Joi~rnal of Controlled Release, 11 (1990) 61-78 61

Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands

DELIVERY OF VITAL DRUGS TO THE BRAIN FOR THE TREATMENT OF BRAIN

TUMORS*

Nigel H. Greig**, Shigeru Genka and Stanley I. Rapoport

Laboratory of Neurosciences, Nationa/ Institute on Aging, Nationa/ Institutes of Hea/th, Bethesda, MD 20892 (U.S.A.)

Key words: blood-brain barrier; brain tumor treatment; cancer chemotherapy; prodrugs; chlorambucil

The brain entry of many clinically valuable drugs is restricted by the blood-brain barrier, even
during pathological conditions when the integrity of this barrier may become partially reduced.
In this article, we describe the factors which, combined, determine the amount of a drug that
enters and is maintained in brain following its systemic administration. These factors include,
(i) the plasma concentration-time profile of the drug, (ii) its binding affinity and binding off-rates
to plasma proteins and tissues, (iii) the permeability of the drug at the blood-brain barrier, and
(iv) the rate of cerebral blood flow. We describe how drug uptake into brain changes during path-
ological conditions, specifically in the presence of a brain tumor. The problems of brain tumor
treatment (surgery, radiation therapy and chemotherapy) are reviewed and the delivery of drugs
into microenvironments within brain tumors and surrounding brain are described. Finally, we
report our studies on two techniques to enhance the uptake of drugs, biological response modifiers
and proteins such as monoclonal antibodies into brain and brain tumors. The first involves the
transient and innocuous opening of the blood-brain barrier by a 20 second infusion of a hyperos-
motic sugar solution, either mannitol or L-arabinose, into the carotid or vertebral arteries. This
allows the brain uptake of a compound to be increased by up to lO0-fold. We describe studies
undertaken to characterize this technique prior to its clinical use, and its value, when combined
with non-neurotoxic anticancer agents, in the treatment of patients with primary and metastatic
brain tumors. The second technique to improve delivery of drugs into brain involves the chemical
modification of specific moieties on drugs. We describe our studies with the anticancer alkylating
agent chlorambucil, in the development of lipophilic derivatives of potential clinical value for brain
tumor treatment.

INTRODUCTION classes of therapeutic agents which can interact

Recent advances in the fields of pharmacol-
ogy and molecular biology have led to a greater
understanding of disease processes at the mo-
lecular level, allowing the development of new
*Paper presented at the Fourth International Symposium
on Recent Advances in Drug Delivery Systems, Salt Lake
City, UT, U.S.A., February 21-24, 1989.
**To whom correspondence should be addressed.
with specific intracellular a n d / o r extracellular
targets. A number of drugs, peptides, biological
response modifiers, and monoclonal antibodies
and fragments thereof are presently available
and have proven of value to inhibit a variety of
malignant and infectious diseases, and rectify
neurotransmitter and enzyme imbalances in
tissue culture systems. However, their thera-
peutic efficacy in vivo frequently is diminished

0168-3659/90/$03.50 1990 Elsevier Science Publishers B.V.

62
or prevented by their inability to reach and
maintain active concentrations at the disease
site for an appropriate period of time. All too
often, functional groups on the agent, generally
polar in nature, that are essential for its inter-
action with or at the disease target to initiate a
pharmacological response, lead to (i) inade-
quate absorption of the agent from its site of
administration, (ii) poor access across biologi-
cal membranes to the disease target, and/or (iii)
vulnerability of the agent to rapid metabolic
degradation, and hence inactivity. It is unfor-
tunate that drug delivery is seldom considered
during drug design except as an afterthought.
The application of controlled drug delivery
concepts should be undertaken early during the
clinical development of therapeutic agents.
The need for effective delivery of a therapeu-
tic agent to its in vivo target is self-evident.
There are few more obvious cases of where in-
adequate pharmacokinetics can limit drug
therapy than when a disease is located within
the central nervous system (CNS). This prob-
lem is commonly encountered in patients with
acute cerebral bacterial or viral infections, as
well as with developmental or neurodegenera-
tive diseases, such as Parkinson's, Hunting-
ton's or Tay-Sach disease. However, possibly
the most extreme cases are encountered by neu-
rooncologists in treating patients with brain tu-
mors [ 1,2 ]. The limitations governing uptake
of therapeutic compounds into the brain and
brain tumors, together with techniques devel-
oped to circumvent these limitations, will
therefore be discussed in this manuscript.
BRAIN TUMORS: INCIDENCE A N D
TREATMENT
Primary neoplasms of the brain are the sec-
ond leading cause of cancer mortality in chil-
dren and represent the sixth leading cause in
the population over 20 years of age. They occur
at an annual rate of approximately 4.5 cases per
100,000 of the population [3]. Their age-spe-
cific incidence in the elderly is much higher, over
20 cases per 100,000 [4-6 ]. In an analysis of the
most recent data from the National Cancer In-
stitute Surveillance, Epidemiology and End
Results (SEER) program, we found that
whereas the overall incidence of primary malig-
nant brain tumors in most ages has changed lit-
tle between 1973 and 1985, there has been a
dramatic rise in the elderly [7]. Incidence has
risen over these recent dozen years by 2-fold in
both men and women between the ages of 75
and 79 years, by 4-fold in those between 80 and
84 years, and by more than 5-fold in those over
85 years of age. The treatment of primary brain
tumors is therefore a mounting problem, par-
ticularly in the elderly who tend to respond less
well to standard therapy than do younger pa-
tients [8,9].
Metastatic brain tumors have an estimated
annual incidence of 8.5 cases per 100,000 of the
population [2,10]. However, as half of all can-
cers in the United States occur in the 11% of
the population over 65 years of age [11], one
would predict the incidence of brain metastases
in the elderly population to be higher. Posner
[10,12] has reported that up to 24% of all pa-
tients dying from cancer have intracranial me-
tastases, including those of the dura. For par-
ticular types of cancer, such as malignant
melanoma and adenocarcinoma of the lung,
metastatic spread to the brain can occur at an
even greater incidence [ 10,13 ].
Malignant brain tumors are invariably fatal
[8,9,14-16]. They can be comprised of as many
as 1 101 cells, approximately 10 g weight, be-
fore they become clinically symptomatic, de-
pending on their location and the degree of as-
sociated edema. Some gliomas can be
considerably larger, up to 1 1011 cells or 100 g
weight, before becoming clinically evident. Sur-
gery, which is generally undertaken on all pri-
mary malignant brain tumors except when their
location precludes this, and often is only per-
formed on patients with a single metastatic le-
sion, the minority of metastatic brain tumors,
with well controlled extracerebral disease, can

