POP Anca Lucia1, VARLAS Valentin3, SAVU Adina2, NASUI Bogdana Adriana5, NEDEA Eduard4,
1Faculty of Pharmacy”Carol Davila” University of Medicine and Pharmacy, Bucharest (ROMANIA)
2“Floreasca” Clinical Emergency Hospital, Bucharest, (ROMANIA)
3“Filantropia” Clinical Hospital, Bucharest, (ROMANIA)
4Scient R&D Center, Bucharest (ROMANIA), Columbia University (SUA)
5University of Medicine and Pharmacy, Cluj-Napoca, Department of Community Health
*Corresponding author: varlas.valentin@umfcd.ro
Abstract
Background and study premises: The development of multidrug resistance (MDR) to chemotherapics in cancer therapy causes difficulties due to the need to increase the doses of these highly toxic drugs. Investigation of the possibility of reversal of MDR by drug-drug interactions could limit the toxicity of the chemotherapics [1, 2]. Studies indicating that verapamil and ketotifen enhances doxorubicin levels in certain drug resistant tumor cells have led in investigation of the possible use of doxorubicin in combination with verapamil, respectively ketotifen. The aim is to evaluate in the same study the in vivo effects of the toxicity of doxorubicin by adding verapamil, a calcium channel blocker (CCB) and ketotifen, a H1 antihistaminic agent, in mice. The assessment of the toxicity of doxorubicin was done by administration of repeated doses of doxorubicin single and together with verapamil, respectively ketotifen intraperitoneally, in mice. The mice were administered high dose doxorubicin (15 mg/kg injected intraperitoneally), verapamil (25 mg/kg injected intraperitoneally), ketotifen (15 mg/kg injected intraperitoneally) and the combination of doxorubicin and verapamil and combination of verapamil with ketotifen. The in vivo effects of verapamil and ketotifen on (a) doxorubicin-induced animal mortality on a 14 days period compared with controls, verapamil and ketotifen toxicity (b) doxorubicin cardiac damage compared with controls. Our results suggest that verapamil and ketotifen reduce significantly the doxorubicin induced toxicity. Ketotifen is more effective in reducing the mortality of doxorubicin than verapamil, but both significantly reduce the mortality of doxorubicin compared to doxorubicin single, fact confirmed anatomo-pathological.
Keywords: multidrug resistance, doxorubicine, ketotifen, verapamil, cancer therapy
Introduction
The development of multidrug resistance (MDR) to chemotherapics in cancer therapy causes difficulties due to the need of increase of the doses of the chemotherapics. Investigation of the possibility of reversal of MDR by drug-drug interactions could limit the toxicity of the chemotherapics. Studies indicating that verapamil and ketotifen enhances doxorubicin levels in certain drug resistant tumor cells have led in use of doxorubicin in combination with verapamil, respectively ketotifen [3, 4, 5]. The aim is to evaluate the in vivo effects of reducing the toxicity of doxorubicin by adding verapamil, a calcium channel blocker (CCB) and ketotifen, a H1 antihistaminic agent, in mice.
Material and Methods
The assessment of the toxicity of doxorubicin was done by administration of multiple doses of doxorubicin single and together with verapamil, respectively ketotifen intraperitoneally, in mice. We used batches of white male mice weighing 18-22 g. The animals from bio base were kept in laboratory conditions during 2 days for accommodation with the laboratory conditions and fed at fixed intervals. We made 6 batches of 20 healthy mice per batch (marked from A-F), weighing 22 g± 4g. The animals were administered daily doses of doxorubicin solution Adriablastine®) -15 mg/kg injected intraperitoneally, verapamil solution (Cordamil®) – 25 mg/kg injected intraperitoneally, ketotifen solution (H-Ketotifen®) – 15 mg/kg injected intraperitoneally – and the combination of doxorubicin and verapamil and combination of verapamil with ketotifen at the same doses as for the single drug batches, on a period of 14 days. The animals were observed during 14 days following the parameters: lethality, weigh, motor behavior, external appearance: fur, secretions.
The animals were purchased from the Animal Bio-base of the University of Medicine and Pharmacy “Carol Davila”, Bucharest, Romania. With 24 hours before the study the animals were restricted from food during 24 hours and water “ad libitum”. All experiments were performed in compliance with European Communities Council Directive 1986 (86/609/EEC) and Ordinance No. 37 of the Romanian Government from 2nd February 2002. The animals were distributed in six batches of eight animals each.
