).

In summary, hematological toxicity is more pronounced with the shorter

oral regimens, but is still mostly mild and noncumulative, whereas diarrhea is a
severe and intractable side effect of more prolonged daily administration (157). An
analysis of the pharmacokinetic/pharmacodynamic relationships revealed that
the total AUC per course did not differ between the various regimens, and in an
analysis of the time over the threshold concentration of 1 ng/ml, it appeared that
the daily-times-five schedule provided the best systemic exposure and toxicity
profile (227).

In acute leukemia, the maximum-tolerable dose of a daily 30-min i.v.

infusion for 5 consecutive days every 3 weeks was 4.5 mg/m2/day (228). Dose-
limiting toxicity at higher dose levels were a complex of symptoms, comprising
high fever, rigors, precipitous anemia, and hyperbilirubinemia. Although the
precise etiology of these adverse effects was not known, it was believed that high
doses of topotecan had induced an acute hemolytic reaction (228).

C. ANTITUMOR EFFICACY

The dosage regimen of topotecan approved for clinical use is 1.5 mg/m2/day
given as a 30-min i.v. infusion daily for 5 days every 3 weeks. Dose-related,
reversible, and noncumulative myelosuppression is the most important side
effect of topotecan (229). Neutropenia – the nadir is usually approximately 9 days
after the start of the treatment and the median duration is approximately 7–10 days
– occurred more frequently and is often more severe than thrombocytopenia. Also,
neutropenia was more severe in heavily pretreated patients compared
with minimally pretreated patients (229). Besides myelosuppression, stomatitis
(24–28% of patients) and late-onset diarrhea (40%) were noted at higher doses
(65,229). Other nonhematological toxicities reported included alopecia (76–82% of
patients), nausea (75–78%), vomiting (53–64%), fatigue (30–41%), and asthenia
(21–22%) (229).

Antitumor activity of topotecan, given as a single agent in various schedules

of administration, was established in a variety of Phase II studies, including
ovarian cancer [overall response rate (OR), 14–38%], SCLC (OR,
39%), NSCLC
(OR,
13%), breast cancer (OR,
10%), myelodysplastic syndrome (MDS)
[complete response rate (CR),
37%], and chronic myelomonocytic leukemia
(CMML) (CR,
27%) (65). Marginal activity was seen in head and neck cancer
26 SOEPENBERG ET AL.

(202,230), prostate cancer (231), pancreatic cancer (210,215,217), gastric cancer

(211,212), esophageal carcinoma (204), hepatocellular carcinoma (213), and
recurrent malignant glioma (216).

In a Phase III study, the daily-times-five i.v. topotecan regimen was

compared with paclitaxel (3-h infusion of 175 mg/m2/day every 3 weeks) in ovarian
cancer. In this disease, topotecan and paclitaxel were equally effective with regard
to response rates, progression-free survival, and overall survival (191). The
median duration of response was 26 weeks (range, 7–84 weeks) for topotecan and
22 weeks (range, 9–67 weeks) for paclitaxel. The respective median times to
progression were 19 weeks (range, <1–93 weeks) and 15 weeks (range, <1–77
weeks), while the median survival was 63 weeks (range, <1–122 weeks) versus
53 weeks (range, <1–130 weeks) (232).

In an open-label, multicenter study comparing the activity and tolerability

of oral versus i.v. topotecan, 266 patients with relapsed epithelial ovarian
cancer after failure of one platinum-based regimen, which could have included a
taxane, were randomized to the two arms (233). Oral versus i.v. doses of topotecan
were administered as 2.3 and 1.5 mg/m2/day, respectively, for 5 consecutive days
every 3 weeks. The principal toxicity was noncumulative myelosuppression,
although moderate to severe neutropenia was less frequently seen in patients
treated with oral topotecan. Furthermore, grade 3 and 4 gastrointestinal toxicities
were seen slightly higher in the oral treatment arm. No difference in response
rates between the treatment arms was reported. Although a small, statistically
significant difference in survival favored the i.v. formulation (58 weeks) versus the
oral formulation (51 weeks) (P ¼ 0.033), in the context of second-line palliative
treatment for ovarian cancer, this outcome has only limited clinical significance
(233). For this reason, oral topotecan could be an alternative treatment modality
in this setting because of its convenience and good tolerability. Its definite place
has to be clarified in further studies.

A Phase III study compared single-agent topotecan with combination

chemotherapy, comprising cyclophosphamide, doxorubicin, and vincristine
(CAV), in 211 patients with SCLC relapsing after first-line chemotherapy (234).
Although response rate, time to disease progression, and overall survival
were similar, palliative efficacy of disease-related symptoms was better with
topotecan (234). In a randomized Phase III trial performed by the Eastern
Cooperative Oncology Group (ECOG), topotecan was compared with best
support of care in patients with extensive SCLC. In this trial, topotecan was
administered as a consolidation therapy after response induction with cisplatin and
etoposide (235). Although topotecan induced a moderate increase in the time
to disease progression, it did not improve survival (235). Finally, similar to the
study in ovarian cancer, oral topotecan was compared to i.v. administration of
topotecan in patients with relapsed and chemosensitive SCLC. The oral
formulation was found to be similar in efficacy with less severe neutropenia and
greater convenience of drug administration (236). Based on these data, topotecan
 CLINICAL STUDIES OF CAMPTOTHECIN AND DERIVATIVES 27

has been approved by the FDA for the treatment of recurrent SCLC in the
USA (74).

Although topotecan has shown some activity against hematological

malignancies, its use for this specific indication has still to be further explored
in research (237). As indicated, the complete remission rate is interesting in
MDSs (37%) and in CMML (27%) (238,239). Of note, the presence of a mutation
of the ras-oncogene seems to predict insensitivity to topotecan treatment in
CMML. In relapsed or resistant multiple myeloma, the overall response
rate was 16% (95% CI, 7–31%). Responses have lasted 70 to 477þ days, with a
median progression-free survival of 13 months and a median survival time of
28 months (240,241).

VI. Considerations of Route of Administration

As mentioned earlier, cytotoxicity of topoisomerase I inhibitors increases

with the duration of exposure. In vitro studies showed that short-term exposures
to high concentrations are less effective than long-term exposure to low
concentrations (29,30). Low-dose, prolonged exposure in vivo studies in animal
models also resulted in less toxicity (242–246). On the other hand, in vitro and
animal models have shown to be poor predictors of clinical efficacy and toxicity for
several reasons, including species differences in drug disposition, tolerability,
and intrinsic differences in tumor sensitivity, and the ascertainment that animal
models are relatively resistant to the myelosuppresive effects of the CPT analogs
(157). As a consequence, various Phase I and II studies have been performed to
focus on low-dose prolonged exposure to or continuous infusion of CPT derivatives
in the treatment of cancer patients (76,170,171,209,220,242,247,248). In addition,
the results of studies in animal models showing that the intragastric administration
of CPTs was effective, added to the fact that the low gastric pH would favor the
active lactone-ring configuration of the CPTs; these data would favor oral drug
application (157).

From a clinical point of view, as long as equivalent safety and efficacy can
be ensured, the majority of patients prefer oral instead of i.v. administration of
chemotherapy, predominantly due to the convenience of administration outside a
clinical setting and avoidance of vascular complications, related to i.v. access,
including catheter-associated infections or potential thrombosis (249,250).

From a pharmacological point of view, the oral route of drug

administration has some disadvantages. Absorption of an oral drug from the
gastrointestinal (GI) tract is a prerequisite for its activity, but this process can be
influenced by several factors. Delays or losses of the drug during absorption may
contribute to variability in drug response or may even result in failure of the
treatment (250). Both anatomical and physiological factors affect the overall rate
and extent of absorption from the GI tract and they influence the precise
quantitative prediction. Ideally, a cytostatic drug should have little interpatient
28 SOEPENBERG ET AL.

variability in absorption and AUC, and even more important, little intrapatient
variability with successive doses (251). As an inverse relationship is demonstrated
between decreasing absolute bioavailability of drugs and the interindividual
variation in bioavailability, it is recommended that caution must be taken in
prescribing oral drugs with low oral bioavailability as the therapeutic index is
narrow and thus, either toxic or subtherapeutic dosing may easily occur (250). For
instance, given the relatively low bioavailability of orally administered topotecan,
ranging from 30% to 40%, and the relatively high variability in the AUC both
between patients (CV, 40–73%) and within the same patient (CV, 25–96%), this
issue of a narrow therapeutic index is clearly demonstrated for the oral
administration of topotecan at its maximum-tolerable dose (166,252).

