Preclinical evaluation of NUDT15-guided thiopurine therapy and its effects on toxicity and antileukemic efficacy.

Thiopurines (eg, 6-mercaptopurine [MP]) are highly efficacious antileukemic agents, but they are also associated with dose-limiting toxicities. Recent studies by us and others have identified inherited NUDT15 deficiency as a novel genetic cause of thiopurine toxicity, and there is a strong rationale for NUDT15-guided dose individualization to preemptively mitigate adverse effects of these drugs. Using CRISPR-Cas9 genome editing, we established a Nudt15-/- mouse model to evaluate the effectiveness of this strategy in vivo. Across MP dosages, Nudt15-/- mice experienced severe leukopenia, rapid weight loss, earlier death resulting from toxicity, and more bone marrow hypocellularity compared with wild-type mice. Nudt15-/- mice also showed excessive accumulation of a thiopurine active metabolite (ie, DNA-incorporated thioguanine nucleotides [DNA-TG]) in an MP dose-dependent fashion, as a plausible cause of increased toxicity. MP dose reduction effectively normalized systemic exposure to DNA-TG in Nudt15-/- mice and largely eliminated Nudt15 deficiency-mediated toxicity. In 95 children with acute lymphoblastic leukemia, MP dose adjustment also directly led to alteration in DNA-TG levels, the effects of which were proportional to the degree of NUDT15 deficiency. Using leukemia-bearing mice with concordant Nudt15 genotype in leukemia and host, we also confirmed that therapeutic efficacy was preserved in Nudt15-/- mice receiving a reduced MP dose compared with Nudt15+/+ counterparts exposed to a standard dose. In conclusion, we demonstrated that NUDT15 genotype-guided MP dose individualization can preemptively mitigate toxicity without compromising therapeutic efficacy.

The protective effect of Capparis ovata on 6-mercaptopurine-induced hepatotoxicity and oxidative stress in rats.

Capparis ovata is a member of Capparidacaeae family has been used in phytomedicine with a lot of positive effects such as an antioxidative, antihyperlipidemic, anti-inflammatory, and antihepatotoxic agent. The aim of this study was to research the protective effect of C. ovata on 6-mercaptopurine (6-MP) induced to hepatotoxicity and oxidative stress in rats. The rats were divided into 4 groups: control, 6-MP, C. ovataovate, and 6-MP + C. ovata. A complete blood count was performed, liver function test and antioxidant enzymes levels such as superoxide dismutase, glutathione peroxidase, catalase, and malondialdehyde were measured in blood before and after a 14-day test period. White blood cell and platelet counts were lower in the 6-MP group than other 3 groups (P < 0.005). Hepatic transaminase levels were higher in 6-MP group than the 3 groups (P < 0.05). Superoxide dismutase, glutathione peroxidase, and CAT levels were lower and malondialdehyde was higher in blood samples in 6-MP group than other 3 groups (P < 0.005). In conclusion, our tests were showed that C. ovata may be useful in patients receiving 6-MP therapy to prevent hepatotoxicity and in order to maintain uninterrupted therapy possibly reducing the risk of relapse. Although additional studies ensure that Capparis does not affect 6-MP antileukemic activity. We believe these results are important contribution to the literature.

Design and in vitro evaluation of branched peptide conjugates: turning nonspecific cytotoxic drugs into tumor-selective agents.

The use of peptide receptors as targets for tumor-selective therapies was envisaged years ago with the findings that receptors for different endogenous regulatory peptides are overexpressed in several primary and metastatic human tumors, and can be used as tumor antigens. Branched peptides can retain or even increase, through multivalent binding, the biological activity of a peptide and are very resistant to proteolysis, thus having a markedly higher in vivo activity compared with the corresponding monomeric peptides. Oligo-branched peptides, containing the human regulatory peptide neurotensin (NT) sequence, have been used as tumor-specific targeting agents. These peptides are able to selectively and specifically deliver effector units, for cell imaging or killing, to tumor cells that overexpress NT receptors. Results obtained with branched NT conjugated to different functional units for tumor imaging and therapy indicate that branched peptides are promising novel multifunctional targeting molecules. This study is focused on the role of the releasing pattern of drug-conjugated branched NT peptides. We present results obtained with oligo-branched neurotensin peptides conjugated to 6-mercaptopurin (6-MP), combretastain A-4 (CA4) and monastrol (MON). Drugs were conjugated to oligo-branched neurotensin through different linkers, and the mode-of-release, together with cytotoxicity, was studied in different human cancer cell lines. The results show that branched peptides are very promising pharmacodelivery options. Among our drug-armed branched peptides, NT4-CA4 was identified as a candidate for further development and evaluation in preclinical pharmacokinetic and pharmacodynamic studies. This peptide-drug exhibits significant activity against pancreas and prostate human cancer cells. Consequently, this derivative is of considerable interest due to the high mortality rates of pancreas neuroendocrine tumors and the high incidence of prostate cancer.

[Phytocorrection of structural changes in the gastrointestinal tract caused by 6-mercaptopurine].