We assessed the in vivo effects of verapamil and ketotifen on (a) doxorubicin-induced animal mortality in a 14 days period compared with controls, verapamil and ketotifen toxicity (b) doxorubicin cardiac and liver tissue damage compared with the association batches and controls. The data was processed with the statistical „t” Student test and ANOVA. The frozen anatomical pieces excised from the animals were prelevated, fixed in LILLY fixator (consisting of formyl, acetic acid, alcohol), included in paraffin and microtomised at 5μ, prepared for anatomical microscopy lamellas with a hematoxylin-eosin coloration, visualized at a trifocal microscope Olympus AX70 at 100X and 200X resolutions, in the anatomo-pathological laboratory.
The assessment of the toxicity on the vegetal cell, of the verapamil, ketotifen, doxorubicin and of the associations verapamil – doxorubicin and ketotifen – doxorubicin we analyzed the inhibition of the growth of the roots of the wheat Triticum aestivum – after 7 days of germination in the solutions of the three substances and associations of doxorubicin with verapamil and ketotifen respectively. A % verapamil aqueous solution and 5 progressive dilutions were used, a % ketotifen solution and dilutions and a % doxorubicin solution was used to perfuse the 24 hours germinated wheat grains in favorable light and temperature solutions.
At 24 hours after the perfusion with the studied solutions one set of roots was microscopically analyzed, with a orcein dye, with a trifocal microscope Labophot 2 at 100X and 200X resolutions.
Results and Discussions
The overall mortality in 14 days of the doxorubicin batch (15 mg/kg injected intraperitoneally) was 100%. The mortality of the doxorubicin and verapamil batch was 80%, 20% lower than in doxorubicin batch. The mortality for the doxorubicin and ketotifen batch was only 60%. The mortality in the control batch was 0%, the verapamil batch had a 70% survival rate, the ketotifen batch had a 80% survival rate. (Table no. I)
Table I. Lethality after 2, 5, 8 and 14 days in the six batches took into study
| Batch | Substance /dose
(mg/kg intraperitoneally) |
Lethality
(%) |
||||
| Drug 1 | Drug 2 | Da y 2 | Day 5 | Day 8 | Day 14 | |
| A | Cordamil® 25 mg/kg/i.p. | – | 0 | 0 | 20 % | 30% |
| B | Cordamil® 25 mg/kgi.p. | Adriablastin a® 15 mg/kg i.p. | 0 | 30 % | 60 % | 80% |
| C | Adriablastina® -15 mg/kg i.p | – | 0 | 100 % | 100 % | 100 % |
| D | H-Ketotifen® 15 mg/kg i.p. | Adriablastin a® 15 mg/kg i.p. | 0 | 20% | 40 % | 40% |
| E | H-Ketotifen® 15 mg/kg i.p. | – | 0 | 0 | 10 % | 20% |
| F | CONTROL – | – | 0 | 0 | 0 | 0 |
Figure no. 1 Lethality after 7 days in the six batches took into study; Figure no. 2 Lethality after 14 days in the six batches took into study (A-Cordamil® 25 mg/kg/i.p., B- Cordamil® 25 mg/kgi.p.+ Adriablastina® -15 mg/kg i.p., C- Adriablastina® -15 mg/kg i.p, D-H-Ketotifen® 15 mg/kg i.p.+Adriablastina® -15 mg/kg i.p. E-H-Ketotifen® 15 mg/kg i.p., F – CONTROL)
The anatomo-pathological sections for the batch treated with doxorubicin showed signs of high toxicity for the heart, with vacuolated dystrophy of the myocytes and hypertrophy, interstitial hematic infiltrates. The liver showed signs of fulminant hepatitis with intense dystrophy of the hepatic architecture, low chronic inflammatory infiltrate and high level of focal hepatocyte necrosis, micro vesicular steatosis at the level of second and third acinar area – intense drug toxicity- sinusoid capillary stasis, hepatocyte apoptosis, dysplasia and frequent “mattified glass” cells (Figure no. 3).
Figure no 3. Histopathological changes in the animals treated with doxorubicin. Cardiac and liver sections, optical microscopy, A- heart 100X, B-liver 100X , C-liver 200X (Batch C)
For the batches with the same dose of doxorubicin associated with verapamil respectively ketotifen, the heart tissue presented a medium degree of toxicity, with the same signs of myocyte hypertrophy and vacuolization. At the liver level the hepatic architecture was maintained with low chronic inflammatory infiltrate, focal hepatocyte necrosis, reduced micro vesicular steatosis and sinusoidal stasis (Figure no. 4, 5).