The variability of pharmacokinetics of orally administered anticancer drugs

can among other things be explained by the affinity for drug-transporting proteins
expressed in the intestinal epithelium and directed toward the gut lumen (250).
Currently, three major classes of drug pumps, including P-glycoprotein (ABCB1),
multidrug resistance-associated protein (MRP1 or ABCC1) and its homolog
MRP2 (also known as cMOAT or ABCC2), and BRCP (synonymous for
MXR, ABCP1, or ABCG2), have been characterized, that may play a role in
mediating transmembrane transport of anticancer agents, including irinotecan
and topotecan (250). The abovementioned proteins belong to the large superfamily
of ATP-binding cassette transporters that are found in almost all prokaryotic
and eukaryotic cells. The characteristic tissue distribution of these drug
transporters strengthens the indication that they play an important role in
detoxification and protection against xenobiotic substances (250). In vivo studies
with genetic knockout of murine P-glycoprotein genes revealed that the intestinal
absorption of various anticancer drugs, including paclitaxel and topotecan,
was increased (181,250). These experimental studies led to the development of
clinical trials of anticancer drugs modulated by co-administration of inhibitors
of P-glycoprotein and BCRP. Recently, a proof-of-concept study in 16 patients
with solid tumors was reported in which topotecan was administered in the
presence and absence of GF120918, a potent inhibitor of BRCP and P-glycoprotein
(253). This study showed that the co-administration of the inhibitor of the drug
transporters significantly increased the systemic exposure of oral topotecan, with
the mean AUC of total topotecan increasing from 32.4  9.6 mg h/l without co-
administration of GF120918 to 78.7  20.6 mg h/l with co-administration of
GF120918 (P ¼ 0.008). Furthermore, the apparent oral bioavailability increased
significantly from 40% to 97.1% (P ¼ 0.008) (253). Interpatient variability of
the apparent bioavailability was 17% without versus 11% with co-administration
of the inhibitor.

There is no doubt that, in order to approach the conceptual starting

point of prolonged exposure to a minimum concentration of the drug for
optimizing therapeutic efficacy, daily oral dosing would be preferable to
continuous i.v. infusion. But, up till now Phase I and II studies did not show
superior efficacy after prolonged exposure of topotecan (172,220,222,253,254).
 CLINICAL STUDIES OF CAMPTOTHECIN AND DERIVATIVES 29

Actually, the assumptions about the importance of prolonged exposure are being
reconsidered (254).

VII. Investigational Derivatives

A. 9-SUBSTITUTED CAMPTOTHECINS

9-Aminocamptothecin (9-AC) is a semisynthetic CPT derivative which

showed outstanding preclinical activity against a wide spectrum of tumor types,
including those of breast, colon, lung, prostate, and melanoma (255). In clinical
trials, the drug has been very extensively studied using two different formulations
based on the use of dimethylacetamide/polyethylene glycol 400 or a colloidal
dispersion preparation, which enhances solubility and stability. Clinical Phase I
investigations have been conducted using a variety of i.v. administration schedules,
including a 30-min infusion given daily for 5 days every 3 weeks (256), and
more prolonged infusion schedules using 24-h (257), 72-h (258–261), 120-h (262), or
7-day continuous dosing repeated every 4 weeks (263). In addition, trials
have evaluated the usefulness of delivering the agent intraperitoneally (264) or
orally (265–267).

All of the studies report neutropenia as the DLT, while thrombocytopenia

is also frequent and sometimes severe. Gastrointestinal toxicity is the second
most reported, though not dose limiting. Other toxicities are considered mild to
moderate. Numerous multi-institutional Phase II studies have been conducted in
several disease types, and overall, 9-AC shows only very modest single-
agent activity with the prolonged (72- or 120-h infusion) regimens. These
include trials in breast cancer (268), colorectal cancer (269–272), glioblastoma
(273), head and neck cancer (274), lymphoma (275,276), and NSCLC (277,278).
Therefore, its further evaluation does not seem to be indicated. It has been
suggested that the lack of clinically relevant antitumor efficacy relates to substantial
inactivation of the agent due to the unfavorable lactone/carboxylate ratio
in patients (279,280). In addition, using preclinical studies it has been shown
that solid-tumor xenografts were highly sensitive to 9-AC therapy, but the systemic
exposure required for antitumor effects was in excess of that achievable clinically
in patients (281).

Because of the poor aqueous solubility and poor antitumor activity of 9-AC
clinically, some major efforts have been put into the design and synthesis of more
derivatives that could be alternatives to the parent drug, including an N-(2-
hydroxypropyl)methacrylamide copolymer conjugate (282). Some of the synthe-
sized compounds have shown only marginal improvements in solubility or are too
unstable to allow administration in a clinical setting. In addition to solving
solubility issues, linkage of specific carriers to 9-AC offers the possibility of targeted
drug delivery that may enhance the therapeutic index. The potential targeting of
9-AC to tumor cells has been investigated with the use of enzyme-activatable
prodrugs, which include a 9-AC-glucuronide (283) and a 20-carbonate-linked
30 SOEPENBERG ET AL.

derivative (284) designed for activation by tumor-associated beta-glucuronidase

and plasmins, respectively.

To date, only one approach in prodrug design has yielded an agent that
has progressed to clinical evaluation, i.e., the 9-nitro derivative of CPT [i.e.,
9-nitrocamptothecin (9-NC; RFS 2000; rubitecan)], which acts as a partial prodrug
of 9-AC (285,286). 9-NC has a nitro group in the C-9 position and is highly
insoluble in water, and was initially identified as a precursor in the semisynthetic
production of 9-AC. Since nearly all human cells are able to convert 9-NC to
9-AC, including tumor cells, it has been proven difficult to identify whether 9-NC-
mediated antitumor activity is directly associated with the parent drug alone or
with 9-AC alone, or the combination of both (287).

Phase I clinical evaluation of 9-NC has been focused on oral administration

in a daily-times-five per week regimen, either given alone (288,289) or in
combination with cisplatin (290). More recently, attempts have been made to
administer the drug as an aerosolized liposomal preparation (290,291). Preliminary
evidence generated in Phase II clinical trials with the oral formulation suggests
that 9-NC has moderate activity in the treatment of advanced pancreatic cancer
(292,293) and refractory ovarian cancer (294). An extensive Phase II clinical
program is currently being conducted in Europe to test the efficacy of this agent
against various malignant diseases. Thus far, oral 9-NC has been shown to be
inactive against metastatic colorectal cancer (295), advanced glioblastoma
multiform (296), and cutaneous or uveal melanoma (297).

B. EXATECAN MESYLATE (DX-8951f )

The hexacyclic camptothecin analog exatecan mesylate (DX-8951f) ([1S,9S]-

1-amino-9-ethyl-5-fluoro-1,2,3,9,12,15-hexahydro-9-hydroxy-4-methyl-10H,13H-
benzo[de]-pyrano[30 ,40 :6,7]-indolizino[1,2-b]quinoline-10,13-dione monomethane
sulfonate [salt], dihydrate) is a synthetic derivative with an amino group at C-1
and a fluorine atom at C-5. The compound has increased aqueous solubility in
comparison with other CPT analogs. As exatecan does not require enzymatic
activation, interindividual variability in efficacy and side effects might be reduced
as compared to some prodrug analogs (298). The anhydrous free-base form of the
drug is referred to as DX-8951. The lactone form of DX-8951 is hydrolyzed into
an open-ring hydroxy-acid form, comparable with most other CPTs. Similarly,
the lactone and hydroxy-acid form coexist in solution according to a reversible
pH-dependent equilibrium, i.e., DX-8951 is presented by its lactone form at acidic
pH and by its hydroxy-acid form in neutral and basic pH (299,300).

