Pharmacogenetics of anticancer drugs

Much progress has been made in treating human malignancies and there are now multiple treatment options with similar efficacy for nearly every type of cancer. However, the narrow therapeutic index of most chemotherapeutic agents and the severe consequences of undertreatment or overdosing have led to research molecular predictive factors of the toxicity and efficacy of cancer treatments. Genetic factors affecting drug metabolism and transport partly explain interindividual variability in drug response. Pharmacogenetic focuses on the molecular mechanisms involved in drug response, and its ultimate goal is the optimisation of the treatments, that combines the optimal efficacy and the minimal risk of severe side effects. Polymorphisms in genes encoding specific drug-metabolising enzymes can result in individuals in the general population being characterised as low, rapid or even ultra-rapid metabolisers. Phenotyping and genotyping tests are now available that determine or predict the metabolic status of an individual and, thus, enable the evaluation of risk of drug failure or toxicity. Some clinical applications of pharmacogenetics (5-FU, irinotecan, thiopurines) have already been developed in routine medicine resulting in significant improvement in patient treatment. The clinical validation of an increasing number of pharmacogenetic tests, as well as the development of new highly efficient technologies for genotyping (real-time PCR, DNA chips...) should further promote pharmacogenetics in clinical practice and lead to the development of a patient-tailored drug therapy.     

Genotoxic classification of anticancer drugs

The effects of bacterial DNA excision repair on the mutagenic and lethal actions of 17 injectable anticancer drugs have been used to classify them into three levels of potential risk to medical personnel who are involved in their preparation and administration.     

Synthesis and cytotoxicity studies of new morpholino-functionalised and N-heteroaryl-substituted titanocene anticancer drugs

From the carbolithiation of 6-morpholino fulvene (3) and different lithiated nitrogen containing heterocycles (2-N-methylimidazolyl, 2-N-(N,N-dimethylamino)methyl-imidazolyl, and 2-N-methylindolyl), the corresponding lithium cyclopentadienide intermediate (4a-c) was formed. These three lithiated intermediates underwent a transmetallation reaction with TiCl(4) resulting in morpholino-functionalised titanocenes 5a-c. When these titanocenes were tested against LLC-PK cells, the IC(50) values obtained were of 24, 36, and 41 microM respectively. The most cytotoxic titanocene in this paper (5a) with an IC(50) value of 24 microM is found to be almost ten times less cytotoxic than cis-platin, which showed an IC(50) value of 3.3 microM when tested on the epithelial pig kidney LLC-PK cell line, and approximately 2 times less cytotoxic than its dimethylamino-functionalised analogue. Encouragingly however, the IC(50) value obtained for titanocene 5a is approximately 100 times better than titanocene dichloride itself.     

The synergistic effects of hyperthermia and anticancer drugs on induction of apoptosis

We studied the synergistic effects of hyperthermia and anticancer drugs on induction of apoptosis in lung cancer cells (LK-2 and LU-65A) using in situ end-labeling of DNA, the DNA fragmentation assay, and transmission electron microscopy. A few apoptotic cells were detected only when both cell lines were heated at relatively high temperature (44 degrees C). Moderate numbers of apoptotic cells were observed when both cell lines were incubated with high concentrations (30 or 40 microM) of anticancer drug. Compared with hyperthermia or anticancer drug alone, the combined treatment induced many apoptotic cells in both cell lines, even in the cells treated with lower concentrations (6 or 8 microM) of anticancer drugs following mild hyperthermia (43 degrees C). In regard to kinetics of apoptotic cells induced by treatment, the maximum induction of apoptosis by the combined treatment was higher than that of hyperthermia or anticancer drug alone in both cell lines, although the time of the peak of apoptotic index differed among the three treatments. Therefore, "hyperthermo-chemotherapy" may reduce the required dosage of anticancer drug and decrease the temperature of hyperthermia on induction of apoptosis.     

