British Journal of Pharmacology (2003) 140, 1201-1210. doi: 10.1038/sj.bjp.0705554
Published online 03 November 2003
Alkylglycerol opening of the blood-brain barrier to small
and large fluorescence markers in normal and C6 gliomabearing
rats and isolated rat brain capillaries
Bernhard Erdlenbruch1, Mehrnaz Alipour1, Gert Fricker2, David S Miller3, Wilfried Kugler1,
Hansjörg Eibl4 and Max Lakomek1
1. 1Kinderklinik der Universität at Göttingen, Robert-Koch-Str. 40, D-37075 Göttingen,
2. 2Institut für Pharmazeutische Technologie und Biopharmazie, Im Neuenheimer Feld
366, D-69120 Heidelberg, Germany
3. 3National Institutes of Environmental Health Sciences, National Institutes of Health,
Research Triangle Park, NC 27709, U.S.A.
4. 4Max-Planck-Institut für Biophysikalische Chemie, Am Fa berg, D-37077 Göttingen,
Correspondence: Bernhard Erdlenbruch, Kinderklinik der Universität at Göttingen, Robert-
Koch-Str. 40, D-37075 Göttingen, Germany. E-mail: firstname.lastname@example.org
Received 14 July 2003; Revised 23 September 2003; Accepted 24 September 2003; Published
online 03 November 2003.
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1. The blood-brain barrier (BBB) represents the major impediment to successful
delivery of therapeutic agents to target tissue within the central nervous system.
Intracarotid alkylglycerols have been shown to increase the transfer of
chemotherapeutics across the BBB.
2. We investigated the spatial distribution of intracarotid fluorescein sodium and
intravenous lissamine-rhodamine B200 (RB 200)-albumin in the brain of normal and
C6 glioma-bearing rats after intracarotid co-administration of 1-O-pentylglycerol (200
mM). To elucidate the mechanisms involved in the alkylglycerol-mediated BBB
opening, intraluminal accumulation of fluorescein isothiocyanate (FITC)-dextran
40,000 was studied in freshly isolated rat brain capillaries using confocal microscopy
during incubation with different alkylglycerols. Furthermore, 1-O-pentylglycerolinduced
increase in delivery of methotrexate (MTX) to the brain was evaluated in
3. Microscopic evaluation showed a marked 1-O-pentylglycerol-induced extravasation of
fluorescein and RB 200-albumin in the ipsilateral normal brain. In glioma-bearing
rats, increased tissue fluorescence was found in both tumor tissue and brain
surrounding tumor. Confocal microscopy revealed a time- and concentrationdependent
accumulation of FITC-dextran 40,000 within the lumina of isolated rat
brain capillaries during incubation with 1-O-pentylglycerol and 2-O-hexyldiglycerol,
indicating enhanced paracellular transfer via tight junctions. Intracarotid coadministration
of MTX and 1-O-pentylglycerol (200 mM) in nude mice resulted in a
significant increase in MTX concentrations in the ipsilateral brain as compared to
controls without 1-O-pentylglycerol (P<0.005).
4. In conclusion, 1-O-pentylglycerol increases delivery of small and large compounds to
normal brain and brain tumors and this effect is mediated at least in part by enhanced
permeability of tight junctions.
Alkylglycerol, blood-brain barrier, brain tumor, drug delivery, fluorescence markers,
methotrexate, rat brain capillaries, tight junction, confocal microscopy
BBB, blood-brain barrier; BSA, bovine serum albumin; BW, body weight; FITC, fluorescein
isothiocyanate; FPIA, fluorescence polarization immunoassay; MTX, methotrexate; RB 200,
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The brain capillary endothelium plays a key role in the pathophysiology of various diseases of
the CNS such as inflammatory disorders, tumors, ischemia, and seizures (Pardridge, 1998).
The mechanisms of functional regulation and transport at the blood-brain barrier (BBB) are
not well understood, and further investigation is needed to clarify both how BBB dysfunction
is mediated in disease states and how transport systems at the barrier can be altered for
therapeutic purposes. Currently, successful treatment of many brain disorders seems to be
impossible even though highly active drugs exerting powerful effects at the target site have
been developed. Their efficacy is limited by very low penetration across the BBB (Neuwelt et
al., 1999). Thus, enhancing or targeting drug delivery to the brain has become a major issue in
experimental neurology. Chemical modification of drugs and the use of transport mediating
agents or vector systems are possible strategies for drug targeting to the brain (Smith, 1993;
Huwyler et al., 1996; Reszka et al., 1997; Kroll & Neuwelt, 1998; Jolliet-Riant & Tillement,
1999); however, the clinical benefit of such measures has still to be demonstrated. In contrast
to this, methods to open the BBB such as hyperosmotic disruption of the BBB or bradykinin
receptor-induced increase in barrier permeability have already been shown to be effective in
the treatment of experimental brain tumors (Blasberg et al., 1990; Nomura et al., 1994; Elliott
et al., 1996; Matsukado et al., 1996; Kroll et al., 1998). Chemotherapy of malignant brain
tumors in conjunction with osmotic opening of the BBB has been advanced to the stage of
clinical trials and tumor response has been documented in both adults and pediatric patients
(Gumerlock et al., 1992; Dahlborg et al., 1996; 1998; Doolittle et al., 2000).
The inadequate drug delivery across the BBB is a major factor that explains the poor response
rates of chemosensitive brain tumors (Siegal & Zylber-Katz, 2002). Consequently, there is a
need for new maneuvers designed to overcome the limited access of anticancer agents to the
brain and to brain tumors. Recently, the transfer of a variety of chemotherapeutic drugs across
the BBB was shown to be increased dramatically by intracarotid drug administration in the
presence of short-chain alkylglycerols (Erdlenbruch et al., 2000; 2002). The intracarotid
injection of alkylglycerols resulted in a concentration-dependent accumulation of the
coinjected drugs within the brain. The effect was rapidly reversible and variations in the
chemical structure of the alkylglycerols allowed for modulation of the extent of increased
barrier permeability (Erdlenbruch et al., 2003). Of particular importance is the fact that there
were no signs of toxicity in long-term experiments using intracarotid 1-O-pentylglycerol and
2-O-hexyldiglycerol in rats (Erdlenbruch et al., 2003). In view of their low toxicity and the
potent and well-controllable effects, intracarotid alkylglycerols are thought to be a very
promising principle to facilitate the transport of therapeutics across the BBB. However, little
is known about the distribution of the delivered drugs within the hemispheres and whether
large compounds such as proteins will enter the brain after intracarotid alkylglycerols.
Furthermore, there are only marginal insights into the mechanisms involved in alkylglycerolmediated
BBB opening. Finally, nude mice represent the species mostly used for the
treatment of experimental human brain tumor xenografts, but alkylglycerols were not
administered in nude mice so far.
Therefore, the purpose of the present study was (a) to estimate the 1-O-pentylglycerolmediated
increase in the penetration of small and large fluorescence markers into the brain of
both normal and glioma-bearing rats and to investigate the spatial distribution of the different
markers within the brain, (b) to elucidate the mechanisms of action of the alkylglycerols by
studying the accumulation of fluorescein isothiocyanate (FITC)-dextran 40,000 (40 kDa) in
freshly isolated rat brain capillaries using confocal microscopy and quantitative image
analysis during incubation with different alkylglycerols, and (c) to demonstrate feasibility and
effectivity of the permeabilizing effect of 1-O-pentylglycerol in nude mice by intracarotid coadministration
with methotrexate (MTX).
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The synthesis of 1-O-pentylglycerol and 2-O-hexyldiglycerol has been described in detail
elsewhere (Erdlenbruch et al., 2000). Purity of the products was assessed by HPLC and was
above 99% in all experiments. Depending on the binding site of the alkyl group, up to 1% of
the respective 1-O- or 2-O-positional alkyl isomers were found. No other by-products were
identified. For the in vivo experiments, 1-O-pentylglycerol was used as the prototypical
compound of the alkylglycerols and because long-term in vivo studies have demonstrated a
lack of toxicity (Erdlenbruch et al., 2003). Incubation with rat capillary preparations was
performed in vitro using 1-O-pentylglycerol and 2-O-hexyldiglycerol, an effective derivative
of the alkyldiglycerol family associated with no toxic effects at therapeutic levels
(Erdlenbruch et al., 2003).
Fluorescence markers of different molecular size were chosen to estimate the permeability of
the BBB in the presence or absence of 1-O-pentylglycerol. In the animal experiments, low
and high molecular fluorescence dyes were administered. Fluorescein sodium (MW 367 Da)
was purchased from Merck (Darmstadt, Germany), and lissamine-rhodamine B200 (RB 200)
and human albumin were obtained from Sigma-Aldrich (Deisenhofen, Germany). Fluorescein
sodium was dissolved in physiological saline, forming a stock solution of 5%. For intracarotid
use (80 mg kg-1), this solution was further diluted to a final concentration of 2.5% in
accordance with the protocol for the respective treatment groups. RB 200 was coupled to
albumin and purified as described by Klein et al. (1986). FITC-dextran 40,000 (MW 40 kDa,
Sigma-Aldrich) was used for in vitro investigation of alkylglycerol-dependent permeation of
drugs across isolated rat brain microvessels.
