Download 1 Benfotiamine: possibility for implication to cancer chemotherapy

Benfotiamine: possibility for implication to cancer chemotherapy and pain management
Spiro Konstantinova*, Vanya Tsvetkova , Pavel Kraevskyb, Georgi Momekova, Tsvetomira Tsanovaa, M. Karaivanovaa, L.
Kasakovb, M. Vlaskovskac
a
Department of Pharmacology and Toxicology, Faculty of Pharmacy, Medical University Sofia, 2 Dunav St., 1000, Sofia, Bulgaria
Institute of Physiology, Bulgarian Academy of Sciences, Sofia, Bulgaria
c
Department of Pharmacology and Toxicology, Faculty of Medicine, Medical University Sofia, 2 Zdrave St., Sofia, Bulgaria
* corresponding author
b
Summary
Benfotiamine is a lipid soluble thiamine analogue, characterized by its superior bioavailability after oral intake, which is proven to
be highly efficient for alleviation of neuropathic pain as well as of other neuropathy associated symptoms. Considering its possible
usefulness to correct chemotherapy-induced neurotoxicity and to be used as opioid adjuvant in cancer patients, we performed
investigations of the effects of benfotiamine co-administration on tumor cell proliferation and antineoplastic efficacy in vitro of
various cytotoxic drugs (5-FU, gemcitabine, methotrexate, bendamustine, cisplatin and vincristine), as well as on heat induced
nociception, morphine analgesia and tolerance/dependence development in rats. Benfotiamine neither altered the tumor cell
proliferation itself, nor inhibited the efficacy of the antineopolastic agents applied. The only exceptions found were a slight
decrease in the efficacy of cisplatin in SKW-3 leukemic cells and a marginal modulation of tamoxifen effect on MCF-7 breast
carcinoma cells. Benfotiamine induced a significant increase of the intracellular level of reduced glutathione and thereby the
modulation of cisplatin efficacy in terms of apoptosis induction could be explained. Despite of the lack of intrinsic analgesic
activity of benfotiamine against heat-induced nociception, it was found to cause a significant increase of the analgesic efficacy of
morphine. Moreover, benfotiamine co-administration alleviated the abstinence signs and retarded the morphine tolerance
development. Taken together our experimental findings indicate that benfotiamine could have some relevant benefits for cancer
patients, especially for those suffering from neuropathic pain caused by both disease progression and/or neurotoxic chemotherapy.
Key words:
antineoplastic drugs, benfotiamine, morphine analgesia
1. Introduction
The physiologic and biochemical functions of thiamine are well established: being an important co-factor, necessary for the
carboxylation reactions, it plays crucial role in the carbohydrate metabolism, especially in the nervous system [1]. Together with its
classic application for alleviation of symptoms, associated with its depletion or impaired absorption, thiamine is also effective for
the therapeutic management of polyneuropathies of different origin, e.g. associated with chronic alcohol abuse, diabetes mellitus
etc. [19,40]. There is also growing evidence for the intrinsic analgesic and anti-inflammatory effects of thiamine, which is
nowadays widely recommended as analgesic adjuvant for the treatment of painful conditions, such as lumbago, sciatica, trigeminal
neuralgia, facial paralysis and optic neuritis, all characterized with involvement of neuropathic, rather than nociceptive pain
mechanisms [14,30,33]. The usefulness of thiamine in neuropathic pain relief is dose-dependent and generally the possibilities for
increasing the dose intensity are restrained by its limited absorption after oral intake [19,28]. This fact led to investigation of lipidsoluble thiamine analogues and different pharmacokinetic studies show, that the allithiamine derivative benfotiamine reaches the
most suitable for therapeutic purposes tissue concentrations and thus is considered as the main thiamine alternative for oral intake
[19,28]. Due to the well established efficacy of benfotiamine as a therapeutic and preventive agent in chronic neurologic
conditions [21,39] an attractive field for clinical application in cancer patients, taking into account the fact, that several important
antineoplastic agents, such as platinum complexes and Vinca alkaloids cause peripheral neuropathy [9,37]. The pain management
in cancer patients is usually based on the use of opioid analgesics with conventional adjuvants in compliance with the World
Health Organization -recommended analgesic ladder [35,36]. The analgesic ladder is suitable guide for choice of suitable analgesic
drug in patients with nociceptive pain, but is not quite relevant for the pain management when neuropathic mechanisms are
involved, e.g. in cancer patients [3,35]. Despite of the possible benefits of benfotiamine as analgesic adjuvant, and for relief of
chemotherapy-induced neurotoxicity, its application in cancer patients remains an open question, due to the eventual stimulation of
tumor growth and possible interference with the main effect of the cytoreductive therapy [10]. Considering the fact that there is
generally lack of data for benfotiamine in this field in the present study we focused on: a) elucidation of the effects of benfotiamine
and thiamine on tumor growth and cytotoxicity of diverse antineoplastic drugs; b) investigation of the effects of benfotiamine and
thiamine on morphine analgesia and development of tolerance/dependence.
2.