at best remove up to 99% of the tumor mass.
This would reduce pressure on the surrounding
brain tissue and alleviate symptoms by the re-
moval of proliferating, quiescent and dead tu-
mor cells. However, due to the infiltrative na-
ture of most brain tumors [13,17,18], and to
the difficulty of differentiating normal from
malignant tissues, between 1 X l0 s and 1 X 109
tumor cells would remain. Consequently, sur-
gery alone yields only modest results, increas-
ing the median survival of glioma patients by
approximately 17 weeks [8,14,19 ].
Postoperative radiation therapy may kill up
to 99% of the remaining cells, leaving between
I X 106 and 1 X 107 tumor cells, with tumor re-
growth inevitable in the absence of further
treatment. The combination of surgery and ra-
diation therapy increases the median survival
of patients with glioma to approximately 37.5
weeks [9,15 ]. To further reduce the residual tu-
mor cell number towards an eventual cure (i.e.,
less than 1 X 104 cells) one must rely on chem-
otherapy, potentially the most promising ap-
proach to brain tumor therapy, and immuno-
therapy.
In vitro studies have demonstrated that cell
kill by most anticancer agents follows first-or-
der kinetics (i.e., a fraction of the total tumor
cells is killed rather than a specific number)
[20-22]. In theory, the desired remaining log
kill of tumor cells can be achieved by a combi-
nation of active anticancer drugs a n d / o r re-
peated administration. However, to maximize
the effect of chemotherapy in vivo, an appreci-
ation of the cell kinetics, pathophysiology and
location of the tumor is essential, in addition to
an understanding of the physicochemical char-
acteristics and pharmacokinetics of the drugs,
including the effects of dose, route of adminis-
tration and schedule.
63
TUMOR CELL KINETICS A N D THE BLOOD-
BRAIN AND BLOOD-BRAIN TUMOR
BARRIERS
Extensive studies by Hoshino and colleagues
[ 23-26 ] have elucidated the cell kinetic param-
eters of malignant brain tumors. Glioblasto-
mas, for example, which are the most common
primary malignant brain tumors in the elderly,
are the most aggressive form of glioma and are
lethal, have a growth fraction of only 30% to
40%. Therefore, the majority of their cells are
nonproliferating. Further, they have a cell loss
of between 80% to 90%, which is greater than
most extracerebral solid tumors. Hoshino and
colleagues have reported that the cell cycle time
of glioblastomas is approximately 2 to 3 days,
and that the duration of their S-phase is be-
tween 4.4 and 10.5 h. Such cell kinetic data have
immediate relevance to brain tumor chemo-
therapy, as it can be predicted that only ap-
proximately 10% of the proliferating pool of
cells would be sensitive to a cell cycle-depen-
dent anticancer drug, and only by maintaining
therapeutic drug levels for 2 or 3 days might it
be possible for such an agent to kill up to 30%
of tumor cells. Due to the slow removal of dead
cells from brain tumors and to recruitment of
cells from the nonproliferating pool, this would
not significantly reduce the diameter of a tumor
as measured with X-ray computerized tomog-
raphy or magnetic resonance imaging. Con-
versely, cell cycle-independent anticancer drugs
have a cell kill capacity that is not limited by
the tumor growth fraction and the cell cycle time
[20-22].
As a consequence of the limited log cell kill
capacity of a single anticancer agent, particu-
larly a cell cycle-dependent one, in addition to
the heterogeneity of subpopulations of neoplas-
tic cells within tumors, which may either be in-
sensitive to specific anticancer drugs or may
develop resistance mechanisms [27-29], com-
binations of drugs that may be synergistic or do
not possess overlapping toxicities often are ad-
ministered to maximize tumor cell kill
64
[21,30,31]. Such combinations have proved to
be valuable in treating leukemias and extracer-
ebral solid tumors, such as carcinoma of the
breast and ovary [20-22 ]. However, for tumors
sequestered within the brain, delivering and
maintaining therapeutic drug concentrations at
critical sites of high cell proliferation, have
proved difficult [ 1,32,33 ].
Most anticancer drugs, with the exception of
the nitrosoureas and procarbazine (which
undergoes rapid autoxidation to its lipophilic
azo-derivative in vivo) are hydrophilic in na-
ture and do not readily cross the blood-brain
barrier [2]. The barrier is located at the cere-
bral capillaries and is formed by a continuous
layer of endothelial cells that are joined by con-
tinuous belts of tight junctions [34,35 ]. We have
recently reviewed the state of this barrier in both
primary and metastatic brain tumors [13 ]; al-
though its integrity may be compromised in
some brain tumors, tumor capillaries still pro-
vide a significant diffusion restriction to drug
uptake.
The histology and vascularization of most
brain tumors often are as heterogeneic as the
neoplastic cells themselves. Sinus-like as well
as tortuous, glomeruloid-like capillaries that
have abandoned their normal linear pattern
often are present in a brain tumor, particularly
within the central region [17,18,36]. The alli-
ance between, and dependence of tumor cell
survival on a patent blood supply results in areas
of necrosis in central regions where the tumor
has outgrown its supply of nutrients and oxy-
gen, and areas of high proliferation in periph-
eral regions where tumor cells invade along
nerve tracts and along the perivascular space of
capillaries into adjacent normal brain [17,37].
Electron microscope and permeability studies
have shown that areas of barrier breakdown,
when they occur, are generally within the tu-
mor center and not in the peripheral margin or
in the surrounding brain, where blood vessels
are normal in structure [38-41]. Further, due
to tumor-associated barrier breakdown and to
the lack of a lymphatic drainage system in the
brain, cerebral edema and cysts often are asso-
ciated with large primary and metastatic brain
tumors [13,17,18].
Regional differences in tumor capillary den-
sity, blood-brain tumor barrier integrity and
blood flow, from a pharmacokinetic viewpoint,
and differences in the number of proliferating
cells, from a cell kinetic viewpoint, should lead
to different pharmacological microenviron-
ments within a brain tumor during systematic
therapy [33 ].
DRUG PERMEATION INTO BRAIN A N D
BRAIN T U M O R S
The factors that govern the uptake of a drug
across the normal blood-brain barrier and de-
termine its time-dependent concentration
within the brain following its systemic admin-
istration have been examined by a number of
investigators [42-45], including the authors
[1,46,47]. In summary, our studies have dem-
onstrated that the cumulative effects of four
major factors determine the ultimate delivery
of a drug and its time-dependent concentration
within the brain. The first is the time-depen-
dent plasma concentration profile of the com-
pound. This is related to its route of adminis-
tration, its distribution and elimination
processes. The second is the binding of the agent
to plasma constituents and tissues, and binding
off-rates from them [48]. Recent studies have
challenged the "free drug hypothesis", that only
the free dialyzable fraction of an agent is at lib-
erty to penetrate the blood-brain barrier,
whereas the remainder is restrictively bound to
plasma proteins. Studies with palmitate, phen-
ytoin, diazepam and other benzodiazepines have
demonstrated that these agents, unlike others
such as billirubin and phenobarbital, are not re-
strictively bound to plasma constituents and
that their extraction from blood to brain is
greater than, and independent of, their free
plasma concentrations [48-51]. Such a phe-
nomenon is well described in the liver and kid-
 65