MTX was purchased from Onco-Hexal AG (Holzkirchen, Germany) and a dose of 5 mg kg-1
body weight (BW) was given to nude mice. Control animals received intra-arterial MTX
without 1-O-pentylglycerol. In these experiments, MTX was diluted with physiological saline
to the desired final volume (100 l). For intracarotid coinjection with 1-O-pentylglycerol, a
mixture of alkylglycerol and MTX was diluted with water. The concentration of 1-Opentylglycerol
in the final solution was 200 mM and the osmolality of the injected solutions
ranged from 421 to 448 mosm kg-1. Within this range of osmolality, no permeability changes
due to osmotic effects have to be expected.
Male Wistar rats and male nude mice were kept under conventional controlled conditions
(22°C, 55% humidity, and day-night rhythm) and had free access to a standard diet (V1534-
000, Fa. sniff, Soest, Germany) and tap water. The nude mice were purchased from Charles
River Laboratories (CD®-1 nude mice, Crl:CD1®-nu, Sulzfeld, Germany). The experiments
were carried out in accordance with the German Law on the Protection of Animals.
The differential permeability of the BBB in the absence and presence of 200 mM 1-Opentylglycerol
was investigated in tumor-free rats (n=19) and in rats bearing C6 gliomas
(n=12) using small and large fluorescence markers. The delivery of MTX to the brain of
tumor-free nude mice was evaluated in the absence and presence of 1-O-pentylglycerol
(n=12). MTX was chosen for these experiments because (a) it is used in different treatment
protocols of pediatric brain tumors, (b) it is known to exhibit poor penetration into the CNS
after both intravenous and intra-arterial administration (Neuwelt et al., 1998; Erdlenbruch et
al., 2000), and (c) the effect of alkylglycerols on the transfer of MTX into the CNS has been
thoroughly investigated in rats (Erdlenbruch et al., 2003).
Wistar rats weighing 180-220 g received intraperitoneal ketamine/xylazine (90 g/7.5 g
per g BW) and 1 105 C6 cells were inoculated into the right putamen as described previously
(Erdlenbruch et al., 1998). Briefly, rats were placed in a stereotaxic instrument (David Kopf
Instruments, Tujunga, CA, U.S.A.) and 10 l of a suspension of C6 cells in RMPI 1640
medium was injected using a 10- l Hamilton syringe with a 26-gauge needle 1 mm anterior
and 3 mm lateral to the bregma, and 5 mm deep to the dural surface. Tumors were allowed to
grow until first signs of manifestation (first day of weight loss or second day of no weight
gain, 14 2 days after tumor implantation). At this time, large tumors were found with only
minor variability in tumor size (Figure 4).
Tissue fluorescence of fluorescein sodium and RB 200-albumin in C6 glioma-bearing rats.
Dyes were injected either in the absence (a) or presence (b) of intracarotid 1-O-pentylglycerol
(200 mM). Dye extravasation in tumors without alkylglycerol treatment (a) and high
fluorescence intensity in both tumor and surrounding ipsilateral brain in the presence of 1-Opentylglycerol
(b). Note: Four times shorter exposure time in (b) to avoid overexposure of the
image. Photographs are representative of six experiments within each group.
Intra-arterial drug administration
The intracarotid administration of the drugs to rats was performed as described previously
(Erdlenbruch et al., 2003). In brief, rats were anesthetized with intraperitoneal pentobarbital
(50 mg kg-1 BW) followed by intravenous injections. Body temperature was maintained at
37.5°C, and arterial blood pressure and heart rate were monitored by a Statham transducer
(Gould, Oxnard, CA, U.S.A.) throughout the experiments via the left femoral artery. The right
external carotid artery was cannulated and the microcatheter system was filled with blood in a
retrograde manner. Fluorescein sodium (80 mg kg-1 BW) or MTX (5 mg kg-1 BW) was
injected into the right internal carotid artery either in the presence (simultaneously) or in the
absence of 1-O-pentylglycerol (200 mM, mean dose 90 10 mg kg-1 BW). All solutions were
heated to 37°C and sterile filtered immediately before administration. In rats, a total volume
of 1.2 ml consisting of 800 l drug solution followed by rinsing with 400 l of isotonic
saline was injected with a flow rate of 6 ml min-1 using a Hamilton dispenser (Microlab,
Hamilton Bonaduz, Switzerland). During the injection, the common carotid artery was
clamped. RB 200-albumin (200 l per 100 g BW) was administered intravenously 3 min
before the intracarotid bolus of fluorescein sodium. RB 200-albumin was given to stain
intravascular space as well as to assess the permeabilizing effect of 1-O-pentylglycerol on
large compounds. A simultaneous intracarotid co-injection of both fluorescent dyes was not
performed, since the viscosity and the volume of the infusate would be too high. At 5 min
after the intracarotid administration of fluorescein, the brains were rapidly removed and
frozen in isopentane (-50°C).
The nude mice were anesthetized by intraperitoneal ketamine/xylazine (75 mg per kg BW/5
mg per kg BW). The surgical procedure was the same as described for the rat experiments;
however, the trachea and the femoral artery were not cannulated. A special fine and bent glass
catheter was used to cannulate the external carotid artery. MTX (5 mg kg-1 BW) was
administered simultaneously or without 1-O-pentylglycerol (200 mM, 105 29 mg kg-1 BW).
A volume of 100 l of drug solution was injected followed by rinsing with 40 l of isotonic
saline (total volume 140 l). At 5 min after the MTX administration, mice were perfused with
Ringer's solution via the left ventricle, the brains were rapidly removed and stored at -20°C
until further analysis.
For histology, the frozen brains were sectioned into 7 m slices. Either sagittal or coronal
serial sections were made and air dried. Evaluation of the sections was performed using
fluorescence microscopy (Zeiss Universal, Zeiss Göttingen, Germany). Na-fluorescein and
RB 200 were visualized by use of filter combinations as described by Klein et al. (1986). The
histological preparations were assigned to the respective anatomical planes according to the
stereotaxic atlas from König & Klippel (1963). Serial coronal planes were used for
quantitative interindividual comparison of the fluorescence intensity of the brain. The
reference plane (zero) for coronal sections was a plane through the interaural line and
distances were given from this plane to identify the respective section levels (König &
Klippel, 1963). Fluorescence intensity was measured semiquantitatively using a highresolution
black and white camera (Kappa, CF8/1 DXC, Gleichen, Germany) and
computerized image analysis.
Analysis of MTX concentrations
The concentrations of MTX in the brain tissue were determined separately in the right
hemisphere (ipsilateral to the injection), in the left hemisphere (contralateral), and in the
cerebellum (including brain stem) as described previously (Erdlenbruch et al., 2000). In brief,
organs were minced and homogenized in alkaline medium (NaOH 0.1 M, total volume 0.8 ml,
pH=12-13). After neutralization with hydrochloric acid, MTX concentrations were
determined by fluorescence polarization immunoassay (FPIA; Jolley et al., 1981). The FPIA
reagent systems were purchased from Abbott Laboratories, IL, U.S.A., and analyses were
performed according to the operation manuals. Calibration curves for tissue concentrations of
MTX were established for each assay. Values are given as pmol mg-1 wet weight.
In vitro experiments
Rat brain capillaries were isolated as described by Miller et al. (2000). In brief, capillaries
(3-6 animals per preparation) were isolated using a modification of the procedure of
Pardridge et al. (1985). All steps were carried out at 4° C in pre-gassed (95% O2/5% CO2)
solutions. Keeping the tissue on ice and in well-gassed buffers was essential for preservation
of transport function. Pieces of grey matter were gently homogenized in three volumes (v w-1)
of buffer A (103 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO4,
15 mM HEPES) and, after addition of dextran (final concentration 30%), the homogenate was
centrifuged at low speed. The resulting pellet was resuspended in buffer B (buffer A
supplemented with 25 mM NaHCO3, 10 mM glucose, 1 mM Na-pyruvate, and 0.5% (w/v)
bovine serum albumin (BSA)) and then filtered through a 200 m nylon mesh. The filtrate
was passed over a glass bead column and, after washing with 500 ml buffer, the capillaries
adhering to the beads were collected by gentle agitation. Capillaries were centrifuged, the
pellet resuspended in ice-cold, gassed, BSA-free Krebs-Henseleit buffer and immediately
used for transport experiments.
Confocal microscopy was performed as described (Miller et al., 2000). Briefly, capillaries
were transferred to a covered Teflon incubation chamber containing 1.5 ml of pre-gassed
Krebs-Henseleit medium with 1 M FITC-dextran 40,000 in the absence or presence of 1-
O-pentylglycerol or 2-O-hexyldiglycerol at concentrations up to 20 mM. The chamber floor
was a 4 4 cm glass coverslip to which the capillaries adhered and through which they could
be viewed. All experiments were conducted at room temperature (18-20°C). The chamber
was mounted on the stage of a Zeiss LSM 5 Pascal inverted confocal laser scanning
microscope and viewed through a 63 or 40 water immersion objective. The 488-nm laser
line, a 510-nm dichroic filter, and a 515-nm long-pass emission filter were employed. Low
laser intensity was used to avoid photobleaching of the dyes. With the photomultiplier gain
set, tissue autofluorescence was undetectable. Capillaries were first viewed under reduced
transmitted light illumination. A field containing 2-5 capillaries was selected and a confocal
fluorescence image was obtained. Capillaries contained mostly unbranched segments at least
100 m in length and were 5-8 m in diameter at pixel resolutions 0.2 m pixel-1.