Exatecan showed superior and a broader spectrum of antitumor activity in

vitro and in vivo in comparison with some other CPT analogs tested (298,301–306).
Comparable with other CPT derivatives, exatecan is metabolized by CYP3A4 and
CYP1A2, resulting in the formation of at least two hydroxylated metabolites
 CLINICAL STUDIES OF CAMPTOTHECIN AND DERIVATIVES 31

referred to as UM-1 and UM-2. The antitumor activity of these metabolites is

much less potent than the parent compound itself (299,307,308).

Phase I clinical studies included DX-8951f administered as a 30-min i.v.

infusion once every 3 weeks, as a 30-min i.v. infusion daily for 5 days every 3 weeks,
as a 24-h continuous i.v. infusion every 3 weeks, and as a weekly 24-h i.v. infusion
3 of every 4 weeks (309–314).

Reversible, noncumulative, and dose-related neutropenia was the DLT

in all five schedules (309–313). With a 24-h continuous infusion every 3 weeks,
thrombocytopenia was an added DLT in heavily pretreated patients (310).
Neutrophil and platelet count nadirs occurred between days 10 and 15, with
recovery by day 22. Nonhematological toxicities included mild to moderate
gastrointestinal toxicity (nausea, vomiting, stomatitis, diarrhea), fatigue, asthenia,
and alopecia (309–313). Transient and reversible liver dysfunction was also
observed, and in a Japanese study this event was dose limiting at the dose of
6.65 mg/m2 (309,312).

In advanced leukemia, stomatitis was dose limiting (314). Remarkably, the

MTD in leukemia (at 0.9 mg/m2/daily for 5 days) is almost double the one in solid
tumors (314).

For Phase II clinical trials, the 30-min infusion regimen with daily admi-
nistration for 5 consecutive days every 3 weeks was selected because this schedule in
Phase I studies showed mostly antitumor activity.

Exatecan was already tested in a Phase II study program in a variety of

malignancies, including NSCLC, pancreatic cancer, ovarian cancer, and colorectal
cancer, and Phase III studies are currently ongoing. Only preliminary results of
these Phase II studies are available, and the Phase III studies have yet not been
reported (242,315).

C. DIFLOMOTECAN (BN80915)

Diflomotecan (BN80915) (5-ethyl-9,10-difluoro-4,5-dihydroxy-5-hydroxy-

1H-oxepino[30 ,40 :6,7]indolazino[1,2-b]quinolone-3,15[13H]dione) belongs to the
class of fluorinated homocamptothecins. Homocamptothecins are synthetic,
water-insoluble CPT analogs with a stabilized lactone ring due to modification
of the naturally occurring six-membered -hydroxylactone ring into a seven-
membered -hydroxylactone ring by insertion of a methylene spacer between the
alcohol and the carboxyl moieties (242). Lactones are cyclic carboxylic esters and are
rather unstable molecules (316). The inductive effect from the electronegative
oxygen of the adjacent hydroxyl group causes higher reactivity of the carboxyl
group of CPTs. By inserting a methylene spacer between the carboxylic and
alcoholic functions of the E-ring, it was believed that the electronic influence of
the hydroxyl group was removed (316). The alcohol moiety was seen as an important
32 SOEPENBERG ET AL.

structure for stabilizing the cleavable complex, because neither dehydroxycamp-

tothecin nor the non-natural enantiomer of CPT is biologically active (316). Since
a one-carbon ring expansion is chemically termed a homologation, these new
lactone- or E-ring modified compounds were named homocamptothecins (317).

In comparison with most other CPTs, which show rapid hydrolysis of the
lactone moiety until a pH- and protein-dependent equilibrium has been reached,
homocamptothecins display a slow and irreversible hydrolytic lactone-ring opening
(318). This key feature of irreversibility of E-ring opening may lead to reduced
toxicity (316). After 3 h incubation of camptothecin and homocamptothecin in
human whole blood at 37  C, the fraction present in the lactone form was 6% in the
case of camptothecin and 80% in the case of homocamptothecin. Besides the slower
ring opening of homocamptothecin, this difference is also due to a higher affinity of
homocamptothecin for red-blood cells (316). Indeed, the homocamptothecins were
shown to be more stable than camptothecin, were a highly potent inhibitor of
cell growth with superior topoisomerase I-inhibitory activity as compared
to camptothecin, and changed the sequence specificity of the drug-induced
DNA cleavage by topoisomerase I (319–321). In vitro studies revealed that the
fluorinated homocamptothecins showed the best antiproliferative activity in the
A427 human lung carcinoma cell line in comparison with other homocamptothecin
compounds (316).

Diflomotecan, one of the fluorinated homocamptothecin derivatives, has

entered Phase I clinical testing. Oral diflomotecan administered once daily for
5 days every 3 weeks was limited by dose-dependent myelosuppression (317). Other
toxicities observed were gastrointestinal (i.e., mild nausea and vomiting), alopecia,
and fatigue. The recommended dose for Phase II studies is 0.27 mg once daily for
5 days every 3 weeks. The i.v. studies have yet not been published.

Oral diflomotecan exerts a linear, dose-independent, pharmacokinetic

profile over the higher dose-range studied with high inter- and intrapatient
variability. It was also reported that flat dosing of oral diflomotecan resulted in the
same variation in AUC as dosing per square meter would have done, as already
established for many other cytotoxic agents (322). In the future, population
pharmacokinetic studies might enable to reduce the interpatient variability. But, as
long as these models are not available, the more convenient flat dosing of
diflomotecan is as accurate as the more complex dosing per body surface area. The
oral bioavailability of diflomotecan at the recommended dose was 67.1% which is
much better than for other oral topoisomerase I inhibitors such as topotecan
(F ¼ 30–44%), lurtotecan (F ¼ 12–21%), and 9-AC (F ¼ 48.6%) (166,252,265,323).

D. LURTOTECAN (GI147211 or GG211)

Like DX-8951f, lurtotecan (also known as GI147211 or GG211) is a

hexacyclic CPT analog currently under clinical investigation as an anticancer drug.
Lurtotecan is a water-soluble, totally synthetic derivative with a dioxalane moiety
 CLINICAL STUDIES OF CAMPTOTHECIN AND DERIVATIVES 33

between C-10 and C-11 (324). This agent has been evaluated clinically in various
Phase I and II trials using a 30-min i.v. infusion given daily for 5 consecutive days
(325,326), and as a 72-h (327) or 21-day continuous i.v. infusion (328). The DLT in
all schedules was myelosuppression, including severe neutropenia and thrombo-
cytopenia. Nonhematological toxicities were various and only mild to moderate. In
Phase II trials, lurtotecan has shown modest activity in breast cancer, colorectal
cancer, NSCLC (329), and SCLC (330). Overall, these data suggest that the
hematological-toxicity profile and antitumor activity closely resemble that observed
with topotecan, which remains the leading CPT analog for salvage treatment of
lung cancer.

Because the oral bioavailability of lurtotecan was previously shown to be

highly variable and as low as 10% (323), alternative ways of drug administration
are currently being developed, including a new liposomal formulation (OSI-211;
also known as NX 211). Preclinical data have been generated demonstrating that
this unilamellar liposomal formulation of lurtotecan has significant therapeutic
advantage over the free drug, showing increased antitumor activity in xenograft
models, which is consistent with increased systemic exposure and enhanced tumor-
specific delivery of the drug (331). Based on these exciting data, a Phase I clinical
trial has been performed with OSI-211 given to cancer patients as a 30-min infusion
in a once every 3-week regimen (332). As expected, the DLTs in this trial were
neutropenia and thrombocytopenia, and pharmacological findings seem to
corroborate the preclinical profile of this agent. Indeed, the clearance of total
lurtotecan following administration of OSI-211 was approximately 25-fold slower
than that of the free drug, which might prove to be beneficial for pharmacodynamic
outcome of treatment.