Materials and methods:
2.1. Drugs and chemicals.
Agarose, ethanol, formic acid, 2-propanol, methanol, EDTA, ethidium bromide, sodium chloride, Tris hydrochloride, Triton® X100, L-glutamine, DMSO, and 5,5’-dithio-bis (2-nitrobenzoic acid) (DTNB) were purchased from AppliChem GmbH, Darmstadt,
Germany. Fetal calf serum (FCS), powdered RPMI 1640 medium, non-essential amino-acids cell culture supplement and naloxone
were purchased from Sigma – Aldrich GmbH, Steinheim, Germany. Pure swine insulin was used for preparing the complete
growth medium for MCF-7 cells. The tetrazolium salt3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was
supplied from Merck, Darmstadt, Germany. Benfotiamine and thiamine hydrochloride were kindly provided by Wörwag Pharma
GmbH, Böblingen, Germany. Morphine hydrochloride was supplied from.Sofarma Co., Sofia, Bulgaria. All cytotoxic drugs
exploited were used as the commercially available sterile dosage forms for clinical application, with the only exception of
bendamustine hydrochloride which was kindly provided as pharmaceutical grade substance from Ribosepharm GmbH, Munich,
Germany.
1
2.2. Cell lines and culture conditions
The procedures of cell culture maintenance, drug stock solution preparation and treatment were carried out in a ‘Heraeus’ Laminar
flow cabinet (Kendro GmbH, Hanau, Germany). All human tumor cell lines involved in this study were obtained from the German
Collection of Microorganisms and Cell Cultures (DSMZ GmbH, Braunschweig, Germany). The panel of malignant cell lines
consisted of the lymphoid SKW-3 (DSMZ №: ACC 53, cell type: human T-cell leukemia, origin: established from the peripheral
blood of a 61-year-old man with T-cell chronic lymphocytic leukemia in 1977, doubling time of ca. 30-40 hours, cytogenetics:
human near diploid karyotype with 4% polyploidy); the myeloid HL-60 (DSMZ №: ACC 3, cell typе: human acute myeloid
leukemia, origin: established from the peripheral blood of a 35-year-old woman with acute myeloid leukemia in 1976, doubling
time of ca. 25 hours, cytogenetics: human flat-moded hypotetraploid karyotype with hypodiploid sideline and 1.5% polyploidy);
the resistant variant HL-60/Dox cell line, characterized by the expression of MRP1(multidrug resistance-associated protein), which
confers drug resistance; and the breast cancer cell line with estrogen receptor expression MCF-7 (DSMZ №: ACC 115, cell typе:
human breast adenocarcinoma, origin: established from the pleural effusion of a 69-year-old Caucasian woman with metastatic
mammary carcinoma after radio- and hormone therapy in 1970, doubling time of ca. 50 hours (range 30-72 hours), cytogenetics:
human hypotetraploid karyotype with 8 % polyploidy, and without estrogen receptor); Leukemic cells were grown as suspension
cultures in controlled environment – cell culture flasks at 37oC in an incubator 'BB 16-Function Line' Heraeus (Kendro, Hanau,
Germany) with humidified atmosphere and 5% CO2. Cells were kept in log phase by supplementation with fresh medium after
removal of cell suspension aliquots, two or three times a week. For all cell lines RPMI-1640 liquid medium supplemented with 10
% FBS and 2 mM L-glutamine was used. HL-60/Dox cells were maintained in medium containing 0.2 M doxorubicine. The
mammary carcinoma cell line MCF-7 was grown as monolayer culture and was reset by trypsinisation two times a week. It was
maintained in RPMI-1640 medium supplemented with 10% FBS, non-essential amino acids, 1mM sodium pyruvate and 10 g/ml
insulin.
2.3. Drug solutions, treatment and cytotoxicity determination
Stock solutions of the antineoplastic agents and thiamine were prepared in purified water and after antibacterial filtration these
were consequently diluted with RPMI-1640 medium to yield the final concentrations. Benfotiamine stock was prepared in 0.5M
NaOH and was neutralized to pH 7.4 via addition of 5% HCl solution.
Cells were seeded into 96-well plates (100 µl/well at a density of 1105 cells/ml) and exposed to various concentrations of the
antineoplastic agents and/or benfotiamin or thiamine for 72 h. Cell survival was determined with the MTT dye-reduction assay as
described by Mosmann [32], with some modifications [26]. Briefly, after the incubation with the test-compound, MTT-solution (10
mg/ml in PBS) was added (10 µl/well). Plates were further incubated for 4 h at 37o C and the formazan crystals formed were dissolved by
adding 100 µl/well of 5% formic acid in 2-propanol. Absorption was measured on an ELISA reader (Uniscan®Titertek, Helsinki, Finland)
at 540 nm. For each concentration at least 8 wells were used. As a blank solution 100 µl RPMI 1640 medium with 10 µl MTT stock and
100 µl 5% formic acid in 2-propanol was used.