Transmitted light and fluorescence micrographs showed the endothelium to be 1-1.5 m
Fluorescence intensities were measured from stored images using NIH Image 1.61 or Scion
Image software as described previously (Miller, 1995). Owing to microscope adjustments and
focusing the capillaries selected, the first image could be made 4 min after starting incubation.
Since the appearance of FITC-dextran 40,000 within the capillary lumen was mediated by
passive diffusion, equilibrium has to be expected at maximum effects. The background
fluorescence intensity was subtracted and the average pixel intensity for each area was
calculated. The value assigned to a capillary was the means of all selected areas. As there are
uncertainties in relating cellular fluorescence to the actual concentration of an accumulated
compound in cells and tissues with complex geometry (Sullivan et al., 1990; Miller &
Pritchard, 1991), data are reported here as average measured pixel intensity rather than
estimated dye concentration.
Mean values s.d. are presented unless otherwise indicated. For statistical analyses, one-way
analysis of variance (ANOVA) was used.
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In vivo experiments
To investigate the spatial distribution of small and large fluorescence markers in normal
tumor-free rats, serial sections of the brains were analyzed at defined planes. In the absence of
1-O-pentylglycerol (control animals, n=5), fluorescein sodium and RB 200-albumin were
detected only within the lumina of the cerebral vasculature and no differences in fluorescence
intensity were registered between the ipsilateral and the contralateral hemisphere (Table 1 ).
Intracarotid injection of 1-O-pentylglycerol resulted in a marked transfer of both fluorescein
and RB 200-albumin into the brain tissue ipsilateral to the injection (n=14, Figures 1 and 2).
Almost no extravasation of the fluorescent dyes was found in the contralateral hemisphere.
The ratio of ipsilateral to contralateral fluorescence intensity in the coronal sections was
chosen to quantify the 1-O-pentylglycerol-induced increase in BBB permeability. The mean
ratio for fluorescein amounted to 6.45 1.4 and for RB 200-albumin to 2.66 1.0 (Table 1).
The increase in the fluorescence intensity of fluorescein was considerably stronger than of the
intravenously administered high molecular RB 200-albumin. Since the two fluorescent
probes were not administered at the same site and circulation times were different, the
fluorescence intensities could not be compared. Intraindividual regional differences in the
extent of ipsilateral dye extravasation became apparent by analyzing serial sections from the
entire brain (Figure 3). Contralateral tissue fluorescence was low in all brain areas (Figure 3).
As visualized in Figures 1 and 2, extravasation of the fluorescence markers was slightly more
prominent in the cortical regions. Only small interindividual variations were found when
comparing the pattern of the 1-O-pentylglycerol-induced increase in fluorescence intensity of
both of the fluorescent markers (Table 1).
Spatial distribution of tissue fluorescence of small and large fluorescence markers
(fluorescein sodium and RB 200-albumin) in normal rat brain in the absence (a) or presence
(b) of intracarotid 1-O-pentylglycerol (200 mM): coronal sections. Serial coronal sections of
frozen brains were obtained and observed by fluorescence microscopy (left panel: FITC; right
panel: RB 200-albumin). Fluorescence strictly restricted to the brain vasculature (a) and 1-Opentylglycerol-
induced increase in tissue fluorescence of the ipsilateral hemisphere (b).
Boxes: Magnification of the respective brain region. Representative sections from one of
Spatial distribution of tissue fluorescence of small and large fluorescence markers
(fluorescein sodium and RB 200-albumin) in normal rat brain in the presence of intracarotid
1-O-pentylglycerol (200 mM): sagittal sections. Serial sagittal sections of frozen brains were
obtained and observed by fluorescence microscopy (left panel: FITC; right panel: RB
200-albumin). Lack of tissue fluorescence in the contralateral hemisphere (a) and increased
fluorescence in the ipsilateral hemisphere after intracarotid 1-O-pentylglycerol (b).
Representative sections from one of seven experiments.
Fluorescence intensity of fluorescein sodium and RB 200-albumin in serial coronal brain
sections after co-administration with 1-O-pentylglycerol in normal rats. RB 200-albumin was
administered intravenously 3 min before an intra-arterial co-injection of 1-O-pentylglycerol
(200 mM) and fluorescein sodium via the internal carotid artery. Serial coronal sections were
obtained at defined planes anterior to the interaural plane. Fluorescence intensity was
determined separately in the ipsilateral and contralateral hemisphere using a computerized
camera system. (a) Fluorescence intensity of fluorescein sodium and RB 200-albumin in the
different brain regions. (b) Values depicted represent the ratio of fluorescence intensity
ipsilateral to contralateral of fluorescein sodium (circles) and RB 200-albumin (triangles).
Table 1 - Ratio of fluorescence intensity ipsilateral to contralateral hemisphere after intravenous RB
200-albumin and intracarotid fluorescein sodium in the presence or absence of intracarotid 1-Opentylglycerol.
In contrast to the brain of tumor-free rats, basal vascular permeability of intracerebral C6
tumors was increased as demonstrated by intratumoral extravasation of the fluorescent
markers in the absence of 1-O-pentylglycerol (Figure 4a). The intracarotid co-injection with
1-O-pentylglycerol resulted in a conspicuous additional transfer of fluorescein sodium and
RB 200-albumin to both tumor tissue and surrounding ipsilateral brain (Figure 4b). Owing to
the excessive increase in fluorescence intensity within the tumors, overexposure of slices
showing parts of the tumor was observed even if exposure times were substantially reduced.
In view of the high brightness in tumor tissue after 1-O-pentylglycerol, variations in the
conditions for the computerized imaging and the analysis of fluorescence intensity (e.g.
exposure times and additional filters) were necessary to obtain usable results. Therefore, no
statistical evaluation of tissue staining was feasible in the tumor experiments.
In an additional series of experiments, intracarotid MTX was given in the presence or absence
of 200 mM 1-O-pentylglycerol to tumor-free nude mice. After intracarotid administration of
MTX without alkylglycerols, low tissue concentrations were found in the brain parenchyma.
MTX delivery to the brain was markedly increased by 1-O-pentylglycerol. The increase of
MTX concentrations was found predominantly in the right hemisphere ipsilateral to the bolus
MTX transfer to different regions of the brain of tumor-free nude mice after intracarotid
administration of 1-O-pentylglycerol. MTX (5 mg kg-1) was given to nude mice (n=12) in the
absence or presence of 1-O-pentylglycerol (200 mM). Right: right hemisphere; left: left
hemisphere; cerebellum: cerebellum and brain stem. Concentrations given are means s.d.;
*P<0.05, right versus left and right versus cerebellum (ANOVA).
In vitro experiments
The permeation of FITC-dextran 40,000 across the walls of freshly isolated rat brain
capillaries was measured using confocal laser scanning microscopy. Figure 6 shows
representative experiments using control and 1-O-pentylglycerol-exposed capillaries. In the
control capillary, little change in fluorescence was observed after 20 min. In contrast, 1-Opentylglycerol-
exposed capillaries showed rapid increases in fluorescence. We used
quantitative image analysis to measure dextran permeation into capillary lumens. Figure 7a
shows little change in luminal fluorescence of control capillaries even after 2 h incubation in
FITC-dextran-containing buffer. In contrast, capillaries exposed to 2 or 10 mM 1-Opentylglycerol
showed a steady increase in luminal fluorescence over 30 min. Addition of 1-
O-pentylglycerol to control capillaries elicited a rapid and sustained increase in luminal
fluorescence (Figure 7b). The effects of both 1-O-pentylglycerol and 2-O-hexyldiglycerol
were concentration dependent (Figure 7c), with concentrations as low as 0.2 mM significantly
increasing luminal fluorescence after 20 min exposures. From these dose-response data, 1-Opentylglycerol
appeared to be slightly more effective. Together, the data indicate that
alkylglycerols caused a rapid and concentration-dependent increase in capillary permeability
to a marker of paracellular permeation.
Incubation of freshly isolated rat cerebral capillaries with 1 M FITC-dextran 40,000 in the
absence and presence of 10 mM 1-O-pentylglycerol. Owing to microscope adjustments and
focusing the capillaries selected, the first image could be made 4 min after starting incubation.
Whereas only negligible amounts of the paracellular marker compound in capillary lumens
can be seen in control capillaries (left panel), a clear accumulation of FITC-dextran 40,000
was observed within 20 min in the lumina of capillaries incubated with 1-O-pentylglycerol
(right panels). Arrows indicate the point on which the confocal microscope was focused.
Effect of alkylglycerols on permeation of FITC-dextran 40,000 across the walls of freshly
isolated rat brain capillaries. (a) Time course of FITC-dextran 40,000 permeation across
individual control and 1-O-pentylglycerol-exposed capillaries. (b) Data from a single
capillary showing the effect of addition of 10 mM 1-O-pentylglycerol. (c) Dose response for
1-O-pentylglycerol and 2-O-hexyldiglycerol. Measurements were made over the first 20 min
after transferring capillaries to chambers containing FITC-dextran 40,000 with 0-2 mM
alkylglycerol. Data are given as mean fluorescence intensity for 3-6 capillaries; variability is
shown as s.d.