VIII. Conclusions

The CPTs are a class of effective anticancer drugs derived from plant
alkaloids that exert their action against DNA topoisomerase I and have been
developed in recent years. This specific mechanism of action and the activity against
a broad spectrum of malignancies perpetuated a stimulus for research and clinical
development of several CPT derivatives with improved physicochemical and
pharmacological properties. At present, two analogs, viz. irinotecan and topotecan
are registered for clinical use for the treatment of first- or second-line therapy in
advanced colorectal cancer and second-line therapy in cisplatin-refractory ovarian
cancer, respectively. Furthermore, in Japan irinotecan is also registered for the
treatment of NSCLC. Other members of this promising class of cytotoxic agents,
including 9-AC, 9-NC, exatecan, diflomotecan, and lurtotecan are in progress of
clinical development to specify their definite place in the spectrum of anticancer
treatment.

Several pharmacokinetic and pharmacodynamic factors, including poor

aqueous solubility, a pH-dependent reversible interconversion between the active
lactone and the inactive carboxylate form, increased lactone stability by
34 SOEPENBERG ET AL.

substitutions at specific sites on the molecule, cellular efflux, mechanism of

resistance, and drug–drug interactions influence the antitumor efficacy and toxicity
profile of these drugs.

The knowledge from preclinical studies that topoisomerase I inhibitors

showed S-phase-specific cytotoxicity led to the development of protracted schedules
of administration or prolonged exposure of the drug. Although this concept
established greater tumor efficacy compared with bolus administration in
experimental studies, the optimal treatment and dosing regimens such as chronic
oral delivery, continuous i.v. infusion, liposomal encapsulation, or polymerized
drug formulations have to be crystallized in further clinical investigations. So far,
current insights derived from Phase II clinical trials suggested that prolonged drug
exposure may be at least as effective and less toxic than more conventional
schedules of intermittent administration of higher doses given as short bolus i.v.
infusion. For a variety of reasons, oral regimens of anticancer drugs are more
convenient for continuous drug administration, and, in general, more preferred by
patients. But, the use of oral CPTs has to overcome some disadvantages. These
include maintenance of stability of the drug in the low-pH environment of the
stomach, absorption through the biochemical barrier of efflux pumps, i.e., drug
transporters, and modulation by cytochrome P-450 isoenzymes in the epithelium of
the intestine, the first-pass effect in the liver, and the excretion in the bile, all
influence the relatively low, and highly variable, bioavailability of the CPTs. In
addition, their narrow therapeutic index offers a potential risk of an accidental
overdose and consequently unpredictable excessive toxicity, or suboptimal
exposure and hence lower efficacy.

The biotransformation of the CPTs is a complex process, in which the

hepatic metabolism significantly contributes to the elimination of the drug.
Additionally, the pharmacokinetics of the CPTs can easily be altered by
comedication that modulates the cytochrome P-450 isoenzymes. Furthermore,
new insights of the genetic polymorphisms of the UGT1A1 enzyme, which
detoxified SN-38 to SN-38 glucuronide (SN-38G), may lead to more individualized
dosing of irinotecan guided by pharmacogenetics to overcome the variability in
SN-38 glucuronidation, a major determinant of the severity of late diarrhea.

What will the future bring in the development of the CPTs? Efforts may
focus on enhancement of the antitumor efficacy of the CPTs by combining these
agents with other anticancer drugs, biological modifiers, or radiotherapy.
Especially if these treatment modalities can modulate cell-cycle checkpoints
which promote CPT activity. Furthermore, a new generation of CPTs with a
broader spectrum of cytotoxicity or greater chemical stability of the lactone ring,
e.g., exatecan and diflomotecan, respectively, and with more predictable side effects
are now in clinical development. Also, protracted exposure to the drug can be
achieved by using polymerized or macromolecular CPTs with prolonged half-lives.
In addition, these macromolecular drugs penetrate in tumor tissue better due to
their enhanced permeability and retention characteristics, and have lower systemic
 CLINICAL STUDIES OF CAMPTOTHECIN AND DERIVATIVES 35

toxicity associated with the free drug. Further investigations are required to
demonstrate if these drugs show better antitumor activity.

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Exploring the Variety of Random
Documents with Different Content
 CHAPTER VII.
THE WILES OF A SCHEMER.