Poisoning caused by neuroleptics and other related drugs

Neuroleptics (phenothiazines) and related medicaments such as haloperidol and metoclopramide, are frequent cause of intoxication. The main manifestations are extrapyramidal symptoms. In most cases the intoxications have an acute evolution and the large doses may be complicated with shock, coma and ventricular fibrillation; persistent hyperthermia brings poor prognosis. Their chronic use, even at therapeutic doses, may produce late dyskinesia, difficult to manage. Diphenhydramine is particularly useful to treat the acute intoxication; this could be started with slowly intravenous injected 1 mg/kg until symptoms disappear, and then continue per os for a minimum of these drugs. Its improper use and abuse are due to therapeutic mode.     

Thermosensitive hydrogels based on polypeptides for localized and sustained delivery of anticancer drugs

Thermosensitive hydrogels based on poly(γ-ethyl-l-glutamate)-poly(ethylene glycol)-poly(γ-ethyl-l-glutamate) triblock copolymers (PELG-PEG-PELG) were prepared for localized and sustained delivery of anticancer drugs. The polypeptide-based hydrogels showed much lower critical gelation concentration than the traditional polyester-based hydrogels. In vivo biocompatibility studies revealed that the in situ formed gels in the subcutaneous layer last for ∼21 days, and H&E staining study suggested acceptable biocompatibility of our materials in vivo. Then the hydrogels were tried as injectable implants to encapsulate antitumor drug, paclitaxel (PTX), to assess the in situ anti-tumoral activity using liver cancer xenograft model. The results demonstrated that the PTX-incorporated hydrogels could efficiently suppress the tumor growth, and did not result in obvious damage to normal organs. Therefore, the polypeptide-based thermosensitive hydrogels designed in the present study have great potential to serve as an effective platform for localized anti-cancer drug delivery.     

ICH S9: Developing anticancer drugs, one year later

International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) S9 is the first international regulatory guidance document devoted to a specific therapeutic area, highlighting its importance in harmonizing regulatory expectations for the nonclinical development of therapeutics designed to treat advanced cancer. ICH S9 successfully outlines the core requirements for the nonclinical development of novel oncology therapeutics, and the unique risk-benefit considerations for expediting drug development to treat these most grievous diseases. However, development companies and regulatory agencies have had limited opportunity to apply this guidance, so our collective experience in developing therapeutics under the guidance is limited. Discussed here are several key areas of ambiguity in the guidance, including identification of the patient population, selection of the initial safe dose for first-in-human trials, and requirements for nonclinical recovery data.     

DNA damage and repair in human lymphocytes exposed to three anticancer platinum drugs

Cisplatin is a widely used anticancer drug, but its application is limited due to severe side effects. To reduce these effects, many other platinum drugs have been synthesized. In the present work comparative analysis of the toxicity of cisplatin, oxoplatin, and a conjugate (NH(3))(2)Pt(SeO(3)) (Se-Pt) in terms of cell viability, DNA binding, and DNA damage and repair in human lymphocytes was performed using the Trypan blue exclusion test, atomic absorption spectroscopy, and the comet assay, respectively. Cisplatin and oxoplatin did not cause a significant change in the viability of the lymphocytes even at the highest used concentration (750 microM), but the conjugate dramatically diminished viability at 100 microM only about 60% of the lymphocytes were viable (P < 0.05), and at 750 microM, less than 20% (P < 0.001). Se-Pt bound to isolated DNA was about 100 times weaker than the remaining two compounds; the binding of cisplatin was about 30% stronger than oxoplatin. Cisplatin and oxoplatin formed crosslinks with DNA in lymphocytes, whereas the conjugate induced DNA strand breaks. The lesions evoked by cisplatin and oxoplatin were slowly removed, but damage induced by Se-Pt was not repaired after 5 h even at a drug concentration of 10 microM. Severe cytotoxic and genotoxic effects exerted by Se-Pt in normal human lymphocytes preclude its intravenous application in cancer therapy. Teratogenesis Carcinog. Mutagen. 20:119-131, 2000.     