ney for the elimination of such compounds as
lidocaine, propranolol, and penicillins [52-54].
The third factor is the permeability of the
blood-brain barrier to the agent. Our studies
have demonstrated a linear relationship be-
tween the cerebrovascular permeability of a
compound and its lipophilicity, as determined
by its octanol:water partition coefficient, P
(Fig. 1) [46,47,55 ]. Specific exceptions to this
occur when an agent shares a facilitated trans-
port system, such as the anticancer drug mel-
phalan that shares the large neutral amino acid
transport system at the blood-brain barrier
[56], and the anti-HIV agent azidothymidine,
AZT, that probably shares the thymidine
transport system at the choroid plexus [57 ], and
secondly when a lipophilic agent, such as the
anticancer agents vincristine and vinblastine,
is endowed with several highly polarized func-
tional groups and an unusual geometry [58,59 ].
In the first case, utilization of a facilitated
transport system will augment the brain uptake
of a water-soluble agent, depending on compe-
tition for the binding sites, whereas in the sec-
ond case, repulsive forces between charged
functional groups on a lipophilic compound and
the lipid molecules of the cerebral capillary
membrane will restrict brain uptake of drug. Fi-
nally, the fourth factor is local cerebral blood
flow.
In normal brain, transfer across the blood-
brain barrier of a lipophilic drug, having an oc-
tanol:water partition coefficient exceeding 1.0
(log P > 0 ), i.e. equal partition, is rapid and rate
limited by the rate of local blood flow. A lipid-
insoluble, polar drug, whose octanol: water par-
tition coefficient is less than 0.1 (log P < - 1 )
is restricted from entering the brain by its
permeability across the blood-brain barrier, in-
dependently of blood flow. For compounds
whose octanol:water partition coefficients lie
between these limits, a combination of blood
flow and cerebrovascular permeability deter-
mines their brain uptake. In each case, main-
tenance of high plasma concentrations maxi-
mizes distribution from blood to brain. This is

lx10-2

lx10-3 /
/
/ ~ CCNU
/ ~ BCNU
lx10-4 Caffeine 0,4~ Antipyrine
/ "X"
Procarbazine~P C N U Spirohydantoin
Propylene Glycol _t,./'X~ Metronidazole Mustard
lxl0-S 7JMisonidazole
Ethylene _ / ' ~ r-torafur
Glycol /w Acetamide
Glycerol / ~5FU
Curare / / "~ Dianhydrogalacticol
lx10-6 Thiourea
N-Methyl Urea//" Formamide
icotinamide ~ / ~ Vincristine
/ ~ "~ Methot rexate
1x 10-7 ~-Mannitol/0
,/ Arabinose
/ .~ Epipodophyllotoxin
~ / ~ Sucrose
lx10 -8 / I I I I I I I
lx10 -4 lx10-3 lx10 -2 lx10 -1 1 10 100 1000
OCTANOL/WATER PARTITION COEFFICIENT