Jode Lenning was alone in the tent, which had been erected for
his use, when Mingo, a Mexican distance runner, who belonged to
the G. H. A. C., thrust his head through the flap and announced that
Colonel Hawtrey had arrived in camp.
Lenning, at the moment, had his back to the opening and was
wrapping a long, flat package in his handkerchief.
“What?” he gasped, throwing a startled look over his shoulder at
Mingo.
The other repeated his announcement.
“The devil!” gulped Lenning, in a flurry. “He’s found out what
happened at the house, and put for here on the jump. Now for
merry blazes, and a little slick work by yours truly.”
His hand shook a little as he crowded the handkerchief-wrapped
package into the breast of his Norfolk jacket; then, getting up, he
hurried out of the tent and ran to meet the tall man with the gray
mustache.
“Ah, my boy!” exclaimed Colonel Hawtrey, making no effort to
conceal the pleasure the meeting gave him. “You’re looking fit, I
must say, so there’s not much use asking how you feel.”
“Fine as silk, uncle,” said Lenning, clasping the colonel’s hand.
“How did you find everything at the mines?”
“The mines are all right,” was the answer, “but it was something I
discovered after I got home this morning that has rather shaken me.
Take me to a place where we can be by ourselves and talk.”
“My tent will fill the bill.” They walked together in the direction of
Lenning’s headquarters. “Was that Hawkins I saw leading away the
horses?” Lenning asked.
“Yes, that was Hawkins.” That there was a load of some sort on
the colonel’s mind was evidenced by his tone and manner. “It’s
possible,” he added, “that I am going to need Hawkins in—er—an
official capacity.”
“This sounds pretty warlike!” exclaimed Lenning.
“I suppose so,” and the old soldier stiffened a little. “I have made
some discoveries, Jode, which will astonish you. They nearly carried
me off my feet. By the way, what started you on this camping trip?”
“I thought it would be a good thing for our eleven,” Lenning
explained. “This Merriwell chap took the Ophir team out into the
hills, and I reckoned we’d follow suit. And, say! We bumped into the
Ophir outfit right here at Tinaja Wells. How’s that for a coincidence?”
“Queer, to say the least,” answered the colonel. “I hope all you
fellows will remember that you are true sportsmen, which is only
another term for gentlemen, and avoid any unpleasantness.”
“You can depend upon us to prove a credit to you, colonel!” said
Lenning, with a fine show of admiration for the erect, soldierly old
fellow beside him. “I have a lease from Struthers, and Merriwell has
one from Packard. Now,” and Lenning laughed, “which of us has the
right of it?”
“That’s hard to tell, my boy, until the lawsuit is decided. What sort
of a character is young Merriwell? Anything like his father?”
“I don’t know much about his father, sir; but young Merriwell
seems to be trying to make himself the whole thing. Of course,”
Lenning added, “I tried to smooth matters over, and it looks as
though I had succeeded. As you see, we’re both camped on the
same ground.”
“I’ll have a talk with Merriwell myself, and see what I can do with
him. All that, however, must wait on the important business that
brings me here. I have never had anything make such an impression
on me. Is this your tent, Jode?”
“Yes, uncle. Walk inside and make yourself comfortable.”
When Colonel Hawtrey had seated himself comfortably on a camp
stool, and Lenning had dropped down facing him on a pile of
blankets, the colonel lighted a cigar—possibly to soothe or cover his
nervousness—and began.
“You remember, Jode,” said he, “that I drew a thousand dollars
from the bank on the forenoon of the day I left town, expecting to
pay it out to Judson for an interest in that promising claim of his.”
Lenning nodded.
“You drew the money,” said he, “and Judson didn’t show up; then
you were called from town in a hurry, and locked up the money in
your safe. I remember all that very distinctly.”
“You knew the combination, and were to give Judson the money if
he called for it.”
“Yes, sir; but he didn’t call.”
“I know that. I had scarcely reached town when I saw him, and
he said he’d be around this afternoon to get the thousand. Then I
went home—and found that I had been robbed!”
“Robbed!” gasped Lenning, starting up.
“Yes, my boy, robbed! Of course, a thousand dollars isn’t very
much to me, but it’s losing the money in such a way as that that
gets under my skin. The safe in my study was open, the window had
been unlocked, and the thousand was gone!”
 “Had the safe been blown open?”
“No. Some one had worked the combination and——”
“Uncle!” exclaimed Lenning, in consternation. “You and I are the
only ones who know the combination. You were away from home,
and I—I——”
The colonel leaned forward and dropped an affectionate hand on
his nephew’s shoulder.
“Tut, tut!” said he brusquely. “You know I trust you as I would
myself. There is some one else who knows the combination, and
who at one time had as free access to that safe as you or I. I refer
to—to your half brother, Darrel.”
“But Ellis perished in that train wreck!”
“Supposed to, but I have always had a feeling that there might be
some mistake. That graceless young scamp wasn’t born to shuffle
off in any such way as that. What I should have done, I suppose,
was to have the combination changed. But I did not. This is the
result.”
“I wouldn’t be in too much of a hurry to judge Ellis, Uncle Al,”
pleaded Lenning. “You’re only working on a theory, you know, and
——”
There was sorrow in the fine old face of the colonel, but over all
was the sternness of an iron will.
“I have evidence,” he interrupted; “much as it grieves me to tell it,
Jode, yet I have evidence which cannot be denied. It is like you, boy,
to plead for the rascal who has disgraced our blood; but, as for me,
I shall not be victimized a second time without making him pay the
penalty. I—— You are pale!” exclaimed the colonel, leaning forward
to stare into his nephew’s face; “and you are trembling, too! What
ails you, Jode? Brace up; don’t take this too much to heart.”
 “I have something to tell you, uncle,” answered Lenning; “but,
first, let me hear your evidence.”
The colonel took a knife from his pocket and handed it to Lenning.
“You recognize that, don’t you?” he asked harshly.
“Why,” murmured Lenning, “it’s the knife you gave Ellis years ago.”
“It is,” was the grim rejoinder, “and I found it under the unlocked
window in my study.”
Lenning seemed stunned and incapable of words.
“But that isn’t all,” preceded the colonel. “I hunted up Hawkins,
who happened to be in town, and together we learned that a fellow
answering Darrel’s description had been in Gold Hill the night before
I got home. He had called on Haff, our club secretary, and asked for
me, and about you. Haff told him that you were camping, with some
of our lads, at Tinaja Wells. Supposing that Darrel had come here,
Hawkins and I secured a couple of mounts and made a quick trip
down the cañon. Have you seen anything of Darrel?”
“Then it’s true, it’s true!” Lenning was muttering, as though to
himself.
“What is true?” demanded his uncle. “Don’t try to shield the
fellow, Jode. Your first duty is to me, not to him.”
“There is a fellow here—Merriwell seems to be looking after him—
who says he is Ellis Darrel.” Lenning spoke with apparent reluctance.
“I believed him to be an imposter. How could I think anything else
after the report we had of that Colorado wreck? The fellow seemed
bent on proving that he was really my half brother, and challenged
me to run a race with him. You see——”
“What folly!” cut in the colonel.
“I’m pretty fast in a sprint, uncle, but El was a shade faster. And
you know he had a queer way about him when he was running. I
think he is counting on that race to make his identity known to me
and the rest of the Gold Hill fellows.”
“We don’t need any proof of his identity, Jode! We can take his
word, and then confront him with this damning evidence of his
rascality!”
Lenning put out his hand and rested it on his uncle’s arm.
“Colonel,” said he, his voice shaking, “let us have this race to-
morrow afternoon. Don’t interfere. There’s a chance that, after all,
the fellow is not Darrel.”
“There’s not a shadow of a doubt, not a shadow!”
“But you needn’t hurry about arresting him, need you? Let’s find
out how far Merriwell will go in trying to shield him. Wait until after
the race; and then—well,” and Lenning drew a long, regretful sigh,
“do what you think you have to—what you think you must.”
“If Darrel knows I am here with Hawkins he may suspect
something, and clear out,” demurred the colonel. “It isn’t well, my
boy, to dally too much with an affair of this kind.”
“Have Hawkins watch him,” suggested Lenning.
“True,” said the colonel, “I could probably do that. It’s impossible,
though, that Young Merriwell is mixed up, in any way, with Darrel’s
wrongdoing. He has been deceived in the fellow. I know of the elder
Merriwell, and a straighter man or a better all-round athlete the
world never produced.”
“I hope young Merriwell is square, and a real chip of the old block,
as I understand his friends mean to suggest when they call him
‘Chip’—but, well, I don’t like the way he has been acting. To-morrow
afternoon, uncle, we may know a lot more about him and about
Darrel, too.”
 “Very well,” said the colonel, though reluctantly, “we’ll leave the
matter, Jode, as you desire.”
“Thank you, sir,” said Lenning gratefully.
Why was Lenning so anxious to have his uncle defer action
against Darrel? Had the packet, wrapped in his handkerchief and
stowed in the breast pocket of his Norfolk jacket, anything to do
with his wish to delay proceedings? In view of what happened later,
this seemed like the logical explanation.
 CHAPTER VIII.
A JOKE—WITH RESULTS.