2.4. DNA fragmentation analysis
DNA isolation and electrophoretic analysis were performed as previously described [26]. In brief exponentially growing SKW-3
cells were treated with 20 M cis-DDP alone, and in combination with either benfotiamine or thiamine (0.5 or 0.1mg/ml) for 24 h.
After the incubation period about 0.5-1 x 106 treated and untreated cells were washed in PBS. Cell pellets obtained were
resuspended in 0.25 ml PBS and lysed in 0.5 ml lysis buffer (0.5% Triton X-100, 20 mM Tris HCl, 1 mM EDTA, pH=7.4). After
centrifugation (13 000 rpm for 20 minutes) the supernatants were transferred into fresh tubes. For precipitation of DNA, NaCl
solution (0.187 ml, 6M) and 2-propanol (0.937 ml) were added to each sample. After mixing samples were kept at –20oC overnight
and were spun at 13 000 rpm for 20 min. In addition DNA was washed with 1 ml 70% ethanol, DNA pellets were air dried and
dissolved in 20 µl distilled water. Probes were analysed by electrophoresis followed by ethidium bromide staining. DNA ladder
was visualized and photographed, using an UV-transilluminator with CCD camera (UVPBioDoc-It TM System, a1-Biotech GmbH,
Martinsried, Germany).
2.5. GSH (reduced glutathion) cellular levels determination
For the determination of the GSH levels a spectrophotometric assay was used as described by Sedlak and Lindsay [38] with some
modifications [39]. In brief: about 2-5X105 cells per sample were washed in 1 ml PBS and centrifuged at 6000 rpm for 5 minutes.
Cell pellets were lysed in 100 l 1 % solution of Triton X-100 in 0.2M EDTA at 4C for 5 minutes. Proteins were precipitated
through the addition of 20 l 20% (v/w) trichloroacetic acid. The volume of each sample was then adjusted to 200 l with distilled
water and following centrifugation at 14500 rpm for 10 minutes the supernatants were assayed for GSH content. Supernatant
aliquots were transferred into 96- well microplates (100l per well) together with 160 l Tris buffer (0.4M, pH 8.9) and 4l of 5,5dithio-bis(2-nitrobenzoic acid) 3.4 mg/ml solution in methanol. The color reaction was measured at 405 nm on an
Uniscan®Titertek ELISA reader (Helsinki, Finland).
2.6. Animals
All experiments were carried out on male Wistar rats weighing 180 –200 g at the start of the experiments. The rats were housed in
plastic cages, 4-5 rats per cage, with sawdust bedding, food and water available ad libitum. The experiments were carried out
between 0900 and 1200 h. The analgesic effects of morphine, benfotiamine and thiamine were tested with hot plate (HP) (Ugo
Basile, Italy). The temperature of the hot plate apparatus was maintained at 51 ± 1oC. The latency to licking a hind paw was
measured with accuracy of 0.1 s. Prior to drug administration all rats were tested on the HP and 2 trials/day for 7 to 10 days in
2
order to obtain a stabile control response level. Rats were removed from the hot plate if they did not respond for 30 s in order to
avoid tissue damage.
The experimental protocols had been approved by the Ethics Committee of Institute of Physiology of the Bulgarian Academy of
Sciences.
2.7. Assessment of analgesic activity of morphine, benfotiamine and thiamine
Following two pre-drug control trials and after training for 10 days, rats were divided in the following groups: Group 1 (G1)control (vehicle treated); G2- morphine (Mo) 2.5 mg/kg s.c.; G3 -5 mg/kg Mo s.c.; G4 –10 mg/kg Mo; G5 –benfotiamine 10
mg/kg p.o. per sondam; G6 – benfotiamine – 50 mg/kg p.o; G7 – benfotiamine –100 mg/kg p.o.; G8 – 5 mg/kg Mo s.c plus
benfotiamine 10 mg/kg p.o; G9- 5 mg/kg Mo s.c plus benfotiamine 50 mg/kg p.o; G10 -5 mg/kg Mo s.c plus benfotiamine 50
mg/kg p.o; G11 - 5 mg/kg Mo s.c plus thiamine 10 mg/kg p.o; G12 - 5 mg/kg Mo s.c plus thiamine 50 mg/kg p.o; G13- 5 mg/kg
Mo s.c plus thiamine 100 mg/kg p.o;
The response on the hot plate was tested 30, 60, 90 and 120 min after morphine administration. In the combinations morphine was
injected 30 min after oral aministration of benfotiamine or thiamine. Each group contained 8-10 rats.
2.8. Development of tolerance to morphine: Modulation by benfotiamine and thiamine
After two pre-drug control trials after 7-10 days of training, all rats were injected with 5 mg/kg Mo s.c. between 0800 and 0900h
and their response was tested 30, 60, 90 and 120 min after morphine administration. A second dose of morphine was administered
at 1800-1900 h, but the animals were not tested for analgesic activity. Rats were divided in the following groups: G14 – chronic
treatment with Mo two times a day; G15 – every morning, 30 min before morphine injection rats were treated with benfothiamine
10 mg/kg p.o and G15– every morning, 30 min prior to morphine injection rats were treated with thiamine 10 mg/kg p.o. Such
teratment schedule was performed for a total of 11 days. Hot plate latencies were measured on day: 1, 3, 5, 7, 9 and 11.