Fig. 1. Relationshipbetweenoctanol/waterpartition coefficientand cerebrovascularpermeabilityof a variety of compounds

(asterisk designatesan anticancer drug).
66
particularly true for lipophilic agents which will
readily diffuse out of brain into blood should
plasma levels decline below their steady-state
brain/plasma ratio, unless drug is bound or me-
tabolized by brain cells [ 1,47 ].
As a consequence of the described heteroge-
neity of vascular integrity, and hence permea-
bility in brain tumors, as well as differences in
blood flow and intercapillary distances, trans-
capillary exchange and distribution of drugs
from blood to brain tumors is less predictable
than in normal brain. In small growing lesions
which have an intact barrier, lipophilic drugs
but not water-soluble drugs are likely to reach
significant and, depending on the peak plasma
level, therapeutic concentrations. In larger tu-
mors, however, this may be different. In the core
of a large tumor, which would represent a low
density area on a CT scan, there is frequently
low blood flow and necrosis, a high intercapil-
lary distance and a variably intact blood-brain
tumor barrier [17,60,61]. Delivery of a lipo-
philic agent to this area will be less than to nor-
mal brain, particularly to cells distant from the
capillary. Conversely, because of barrier de-
fects, delivery of a water-soluble agent may be
greater than to normal brain if blood flow is sig-
nificant, but, due to the large intercapillary dis-
tance, may still result in subtherapeutic levels.
Drug delivery to a tumor area similar to one that
would enhance on a CT scan, with a relatively
normal blood flow and a variably intact barrier,
would be approximately similar to delivery to
normal brain for a lipophilic agent, but greater
for a polar drug due to barrier breakdown, al-
though surrounding brain may act as a diffu-
sion sink and reduce tumor drug levels [62].
Finally, delivery of a lipophilic or water-soluble
drug to the periphery of a brain tumor, which
contains the greatest tumor burden and has
blood flow and permeability characteristics
similar to those of normal brain, can be pre-
dicted to be similar to normal brain. Studies by
Levin and colleagues [33,63] show that the
permeability of the brain immediately sur-
rounding a tumor, into which malignant cells
may have invaded, has a permeability that is
lower than normal brain, and thus delivery of
water-soluble drugs to this area would be re-
duced concomitantly.
In summary, lipophilic drugs are able to read-
ily penetrate the blood-brain barrier and, due
to high cerebral blood flow, approximately 20%
of cardiac output, to reach high concentrations
in brain and brain tumors, except in regions
were blood flow is dramatically reduced. Lipo-
philic drugs, however, have a relatively short
diffusion distance into brain and brain tumors
before intracellular uptake occurs and this can
limit drug delivery in regions of high intercap-
illary distance. Conversely, water-soluble drugs
are restricted from entering brain and signifi-
cant areas of high cell proliferation in brain tu-
mors by their low permeability across cerebro-
vascular endothelial cells. In areas of reduced
barrier integrity, however, significant amounts
can accumulate in brain tumors. They will have
a longer diffusion distance and, therefore, may
be diluted as a consequence of the sink effect of
normal brain.
The lipophilic nitrosourea carmustine,
BCNU, is the primary anticancer agent used in
brain tumor chemotherapy [8,9,15,16,64]. Its
combination with surgery and radiation ther-
apy, however, has increased median survival of
patients with glioma to only 48.5 weeks [9,15 ].
It is unfortunate that most other anticancer
agents are water-soluble and, hence, that their
combination with the nitrosoureas has not pro-
longed patient survival [2,16]. In the absence
of significant toxicity, investigations have dem-
onstrated that increasing the concentration-
time product of a drug at the site of a tumor
even by only two-fold may increase fractional
cytoreduction in a sensitive tumor by three- to
ten-fold or more [65].
In order to increase the arsenal of valuable
anticancer agents available to the oncologist for
the treatment of brain tumors, we have ex-
plored two avenues to increase the time-con-
centration product of therapeutic drugs
throughout tumors and the surrounding brain