The limited access of potentially helpful therapeutics into the CNS resulting from the
presence of the BBB emphasizes the importance of developing strategies for overcoming the
BBB. Only a limited number of approaches to increase the transfer of drugs to the brain have
been used in clinical studies so far (Cornford & Hyman, 1999). Osmotic opening of the BBB
by intracarotid infusion of hypertonic mannitol solution has been reported to increase the
delivery of water-soluble drugs, peptides, antibodies, and viral vectors to the brain (Rapoport,
2000). This technique is used in conjunction with intra-arterial chemotherapy to treat human
primary CNS lymphomas or high-grade malignant gliomas, and improved survival has been
reported (Dahlborg et al., 1996; 1998; Doolittle et al., 2000; McAllister et al., 2000).
However, barrier opening has been shown to last for 6-8 h (Siegal et al., 2000), and
treatment-related toxicity (Roman-Goldstein et al., 1991; Gumerlock et al., 1992; Williams et
al., 1995; Siegal & Zylber-Katz, 2002) as well as a number of methodological difficulties, for
example, catheter access, optimal flow rate, thrombotic complications, and choice of
anesthetic (Gumerlock & Neuwelt, 1990; Mortimer et al., 1992; Rapoport, 2000), has
prevented the widespread use of this technique so far. The administration of the bradykinin
B2 receptor agonist RMP-7 represents a biochemical method to open the BBB in patients with
malignant brain tumors (Gregor et al., 1999; Emerich et al., 2001; Warren et al., 2001). RMP-
7-mediated barrier permeabilization is almost restricted to the blood-brain tumor barrier
(Nomura et al., 1994; Matsukado et al., 1996) and only a very modest increase in drug
transfer to the tumor was achieved in animal models (Kroll et al., 1998). In view of these
difficulties, other strategies are in demand for the delivery of neuropharmaceuticals across the
Intracarotid short-chain alkylglycerols have been reported to induce a strong and transient
increase in the transport of chemotherapeutic drugs to the ipsilateral hemisphere (Erdlenbruch
et al., 2003). One goal of the present study was to evaluate the extent and local distribution of
alkylglycerol-mediated delivery of large compounds to the brain. Staining of brain
parenchyma with albumin-bound RB 200 demonstrated that even proteins could enter the
brain when administered in conjunction with 1-O-pentylglycerol. Thus, opening of the BBB
using alkylglycerols permits brain delivery of drugs with a wide range of molecular size.
From preliminary data using intracarotid globulin coupled fluorescence markers, it was
inferred that even larger proteins could be transferred to the brain. In earlier experiments
using chemotherapeutic drugs of different molecular size, it was shown that the amount of
drug delivered to the CNS using 1-O-pentylglycerol decreased with increasing size of the coinjected
compounds (Erdlenbruch et al., 2000). The higher extravascular fluorescence
intensities of small marker substances such as fluorescein sodium concur with the fact that 1-
O-pentylglycerol-mediated enhancement of drug delivery to the brain depends on the
molecular size of the injected drug. Furthermore, within the same brain section, tissue staining
with fluorescein was more homogenous compared with RB 200-albumin, which exhibited a
more spot-like, patchy extravasation predominantly around the vessels. This difference in the
pattern of fluorescence can be explained by weaker and slower penetration of the large
albumin-linked RB 200 into the brain parenchyma. From recent barrier experiments using
different chemotherapeutics in rats, it was already assumed that both high molecular size and
high polarity of the co-administered drugs are associated with lower CNS penetration,
because 1-O-pentylglycerol-induced accumulation of vancomycin and gentamicin within the
brain was significantly lower than that of cisplatin and MTX (Erdlenbruch et al., 2000).
There was little interindividual variation in the regional distribution of the fluorescence
markers in normal animals. This provides further evidence of the reliability of barrier opening
by 1-O-pentylglycerol in the normal unchanged brain. Within the C6 gliomas, baseline
permeability was heterogeneous. Increased vascular permeability of brain tumors has been
well described in the literature (Hiesiger et al., 1986; Inoue et al., 1987; Neuwelt et al., 1998)
and may account for some tumor responses to chemotherapy. In the present study, there was a
strong increase in tissue fluorescence in both tumor tissue and surrounding ipsilateral normal
brain reflecting similar permeabilizing effects of 1-O-pentylglycerol at the blood-brain tumor
barrier and the intact barrier. Due to the high fluorescence intensity of the tumor tissue after
administration of the fluorescent markers in the presence of 1-O-pentylglycerol, the exposure
time had to be shortened substantially to avoid overexposure of the images. Therefore, no
quantification of the increase in tissue fluorescence within the different brain regions could be
performed. In earlier studies, however, the increase in drug delivery to brain tumor tissue and
ipsilateral cortex after intracarotid administration of 1-O-pentylglycerol was analyzed using
different chemotherapeutic drugs (Erdlenbruch et al., 2000; 2002). The 1-O-pentylglycerolmediated
increase in the transfer of methotrexate to tumor tissue was approximately as high as
to the surrounding ipsilateral tumor-free brain (18-fold in the tumor as compared to 28-fold in
the surrounding brain). Thus, the use of alkylglycerols in conjunction with intra-arterial
chemotherapy enables enhanced access of anticancer drugs to the tumor mass and to
infiltrative malignant cells at the tumor edge.
This contrasts with the effects observed after osmotic BBB disruption, because mannitol has
been reported to increase drug delivery predominantly to the normal brain rather than to the
tumor itself, resulting in a reversal of the tumor-to-cortex permeability relationship (Hiesiger
et al., 1986; Inoue et al., 1987; Shapiro et al., 1988; Barnett et al., 1995; Neuwelt et al.,
1998). As the present study allowed no quantification of the 1-O-pentylglycerol-mediated
increase in tumor uptake of the fluorescent markers, further studies using both mannitol and
alkylglycerols are needed to compare the increase in drug transfer to different tumor areas and
to the surrounding normal brain. Intra-arterial chemotherapy of brain tumor-bearing animals
in conjunction with BBB opening by hyperosmolar mannitol or 1-O-pentylglycerol will
clarify whether alkylglycerols offer any superiority over mannitol.
Since large molecular weight agents were also transported across the BBB, and in view of
both the potential for exact regulation of barrier opening and the lack of long-term toxicity of
short-chain alkylglycerol derivatives (Erdlenbruch et al., 2003), other applications also appear
to be of great promise. Therefore, it is noteworthy that increased drug transfer in the presence
of alkylglycerols was easily reproducible in nude mice. The 1-O-pentylglycerol-mediated
MTX accumulation found in tumor-free nude mice was less marked than in normal Wistar
rats (Erdlenbruch et al., 2003) indicating that the permeabilizing effect of alkylglycerols may
differ between different species. Improved transit of specific brain-targeted compounds to the
brain tissue will be of great interest in the next few years because new and effective
neuropharmaceutics have been designed recently.
The use of the optical sectioning capabilities of confocal microscopy allowed us to develop a
procedure that can provide new insights into the mechanisms of alkylglycerol-associated
increase in transendothelial drug transport. The selective intraluminal accumulation of the
fluorescent markers indicated that permeation of the drugs was mediated by enhanced
permeability of the zonulae occludens. Relevant transcellular transport could be excluded due
to the lack of intracellular labeling of the endothelial cells. Even at high magnification there
was no evidence for a transcytotic pathway of the marker. Incubation of low concentrations of
alkylglycerols with synthetic membranes consisting of dipalmitoylglycerophosphocholine or
dimyristoylglycerophosphocholine resulted in an impressive and concentration-dependent
decrease in phase transition temperature (data not shown). From these data, alkylglycerolinduced
fluidization of biological membranes was hypothesized, possibly acting via changes
in tight-junctional integrity. This effect, however, appears to be short-lasting, because
baseline permeability of the barrier was restored within a few minutes (Erdlenbruch et al.,
2000; 2003). Furthermore, no clinical or neuropathological alterations were found 2 and 4
weeks after intracarotid 1-O-pentylglycerol treatment (Erdlenbruch et al., 2003). Ongoing
experiments focusing on functional alterations of tight junction proteins will contribute to
further clarify the mechanisms involved in the alkylglycerol-mediated increase in BBB
In summary, a strong increase in delivery of fluorescence markers of different molecular
weight to both normal brain and brain tumors was demonstrated in rats by intracarotid coadministration
of 1-O-pentylglycerol. Increased drug transfer across the BBB was also
observed after intracarotid 1-O-pentylglycerol in nude mice. The permeabilizing effect of the
alkylglycerols is mediated at least in part by enhanced permeability of the tight junctions.
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Leukemia and Lymphoma (Hodgkin's and Non-Hodgkin's Disease)
Leukemias are cancers of the blood-forming organs, and lymphomas are cancers of the lymphatic
tissues. In general, leukemias and lymphomas respond well to the conventional treatment methods of
chemotherapy and radiation therapy. Because there are many different types of these cancers,
treatment is based on the specific diagnosis of the disease.