It was shown that the course use of Ventrofit, a complex plant remedy, normalized the structural and functional integrity of the stomach and thin intestine mucous membranes damaged by the use of 6-mercaptopurine.

The nucleoside and nucleotide mixture (OG-VI) rescues intestinal-like epithelial cells from the cytotoxicity of chemotherapeutic agents.

Immune cells and cells undergoing rapid turn-over can obtain exogenous nucleotides via salvage synthesis. We evaluated whether or not the balanced nucleoside and nucleotide mixture OG-VI, could rescue intestinal epithelial-like Caco-2 cells from the cytotoxic effects of several chemotherapeutic agents, in the presence and absence of glutamine (Gln). Cells were exposed to 5-fluorouracil (5FU), methotrexate (MTX) or 6-mercaptopurine (6MP), after which proliferation and cell cycle analyses were performed. Following exposure to the chemotherapeutic agents, we observed that cells treated with OG-VI proliferated well, whereas those without the supplement did not proliferate. Furthermore, following treatment with either 5FU or MTX, we observed that the number of cells in the G0/G1 phase decreased and those in the S phases increased. However, these cell cycle alterations were prevented by the addition of OG-VI. With the exception of 6MP-treated cells, we did not observe any effects on proliferation or cell cycle regulation that could be ascribed to the presence of Gln. Thus, we have demonstrated that OG-VI rescues cells from the cytotoxic effects of several chemotherapeutic agents.

Modification of the clastogenic activity of X-ray and 6-mercaptopurine in mice by prefeeding with vitamins C and E.

The effect of a 30-d pretreatment with vitamin C (L-ascorbic acid) in the drinking water and vitamin E (all-rac-alpha-tocopherol) in the diet on the clastogenic activity induced by X-rays and 6-mercaptopurine was investigated in female ICR/Jcl mice by the bone-marrow micronucleus test. Prefeeding with vitamin E-deficient diets led to a significant decrease in serum vitamin E concentration and to an enhancement of micronucleus formation by X-rays in bone marrow cells. Although dietary supplementation with vitamin E significantly increased the vitamin E concentration in serum, it did not affect the frequency of X-ray-induced micronuclei. Treatment with a high level of vitamin C in drinking water was effective in protecting against micronucleus formation by X-rays. The increase in micronucleus frequency in the vitamin E-deficient mice compared with the mice fed vitamin E-normal diets was no longer observed when a high level of vitamin C in drinking water was given simultaneously. The most efficient protective action against X-rays was observed when vitamin E-supplemented diets and a high level of vitamin C in drinking water were used together as a pretreatment. Any combination of the vitamins did not affect the micronucleus induction by 6-mercaptopurine.

Ocular pharmacokinetics of subconjunctivally versus intravenously administered 6-mercaptopurine.

The levels of 6-mercaptopurine (6-MP) were measured in the anterior chamber aqueous, the vitreous and the serum of rabbits 0.5, 1, 2, 4, 8 and 12 hours following subconjunctival or intravenous injection of 10 mg/kg in 0.5 mL of saline, the maximum tolerated dose as determined experimentally. The mean peak concentrations of 6-MP in the aqueous and the vitreous respectively of the subconjunctivally injected eyes were 15 and 10 times those achieved in all the eyes following intravenous administration. The bioavailability of the drug over 12 hours was 21.67 micrograms/h in the aqueous and 22.22 micrograms/h in the vitreous following subconjunctival administration but only 1.47 and 3.50 micrograms/h respectively following intravenous administration. The serum concentration of 6-MP following subconjunctival injection was about half that following intravenous administration.

[Culture of mouse embryo for the toxicological evaluation of drugs in vitro. (1). Establishment of in vitro culture system and effect of 6-mercaptopurine on development of early stage embryo].

The basal conditions for culture of early mouse embryo in vitro were examined for drug evaluation. Among the media tested, BMOC-III was most suitable for mouse embryo culture. Changing the medium at 1 or 3 hr after culture was harmful to the survival and/or development of early staged embryos, whereas at 12 or 24 hr after culture, changing of the medium showed slight effect on embryo development. Eight-cell staged embryos were more resistant to changing medium that two-cell staged ones. Preliminary work using a suitable culture system established that exposure to 6-mercaptopurine for 12 or 24 hr resulted in a prolonged developmental rate at each stage. The effects showed a dose-dependent relationship with the 24 hr exposure. It was suggested that inhibition of the developmental rate during exposure resulted from a direct toxic effect on the cell and that the inhibition after exposure resulted from metabolic toxicity.

Screening of cytotoxic drugs for residual bone marrow damage.

Several cytotoxic drugs were tested for their ability to produce permanent residual damage to the bone marrow. A short course of the drug was given to BALB/c female mice, and the numbers of various types of bone marrow cells were determined at least two months later. Evidence of residual damage was found after administration of busulfan and 1,3-bis(chloroethyl)-1-nitrosourea, but not after administration of cyclophosphamide, 5-fluorouracil, 6-mercaptopurine, methotrexate, or vinblastine.