they invade. We chose compounds of proven
clinical benefit whose pharmacological proper-
ties, including toxicities, are known. Further,
as these compounds are normally restricted
from entering the brain, we carefully monitored
their toxicity when delivered to the brain.
Our first approach has been to reversibly and
without damage increase the cerebrovascular
permeability of the blood-brain barrier to spe-
cific water-soluble agents, and thereby allow
their greater entry into brain. Our second ap-
proach has been to modify specific drugs them-
selves, to increase their lipophilicity and, hence,
brain penetration. Other techniques reported to
augment brain uptake of drugs have been re-
ported by Greig [1,2,67]. Additionally, drugs
have recently been placed within the brain in
biodegradable polymers for the treatment of
primary brain tumors [66]. However, further
studies are required before the efficacy of intra-
cerebral polymer-drug complexes is established.
BLOOD-BRAIN BARRIER M O D I F I C A T I O N
A N D THE O S M O T I C TECHNIQUE
The blood-brain barrier maintains the hom-
eostatic environment of the brain, allowing it
to function relatively independently of large
systematic fluctuations of compounds that have
a central action and/or are required for normal
brain function [42,44,46]. In addition, the bar-
rier protects the brain against toxic systemic
substances. Although the barrier is notably re-
sistant to many chemical and physical insults,
a variety of techniques have been reported to
innocuously and reversibly increase its perme-
ability. We recently reviewed some of these
[1,2,67 ], many remain unsubstantiated and
several have been refuted. One method, the os-
motic opening technique, has been well char-
acterized and is in clinical use.
Rapoport [46,68-71] undertook extensive
studies to determine which compounds could
induce reversible barrier opening without tox-
icity. Examination of a wide variety of agents
67
of differing physicochemical characteristics
demonstrated that the carotid administration
of a hyperosmotic solution of the sugars man-
nitol or L-arabinose consistently and safely
produced barrier opening throughout the cere-
bral capillary network supplied by the infused
artery. An osmotic threshold of 1.6 osmolal was
required over an infusion period of at least 20
seconds. Barrier opening was reversed com-
pletely within 4 h, initially closing to larger
rather than to small compounds [72,73].
Morphological studies utilizing the electron-
dense protein horseradish peroxidase as well as
ionic lanthanum, both of which are normally
impeded from entering the brain by the tight
junctions between cerebral capillary endothe-
lial cells, demonstrated that the increase in cer-
ebrovascular permeability resulted from an os-
motic-induced shrinkage of the endothelial
cells, which caused a subsequent widening of
intercellular tight junctions [35,74,75]. The
gradual rehydration of the endothelial cells after
the osmotic load slowly restores barrier integ-
rity and accounts for the size-dependent clo-
sure. Extensive studies in monkeys, dogs, rats
and mice have demonstrated that the proce-
dure is safe provided that (i) the hyperosmotic
sugar is filtered directly prior to use, (ii) the
cerebral circulation is not compromised and
(iii) the technique is not combined with the co-
delivery of a neurotoxic agent [32,69,76-81 ].
Administration of a therapeutic water-solu-
ble agent, directly following osmotic barrier
opening, results in its significant accumulation
in brain. Two examples are the anticancer an-
timetabolite methotrexate (molecular weight,
MW, 454 daltons) [ 77,82,83 ] and the naturally
occurring protein interferon-~ (MW approxi-
mately 19,500 daltons) [84]. Methotrexate is
widely used in the treatment of acute lymphob-
lastic leukemia, non-Hodgkin's lymphoma,
breast, bronchogenic, testicular and choriocar-
cinoma [20,21], whereas interferons are pres-
ently being tested against a variety of cancers,
including brain tumors [85,86]. Administra-
tion of methotrexate directly after osmotic
68

opening increases its level in brain by 7-fold.
An additional 7-fold is achieved by its admin-
istration into the artery previously infused with
the hyperosmotic arabinose [68,83]. Similarly,
as shown in Fig. 2, brain concentrations of in-
terferon were increased by osmotic opening
from 0.003% to 0.18% per gram of the total ad-
ministered dose, 60-fold [84 ].
Not only are brain levels of drugs increased
but due to barrier reclosure and the trapping of
agent in brain they remain there longer and
their duration of action is thereby extended.
This is of particular importance for a cell cycle-
dependent drug like methotrexate. The sys-
temic disappearance of methotrexate is bi-
phasic with an initial half-life of 45 min [87].
Its half-life in brain after osmotic barrier open-
ing is extended to 4.8 h [82 ], over 6-fold longer,
and is similar to that in CSF, 4.5 h, following
its intraventricular administration [88,89]. In-
fusion of methotrexate into CSF has proved of
significant clinical value, with acceptable tox-
icity, in the treatment of CSF-sequestered leu-
kemia [88,90 ]. Similarly, concentrations of in-
terferon are maintained in brain for many hours
following osmotic barrier opening despite its
relatively rapid systemic disappearance, plasma
half-life approximately 70 min [91 ]. Our stud-
I0000
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O, 09'IOTI[ OPENEOgRAIN
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ies showed that the rates of efflux from brain of
methotrexate and interferon-c~, following en-
hanced entry with the osmotic technique, are
slower than that of sucrose, a nonmetabolizable
marker of the extracellular space, indicating
cellular uptake or binding of both agents
[82,84]. Methotrexate is known to be trans-
ported into tumor cells, where it forms polyglu-
tamate conjugates, whereas interferons are
known to bind and become internalized in cells
[22,92]. As a consequence of a greater perme-
ability at the blood-brain barrier and an ex-
tended half-life in brain, the time-dependent
concentration integrals of both agents are dra-
matically increased in brain. As shown in Fig.
3, the concentration integral of interferon in
brain, following osmotic opening, is more than
100-fold greater than that in normal brain and
similar to integrals in peripheral tissues whose
capillaries do not restrict drug delivery [84,91 ].
Studies in experimental intracerebral tu-
mors have similarly demonstrated that osmotic
barrier opening significantly increases delivery
of water-soluble drugs to tumors in areas of
complete or partial barrier integrity [93 ]. How-
ever, in areas of total barrier breakdown, where
water-soluble drugs can readily penetrate, the
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achieved in osmotic opened and normal b r a i n after a 5 m i n between 30 a n d 240 m i n ) in normal a n d osmotic opened
infusion of 2 X 106 U / 1 0 0 g interferonoo~ into rats. T h e con- brain, peripheral tissues a n d plasma after a 5 m i n infusion
centration in osmotic opened brain was significantly greater of 2 X 106 U / g into rats. AUC represents area u n d e r the
t h a n in normal brain, whose levels were minimal ( P < 0.05 ). curve