Figure no 4. Histopathological changes in the animals treated with doxorubicin and verapamil. Cardiac (A) and liver (B) sections, optical microscopy, 100X (Batch B)
Figure no 5. Histopathological changes in the animals treated with doxorubicin and ketotifen. Cardiac and liver sections, optical microscopy, A – heart 100X, B-liver 200X, (Batchd)
Figure no 6. Histopathological changes in the animals treated with verapamil. Cardiac and liver sections, optical microscopy, A – heart 200X, B-liver 100X, (Batch A)
The anatomo-pathological study showed for the verapamil batch, for the heart section myocardia fibrils with vacuolated myocyte degenerescence, interstitial hematic infiltrates, myocytes hypertrophy and vacuolar congestion at a low level. The liver presented degenerative granular-vacuolar lesions, moderate chronic inflammatory infiltrate, nodular focal peri-centro-lobular inflammation, hypertrophy of Kupfer cells, cholestasis. The vessels presented hepatic peliosis, few dysplastic cells, low micro vesicular steatosis, showing a low degree of hepatic toxicity for the verapamil dose took into study. Less degree of changes was visualized for the ketotifen batch (Figure no. 7).
Figure no 7. Histopathological changes in the animals treated with ketotifen. Cardiac and liver sections, optical microscopy, A – heart 200X, B-liver 200X, (Batch E)
The control batch presented at the liver level conserved structures low chronic inflammatory infiltrate, fibrosis and vascular stasis, most probably due to the food regimen. The heart tissue presented very few myocyte hypertrophy and degenerescence.
Figure no 8. Histopathological changes in the control batch. Cardiac and liver sections, optical microscopy, A – heart 200X, B -liver 100X , (Batch F)
Assessment of the toxicity of verapamil, ketotifen, doxorubicin on the vegetal cell
The assessment of the toxicity on the vegetal cell, of the verapamil, ketotifen, doxorubicin and of the associations verapamil – doxorubicin and ketotifen – doxorubicin we analyzed the inhibition of the growth of the roots of the wheat Triticum aestivum – after 7 days of germination in the solutions of the three substances and associations of doxorubicin with verapamil and ketotifen respectively.
Figure no 8. Histopathological changes in the Triticum aestivum batches on the vegetal cells. Sections of the 24 hours germinated root in the studied aqueous solutions of the studied substances, optical microscopy, 200X. (A-doxorubicin (Dr), B-verapamil (Vp), C-doxorubicin and ketotifen, D-ketotifen (Kt) batch, E, F-doxorubicin and verapamil, G- untreated control)
In the doxorubicin batch, the microscopy revealed a completely disorganized tissue architecture, with mattified cells, deformed cell walls; in the verapamil batch are visualized multiple metaphases and prophases, telophase in tropokynesys, signs of unaffected growth processes, with slight signs of toxicity, with hypertrophied nucleoli.
In the ketotifen batch, the cell division is in progress, unaffected, with slight signs of toxicity: hypertrophied nucleoli, semilunar and triangle shaped nucleoli, undulated cell walls, unmodified telophase, anaphase; in the associations batches, doxorubicin and verapamil batch, there are signs of considerable toxicity, with deformed, telophase, prophase, mattified cells, significantly reduced compared with the doxorubicin batch, at the same cumulative doses.
In the ketotifen doxorubicin batch, the telophase is normal, moderate altered prophase, metaphase in tropokynesis, moderate signs of toxicity, disorganized metaphases, anaphase with a retarded chromosome. Compared with the doxorubicin batch, the toxicity of the association verapamil-doxorubicin and ketotifen-doxorubicin at the same cumulative doses was reduced in the anatomo-pathological studies
Conclusions
Our results suggest that verapamil and ketotifen inhibit the doxorubicin induced toxicity. Ketotifen is more effective in reducing the mortality of doxorubicin batch (-40%) than verapamil (-20%), but both significantly reduce the mortality of doxorubicin batch compared to doxorubicin treated batch. The toxicity studies on the vegetal cell confirmed the results obtained on the animal cell.
At the histopathologic examination we observed that the animals treated with doxorubicin suffered the most profound toxic changes. At the same dose, the associated treatment of doxorubicin with verapamil and doxorubicin with ketotifen caused a significant reduction of the signs of cardiotoxicity and hepatotoxicity.
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