Correlation between chemosensitivity to anticancer drugs and Bcl-2 expression in gastric cancer

Objective: To investigate chemoresistance of human gastric cancer to chemotherapeutic drugs in vitro and explore the relationship with Bcl-2 protein expression. Methods: Single-cell suspensions were prepared from freshly excised samples of primary gastric cancer, and were separately exposed to taxol (TAX), cisplatin (CDDP), 5-fluorouracil (5-FU), adriamycin (ADM) and mitomycin (MMC) for 48 h. The induction of cell death was confirmed by microscopic analysis of cell morphology. Metabolic activity and the inhibitory rate (IR) of cells were evaluated by MTT assay. Expression of Bcl-2 was determined by immunohistochemistry of gastric cancer tissue samples. Results: The IRs of cancer cells exposed to different chemotherapeutic drugs varied as follows: the IRs for TAX, CDDP and 5-FU were significantly higher than those for ADM and MMC (P < 0.01). Poorly differentiated gastric cancer cells were more sensitive than well-differentiated cells (P = 0.021). The positive rate of Bcl-2 expression was 80%, and Bcl-2 expression was significantly associated with chemoresistance to 5-FU (r_s = 0.265, P = 0.041), ADM (r_s = 0.425, P = 0.001) and MMC (r_s = 0.40, P = 0.002). Furthermore, Bcl-2 expression was strongly associated with lymph node metastasis in gastric cancer (P = 0.009). Conclusion: Overexpression of Bcl-2 may predict a loss of the efficacy of the chemotherapy drugs 5-FU, ADM and MMC in patients with gastric cancer.     

Oxidative stress induced in rat liver by anticancer drugs doxorubicin,paclitaxel and docetaxel

PurposeOxidative stress generated by anticancer drugs in non-targeted tissues, is considered as a significant factor responsible for their severe side effects, e.g. cardiotoxicity, neurotoxicity and hepatotoxicity. Lack of data on the effect of concurrent administration of commonly used anticancer drugs: doxorubicin (DOX), paclitaxel (PTX) and docetaxel (DTX) on normal tissue, prompted us to examine the markers of oxidative stress in the liver of rats treated with these drugs.Material/MethodsMale Wistar rats of average weight 200 g were injected intraperitoneally (i.p.) with 10 mg/kg of body weight (b.w.) of DOX, PTX and DTX. The drugs were given alone or in combinations DOX+taxane. The activities of superoxide dismutase (SOD), catalase (CAT), low molecular weight and total thiols and thiobarbituric acid-reactive substances (TBARS) were estimated.ResultsCombination of two drugs generated greater changes than single agents. Concurrent administration of DOX and PTX increased SOD activity and TBARS, decreased the amount of low molecular weight and total thiols, but did not cause any changes in the activity of catalase. Combination of DOX and DTX induced similar changes except for the activity of catalase, which decreased after the treatment. Of the three drugs only DTX significantly decreased the activity of SOD. However, both taxanes increased the activity of catalase. Although a decrease in concentration of –SH groups, depletion of glutathione and an increase of TBARS were observed after treatment with single drugs, the changes were not statistically significant.ConclusionConcurrent administration of DOX and taxane induced enhanced oxidative stress in comparison to single drugs, which suggests their synergistic prooxidant mode of action in liver.     

Effect of anticancer drugs on the release of interleukin-3 in vitro

In this study several anticancer drugs were tested for their effect on the release of interleukin-3 (IL-3) from concanavalin A (Con A) stimulated mouse splenocytes in vitro. When Adriamycin or vincristine were added to the cultures at non-cytotoxic concentrations, the release of IL-3 was inhibited. However, bleomycin, FK156, FK565 and 4-OOH-cyclophosphamide (the in vitro active analogue of the anticancer and immunosuppressive drug cyclophosphamide) did not alter the release of IL-3 under the same conditions. It was confirmed that cyclosporin A inhibited the release of IL-3 in the same experimental system.     