osmotic technique has little additional effect.
Similarly, enhanced CT studies in humans by
Neuwelt and colleagues [32,80 ] have shown in-
creased penetration of contrast material in
brain tumors and surrounding brain following
osmotic barrier opening with 25% (w/v) man-
nitol. Additionally, the technique has shown the
presence of lesions with intact barriers that were
not previously evident by enhanced CT scan.
A criticism of the osmotic technique is that
it delivers increased amounts of drug to normal
brain as well as to brain tumor [94,95 ], and this
may cause neurotoxicity. Whether or not this
objection is well founded remains to be deter-
mined. It is not possible for the hyperosmotic
infusate to differentiate between normal and
brain tumor capillaries in the infused area. As
described, it also is not possible to distinguish
exactly how far a tumor has invaded into nor-
mal brain [13]. Therefore, from a pharmaco-
kinetic viewpoint, it is advantageous to deliver
increased drug levels to brain surrounding a tu-
mor. First, this will ensure that all tumor cells,
particularly those with the highest growth frac-
tion at the invasive edge, are subjected to cy-
tocidal concentrations, and, second, this over-
comes the "diffusion sink" effect of brain
adjacent to tumor. Finally, clinical studies that
were based on extensive neurotoxicological
studies in animal models and involved indepen-
dent neuropathology [96-98] have combined
methotrexate, activated cyclophosphamide and
procarbazine with the osmotic technique and
have shown that neurotoxicity associated with
these drugs does not occur [32].
Clinical studies utilizing the osmotic tech-
nique have thus far involved over 1500 proce-
dures in more than 110 patients, and have been
extensively reviewed by Neuwelt [32]. As in-
dicated, the technique has proved minimally
toxic. Indeed, the majority of the few reported
toxicities related to the use of contrast agent
required for CT evaluation of barrier opening
and not to the use of the described anticancer
agents. After these initial toxicities, the type of
contrast material was changed. In summary,
69
Neuwelt and colleagues have documented tu-
mor regression in patients with metastatic brain
tumors [99] and have increased the median
survival time of patients with gliomas to 17.5
months [100]. However, best responses have
been associated with their treatment of pri-
mary CNS lymphoma [101]. In a recent anal-
ysis of 29 patients, they reported a median sur-
vival time of 32 months [102]. This is
significantly longer than that of 13.5 months
reported by Massachusetts General Hospital
following their treatment of 61 similar patients
with conventional therapy between 1958 and
1984.
CHEMICAL MODIFICATION OF DRUGS
We recently reviewed how the chemical mod-
ification of a therapeutic compound may over-
come a pharmacokinetic problem associated
with it, such as poor CNS penetration, and en-
hance its brain uptake [67 ]. In theory there are
three approaches by which this can be under-
taken. The first is by the synthesis of lipophilic
analogues of the original active compound. In
this case, chemical groups required to initiate a
pharmacological response must be preserved
and should these prove to be highly ionized at
physiological pH, irrespective of lipophilicity,
the compound may be restricted from entering
the brain, as are vincristine and vinblastine
[59]. The second approach is by the chemical
masking of an essential ionized group with an
unionized moiety, such as an ester or ether, that
hopefully is enzymatically or chemically cleaved
preferentially at the target, thereby endowing
it with site specificity. This is the principle of a
prodrug [103-106]. Finally, the third proce-
dure is by the synthesis of an active compound
which structurally resembles an endogenous
agent that shares a facilitated transport system
at the target organ.
Although a certain degree of lipid solubility
is essential for a centrally acting drug to trans-
verse interceding biological membranes, such as
the blood-brain barrier, an agent that predom-
70
inantly distributes in octanol rather than in
water is also subject to pharmacokinetic short-
comings. First, highly lipophilic compounds are
difficult to solubilize in an innocuous vehicle
for administration. The development of cyclo-
dextrins by Pitha and colleagues [ 107 ] may help
to resolve this problem. Further, lipophilic drugs
must have an appreciable solubility in plasma
and extracellular fluid to reach their target at a
therapeutic concentration. Finally, lipophilic
agents often become heavily and restrictively
bound to plasma constituents. Indeed, studies
by Scholtan [108] have demonstrated a posi-
tive linear relation between lipophilicity and
binding constants of a variety of drugs to hu-
man albumin.
Hansch [109,110] has postulated that there
is an optimal lipophilicity, approximately log
P=2 (octanol/water partition coeffi-
cient=100), for uptake of a compound into
brain. This value agrees with those of several
useful centrally acting drugs (diazepam, log
P=2.8; diphenylhydantoin, log P = 2 . 5 ; chlo-
robutanol, log P = 2.0; chloroform, log P = 1.9).
Evidence suggests that different classes of drugs
may have different optimal log P values de-
pending on their mechanism of action and their
metabolism. An agent whose pharmacological
action requires its binding to a cell surface re-
ceptor would require to maintain high concen-
trations in the extracellular fluid and hence, in-
tuitively, its optimal log P would be predicted
to be less than 2. Conversely, for an agent whose
target is intracellular, a log P value of greater
than 2 would ensure high intracellular locali-
zation. Analogous to the linear relation be-
tween cerebrovascular permeability of a com-
pound and its lipophilicity, one would expect a
similar relation to exist at the extracellular/in-
tracellular level of cells within the brain. This
concept can be applied to brain-directed pro-
drugs that often rely on an extracellular en-
zyme system to regenerate the pharmacologi-
cally active parent compound at a suitable rate.
An excessively high lipophilicity, causing pre-
dominant intracellular sequestration, would
prevent this regeneration, whereas a water sol-
ubility would impede brain delivery. Due to this
and to the low relative abundance of specific
enzyme systems that will cleave prodrugs at
their target, few clinically useful brain-directed
prodrugs have yet been developed [67].
We have undertaken drug modification stud-
ies on the anticancer agent chlorambucil [111-
113]. Chlorambucil is a classical bifunctional,
nitrogen mustard alkylating agent that has
proved of clinical value in the treatment of
chronic lymphocytic leukemia, Hodgkin's dis-
ease and carcinoma of the breast and ovary [20-
22 ]. Several studies have reported that alkyl-
ating agents other than the nitrosoureas (i.e.,
BCNU), particularly classical nitrogen mus-
tards, are amongst the most active compounds
against medulloblastoma and human glioma
xenografts in immune-deprived animals
[114,115]. Further, studies by Schabel and col-
leagues [116] and by Hill [117] suggest there
is a lack of cross-resistance between different
types of anticancer alkylating agents for many
tumors, as cellular mechanisms involved in an-
ticancer alkylating activity, as well as those in-
volved in the development of resistance, are dif-
ferent. Consequently, combinations of alkyla-
ting agents have proved of clinical value, par-
ticularly when combined with autologous bone
marrow transplantation [118]. As chlorambu-
cil is ionized at physiological pH (Fig. 4), the
compound only minimally enters the brain
[ 119 ]. Additionally, it binds to plasma constit-
uents, and this further reduces its brain uptake.
We have described its pharmacokinetics and
reported that its brain/plasma integral concen-
tration ratio is only 0.02 [111,119]. Further, we
have shown that its activity against alkylating
agent-sensitive, brain-sequestered tumors is
minimal. We postulated that masking of the
ionizable carboxylic acid moiety of chloram-
bucil by a lipophilic ester may increase the brain
delivery of the compound and yet maintain its
alkylating activity.
Reaction of chlorambucil with the appropri-
ate alcohol, under azeotropic conditions to re-
 71