Every morning before treatment rats were observed for abstinence symptoms.
2.9. Naloxone- precipitated withdrawal
The animals used to study the development of tolerance were injected with Naloxone 5 mg/kg i.p. on day 11 and were observed
for withdrawal symptoms. Behavioural withdrawal reactions of freely moving rats placed under large glass funnels have been
witnessed through 2 consecutive 10 min observation periods. Score-points of the 10 min observation period as well as aggregate
score points of whole observation time were estimated for every animal to obtain mean values of each group.
2.10. Data analysis
The results from the in vitro cytotoxicity assay were analyzed, using the Student’s t-test with p<0.05 taken as significance level.
The data from in vivo tests were analyzed using both absolute response latency values (increase over pre-drug latency) as well as
per cent of the maximum possible effect (%MPE). %MPE = (postdrug latency-control latency)/ cutoff time – control latency X
100. The results were expressed as mean±SEM and analyzed by two-way ANOVA with level of significance set at p<0.05.
3. Results
3.1. Cytotoxic effects
Both thiamine and benfotiamine were found to not stimulate or inhibit the proliferation of the malignant cells under investigation at
concentration range from 0 to 1 mg/ml (Data not shown).
All antineoplastic agents included in this study caused concentration-dependent cytotoxic effects against all tumor cell lines used.
The generated concentration-response curves allowed extrapolation of the corresponding IC50 values after 72h sole administration
and after co-administration with 0.05 mg/ml benfotiamine or thiamine (Tables 1,2 and 3).
The cytotoxic efficacy of the alkylating agent bendamustine as well as that of the antifolate metothrexate and the antimetabolite
gemcitabine against SKW-3 was not affected by the co-incubation with benfotiamine or thiamine (Table 1). SKW-3 cells treated
with cis-DDP and thiamine or benfotiamine, however, showed significantly lower sensitivity compared to those treated with cisDDP omly, as evidenced by the observed IC50 value increase (19.8 M for cis-DDP, 24.5 M for the combination with
benfotiamine and 25.4 M for the combination with thiamine,Table 1).
The co-administration of benfotiamine or thiamine did not alter the in vitro antineoplastic efficacy of gemcitabine, 5-FU,
vincristine and cis-DDP on myeloid HL-60 cells (IC50 values on Table 2). The MRP1 expressing sub-line HL-60/Dox is known to
export GSH-conjugated drugs and therefore these cells were included in the panel of tumor cells investigated. It is noteworthy that
both benfotiamin and thiamine did not affect the cytotoxic effects of cis-DDP on HL-60/Dox, by the corresponding IC50 values,
extrapolated from the concentration-response curves obtained (approximately 2.1 M for cis-DDP alone, 2.3 M for the
combination with benfotiamine and 2.4 M for the combination with thiamine; Table 2)
The influence of the combinations investigated on MCF-7 breast cancer cells are summarized on Table 3. The antineoplastic agent
investigated, with the exception of tamoxifen only, did not cause 50% inhibition of the malignant cell proliferation of MCF-7 and
therefore for estimation of the efficacy of metothrexate, 5-FU, gemcitabine and vincristine IC20 values were extrapolated. The
combinations with benfotiamine or thiamine were not found to alter their antineoplastic activity. In contrast the cytotoxic efficacy
of tamoxifen was slightly reduced (IC50 values: 14.3 M for tamoxifen only, 17 M for the combination with benfotiamine and
15.9 M for the combination with thiamine; Table 3).
3.2. Effects of benfotiamine and thiamine on the GSH cellular levels
3
Because reduced glutathione (GSH), being the major non-protein thiol in mammalian cells, plays a crucial role in governing the
sensitivity and responsiveness of malignant cells to chemotherapy we carried out investigations for determination of the effects of
benfotiamine and thiamine on GSH levels in HL-60, HL-60/Dox and SKW-3 cells.
GSH-modulation by the investigated agents in HL-60, HL-60/Dox and SKW-3 are shown on Fig. 2. N-acetylcysteine was used as
a positive control due to its well-known GSH-replenishing properties.
Benfotiamine led to a concentration-dependent increase of the GSH level in SKW-3 cells by approximately 35% at the lower
concentration of 0.05 mg/ml and by 80% at the higher concentration of 0.1 mg/ml. Similarly, thiamine caused an elevation of GSH
content in SKW-3, but to a lesser extend (21% at 0.05 mg/ml and 45% at 0.1 mg/ml, respectively). These findings correlate with
the observed alteration of cis-DDP cytotoxicity in the presence of both Vitamin B1. Similar but less pronounced effects of
benfotiamine and thiamine on GSH levels were detected on both HL-60 and its subline HL-60/Dox cells.