In the United States, more than 30,000 new cases of leukemia will be diagnosed in the coming year,
and adult onset of the disease will account for 90% of these cases. Leukemia is not a single disease but
a group of related diseases. There are no specific symptoms for leukemias; instead, symptoms are
more generalized and include fatigue, weakness, unexplained weight loss, and pain. Most cases of
leukemia are found during routine laboratory tests such as a complete blood count (CBC with
Once the initial diagnosis of leukemia is made, further testing includes bone marrow aspiration,
lumbar puncture, and excisional biopsies to determine the specific type of leukemia. When leukemias
are detected, they are not classified by stages because they are systemic diseases and other organs such
as the spleen, lymph nodes, liver, and central nervous system are already involved.
Leukemias are classified into acute and chronic forms. Cancerous cells rapidly reproduce and
accumulate in both forms of the disease, crowding out normal white blood cells. The difference
between the two forms of leukemia is that, in the acute form, bone marrow cells do not reach maturity
and immature cells accumulate. In the chronic form, the cells appear mature but are abnormal and live
longer than normal white cells. If left untreated, the majority of patients with an acute form of the
disease have a life expectancy of 1 year.
Leukemias are further classified according to the type of affected bone marrow cells. The cancer is
myelogenous if the involved blood cells are granulocytes or monocytes. The cancer is lymphocytic if
the affected cells are lymphocytes. Leukemias are divided into four main types: acute myelogenous
(AML), chronic myelogenous (CML), acute lymphocytic (ALL), and chronic lymphocytic (CLL).
There are also several subtypes of these diseases based upon the French-American-British (FAB)
classification system for acute leukemias. Prognosis and treatment are based on the diagnosis of the
type and subtype of the disease.
Leukemias respond well to chemotherapy and radiation therapy, and these treatment methods are often
used in combination. The treatment of leukemia involves the use of a combination of cancer
medications given over a period of time. As a general rule, AML will be treated with high doses of
chemotherapy agents over a short period of time, whereas ALL is treated with lower doses of
chemotherapy over a longer period of time.
Chemotherapy agents attack rapidly dividing cells; however, they also interfere with the production of
white blood cells, thereby exposing the patient to the risk of infection. Medications known as growth
factors increase white blood cell counts and are often given in combination with chemotherapy.
Interferons (IFN) are a group of naturally occurring biologic response modifiers that are sometimes
used in the treatment of chronic leukemias (Aviles 1997). The most commonly used of these
substances is interferon-alpha.
Interferon reduces the growth of cancerous cells, inhibits their replication, and enhances the immune
system's response to the cancer. Interferon appears to be particularly useful when it is used as a
maintenance therapy in patients with minimal residual disease (post-remission) or complete remission.
In addition, all-trans retinoic acid (a vitamin A analogue), when used in combination with Interferon,
may be useful in prolonging the lives of patients with promyelocytic leukemia and other forms of the
disease (Zheng et al. 1996; Sacchi et al. 1997). A cautionary note to the use of this therapy is that the
patient may be at risk for thrombosis (blood clots). However, heparin therapy or the use of certain
nutrients may reduce this risk (see the Thrombosis Prevention protocol).
Other therapies for the treatment of leukemias include stem-cell therapy. Stem-cell therapy involves
removing stem cells from the patient either by bone marrow aspiration or by a procedure called
apheresis (also called peripheral blood stem-cell (PBSC) transplant), when the cells are removed from
the peripheral blood system. Stem cells may be obtained from the patient or from a donor who is a
close tissue match to the patient. In this therapy, high doses of chemotherapy and radiation therapy
destroy the patient's bone marrow, and the collected stem cells are then transplanted into the patient to
restore normal blood cell production. This type of therapy is still in the experimental stage. As a result,
it is very expensive and may not be covered by insurance.
Hodgkin's lymphoma is a cancer of the lymph nodes. The American Cancer Society estimated that
over 7400 new cases of the disease would be diagnosed in 2001. However, Hodgkin's disease has an
overall cure rate of 75% in newly diagnosed cases. Slightly more than half of all newly diagnosed
cases will occur in men.
Although it may affect any lymph tissue, Hodgkin's disease most commonly affects the
supraclavicular, high cervical, or mediastinal nodes. Some patients exhibit no symptoms of the
disease, although others may have fever, night sweats, or weight loss, among other symptoms. Most
patients have one or more slow-growing, enlarged lymph nodes, but because swollen lymph nodes are
more often associated with infections, patients often ignore this symptom. It is important to have any
lymph node more than 1 cm (0.39 inches) in size evaluated by a physician, particularly if the node
enlargement is not associated with infection.
If Hodgkin's disease is suspected, the patient may undergo magnetic resonance imaging (MRI) or
computed tomography (CT) to determine the location(s) of enlarged nodes inside the body and to
detect any abnormalities of the spleen or other organs that may be associated with the disease.
Diagnosis is confirmed by any one of a number of biopsy techniques, including fine needle aspiration,
excisional biopsy, or incisional biopsy. A bone marrow aspiration may also be used to stage the
Once the diagnosis is made, it is important to stage the disease. Staging determines the disease's extent
of involvement. This information is used to plan a treatment program and will affect the survival rate.
Clinical staging consists of a thorough patient history and physical examination, x-rays, and laboratory
tests. Other diagnostic tools for staging include gallium scans and lymphangiograms (a type of x-ray).
Some patients require pathological examination that involves a surgical procedure called a laparotomy
(sometimes referred to as a staging laparotomy). The current staging system for Hodgkin's disease is
the Ann Arbor Staging Classification system. Four stages (I, II, III, IV) of the disease are recognized,
based upon the degree of involvement. Stage I disease is the least serious and Stage IV the most
Hodgkin's lymphoma is treated using a combination of chemotherapy agents. There are two common
chemotherapy combinations: mechlorethamine (Mustargen), vincristine (Oncovin), prednisone
(Deltasone, Meticorten), and procarbazine (Matulane) or Adriamycin, bleomycin (Blenoxane), and
dacarbazine (DTIC). The type of chemotherapy used will depend upon a number of factors, including
the stage of the disease and the patient's age.
Radiation therapy is often used in combination with chemotherapy. Depending on the severity of the
disease, radiation may involve the use of a focused beam of radiation or total nodal irradiation. As
with all types of lymphomas, bone marrow transplantation or peripheral blood stem-cell
transplantation may be considered in patients who do not respond to chemotherapy or radiation
The American Cancer Society estimated that nearly 56,200 new cases of non-Hodgkin's lymphoma
(NHL) would be diagnosed in 2001. NHL is the fifth most common type of cancer in the United
States. The disease is difficult to treat, with an average 1-year survival rate of 70% and a 5-year
survival rate of 51%. Approximately 90% of all non-Hodgkin's lymphomas are diagnosed in adults.
The average age at diagnosis is in the early 40s, and the disease is slightly more common in men than
in women. The risk for the disease increases throughout life. Other potential risk factors for the disease
may include adult-onset diabetes of long duration and a history of previous cancers, according to a
British study (Cerhan et al. 1997). Survival rates for non-Hodgkin's lymphoma are variable, depending
on the type of cell involved and the stage of the disease.
Non-Hodgkin's lymphomas are cancers that also affect the lymphatic system, particularly the
lymphocytes--the cells responsible for maintaining the body's immune system. There are two major
types of lymphocytes: B-cells and T-cells. B-cells are more common and are involved in
approximately 85% of all non-Hodgkin's lymphomas.
Generalized symptoms of the disease include unexplained weight loss, fever, profuse sweating, and
severe itchiness. The disease may affect the lymph nodes close to the body's surface (e.g., in the neck,
groin, or underarm). These nodes become swollen and are usually noticeable to the patient. If lymph
nodes in the abdomen are affected, the patient may experience abdominal swelling resulting from
accumulating fluid or tumor growth. If lymph nodes near the intestines are affected, the patient may
have difficulty with the passage of stools. When the lymphoma originates in the thymus, the growth of
the tumor may block the trachea or the superior vena cava may become compressed, resulting in a lifethreatening
condition known as superior vena cava (SVC) syndrome.
The disease is diagnosed by fine needle aspiration, incisional biopsy, or excisional biopsy. Other
techniques used to assist in the diagnosis include x-rays, CT scans, and bone marrow aspiration.
Because there are a number of different types of malignancies in non-Hodgkin's lymphomas, the types
are classified according to two systems. The Working Formulation classifies these lymphomas based
on prognosis: The categories are low, intermediate, and high-grade. The Revised European American
Lymphoma (REAL) system divides NHL into types according to clinical behavior. The categories are
indolent, aggressive, and highly aggressive. High-grade and highly aggressive tumors are the most
difficult to treat.
Treatment for non-Hodgkin's lymphoma depends on the type of lymphoma (e.g., indolent or
aggressive), the stage of the disease, the age of the patient, and the patient's overall health. As in
Hodgkin's lymphomas, chemotherapy and radiation therapy are used to treat the disease. Bone marrow
transplantation may be considered for patients who do not benefit from other forms of therapy. In one
study, Interferon was found to be an effective treatment for low-grade lymphomas; however,
intermediate- and high-grade tumors did not respond as well. Studies of B-cell non-Hodgkin's
lymphomas also indicated that Interferon-gamma and -alpha might be useful in the treatment of certain
types of the disease (Tourani et al. 1989; McLaughlin 1996).