CHLORAMBUCIL
cleaved too rapidly by plasma esterases, to re-
O
II / ~ /CH z CH,--Cl lease chlorambucil, prior to their significant
HO--C--CHz--CH2--CH2~ - N :
~"--CH2_ CHz_ el entry into brain, and this may explain the poor
therapeutic efficacy of prednimustine in the
3,4 DEHYDROCHLORAMBUCIL treatment of patients with glioblastoma. The
O
II ~ /-'CHz- CH,- CI median survival time of these patients was only
HO--C-- CH--CH =CHz~-(I }~N~ 8.7 months [120], which is not significantly
~CHz--CH 2 Cl
different from that of patients treated with only
surgery and radiation therapy. The longer chain
PHENYLACETIC MUSTARD alkyl esters of chlorambucil (chlorambucil
O
II ~ ~ CHz-CH'-CI hexyl and octyl esters) were more stable in
HO-c- CH,--~,LJ/'~\
~--CHz--eHz--Cl plasma. However, these esters bound restric-
tively to plasma constituents and this mini-
METABOLISM OF CHLORAMBUCIL BY ~J-OXIDATION
mized their brain penetration. None of these
derivatives demonstrated improved anticancer
CHLORAMBUCIL TERTIARYBUTYL ESTER activity, compared to chlorambucil, against
GH3 O brain-implanted tumors in rats [112 ]. These
I II ~ jCHz CHz--Cl
CH, C-O C--CH,--CH,--CH,---~( ) > ~ N results are in accord with attempts by others to
CJH3 ~ / ~ C H z - CH2--CI
deliver methotrexate to the brain [121,122].
Fig. 4. Chemical structures of chlorambucil and of its active Dialkyl esters were synthesized to mask the two
metabolites 3,4-dehydrochlorambucil and phenylacetic ionizable carboxylic esters of the glutamine
mustard (produced as a consequence of chlorambucilfl-ox- moiety of methotrexate. However, short ester
idation), and of chlorambucil tertiary butyl ester.
rapidly regenerated methotrexate and longer
esters bound restrictively to plasma constitu-
move water formed during the reaction, can ents. Thus none achieved higher brain concen-
yield multiple esters. During our initial at- trations than methotrexate. Similarly, linkage
tempts to increase the brain delivery of chlor- of chlorambucil, via its carboxylic acid residue,
ambucil, we analyzed and described the physi- to a dihydropyridine~-pyridinium redox sys-
cochemical properties, plasma and brain tem, suggested by Bodor and Brewster [ 123 ] to
pharmacokinetics, and brain anticancer activ- act as a brain delivery system for water-soluble
ity of 7 lipophilic chlorambucil ester derivatives drugs, proved to be ineffective, as brain concen-
[112]. These included an homologous series of trations did not differ from those achieved after
alkyl esters, from one to 8 carbons length, and chlorambucil administration alone [ 124 ].
three aromatic esters. The latter included the To overcome the shortcomings of our initial
phenylmethyl, phenylethyl and prednisolone drug modification studies, we designed and
ester (prednimustine). All the ester derivatives tested a branched ester derivative, chlorambu-
possessed significant intrinsic alkylating activ- cil tertiary butyl ester (Fig. 4), whose steric
ities which, although less than that of chlor- hindrance reduces its rate of ester hydrolysis
ambucil, were similar or greater than that of the and whose lipophilicity is less than that of the
anticancer drug melphalan which is of signifi- long alkyl ester derivatives [111]. The com-
cant clinical value [20,22]. Additionally, all pound possesses intrinsic alkylating activity
broke down to release chlorambucil alone. None, and breaks down in plasma to release chlor-
however, achieved or maintained significantly ambucil alone. Figure 5 shows the concentra-
greater concentrations in brain, after equimo- tions of chlorambucil and of chlorambucil ter-
lar administration, than did chlorambucil. The tiary butyl ester in plasma and brain of rats,
short alkyl esters and aromatic esters were following equimolar intravenous administra-
72
1000 I
100
i 10
1 ~
i -
.1
0 30 60 90 120
Time (mln)
Fig. 5. Concentrations of chlorambueil and of ehlorambucil tertiary butyl ester in plasma and brain following equimolar
intravenous administration to rats ( 10 mg/kg and 13 mg/kg, respectively).
tion. Chlorambucil disappeared from plasma
with a half-life of 28 min and a low concentra-
tion entered brain. This disappeared from brain
with a half-life of approximately 20 min and
could not be detected after 120 min. Calculated
from the concentration versus time profiles, the
brain/plasma ratio of chlorambucil was only
0.02. Compared to chlorambucil, the plasma and
brain concentration profiles of chlorambucil
tertiary butyl ester were reversed. Chlorambu-
cil tertiary butyl ester initially disappeared rap-
idly from plasma with a half-life of approxi-
mately 2 min; thereafter, steady low concen-
trations were detected up to 120 min. The rapid
plasma decline probably was due to its large
distribution volume, as a consequence of its li-
pophilicity, and plasma and tissue de-esterifi-
cation to chlorambucil. A significant amount of
chlorambucil tertiary butyl ester entered and
remained in brain, probably as a consequence
of intracellular uptake. The peak brain concen-
tration was 3-fold higher than that of chlor-
ambucil and the half-life was twice as long. The
brain/plasma concentration ratio was 33, which
is similar to centrally acting drugs such as the
antipsychotic agent haloperidol [ 125 ].
As chlorambucil undergoes fl-oxidation in
vivo to yield the active alkylating agent phenyl-
acetic mustard, via the transitory metabolite
3,4-dehydrochlorambucil [ 126 ] (Fig. 4 ), its bi-
ological activity derives from the combined ac-
tions and concentrations of each of these active
~ i~asma 1000' -N- p l a s m a
-o,- brain -0- brain
0
E 100'
o
c 10
1'
~ .1 i | | ! i - i I i
150 180 210 240 30 60 90 120 150 180 210 240
Time (rain)
compounds. Similarly, the in vivo biological ac-
tivity of chlorambucil tertiary butyl ester would
be predicted to derive from the combination of
its action and that of its active metabolites. As
a consequence, concentrations of each active
metabolite were quantitated in plasma and
brain following the equimolar administration of
chlorambucil and chlorambucil tertiary butyl
ester in rat, these have been summed to yield
total active compounds and are shown in Fig. 6.
The formation of active metabolites, predomi-
nantly phenylacetic mustard, from chloram-
bucil results in the maintenance of active com-
pounds in plasma for a longer duration. As these
metabolites are ionized, they minimally enter
brain and the brain/plasma concentration ra-
tio of total active compounds derived from
chlorambucil is 0.018. Similarly, the systemic
release of chlorambucil from chlorambucil ter-
tiary butyl ester, probably as a result of back-
diffusion of the ester from systemic lipid stores
into plasma and its enzymatic breakdown there,
increases concentrations of active compounds
in plasma. Due probably to intracellular up-
take, this does not occur in the brain and as
minimal amounts of the ionized metabolites in
plasma enter brain, the brain/plasma concen-
tration ratio of total active compounds derived
from chlorambucil tertiary butyl ester admin-
istration is 0.68. Although this is a decline from
the initial brain/plasma ratio of 33 for chlor-
ambucil tertiary butyl ester alone, it is 35-fold