Hawkins, the deputy sheriff, had not much to say to Merriwell

during their walk from the mesa back to the camp. Hawkins was an
admirer, and in many ways had shown himself a true friend, of
Frank’s; and, out of the kindness of his heart and, without divulging
any secrets, he strove to warn him against Darrel.
“They’re talkin’ a heap, down in the camp,” said Hawkins, “of what
a big hit this Darrel person has made with you. Don’t cotton to him
too strong, Merriwell. He isn’t wuth it.”
“What do you mean?” Frank demanded.
“Between ourselves—the thing not to go any further, you
understand—this Darrel’s nothin’ more than a plain thief.”
“You’re mistaken, Hawkins,” said Frank, with spirit. “I can’t believe
it.”
“Well, son, you’ll have the proof before you’re many hours older.”
“Then I’ll wait for the proof, Hawkins; and it will have to be
copper-riveted before I turn against Ellis Darrel.”
“Jest a warnin’ I’m handing you, Merriwell,” grinned Hawkins. “And
you’re to keep what I said to yourself, mind.”
“Of course, Hawkins. I’m obliged to you for taking all this trouble,
but you’re mistaken, and will find it out. It’s the colonel’s business,
isn’t it?”
“Now, I’m not sayin’ another word,” answered the deputy, “and
maybe I’ve let out more’n I ought to, as it is.”
 That ended the brief conversation, and, while it did not shake
Merriwell’s confidence in Ellis Darrel, nevertheless it left him with
vague forebodings of fresh disaster hanging over the head of the
“boy from Nowhere.”
The members of the rival athletic clubs were carefully avoiding
each other. There was no display of ill feeling, perhaps because the
bad blood had no chance to show itself, or because the presence of
the colonel in the Gold Hill camp was a restraining influence. Be that
as it may, yet the topic of conversation in both camps was the
hundred-yard dash to be run on the following afternoon. The object
of the race, unique in the annals of sport, lent the event a
fascination which nothing else could have done. Until ten o’clock the
affair was discussed by the Ophir fellows, and then, agreeable to
schedule, lights went out and the Ophir lads sought their blankets.
By an arrangement, enforced from the very first night that Frank
and his companions went into camp, a watch of three was posted to
look after the live stock and other property during the night. A trio of
lads went on sentry-go from seven to eleven; when their duty was
finished, they aroused three others to do guard duty from eleven to
three; and these, in turn, awoke three more for the morning watch
from three to seven. On this night, the first to be passed on the flat
with the Gold Hillers, Ballard was one of the three who had the
midwatch of four hours around midnight. Ballard’s post was in the
cañon, just below the flat, where the saddle and pack stock had
been gathered.
He had a lonely vigil for an hour. Somewhere in the neighboring
hills the coyotes were howling—a noise, by the way, not calculated
to soothe a person’s nerves. While Ballard was listening to the
coyotes, and thinking more or less about the next day’s race, he
heard a sound as of some one sliding down the slope from the flat.
Alert on the instant, Ballard started up and peered into the gloom
and listened. Some one was breathing heavily and floundering and
stumbling through bushes and over stones.
 “Can’t be a prowler,” murmured Ballard, “for he’s making too much
noise. I’ll just lay hands on the fellow and make him give an account
of himself.”
Creeping forward, and screening himself as well as he could in the
shadows, Ballard was able to rise up suddenly and seize the
wabbling figure.
“Himmelblitzen!” wheezed a voice. “Oof you peen vone oof der
Inchun shpooks, den I bet you I faint fits righdt on der shpot!
Whoosh!” and the voice died away with a suggestion of chattering
teeth.
“Carrots!” laughed Ballard. “Say, you crazy chump, what are you
fooling around the gulch for at this time of night?”
“Oh, Pallard!” puffed Fritz, in great relief. “Vell, vell, vat a
habbiness! Dere vas t’ings vich ve don’d know till ve findt dem oudt,
hey? I vas looking for you, Pallard, yah, so helup me!”
“Looking for me?” echoed Ballard; “what for?”
“Meppyso I gif you haluf oof dot dreasure oof you go along und
hellup me get him.”
“Oh, blazes!” chuckled Ballard. “I thought you’d got over that
treasure notion, Carrots.”
“Lisden, vonce, und I told you someding.” Fritz dropped his voice
to an explosive whisper. “Vat you dink? Py shiminy, so sure as
nodding I findt me dot shtone mit der gross on. Yah, you bed my
life! It vas so blain as I can’t tell, Pallard. Aber ven I roll avay der
shtone und tig mit der shovel, I hear me some voices oof an Inchun
chief. Dot shkared me avay. Haf you got der nerfs to go mit me to
der blace back, Pallard? I peen shaky all ofer, und my shkin geds oop
und valks on me mit coldt feet, yet I bed you I go back, und I findt
der dreasure. You come, und so hellup me I gif you haluf!”
 The excitement at the Wells, incident to the arrival of the Gold
Hillers and following hard upon the rapid return of Fritz and Silva to
the camp, had temporarily closed the fun Merry and his friends had
had in the cañon. More important events had claimed the attention
of the lads who had participated in the joke, and no one had
explained matters to Fritz or the Mexican. So it chanced that the
Dutchman was still laboring under his delusion.
Ballard wondered whether he had better set Fritz right, or keep
the joke going. He finally decided that the stock would not suffer if
he played out the Dutchman a little, and watched his antics in the
supposedly spook-haunted gulch.
“When an Injun goes to the happy hunting grounds, Carrots,”
remarked Ballard gravely, “it’s just as well not to stir him up. I’d hate
to have a red spook get a strangle hold on me—there wouldn’t be
treasure enough in the whole of Arizona to pay a fellow for an
experience of that kind.”
“Haf you no chincher?” demanded Fritz. “Iss it not vort’ a leedle
shcare chust to load oop mit goldt dot vill make you a rich mans for
life, hey? Vell, I bed you! I t’ink him all oudt, und I arrife py der
gonglusion dot a shpook iss nodding more as a shadow in der sun,
oder der moon. Vat a shpook does makes no odds aboudt der
tifference. Ve go, ve ged der goldt, und ve come back. Dot’s all
aboudt it. I got me a shovel in vone handt, und a glub in der odder.
Mit vone, I tig oop der goldt; mit der odder, I knock ofer der
shpooks. Und dere you vas. Ve shall be gompany mit each odder,
Pallard.”
“I don’t see how I can back out, Carrots,” said Ballard, “the way
you put it up to me. You’re an awful persuader. How much gold is
there?”
“I see it in der tream dot dere iss more as ve can carry, yes.”
“Maybe that dream is just fooling you, Carrots.”
 “You say yourselluf dot treams iss somet’ing, Pallard.”
“Did I? Well, maybe they are something. You go first, will you,
Carrots? I’ve got a weak heart, and if I should run onto a spook
without any warning it would knock me stiff.”
“I vill go fairst,” agreed Fritz, generously and valiantly, “und you
precede. I vill vatch aroundt carefully, und oof ve don’d make some
noises, den meppy der shpooks von’t hear, und ve gif dem der slip.”
Fritz waddled off into the darkness, and Ballard, enjoying himself
hugely, trailed after him. Suddenly, without the least warning, Fritz
dropped the shovel and the club, whirled in his tracks, and took
Ballard in a convulsive embrace.
“Ach, du lieber!” he whimpered. “I hear me someding, py shiminy!
Lisden, vonce, Pallard! Vat it iss, hey?”
“Coyotes,” answered Ballard, in a smothered voice. “Brace up,
Carrots. Don’t lose your nerve.”
“Sooch dreasure hundings I don’d like,” mumbled Fritz, slowly
untangling himself from Ballard and cautiously groping for his shovel
and club. “I vish der plame’ coyotes vould go to shleep. Ach, vat a
nervousness I got all droo me. I shake like I hat some agues. Sooch
a pitzness iss vort’ all der dreasures vat ve findt.”
Suddenly Ballard, clapping a hand over Fritz’s mouth, whispered a
hissing warning for him to keep still, and pulled him out of the
narrow trail and in between a couple of huge bowlders.
“V-v-vat iss der drouple!” inquired Fritz feebly. “You see a shpook
yourselluf, Pallard? I bed you——”
Again Ballard clapped a hand to his companion’s mouth.
“Sh-h-h!” he murmured. “There’s some one coming, right behind
us. Not a word, now; not so much as a whisper.”
 Somehow, Ballard got it into his head that the man who was
following them was Silva. The Mexican, he remembered, was also
mixed up, rather vaguely, with Fritz in the treasure hunting. Ballard
had it in mind to give Silva a bit of a scare, and so make the most of
that midnight experience.
Peering out from their dark retreat, Fritz and Ballard saw a dark
figure gliding toward them along the trail. It was impossible for them
to discover who the man was. He was in a hurry, that was evident,
and a peculiar, musical jingling accompanied him as he came on.
The sound was not loud, but more like a tinkling whisper, and barely
distinguishable.
But Silva—if Silva it was—did not pass the two behind the
bowlders. He halted, so close that Ballard could have reached out
and touched him, went down on his knees, and worked at
something in the dark. Even with the fellow so near, the heavy
gloom successfully hid his identity.
Ballard’s desire for fun was lost in a mighty curiosity. The fellow
took something white from his pocket, and, apparently, pushed it
under a stone; then, rising, he sped away in the direction from
whence he had come.
“Vell, vell!” muttered Fritz. “Vat you t’ink iss dot, Pallard?”
“That’s a conundrum, Carrots. How many fellows are looking for
that treasure of yours, eh?”
“No vone but me und you, Pallard.”
“Wait here for a couple of shakes, Fritz. I want to explore.”
Ballard crept to the place where the mysterious figure had been at
work, groped under a stone, and pulled forth a package wrapped in
something white. Lighting a match, he examined his find. Fritz could
hear him muttering excitedly as the match dropped from his fingers.
“Vat it iss, Pallard?” quavered Fritz.
 “I’ve had enough treasure hunting for one night,” answered
Ballard, in a strange voice. “I’m going back to the live stock, Fritz.
Come on!”
Fritz protested, but Ballard stood firm. Fritz would not continue on
without company, and so they returned to the camp—Ballard with
the white packet snugly stowed in his pocket.
 CHAPTER IX.
THE RACE.