SIGNAL TRANSDUCTION PATHWAY INHIBITION--A NEW TREATMENT APPROACH
Information has emerged offering an entirely new approach to the treatment of leukemias and
lymphomas. Early in the progression of these diseases, many, although not all, have been found to
express certain cytokine chemical messengers or signal transduction pathways previously thought only
to be expressed in human solid tumors as well as certain inflammatory and immunosuppressive
cytokines. The blockade or inhibition of these signal transduction pathways in human solid tumors has
yielded dramatic results. One example is the drug Iressa made by AstraZenca. Iressa has produced
positive results in certain cancers via a blockade of the epidermal growth factor receptor site (EGFR)
found to be over-expressed in many cancers (see the Cancer Adjuvant Therapy protocol for
information on natural supplements that have been shown to inhibit certain signal transduction
Growth, pro-inflammatory, and immunosuppressive cytokines often expressed by leukemias and
Vascular endothelial growth factor (VEGF) is considered essential for cancer cell survival and
angiogenesis (the formation of new blood vessels). High levels of VEGF correlate with shortened
survival in chronic lymphocytic leukemia (Ferrajoli et al. 2001).
Basic fibroblast growth factor (bFGF) is a potent mitogen (growth signal) and is essential for
angiogenesis. Simultaneous elevations in bFGF and VEGF are an independent predictor of a poor
prognosis in non-Hodgkin's lymphoma (Salven et al. 2000).
Hepatocyte growth factor (HGF), also known as a multiple function factor, HGF protects cancer cells
from cytotoxic agents, contributes to the development of chemo-resistance, and stimulates
hematopoiesis (Skibinski et al. 2001). (Hematopoiesis refers to the formation and development of blood
cells occurring primarily in the bone marrow and to a lesser extent the lymph nodes.)
Epidermal growth factor (EGF) is essential to the hyperproliferation of some lymphomas and to
epidermal cells (Courville et al. 1999).
Tumor necrosis factor-alpha (TNF-alpha). TNF-alpha is a proinflammatory cytokine significantly
elevated in all leukemias except for AML and myelodysplastic syndromes (Aguayo et al. 2000).
Interleukin-6 (IL-6) is a pro-inflammatory and immunosuppressive cytokine. Elevations in serum IL-6
correlate with adverse disease features and shortened survival in chronic lymphocytic leukemia (Fayad
et al. 2001).
The lymphomas and leukemias that can over-express these cytokines are:
Disease Cytokines Over-expressed
Hodgkin's disease VEGF, bFGF, HGF
T-cell lymphoma VEGF, EGF
Non-Hodgkin's lymphoma VEGF, bFGF, HGF, TNF-alpha, IL-6
Burkitt's lymphoma HGF, EGF
Chronic myeloid leukemia VEGF, bFGF, HGF, TNF-alpha, IL-6
Acute myeloid leukemia VEGF, bFGF, HGF
Chronic myelomonocytic leukemia VEGF, bFGF, HGF, TNF-alpha
Chronic myelomonocytic leukemia VEGF, bFGF, HGF, TNF-alpha
Acute lymphoblastic leukemia bFGF, HGF, TNF-alpha
Chronic lymphocytic leukemia VEGF, bFGF, HGF, TNF alpha, IL-6
Myelodysplastic syndromes VEGF, bFGF, HGF
ADJUVANT TREATMENTS FOR LEUKEMIA AND LYMPHOMA
Although leukemia and lymphomas respond well to the conventional treatment methods of
chemotherapy and radiation therapy, other potentially beneficial treatments are available. Vesanoid, a
vitamin A analogue, has been approved for the treatment of promyelocytic leukemia. The medication
inhibits cell division and allows cells to reach maturity and function normally. Although Vesanoid is
approved in the treatment of only a specific type of leukemia, it may be beneficial in the treatment of
other types of leukemia (but probably not CLL) and some types of lymphoma (Kerr et al. 2001).
Although vitamin A therapy can help to induce remission in patients with promyelocytic leukemia
(Mann et al. 2001), the duration of the response to the medication is short-lived. Additional therapy
with Vesanoid is often less effective, suggesting that patients may develop some resistance to the
Vitamins A and D3
Research has demonstrated that drug resistance may be overcome by using vitamin A derivatives
(retinoic acid) in combination with other medications, such as vitamin D3 and its analogs (Defacque et
al. 1996; Elstner et al. 1996; Nakajima et al. 1996; Miyauchi et al. 1997; Ohno 1997). Patients with
other forms of leukemia or lymphoma should consult with their physician regarding the potential
benefits of this treatment. If the patient's physician does not recommend Vesanoid for treatment of the
disease because the FDA has not approved the medication for their type of cancer, patients can
consider water-soluble vitamin A as an alternative.
The recommended dose of vitamin A supplementation is 100,000-300,000 International Units (IU)
daily. Monthly blood testing is necessary to monitor vitamin A liver toxicity.
Caution: Prior to considering vitamin A therapy, refer to the symptoms of vitamin A toxicity in
Vitamin D3 and its analogs may induce certain leukemia and lymphoma cancer cells to differentiate
into normal cells. If vitamin D3 supplements are used, the typical dose for cancer patients is 4000 IU a
Monthly blood tests to monitor serum calcium, kidney function, and liver function are necessary to
prevent vitamin D3 toxicity. Although not specifically recommended for patients with chronic
lymphocytic leukemia, vitamins A and D3 may be beneficial because of their effects against a wide
range of cancer cells.
A potentially beneficial adjuvant approach for leukemia and lymphoma uses soy extracts with high
genistein content. Genistein is an inhibitor of protein tyrosine kinase, the enzyme that cancer cells
require in order to replicate. A study conducted to assess the effects of genistein in several types of
cancer showed that protein kinase C activity was inhibited, subsequently retarding the growth of
cancer cells (Carlo-Stella et al. 1996a; 1996b).
Studies suggest that genistein may also enhance the effects of chemotherapy via a blockade of a
number of signal transduction pathways. These are:
Inhibition of the EGF receptor via an interference with the transforming growth factor-alpha (TGF-alpha)
pathway (Bhatia et al. 2001)
Suppression of VEGF, considered essential for cancer cell survival (Mukhopadhyay et al. 1995)
Suppression of bFGF, a potent growth cytokine (Hurley et al. 1996)
The blockade or inhibition of these important signal transduction pathways is dose-dependent, that is,
more is better.
In patients whose tumor cells have mutant p53 oncogenes, the benefits of soy extracts may be even
more significant, since genistein, from soy, has been shown to down-regulate mutant p53 oncogenes.
One key to a tumor's response to treatment is the presence or absence of a functional p53 tumor
suppressor gene, which produces a protein that cells need to undergo apoptosis (i.e., to die) when
damaged. If p53 is functional, cancer cells damaged by radiation or chemotherapy self-destruct.
However, if the genetic changes that lead to cancer also inactivate the p53 gene, which appears to
occur in about half of all human malignancies, the cancer defies treatment.
The presence of mutant p53 genes is determined by pathologic examination of the cancer cells. An
immunohistochemistry test for the presence of mutant or functional p53 can be performed by the
1010 Third Avenue, Suite 203
New York, NY 10021
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If the test for mutant p53 is positive, then soy extract therapy may be very beneficial. The Foundation
realizes that many cancer patients desiring to use soy extracts may not be able to have
immunochemistry testing for mutant p53. Patients may wish to consult their physicians to determine if
mutant p53 was discovered during diagnosis of their disease.
The most concentrated form of soy extract available is Ultra Soy Extract. The recommended dose for
cancer patients is five 700-mg capsules taken 4 times daily.
An extract of the spice turmeric, curcumin is synergistic with the soy isoflavone, genistein, and has a
number of cytokine-inhibiting properties, such as the inhibition of angiogenic signals from tissue-like
bone marrow, as well as the down-regulation of pro-inflammatory cytokines.
Curcumin has also been shown to:
Inhibit induction of bFGF, a potent mitogen (growth signal) and essential in angiogenesis (Arbiser et al.
Inhibit induction of hepatocyte growth factor (HGF), a multiple function cytokine. Over-expression of
HGF is involved in the development of chemo-resistance, protecting cancer cell DNA, and excessive
hematopoiesis (Skibinski et al. 2001).
Increase expression of functional nuclear p53 protein in leukemia cell lines. This increases apoptosis
(cell death) (Kuo et al. 1996; Jee et al. 1998; Pan et al. 2001).
Down-regulate the inflammatory cytokine TNF-alpha in bone marrow stromal cells (Xu et al. 1997).
Based on the multiple favorable mechanisms listed above, higher dose curcumin would appear to be
useful for cancer patients to take.
Concerning curcumin being taken at the same time as chemotherapy drugs, there are contradictions in
the scientific literature. Some studies indicate significant benefit, whereas other studies hint at reduced
benefit or even potential toxicity. One study involving curcumin's concomitant use with the
chemotherapy drug, Irinotecan, indicated potential toxicity (Michaels et al. 2001). Therefore, Life
Extension recommends that curcumin not be taken in combination with this drug. Irinotecan is also
known by the names Camptosar and CPT-11.