1000 ' ~ -14- plasma
-o- brain
:
100
"E
0 30 60 90 120 150 180 210
Time (min)
Fig. 6. Concentrations of total compounds possessing anticancer activity in plasma and brain following equimolar intrave-
nous administration of chlorambucil and chlorambucil tertiary butyl ester to rats (10 mg/kg and 13 mg/kg, respectively).
greater than that derived from chlorambucil
administration. Further, as tertiary butyl es-
ters of drugs are more stable in humans than in
rodents, due to lower human levels of unspe-
cific esterases [127-129], we predict that cir-
culating plasma concentrations of total active
compounds in humans would be lower than in
rats, and therefore the brain/plasma ratio would
be higher.
The dose-limiting toxicity of chlorambucil in
humans is myelosuppression [20-22]. There-
fore, lower circulating concentrations of active
compounds in plasma are likely to be associated
with reduced toxicity. In accord with this, we
have demonstrated that the maximum toler-
ated dose of chlorambucil tertiary butyl ester in
rats is 6-fold higher than that of chlorambucil
and, unlike chlorambucil, the administration of
high doses of chlorambucil tertiary butyl ester
does not cause seizure activity. Finally, chlor-
ambucil tertiary butyl ester, unlike chloram-
bucil, significantly prolongs survival of animals
with brain implanted tumor.
CONCLUSIONS
It is accepted that the blood-brain barrier,
even when partially open as may occur in brain
tumors, reduces the efficacy of a large number
of therapeutically valuable agents. It is encour-
aging that research is focusing on this problem
and in the future it is entirely possible that ap-
73
1000 -N- plasma
-g- brain
E
c: 100,
240 0 30 60 90 120 150 180 210 240
Time (mln~
propriate lipid-soluble drugs will be designed
that will overcome the delivery problems that
we have described. However, for many poten-
tially useful compounds, such as monoclonal
antibodies and drug conjugates, it is probable
that their brain uptake will remain restricted.
Further, what should be done now? We believe
that specific techniques, such as the osmotic
blood-brain barrier opening technique and
simple drug modifications, can be of immediate
clinical value. They represent a possible means
to prolong the survival of patients who face a
dismal prognosis.
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