Most of the forenoon, every day except Sunday, Merriwell, Clancy,

and Ballard had to give up to the “grind.” Professor Phineas
Borrodaile rigidly insisted on certain hours for study and recitation,
and would not temper his discipline even on the day that notable
race was to be run between Lenning and Darrel.
Following breakfast, each camp continued to flock by itself. The
live stock belonging to each party was picketed in widely separated
grazing grounds, so there was no opportunity for Silva and the other
packer to wind up their disagreements in a final clash. Peace
hovered over the region adjacent to Tinaja Wells, but to Merry it
suggested a calm preceding a storm.
Hawkins buried himself among the Gold Hillers, and seemed very
careful not to overstep the “dead line” which had been drawn
between the two camps. Colonel Hawtrey also appeared content to
remain in seclusion among the members of his own party.
About eleven o’clock in the forenoon, Frank and his chums, and
the professor and Darrel overheard a brief address which the old
soldier was making to the young athletes of the Gold Hill club. Only
scraps of the colonel’s little speech floated to the fellows in the Ophir
tent, but what they overheard made a deep impression on them.
“Sports of the right kind, properly indulged in, are of vastly more
benefit to the upbuilding of character, my young friends, than to
your muscles and bodily endurance. Understand me, I do not say
that physical development is of less importance than mental
development. Both of these should proceed hand in hand; but if,
over all, the moral and manly qualities do not grow as they should,
all your training in the class and on the track and field will have been
in vain. Try, my lads, to develop the faculty of being good losers, and
to admire and applaud in others those abilities, natural or acquired,
which you possess, but not in the same degree.”
As these words, spoken in a deep and earnest voice, wafted
themselves from the rival camp, the professor softly clapped his
hands.
“Noble sentiments most nobly expressed, young gentlemen,” he
murmured. “This Colonel Hawtrey must surely be a man of splendid
character.”
“He is,” said Darrel, in a low voice. “The colonel is one of the finest
men that ever lived.”
“Listen!” whispered the professor.
Again the colonel’s words drifted into the rival camp:
“If an amateur athlete is not a true sportsman, which is but
another term for gentleman, he is not fit to compete with other true
sportsmen. Your real gentleman, if you please, has courage; but,
more than that, he is so imbued with the spirit of fair play and so
completely captain of his own soul, that the stings of honorable
defeat leave him unscathed.”
These were fine words, and well calculated to inspire a spirit of
high emprise.
“I hope Jode is taking that in,” whispered Darrel to Merriwell; “but,
I’ll gamble my spurs, he’s going to beat the pistol, just the same.”
Ballard, all that morning, had been preoccupied to an extent that
had drawn some criticism from the professor. The interesting events
of the night, which he had not only kept a secret himself but had
likewise warned Fritz to keep in the background, probably had a
good deal to do with his poor showing at the problems put up to him
by Borrodaile.
 At eleven-thirty, when the studious ones were allowed a breathing
spell before dinner, Ballard hooked onto Merriwell and led him to a
secluded place for a talk. Fritz had to call them three times to “grub
pile,” and when the two finally arrived, their faces were flushed with
excitement, and there was an air about them that suggested
mysterious things.
At two-thirty in the afternoon a general movement set in toward
the mesa. Both camps emptied themselves upon the little plateau,
so that nearly forty spectators assembled to watch the race between
Darrel and Lenning.
The course had already been marked off by Brad, Spink, and
Handy. Beman, for Lenning, had looked it over and pronounced it
O. K. On one side of this course the Gold Hill men were grouped,
and on the other side the fellows from Ophir.
Colonel Hawtrey and Hawkins stood together, and Merriwell, for
the first time, got a good look at the colonel. He was much
impressed with his soldierly bearing, but in his face could be read
sternness and determination—and a sadness which did not, in the
least, diminish the more Spartan qualities.
Bleeker, of Gold Hill, crossed the course and stepped up to
Merriwell.
“There ought to be a judge and a starter, I reckon,” said he. “I
don’t see any need of makin’ this event top-heavy with officials. Do
you?”
“Not at all,” Frank answered. “I’d suggest that Colonel Hawtrey act
as judge of the race.”
“He says he won’t have a thing to do with it.”
“Then how about Hawkins, the deputy sheriff?”
“Suits Lenning to a t, y, ty. Lenning would like to have Beman for
starter.”
 Merriwell was expecting this, and yet it came to him with
something like surprise. It pointed to crookedness on the part of
Lenning—and after that fine talk the colonel had given his fellows
that morning, too!
“Let Beman act as starter, then,” assented Frank, keeping to the
plan broached by Darrel.
Bleeker hurried away to inform Hawkins and Beman of the work
laid out for them; and a few minutes later Darrel and Lenning, in
sprinting costumes, came trotting up from the camp.
Merriwell watched Darrel and the colonel. As the old soldier fixed
his eyes on his discredited nephew, a queer play of emotions
showed in his face. In Darrel’s look was a wistfulness and affection
which caused his uncle to turn abruptly and gaze in another
direction.
Beman, a round-shouldered, lanky chap, stepped out back of the
starting line, pistol in hand.
“All ready, you two?” he called.
Darrel and Lenning answered by stepping to the line. Not a sound
of approval or disapproval went up from the gathered throng.
Silence reigned on the mesa.
“This is about as cheerful as a funeral procession, Chip,” muttered
Clancy.
“Everybody’s mightily interested in the race, for all they have
bottled up their feelings,” Merriwell answered.
“Maybe,” was the skeptical response, “but it takes a lot of rooters
to stir up the enthusiasm. This looks about as sporty as the track
event of a deaf-and-dumb school. That Lenning carries himself well.
He walks with a spring that leads you to think he ‘feels his feet.’ But
I don’t like the cut of his jib a little bit.”
 “Nor I. His eyes are shifty, and his face doesn’t inspire much
confidence.”
“The old colonel is about as hilarious as he would be trying to
hunt up a nephew in the morgue. Whoo! I’ll go dippy in a minute if
somebody doesn’t yell. Guess I’ll tear off a whoop myself.”
He suited his action to the word, but it was a melancholy effort.
No one joined in with him, not even Merry or Ballard. From across
the course, the Gold Hillers gave him a startled look of disapproval.
“Once will do, thanks,” muttered Clancy. “I’m frosted so badly I’ve
got chilblains. Why doesn’t that starter set ’em off?”
The words were hardly out of Clancy’s mouth before Beman
shouted: “On your mark!”
Both sprinters dropped in well-nigh perfect style.
“Set!”
With that word, and the tense preparations of the sprinters for the
start, Merry and Brad began watching Lenning keenly. Merry ticked
off the seconds in his mind—one, two, three—and then intuitively he
sensed the forward plunge of Lenning, coming a fraction of a second
before the crack of the pistol. Lenning had not waited to hear the
pistol, and had got away at the explosion.
“He did it, by thunder!” whispered Brad. “Darrel had the skunk
dead to rights. Eh, Chip?”
“No doubt about it, Brad!”
Further talk just then was out of the question. The first stride of
the race had taken Lenning into the lead, and Darrel, waiting
honorably for the signal to start, was rushing to overhaul his
competitor.
“Dig, you kid from Nowhere!” whooped Clancy. “The race isn’t
done till you breast the tape.”
 “Go to it, Darrel!” Merriwell shouted. “You’ll pass him at the
eighty-yard line!”
“Wow!” yelped Ballard; “I’ll bet the boy from Nowhere gets
Somewhere before he’s many seconds older.”
A murmur went up from the Gold Hill side of the course. The
peculiar form in which Darrel was racing was recognized. Various
little mannerisms connected with his sprinting were recalled. They
were all here, in this clean-cut athlete whom Lenning had declared
an impostor! Gold Hill sentiments, it was plain, were undergoing a
change.
Not the least interested observer in the Gold Hill crowd was the
colonel. He leaned forward, the joy of wholesome sport temporarily
brushing aside the sterner proceedings which were to wait upon the
finish of that hundred-yard dash. The object of that race—the “boy
from Nowhere’s” attempt to prove his identity—did not concern
Colonel Hawtrey. He knew Lenning’s competitor was Ellis Darrel, race
or no race. What flamed up in him, as he gazed spellbound, was a
pure love of track athletics, aroused by a contest that was superb.
In about four seconds after the start the Gold Hillers had loosened
up. There were cries of, “Go it, Darrel!” and, “This looks like old
times, Curly!” which proved that Darrel was already winning the
recognition he coveted, no matter whether he won or lost the dash.
At the eighty-yard line, just as Merry had prophesied, Darrel drew
ahead of Lenning. The latter called on his reserve powers for a final
spurt, but Darrel also had speed in reserve. In ten seconds, or a
trifle more or less, Darrel tore away the tape at the finish, a full
stride in the lead.
A roar went up from all sides. The enthusiasm, which had been
held in check, rushed forth like a tidal wave. A rush was made
toward Darrel, but Hawkins, the deputy sheriff, grim and relentless,
waved the throng back. Stepping to the side of the victor, he
dropped an official hand on his shoulder.
 “Youngster,” said he crisply, “I’m sorry a heap to come down hard
on you at a time like this, but you’re under arrest.”
“Arrest?” echoed Darrel, recoiling. “For what?”
“For openin’ your uncle’s safe an’ stealin’ a thousand in cold cash.
Don’t make a fuss, bec’us’ it won’t do you any good.”
Then, amid the dead hush that fell over the mesa, Darrel’s eyes
sought only one face in all the crowd surrounding him. And that face
was Merriwell’s!
 CHAPTER X.
A HELPING HAND.