Chemotherapy drugs are highly toxic. Whether high-dose curcumin is beneficial or detrimental,
depends on the type and dose of the chemotherapeutic drug used, the kind of cancer cell being
attacked, and the dose of curcumin. Until more definitive information is published, we prefer to err on
the side of caution and recommend that chemotherapy patients wait 3 weeks after their last dose of
chemotherapy before taking high doses of curcumin. A high-dose of curcumin is 3600 mg taken 3
times a day. This high dose is sometimes consumed for 6-12 months and then reduced.
The primary action of green tea is through its catechin, EGCG, which blocks the induction of vascular
endothelial growth factor (VEGF), considered essential in angiogenesis. In vivo studies have shown
the following actions on cancer cells (Jung et al. 2001):
A 58% inhibition of tumor growth
A 30% inhibition of microvessel density
Increase tumor cell apoptosis 1.9-fold
Increased endothelial cell apoptosis threefold
Note: It may be more efficacious to take green tea in capsule form rather than as a brewed beverage as
a cancer adjuvant therapy. An appropriate dose for VEGF blockade would be 5 capsules of the lightly
caffeinated Super Green Tea Extract capsules with each meal. Each capsule provides 100 mg of the
critical anticancer polyphenol called epigallocatechin gallate (EGCG). Caffeine has been shown to
potentiate tea polyphenols, such as EGCG. Because caffeine can keep some people awake at night, it
might be preferable to take 5 decaffeinated Super Green Tea Extract capsules as the evening dose, or
use decaffeinated green tea exclusively if hypersensitive to caffeine.
The down-regulation of inflammatory cytokines is fundamental to the control and eradication of the
disease process. As previously noted, many leukemias and lymphomas over-express the inflammatory
cytokines TNF-alpha and IL-6. Essential fatty acids are derived from sources such as fish, primrose,
and borage oils. The docosahexaenoic acid (DHA) and gamma-linolenic acid (GLA) portions of these
fatty acids have been shown to suppress these dangerous cytokines (Purasiri et al. 1997; De Caterina et
al. 2000). Additionally, the use of GLA and DHA has been shown to improve leukemia's response to
chemotherapy (Liu et al. 2000).
A family of oncogenesknown as rasoften governs the regulation of cancer cell growth. The RAS
family is responsible for modulating the regulatory signals that govern the cancer cell cycle and
proliferation. A class of drugs known as the statins (used to control cholesterol) has also been found to
induce apoptosis (cell death) in cancers that express the rasmutation. Acute myeloid leukemia strongly
expresses the H-Rras mutation and has been found to be highly sensitive to one of the newer statins
called cerivastatin. Cerivastatin has been found to be at least 10 times more potent at inducing
apoptosis in AML than any previous statin drug (Wong et al. 2001).
An interesting study (Inserra et al. 1998) showed that the hormone DHEA (dehydroepiandrosterone)
favorably modulated the immune dysfunction that occurred during murine leukemia retrovirus
infection in old mice. Leukemia is associated with deregulated cytokine production. When leukemic
mice were given DHEA supplements, the loss of the cytokines, interleukin-2 and interferon-gamma
was prevented (Araghi-Niknam et al. 1997). DHEA also suppressed the excessive production of the
dangerous cytokines, interleukin-6 and interleukin-10. This preliminary study indicates DHEA might
be effective in treating the immune dysfunction in those leukemia patients with a DHEA deficiency
(especially older people).
Caution: DHEA is contraindicated in both men and women with certain hormone-related cancers
(please refer to the DHEA Replacement Therapy protocol for complete information on the proper use
of DHEA supplements).
Alpha-lipoic acid (also known as lipoic acid) is a powerful antioxidant that has demonstrated effects
against brain damage, aging, and diabetes. It may also help kill cancerous cells and retard heart
A remarkable study shows how lipoic acid can reverse aging. Researchers at the University of
California at Berkeley took liver cells from aging rats and measured how energized they were, how
many free radicals were present, and how well the cells could recycle vitamin C. The aged rats were 3
times less active than the young ones. Free radicals were 5 times higher, the generation of energy had
plummeted, and the ability to recycle ascorbic acid (vitamin C) was about half. After 2 weeks on lipoic
acid, everything was reversed. Ascorbic acid levels rose, free radicals decreased, and energy levels
took off. Levels of glutathione, an important antioxidant for the liver, were also protected by lipoic
acid (Lykkesfeldt et al. 1998; Hagen et al. 1999).
Dr. Lester Packer is a top authority on antioxidants. Packer and his group at Berkeley published results
from a study on lipoic acid and human cancer cells. For the first time they showed that lipoic acid
activates an enzyme that kills leukemia cells. The enzyme caspase increased 100% with treatment (Sen
et al. 1999). Other research from his laboratory indicates that lipoic acid goads crippled immune cells
(such as those of cancer and AIDS patients) into action (Sen et al. 1997). Among his other research
projects is one showing that lipoic acid suppresses the "cancer gene," c-fos (Mizuno et al. 1995).
Another group, this time at Yale, used lipoic acid and vitamin E succinate with vitamin D3 to make
leukemia cells differentiate (become a normal cell as opposed to a cancer cell). Both antioxidants
needed vitamin D3 to cause this positive effect (Sokoloski et al. 1997).
A big question is whether a person undergoing chemotherapy should take antioxidants such as lipoic
acid. Since generating free radicals is one of the ways chemotherapeutic drugs work, there is concern
that taking antioxidants could keep chemotherapy from working. The jury is still out. Some studies
show that antioxidants ameliorate the toxic effects of chemotherapy without affecting the ability of the
drug to work. Others show that antioxidants reduce the effectiveness of the drugs--at least in cell
culture. It may depend on the type of cancer, the drug used, and the dose of antioxidant. People
undergoing chemotherapy have reported positive effects, but this is something that should be
discussed with an oncologist (refer to the protocols entitled Cancer Chemotherapy and Cancer: Should
Patients Take Dietary Supplements? for additional information about using antioxidants with
Shark Liver Oil
Alkylglycerols were first isolated from shark liver oil by Dr. Astrid Brohult, a physician in Sweden.
Dr. Brohult was treating children with leukemia, with little success. Because white blood cells are
produced in the bone marrow, she started to feed bone marrow from calves to the sick children. The
result of this bone marrow feeding was a marked improvement in the immune systems and white
blood cell counts of the children. Unfortunately, Dr. Brohult was unable to get the children to eat
enough bone marrow to sustain these results, so she set out to find the active ingredient in bone
marrow and isolate it. With the help of her husband, it was determined that alkylglycerols were
responsible for the immune system-enhancing effects. Next, they discovered that alkylglycerols are
found in the livers of cold-water sharks, such as the Greenland Shark. The shark in general has
attracted attention because cancer occurrence is very rare in sharks. The existence of alkylglycerols in
the liver of sharks may be one reason for the natural immunity to cancers.
The biologic effects of shark liver oil include stimulation of blood leukocyte and thrombocyte
production (Le Blanc et al. 1995), as well as the activation of macrophage and antitumor activity.
Other effects include the ability to protect against radiation damage during radiation therapy for
various types of cancer. Alkylglycerols act as a powerful immune system booster against infectious
disease and help give nursing animals, including breast-fed babies, protection against infection until
their own immune systems can fully develop.
In a study published in the Journal of Cell Physiology (February 1999), Wang et al. studied the cell
differentiation-promoting potential of a particular type of alkylglycerol on human colon cancer cells.
The scientists wanted to observe the ability of alkylglycerols to change the biological makeup of
human colon cancer cells. Alkylglycerols were shown to "... promote a more benign or differentiated
phenotype in colon cancer cells." Treatment of the cancer cells with alkyl-glycerols resulted in a
reduction of cellular proliferation and a reduced capacity for cellular invasion. In other words,
alkylglycerols led to lowered cancer cell reproduction and a reduced ability of the cancer cells to
invade healthy cells. The authors concluded that alkylglycerols possess both cancer preventative
properties, as well as cancer treatment effects (Wang et al. 1999).
Shark liver oil has been around for 40 years and has been used as both a preventive and therapeutic
agent. Not only have alkylglycerols been used to treat leukemia, as in the case of the children in
Sweden, but they have also been used to prevent radiation sickness stemming from radiation cancer
treatments. Furthermore, the high level of alkylglycerols that exist naturally within any given tumor
cell has led scientists to postulate that this may be an apparent attempt of the body to control cell
growth. Protein kinase C, an essential step in cancer cell growth, can actually be stopped or inhibited
by alkylglycerols. In addition, it has been suggested that alkylglycerols directly act on the
macrophages (large immune cells that "gobble up" cancer cells). Overall, alkylglycerols are able to
stimulate the macrophage to secrete more than 50 substances concerned directly or indirectly with the
immune system. Some of these substances, the interleukins, are powerful immune system fighters that
interact with lymphocytes (Pugliese et al. 1998; 1999).
Resveratrol, a phytoextract found in grapes and red wine may act as a chemotherapeutic agent and
inhibit the growth of various leukemia and melanoma cell lines.