3.3. DNA fragmentation analysis
The 24 h treatment with cis-DDP led to an obvious oligonucleosomal fragmentation of DNA, isolated from the cytosolic fraction
of SKW-3 cells, as evidenced by the DNA ladder ( Fig.3; lane 2). Benfotiamine at concentrations of 0.05 and 0.1 mg/ml (lanes 3
and 4) and thiamine (at the same concentrations, lanes 5 and 6) clearly reduced the pro-apoptotic activity of cis-DDP after
concomitant application.
3.4. Effect of benfotiamine on heat-induced nociception
The basal latencies were stabile and did not differ between the two groups (Tabl.4). Single treatment with benfotiamine (Table 4)
and thiamine (not shown) at a dose of 10, 50 and 100 mg/kg p.o. did not change the nociceptive threshold, though benfotiamine
tended to increase the basal latency without reaching significance.
3.5. Acute effect of benfotiamine on morphine-induced analgesia
Morphine had a strong analgesic effect verified by substantially increased pain thresholds (Tabl. 5; Fig. 4). The maximum of the
morphine-induced analgesia appeared 30 – 60 min after the injection depicted either as increase in latency (sec) or maximum
possible effect (%MPE) and then progressively declined. The mean basal (pre-drug) latency was 7.2 sec (HP). Benfotiamine 10
mg/kg increased morphine analgesia by 34% (p<0.05) after 30 min and by 31% after 60 min on HP test (Tabl. 5). Thiamine at the
same doses did not increase morphine analgesia (data not shown). Higher doses of benfotiamine did not change morphine
analgesia. Benfotiamine and thiamine increased psychomotor activity of rats and itching. Itching was most pronounced with the
highest dose of benfotiamine and thiamine. This suggested that benfotiamine increased morphine induced liberation of histamine.
3.6. Effect of long-term co-treatment with Benfotiamine on the development of tolerance to morphine analgesia
The dynamic of morphine analgesic action was similar after each application through the chronic treatment, i.e. maximum effect
was reached 30-60 min after injection. The tolerance to the analgesic effect of morphine was easily developed after 3-4 days
morphine treatment, which was evidenced by the substantial decrease of the nociceptive thresholds (Fig. 5a; Fig. 5b). In the group
co-treated with 10 mg/kg benfotiamine analgesic activity remained significantly higher in comparison to morphine until day 7 (Fig.
5a; 5b). Thiamine retarded the development of morphine tolerance, but this effect was observed until day 5 only (Fig. 5a). In
parallel with the changes in the nociceptive threshold, benfotiamine retarded and alleviated significantly irritability, defecation and
diarrhea (Tabl. 6). Co-treatment with benfotiamine and thiamine alleviated the observed decrease in body weight gain during the
treatment with morphine (Tabl. 7).
3.7. Effect of long-term co-treatment with benfotiamine on naloxone precipitated withdrawal
The withdrawal syndrome represents a complex of psychic/emotional and physical/somatic reactions, considered as equivalents of
opioid abstinence. Data showed that naloxone (after 11 days treatment with morphine) precipitated severe withdrawal syndrome.
The body weigh loss reached 17.1 ± 2.14 g in morphine treated group, 11.25 ± 3.0 (p<0.05 ) in benfotiamine and 13.75 ± 3.3 in
thiamine group after 24 h. Significant differences were observed in the behavioural symptoms: in the morphine group dominated
wax figure posture, prostration, rearing, ptosis, grooming and teeth chattering (Tabl. 8). A significant decrease in wax figure
posture, ptosis and grooming were observed in the group treated with benfotiamine (Tab. 8). Prostration was totally diminished,
but escape attempts and defence reaction were more pronounced in the benfotiamine group.
4. Discussion
4.1. Influence of benfotiamine and thiamine on the cytotoxic activity of antineoplastic drugs
The main focus of this study was to assess the effects of benfotiamine and thiamine co-administration on the cytotoxicity of the
DNA-adduct forming agents cis-DDP and bendamustine, the antimetabolites metothrexate, 5-FU and gemcitabine; the mitotic
spindle poison vincristine as well as the antiestrogen tamoxifen since alterations in the cytotoxic activity, could compromise the
main goal of the cytoreductive therapy. The choice of the drugs was done after taking into consideration the type of the tumor cell
lines, in order to be in accordance to the clinical treatment protocols.
Benfotiamine and thiamine were not found to modulate the effects of the antineoplastic agents investigated with the exception of
minor reduction of cis-DDP efficacy in SKW-3 cells and marginal modulation of tamoxifen cytotoxicity in MCF-7 cells. The latter
phenomenon could not be explained by the anti-hormonal properties of the drug only and we could hypothesize the involvement of
additional cytotoxic mechanisms of tamoxifen [20,27]. In order to elucidate some of the mechanistic aspects of the observed
modulation of cis-DDP cytotoxicity we investigated the effects of benfotiamine and thiamine on the GSH cellular content, as well
as on cis-DDP induced apoptosis.