Folding graft copolymer with pendant drug segments for co-delivery of anticancer drugs

A graft copolymer with pendant drug segments can fold into nanostructures in a protein folding-like manner. The graft copolymer is constructed by directly polymerizing γ-camptothecin-glutamate N-carboxyanhydride (Glu(CPT)-NCA) on multiple sites of poly(ethylene glycol) (PEG)-based main chain via the ring open polymerization (ROP). The “purely” conjugated anticancer agent camptothecin (CPT) is hydrophobic and serves as the principal driving force during the folding process. When exposed to water, the obtained copolymer, together with doxorubicin (Dox), another anticancer agent, can fold into monodispersed nanocarriers (with a diameter of around 50 nm) for dual-drug delivery. Equipped with a PEG shell, the nanocarriers displayed good stability and can be internalized by a variety of cancer cell lines via the lipid raft and clathrin-mediated endocytotic pathway without premature leakage, which showed a high synergetic activity of CPT and Dox toward various cancer cells. In vivo study validated that the nanocarriers exhibited strong accumulation in tumor sites and showed a prominent anticancer activity against the lung cancer xenograft mice model compared with free drugs.     

Separation methods for anthraquinone related anti-cancer drugs

The quinoid anthracycline-related anti-cancer agents represent an important group of anti-tumour drugs with a wide spectrum of activity. We review here some of the separation techniques used for the analysis of anthracyclines and related compounds. In this review we have covered a range of compounds from the early anthracycline antibiotics such as doxorubicin to the more recent anthracenediones and anthrapyrazoles such as mitoxantrone and losoxantrone, respectively. We also include novel compounds such as AQ4N and C1311, both awaiting clinical trial. Separations of the anthraquinone related anti-cancer agents are predominantly by HPLC. These separation techniques have been used for a variety of applications including drug stability, protein binding and therapeutic drug monitoring as well as detailed pharmacokinetic and metabolic studies. Pharmacokinetics, and therefore drug analysis, plays a central role in both the development of new agents and also leads to a better understanding of clinically established agents in this class. Sample preparation and extraction methods including solid-phase and liquid-liquid extraction have also been highlighted. Many anthraquinone related compounds are highly coloured and fluoresce. They are suitable for a range of detection methods including UV-Vis, electrochemical and fluorescence. The methods described are used for sometimes complex separations that are needed for the evaluation of such compounds in biological samples.     

Quantitation of differential sensitivity of human-tumor stem cells to anticancer drugs.

With a direct in vitro tumor-colony assay developed to measure sensitity of human-tumor stem cells to anticancer drugs, we performed 32 retrospective or prospective clinical studies in nine patients with myeloma and nine with ovarian cancer treated with standard agents that were tested in vitro. The results were clearly correlated (P is less than 0.00001). Unique patterns of sensitivity and resistance to the six drugs tested were observed for individual patients. In eight cases of myeloma and three of obarian carcinoma in vitro sensitivity corresponded with in vivo sensitivity whereas in one case of myeloma it did not. In vitro resistance correlated with clinical resistance in all five comparisons in myeloma and all 15 in ovarian cancer. We conclude that this assay shows sufficient promise to warrant larger-scale testing to determine its efficacy for selection of new agents and individualized cancer chemotherapy regimens.     

Experimental Study of Ultralow-Dose Antibodies to Cyclophosphamide on Cyclophosphamide Myelotoxicity

The possibility of using ultralow-dose cyclophosphamide for reducing the myelotoxicity of cyclophosphamide, injected in the maximum permissible dose, was studied in mice. Combined treatment by the cytostatic and its ultralow-dose preparation led to a lesser suppression of the erythroid, lymphocytic, and particularly granulocytic hemopoiesis stems. This effect is explained by stimulation of the secretory activity of hemopoiesisinducing microenvironment and hence, of the functional activity of granulocytopoiesis under the effect of ultralow-dose cyclophosphamide. Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 147, No. 3, pp. 295–299, March, 2009