The explosion of a bomb could not have caused greater

consternation among the throng on the mesa than that official action
of the deputy sheriff. Hawtrey, erect and with a soldierly stride,
passed out of the stunned crowd and placed himself beside Hawkins.
Merriwell, giving Darrel a reassuring look, also advanced. He had a
sweater on his arm, and began pulling it over Darrel’s head and
shoulders.
“You’d better keep out of this, Merriwell,” Hawkins murmured in
Frank’s ear. “I warned you. The kunnel means biz, and no mistake.”
“So do I,” Frank answered, with a flash of his dark eyes. “Keep
your nerve,” he added, in a low tone to Darrel; “we’ve got a few
cards of our own to play.”
“You are Frank Merriwell?” inquired Colonel Hawtrey, leveling his
gaze at Frank.
“Yes, colonel.”
“The son of Frank Merriwell, of Bloomfield, and the T-Bar Ranch,
in Wyoming?”
“Yes.”
“You are also seeking to befriend this misguided young man,
here?”
“I am Darrel’s friend,” said Merry, with spirit, “right from the drop
of the hat.”
 “Then, my lad, your father will some time hear of it with regret.
What Hawkins said is the truth. This fellow opened my safe and took
from it a thousand dollars in cash night before last. I have the
proof.”
“Pardon me, colonel,” returned Frank respectfully, “but inasmuch
as I am Darrel’s friend, will you let me handle this case for him in my
own way?”
“If you mean to defend him,” frowned Hawtrey, “you will have
your trouble for your pains. He has no defense!”
“Will you let me try and see if I cannot make one, and one that
will command your attention and best judgment?”
“Sufferin’ centipedes, Merriwell!” broke in Hawkins. “I never
reckoned you’d be tryin’ to save the scalp of a plain, out-and-out
thief!”
The white ran into Darrel’s face and his hands clenched. Merry laid
a soothing hand on his arm.
“This isn’t a time for any snap judgments, Hawkins,” said Frank.
“First,” and he turned to the Gold Hillers, “I want to ask if this boy
from Nowhere has proved that he is Ellis Darrel, of Gold Hill?”
“Yes!” came a chorus of responses.
Merry partly turned to face Lenning. The latter, a sneering smile
on his dark face, was standing at a little distance, keenly alive to
everything that was said and done.
“How about you, Lenning?” queried Frank.
“He’s my half brother, all right,” was the answer. “I reckon there’s
not a shadow of doubt about that.”
“You agree, too, colonel?”
 “I knew the fellow was Darrel before the race,” answered Hawtrey.
“If he had proved to be an impostor, this accusation of theft might
not have carried. Now it is absolutely proven—ab-so-lutely.”
“Darrel has been accused here, before all his old friends,” Frank
continued, marshaling all his wits to acquit himself creditably of the
task of clearing Darrel, “and it’s only a fair shake that he should be
proven innocent before them. Colonel, will you please tell us of the
robbery, and show your proofs?”
Hawtrey was visibly annoyed. Nevertheless, he was a great stickler
for fair play, and he had to acknowledge that the position taken by
Merry was logical.
“I have been away from Gold Hill for a week,” said he, “visiting
some of my mining properties. Before I went, I drew a thousand
dollars in cash from the bank to pay to a man from whom I was
purchasing an interest in a ‘prospect.’ I was called from town
hurriedly, before the payment was made. The money was locked up
in the safe in my study, at home. Jode, here, who knows the
combination of the safe, was to pay over the money if the man
presented himself during my absence. The man did not come, and
Jode started off on this camping trip, three days ago. When I
reached home yesterday morning, I found the window of my study
unlocked, the safe door swinging open, the thousand dollars gone,
and this knife lying under the window, inside the room. Hand the
knife to Darrel, Merriwell, and see if he recognizes it.”
The colonel seemed averse to having any direct dealings with
Darrel. He gave the pocketknife to Frank, and the latter presented it
to Hawkins’ prisoner.
“It’s mine,” admitted Darrel huskily.
“Haff, an official of our athletic club, told Hawkins and me,” the
colonel proceeded, “that a fellow answering Darrel’s description had
been in town the night before I got home, that he had made
inquiries about me, that he had told the fellow I was away from
home, and that Jode was off on a camping trip, and that Darrel
started down the cañon to join the Gold Hill campers. Hawkins and I
got horses and hurried on to Tinaja Wells. Ask Darrel, Merriwell, if
he denies being in my house night before last?”
“No, colonel,” spoke up Darrel, without waiting for Merriwell to put
the question, “I do not deny it. I was there. I pushed open the sash
lock with this knife, and went in through your study and up to my
old room. I had the key to my room—have had it in my pocket for a
year. All I wanted to get was my running suit. After I had taken that,
I locked up the room and left by the window. Naturally, I could not
relock the window from the outside. That’s all, sir. I did not tamper
with your safe.”
A sneer of incredulity crossed Lenning’s face. It faded into a
sorrowful look, however, as the colonel gave him a swift glance.
“You admit being in the house,” said the colonel harshly, “so why
not admit the rest of it?”
“Because it is not the truth,” Darrel answered, with spirit.
“Did you know the combination of the safe, Darrel?” asked Frank.
“Yes—that is, if it hasn’t been changed during the past year.”
“It hasn’t,” put in the colonel. “That was my fault, I suppose.”
“Then, three of you knew the combination,” went on Frank,
“yourself, colonel, and Darrel and Lenning.”
“That is the way of it.”
The crowd on the mesa was listening with absorbed attention to
the talk which was going forward over the hapless head of the “boy
from Nowhere.” Nearly all, perhaps, felt that Darrel’s admission that
he had gone to the house for his running suit was a trivial excuse to
cover a design on the safe. Dark looks were thrown at Darrel, and
only here and there was anything bordering on sympathy shown for
him.
“Now,” said Frank, keeping the points he wanted to make well in
mind and working toward them with all the skill he could muster,
“you said, colonel, that Lenning and his camping party left Gold Hill
three days ago?”
“Yes.”
“Less than half a day would be required to make the trip from
Gold Hill to Tinaja Wells, for a mounted party with pack animals.
How does it happen, then, that the Gold Hillers only reached the
Wells yesterday afternoon?”
Colonel Hawtrey seemed puzzled. He turned to Lenning.
“Explain that, will you, Jode?” he requested. “Why didn’t you
reach the Wells day before yesterday?”
“Well, sir,” Lenning answered, “we were about halfway between
town and Tinaja Wells when we found out that Merriwell and his
crowd were camped at the place we wanted.”
“Ah! And what did you do then?”
“I had the boys make temporary camp in a side cañon while I—er
—went back to Gold Hill.”
“That,” said Frank, “would bring you in Gold Hill night before last—
the night of the robbery?”
Lenning reddened and looked confused.
“Why,” he faltered, “I reckon it would.”
“What was your business in Gold Hill, Lenning?”
“I don’t know,” snapped Lenning, “that you’ve got any call to
pump me.”