4
Interestingly, both thiamine and benfotiamine caused concentration-dependent increase of the GSH levels in SKW-3, HL-60 and
HL-60/Dox cells. The elevated levels of GSH in SKW-3 cells correlate well to the observed cis-DDP cytotoxicicity reduction. It is
well established, that cis-DDP elicits its cytotoxic effect after exchange of the labile chlorine ligands to water molecules, thus
yielding highly electrophillic mono- and di-aqua-complexes, which in turn react with DNA-bases (predominantly the N7 position
of guanine), forming inter- and intra-strand cross-links [9,37]. GSH, being the most abundant low-molecular weight thiol in
mammalian cells appears to be a major component of the cellular antioxidant defense system, effective against reactive oxygen
species (ROS) and electrophilic cytotoxins such as cis-DDP and alkylating agents [2,8,17,25,37]. It is well known thet GSH reacts
with cis-DDP to form less cytotoxic GSH-adducts, spontaneously or via the glutathione-S-transferases. Thus the cellular DNA is
protected from Pt-adduct formation [2,25]. It is also described, that GSH is capable to react with Pt-DNA mono-adducts, and so
blocks the cross-linking formation [2,25]. Different studies reveal that a number of cis-DDP-resistant cell lines are characterized
with elevated levels of GSH and or glutathione-S-transferases expression [2,25]. Some authors claim that GSH level plays an
essential role in the mechanism of cisplatin resistance, but it is also reported that butionylsulfoximine (BSO) an inhibitor of the
rate-limiting reaction of GSH cellular synthesis did not augment the cytotoxicity of cis-DDP cytotoxicity [2].
In contrast to the modulation of cis-DDP cytotoxicity against SKW-3 cells there was practically no difference in the response of
HL-60 and HL-60/Dox cells to cis-DDP with or without benfotiamine/thiamine co-administration. The data for HL-60/Dox are
quite interesting, because these cells express the MRP-1 transporter, a member of the ATP-binding cassette proteins such as Pgp
170, known to expel antineoplastic agents out from the cell and so to render resistant malignant cells [23]. MRP-1 does not expel
the antineoplastic agents per se, but their glutathione, glucuronic acid or sulphate conjugates only [2,23,25]. It is noteworthy, that
the actual role of MRP-1 in cisplatin resistance is not finally pointed out [2,25]. While some investigations suggest, that MRP-1
could expel cisplatin-GSH conjugates from tumor cells [2,23], there are also some recent studies indicating that this transporter
fails to do so [24]. Our experimental data show that despite of the benfotiamine/thiamine induced alterations of GSH content, there
is generally no difference between the IC50 values for cis-DDP on HL-60 and HL-60/Dox.
In order to elucidate further the observed benfotiamine/thiamine- induced modulation of cis-DDP activity we assessed the cisDDP induced apoptosis in SKW-3 cells. As evidenced by the results obtained from the DNA-fragmentation assay, both
benfotiamine and thiamine co-administration resulted in less eminent DNA-laddering phenomenon.
4.2. Influence of benfotiamine or thiamine on pain formation, opioid nalgesia and tolerance/dependance development
Benfotiamine and thiamine had no effect on the baseline heat-induced nociception. In line with this lack of analgesic activity by
thiamine on hot plate test has been recently observed [14]. Most experimental data concerning analgesic activity of thiamine are
controversial and very much depending on the model used. Thiamine showed analgesic activity upon long term administration in
nociception induced by i.p. injection of acetic acid and formaldehyde hind paw edema [14]. An enhancement of analgesic activity
of morphine and paracetamol was observed after repeated application of vitamins B1, B6 and B12 [24]. Benfotiamine, but not
thiamine showed significant increase of morphine analgesia on hot plate test after 30 and 60 min. This effect was observed at a
dose of 10 mg/kg. Intrathecal superfusion of vitamins B1, B6 and B12 produced dose-dependent depression in the responses
evoked by noxious skin heating of hind foot, but not of spontaneous activity in dorsal horn neurons [15]. The therapeutic effect of
B vitamine compounds in pain management may involve a suppression of nociceptive transmission at the spinal level [15].
Pretreatment with B vitamins significantly enhanced the afferent inhibition of heat noxious stimuli probably due to an increase in
the synthesis of inhibitory neurotransmitters in central neurons [16]. Lack of potentiation of morphine analgesia by single thiamine
injection has been reported on p-benzoquinone (PBQ) induced abdominal constriction test [1]. Our data indicate, that long term cotreatment with benfotiamine resulted in a significant retardation of the development of tolerance to morphine analgesia. This effect
was better observed with benfotiamine, in comparison to thiamine. Benfotiamine alleviated physical dependence during the
treatment with morphine (Tabl. 5, 6). Other investigations have also shown superior analgesic activity of long-term (7 days)
application of thiamine [14]. In our hands naloxone precipitated abstinence showed that benfotiamine significantly diminished wax
figure posture and prostration, which are equivalents of the most severe abstinence syndrome. This effect could be partially
explained by the antioxidant effect of thiamine [18]. The better efficacy of benfotiamine in comparison to thiamine is most
probably due to the pharmacokinetic advantages [22,28].