Resveratrol is a plant polyphenol found in grapes and red wine. A study published in the journal Blood
indicates that resveratrol effectively inhibits acute myelogenous leukemia (AML) cells in vitro through
several differentiating properties: blocking activation of nuclear transcription factor NF-kB, inhibiting
proliferation, causing S-phase arrest, and inducing apoptosis. This suggests that resveratrol may have a
role as a therapeutic agent in the treatment of AML ( Estrov et al.2003)
Asou et al. studied the in vitro activity of resveratrol on acute myeloid leukemia by examining its
effect on proliferation and differentiation in various cell lines and in fresh samples of 17 AML
patients. Used alone, resveratrol inhibited the growth of all AML . The authors concluded that
resveratrol inhibits proliferation and induces differentiation of myeloid leukemia cells ( Asov et al.
Niles et al. examined the effect of resveratrol on the growth of two human melanoma cell lines. They
found it inhibited growth and induced apoptosis in both cell lines with one (A375) being more
sensitive. The authors concluded that resveratrol may be effective as either a therapeutic or
chemopreventive agent ( Niles et al. 2003.)
From the in vitro studies cited above, an appropriate human dosage cannot be extrapolated.
Monthly Blood Markers
Because all cancer therapies produce individual responses based on factors such as the type of disease,
patient's age, and the presence of other diseases, the Foundation recommends monthly blood markers
and other diagnostic testing to monitor the benefits of any supplemental therapies. The results of these
tests provide critical information to evaluate the effectiveness of nonconventional therapies. If tumor
indicators do not decrease after the initiation of any nonconventional therapy, patients should
discontinue their use and seek other alternatives immediately.
Inhibiting Protein-Tyrosine Kinase with Gleevec
In the various cancer protocols discussed in this book, references are made to nutrients like curcumin,
genistein, and tocopherol succinate that function as protein-tyrosine kinase inhibitors. Because
tyrosine kinases induce hyperproliferation of cancer cells, inhibiting these kinases has been shown to
slow cancer cell propagation.
A drug called Gleevec (formerly known as STI571) is a protein-tyrosine kinase inhibitor that
specifically interferes with the Bcr-Abl tyrosine kinase--the typical chromosomal abnormality seen in
chronic myeloid leukemia (CML). Gleevec inhibits proliferation and induces apoptosis in Bcr-Abl cell
lines as well as fresh leukemic cells from "Philadelphia chromosome positive" chronic myeloid
leukemia (CML). Gleevec may also inhibit growth of other types of cancer cells.
Gleevec (imatinib mesylate) was first made available to patients with chronic myeloid leukemia
(CML) in May 2001 after the results of exciting clinical studies were released in Europe. Gleevec is
indicated for the treatment of patients with Philadelphia chromosome positive (Ph+) chronic myeloid
leukemia (CML) in blast crisis, accelerated phase, or in chronic phase after failure of interferon-alpha
The effectiveness of Gleevec is continuously being evaluated for efficacy, though it is now an FDAapproved
drug. To read about the latest findings on Gleevec, log on to a special website
It is interesting to note that a drug that functions along a similar mechanism as certain dietary
supplements was put on the FDA's "fast-track" for approval.
Leukemia, Hodgkin's lymphoma, and non-Hodgkin's lymphoma generally respond well to
conventional therapies. There are many different types of these diseases; therefore, chemotherapy and
radiation therapy are individualized. Patients who do not respond well to chemotherapy and radiation
therapy may benefit from other treatments such as bone marrow transplantation or a peripheral blood
stem-cell transplant. In addition to conventional treatment, there are a number of alternative therapies
available. Patients with certain types of leukemia or lymphoma may derive beneficial effects from
Vesanoid, vitamin A, vitamin D3, curcumin, green tea, and soy extracts. It is imperative that patients
have regular monitoring of tumor markers (or tumor size) to assess the usefulness of any treatment.
Consult your hematologist or oncologist prior to initiating alternative treatments.
1. Early diagnosis and treatment of leukemias and lymphomas are essential. Symptoms of leukemia and
lymphoma are generalized and include fatigue, weight loss, fever, and night sweats. In Hodgkin's and
non-Hodgkin's lymphomas, swollen lymph nodes may be present.
2. Diagnosis of the specific disease may include MRI scans, CT scans, and biopsy.
3. Chemotherapy and radiation therapy are usually used in combination to treat these diseases. The
actual course of therapy depends on the specific type of disease.
4. Interferon-alpha, a biologic response modifier has been proven effective in the treatment of some
leukemias and low-grade lymphomas.
5. Patients who do not respond to chemotherapy and radiation therapy may be considered for peripheral
blood stem-cell transplants or bone marrow transplants.
6. Vesanoid, a vitamin A analog, has proven effective in patients with chronic promyelocytic leukemia and
may be beneficial for other types of cancers. For chronic myeloid leukemia (CML), ask your doctor
7. Water-soluble vitamin A may provide a useful alternative to Vesanoid for some cancer patients. The
recommended dosage of this vitamin is 100,000-300,000 IU daily.
CAUTION: Monthly blood tests are necessary to avoid vitamin A toxicity.
8. Vitamin D3 and its analogs may induce differentiation of cancer cells into normal cells in certain types of
lymphomas and leukemias. A high dose to consider is 4000-6000 IU daily.
CAUTION: Serum calcium, kidney function, and liver function should be monitored monthly to avoid
vitamin D toxicity.
9. Curcumin may induce cancer cell death via a blockade of various signal transduction pathways. The
recommended daily dosage is four 900-mg capsules 3 times daily with food, taken 2 hours apart from
CAUTION: Patients with biliary tract obstruction should not take curcumin. High doses of curcumin may
induce NSAID-like side effects in the stomach.
10. Green tea extract providing high amounts of epigallocatechin gallate (EGCG) suppress VEGF and other
growth factors used by cancer cells to escape regulatory control. An appropriate dose for VEGF
blockade would be 5 capsules of the lightly caffeinated Super Green Tea Extract capsules with each
meal. Each capsule provides 100 mg of the critical anticancer polyphenol called EGCG. Caffeine has
been shown to potentiate tea polyphenols, such as EGCG. Because caffeine can keep some people
awake at night, it might be preferable to take 5 decaffeinated Super Green Tea Extract capsules as the
evening dose, or use decaffeinated green tea exclusively if hypersensitive to caffeine.
11. Patients who are positive for mutant p53 oncogenes may receive substantial benefits from the use of
soy extracts. Soy extract high in genistein, such as Ultra Soy Extract, may inhibit cancer cell growth for
a number of types of cancer. Recommended daily dosage of Ultra Soy Extract is five 700-mg 40%
isoflavone extract capsules taken four times a day.
12. DHEA replacement therapy may be considered. Blood testing is recommended prior to and during
therapy (refer to DHEA Replacement Therapy for more information).
13. Alpha-lipoic acid may help activate the enzyme caspase, which kills leukemia cells. It may also
suppress the cancer gene C-FOS. People on a chemotherapeutic regimen should discuss the use of
alpha-lipoic acid with their oncologist before taking this supplement. Typical doses of alpha-lipoic acid
for cancer patients are 500 mg twice a day.
14. Shark liver oil functions via several mechanisms to suppress cancer growth, enhance immune function,
and protect against radiation damage. We recommend five or six 1000-mg capsules (containing 200 mg
of alkylglycerols each) daily for a period not to exceed 30 days.
CAUTION: At no time should the maximum recommended dose of shark liver oil be exceeded. In the
case of chronic use, more than 30 consecutive days, a possible, albeit rare, side effect known as
thrombocythemia (excess thrombocytes) can occur, leading to a tendency for the blood to clot. This
condition is easily diagnosed with a blood test and reversed with lower dosages, the addition of a lowdose
aspirin (81 mg daily), or omega-3 fatty acid supplementation. Consult with your physician if thrombocythemia
is a consideration or if you are using shark liver oil for the treatment of serious disease
states. Other than the rare instance of blood clotting at chronic high doses, the alkylglycerols found in
shark oil are remarkably nontoxic.
15. GLA/DHA may be taken for the suppression of inflammatory cytokines. Super GLA/DHA is derived from
borage oil and marine lipid concentrate. The suggested dose is 6 softgels daily.
16. Resveratrol has been shown to act as a chemotherapeutic agent in vitro on certain leukemia cell lines.
Although as a therapeutic agent, a dosage has not been established, 1 20-mg capsule daily of
resveratrol provides multiple health benefits.
Note: At this juncture, the hormone melatonin is not recommended in the treatment of lymphoma and
leukemia. Patients should avoid the use of this product until more information is available. If patients
do choose to use melatonin, monthly blood testing for tumor markers should be closely monitored to
determine if melatonin is promoting leukemic or lymphatic cell proliferation.
This information (and any accompanying printed material) is not intended to replace the
attention or advice of a physician or other health care professional. Anyone who wishes to
embark on any dietary, drug, exercise, or other lifestyle change intended to prevent or treat a
specific disease or condition should first consult with and seek clearance from a qualified
health care professional.
The information published in the protocols is only as current as the day the book was sent to
the printer. This protocol raises many issues that are subject to change as new data emerge.
None of our suggested treatment regimens can guarantee a cure for these diseases.
- Casos clínicos