Thiamine plays an important role in acetyl coenzyme A synthesis, which is an active component for acetylcholine synthesis [18],
which could contribute to the observed phenomena since acetylcholine is involved in the mechanism of narcotic analgesic effects
via stimulation of guanylyl cyclase similar to morphine [13]. Thiamine and its phosphorylated esters appear to play an essential
role in nerve conduction and excitation at the molecular level, quite independently of their co-enzymatic activity [11]. Thiamine
triphosphate regulates large conductance chloride channels [7], serves as a phosphate donor for the phosphorylation of synaptic
proteins [34] and modifies ion transport, especially the sodium transport [5]. Misra et al. [31] reported naloxone-dependent
increase of thiamine content in the cortical hemisphere, cerebellum and the brain stem by 21%, 44%, and 29% respectively induced
by morphine. It has also been shown that subacute treatment of rats with B vitamins (B1, B6 and B12) increased the sensitivity to
analgesic effect of morphine most probably by interfering with the serotoninergic transmitter system [6,12]. Moreover thiamine
could produce antinociception by the activation of guanylyl cyclase [1]. Development of tolerance and dependence to morphine
includes adaptive changes in various transmitter systems, including cholinergic, serotoninergic, adrenergic, glutamatergic and
nitroxidergic [3]. The interference of benfotiamine with synthesis and release of neurotransmitters or receptor proteins, ion
conductance mentioned above could explain the observed by us retardation of morphine tolerance and alleviation of physical
dependence. In conclusion, data presented in this study suggest that benfotiamine is a beneficial adjuvant in long term opioid
therapy.
This is the first investigation to show that benfotiamine did not elicit any stimulatory effect on malignant cell proliferation, but
retarded development of tolerance to morphine analgesia and alleviated physical dependence, which could have an important
clinical implication for the treatment of cancer pain.
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5.
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6
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Acknowledgments
This study was financially supported by Wörwag Pharma GmbH, Böblingen, Germany. The authors gratefully acknowledge the
excellent technical assistance of Mrs Theodora Atanassova.
Table 1. Effects of bendamustine, cis-DDP, metothrexate and gemcitabine administrated alone, and with benfotiamin or thiamine
after 72h on SKW-3 cells
Drug
IC50 value
Alone
+ benfotiamin
+ thiamine
bendamustine
43.10 (40.21-45.99)
42.08 (35.13-47.86)
44.22 (41.33-46.54)
cis-DDP
19.75 (16.97-21.72)
24.47 (22.7-27.55)
25.42 (23.84-27.45)
metothrexate
3.61 (3.42-3.70)
3.60 (3.45-3.84)
3.63 (3.35-3.73)
gemcitabine
1.50 (1.46-1.53)
1.57 (1.45-1.78)
1.52 (1.36-1.64)
Table 2. Effects of 5-FU and vincristine administered alone, and with benfotiamin or thiamine after 72h on HL-60 cells
Drug
IC50 value
Alone
+ benfotiamin
+ thiamine
5-FU
25.41 (22.99-29.16)
24.63 (22.79-26.79)
25.46 (24.21-27.31)
vincristine
0.107 (0.095-0.111)
0.096 (0.090-0.097)
0.102 (0.093-0.111)
gemcitabine
0.87 (0.69-1.3)
0.92 (0.54-1.41)
0.95 (0.72-1.27)
cis-DDP
2.49 (2.11-2.70)
2.307 (2.06-2.59)
2.01 (1.86-2.81)
cis-DDPb
2.10 (1.70-2.29)
2.33 (2.15-2.50)
2.38 (2.212-2.513)
b-results obtained after treatment on HL-60/Dox cells with induced resistance to anthracyclines.
Table 3. Effects of metothrexate, 5-FU, gemcitabine, vincristine and tamoxifen administered alone, and with benfotiamine or
thiamine after 72h on MCF-7 cells
Drug
IC20 values (IC50 for tamoxifen)
Alone
+ Benfotiamine
+ Thiamine
metothrexate
5.62 (4.89-6.11)
5.46 (3.65-8.77)
6.124 (4.83-9.18)
5-FU
5.409 (4.81-6.22)
5.63 (4.69-6.79)
5.55 (4.64-6.15)
gemcitabine
0.097 (0.053-0.168)
0.130 (0.062-0.201)
0.130 (0.062-0.201)
vincristine
0.0028 (0.0023-0.0035)
0.003 (0.0023-0.0039)
0.003 (0.0026-0.0035)
tamoxifen
14.34 (13.96-14.65)
16.99 (15.88-18.08)
15.88 (15.27-16.78)
Tabl. 4. Effect of different doses of benfotiamine on heat-induced nociception (Hot plate test)
Control
Benfotiamine 10
Benfotiamine 50
Benfotiamine 100
mg/kg
mg/kg
mg/kg
Basal latencies
(sec)
Increase over basal
(Δ)
30 min
60 min
90 min
120 min
7.2 ±0.7
8.3 ±1.0
7.2 ± 0.7
6.3 ± 0.74
1.19 ±0.9
2.53 ±1.7
1.74 ± 0.7
0.8 ± 0.4
2.71±0.72
2.47± 0.5
1.15 ± 0.6
0.90±0.72
3.59±0.41
3.83±0.36
2.7±0.25
1.2±0.8
1.61±0.22
2.85±0.63
0.94 ± 0.47
1.48±0.52
7
Tabl.5. Effect of different doses of benfotiamine on morphine-induced analgesia (hot plate test)
benfotiamine 100
benfotiamine 50
morphine 5 mg/kg benfotiamine 10
mg/kg +
mg/kg +
mg/kg +
Morphine
Morphine
Morphine
Basal latencies
7.19 ±0.69
7.81 ± 1.1
6.5 ± 0.64
7.20 ± 0.25
(sec)
Increase over
basal (Δ)
30 min
60 min
90 min
120 min
5.81±0.95
10.99± 0.91
9.01 ± 2.0
8.03 ± 0.35
8.87± 1.3*
14.36 ± 1.66*
12.61 ± 1.3
9.64 ±0.69
6.94±1.2
12.26±1.98
10.7±2.43
6.08±1.14
Table 6. Effect of co-treatment with benfotiamine on morphine dependence
Dominating
morphine
benfotiamine + morphine
symptoms
Day 4
Day 11
Day 4
Day 11
7.25±1.42
13.86±1.8
9.95 ± 2.37
6.63±1.48
thiamine + morphine
Day 4
Day 11
Irritability
1.25±0.25
(8/8)
2.25 ±0.3
(8/8)
0**#
0.75±0.41**
(3/8)
0.5±0.2**
(4/8)
1.13±0.3
(6/8)
Screaming
upon touch
1.0±0.28
(6/8)
1.88±0.23
(8/8)
0.25±0.16*
(1/8)
0.5 ±0.33*
(2/8)
0.63±0.32
(3/8)
0.88±0.3*
(5/8)
Defecation
(mean № of
feces)
1.62±0.26
(8/8)
3.0±0.27
(8/8)
0.13±0.13**
(1/8)
1.13±0.46 *
(7/8)
1.12±0.5
(4/8)
2.5±0.5
(7/8)
0/8
2/8*
2/8
6/8
0/8
2/8*
Diarrhoea
№ of rats
*p<0.05 versus morphine; **p<0.01 versus morphine; #p<0.05 versus thiamine
Table 7. Effect of benfotiamine on the body weight gain during morphine treatment
Day after beginning Control
Morphine
Benfo 10 mg/kg +
Thia 10 mg/kg +
of treatment
(Δ) gr
(Δ) gr
Mo
Mo
(Δ)gr
(Δ) gr
Day 5
10.37 ±2.39
10.0 ±3.1
11.25 ± 2.9
8.57 ± 1.4
Day 11
11.25 ± 3.5
5.7 ± 2.2*#
13.75±3.75
12.5 ± 3.13
Values represent increase of the body weight versus first day of treatment (Δ); Body weight before treatment was: Control:
291.4±11.2; Morphine 301.4 ± 12.2; Morphine + Benfotiamine 286.3 ±8.22; Morphine + Thiamine 290± 8.23;
* p<0.05 versus control; #p<0.05 versus co-treatment with Benfotiamine
Tabl. 8. Effect of Benfotiamine on naloxone-precipitated withdrawal
Symptoms
Morphine
Benfotiamine
Ptosis
3.9 ± 0.99 (6/7)
1.12 ± 0.23 (7/8)*
Sniffing
2.57 ± 0.97 (6/7)
1.0 ± 0.38 (4/8)
Grooming
2.71±0.54 (6/7)
2.0 ± 0.5 (6/8)
Teeth chattering
3.71 ± 1.34 (6/7)
2.35 ± 0.16 (8/8)
Escape attempts
1.29 ± 0.64 (3/7)
2.25 ± 0.49 (7/8)
Writhing
3.29 ± 0.52 (7/7)
1.5 ± 0.2 (4/8)*
Rearing
3.86 ± 1.62 (5/7)
2.88 ± 0.52 (7/8)
Wax figure posture
4.14 ± 1.08 (7/7)
1.63 ± 0.46(6/8)*
Defence/aggresion
0
1.87 ± 0.55 (6/8)**
Penile licking
2.29±0.86 (6/7)
1.25 ±0.31 (6/8)
Prostration
3.71 ± 0.42 (7/7)
0**
Diarrhoea
2.0 ± 0.38 (7/7)
2.12 ± 0.35 (6/8)
Body weight loss after 24 17.1 ± 2.14
11.25±3.0*
h (∆g)
*p<0.05 versus morphine; **p<0.01 versus morphine
Thiamine
3.37 ± 0.62 (8/8)
1.87 ± 0.35 (8/8)
2.25 ± 0.25 (8/8)
3.12 ± 0.3 (8/8)
1.38 ± 0.46 (6/8)
1.62 ±0.18 (6/8)*
2.12 ±0.23 (8/8)
1.88 ± 0.3 (8/8)*
0.88 ± 0.35 (5/8)*
1.88 ± 0.3 (8/8)
1.25 ± 0.5* (5/8)
2.12 ± 0.23 (6/8)
13.75±3.3
8