Deoxynucleoside Therapy for Thymidine Kinase 2-Deficient Myopathy.

OBJECTIVE: Thymidine kinase 2, encoded by the nuclear gene TK2, is required for mitochondrial DNA maintenance. Autosomal recessive TK2 mutations cause depletion and multiple deletions of mtDNA that manifest predominantly as a myopathy usually beginning in childhood and progressing relentlessly. We investigated the safety and efficacy of deoxynucleoside monophosphate and deoxynucleoside therapies. METHODS: We administered deoxynucleoside monophosphates and deoxynucleoside to 16 TK2-deficient patients under a compassionate use program. RESULTS: In 5 patients with early onset and severe disease, survival and motor functions were better than historically untreated patients. In 11 childhood and adult onset patients, clinical measures stabilized or improved. Three of 8 patients who were nonambulatory at baseline gained the ability to walk on therapy; 4 of 5 patients who required enteric nutrition were able to discontinue feeding tube use; and 1 of 9 patients who required mechanical ventilation became able to breathe independently. In motor functional scales, improvements were observed in the 6-minute walk test performance in 7 of 8 subjects, Egen Klassifikation in 2 of 3, and North Star Ambulatory Assessment in all 5 tested. Baseline elevated serum growth differentiation factor 15 levels decreased with treatment in all 7 patients tested. A side effect observed in 8 of the 16 patients was dose-dependent diarrhea, which did not require withdrawal of treatment. Among 12 other TK2 patients treated with deoxynucleoside, 2 adults developed elevated liver enzymes that normalized following discontinuation of therapy. INTERPRETATION: This open-label study indicates favorable side effect profiles and clinical efficacy of deoxynucleoside monophosphate and deoxynucleoside therapies for TK2 deficiency. ANN NEUROL 2019;86:293-303.

Role of antiviral treatment for HCC prevention.

Chemoprevention of hepatocellular carcinoma (HCC) is listed as a yet highly debated long-term benefit of a successful treatment of patients with chronic viral hepatitis. In the hepatitis B virus (HBV) arena, the retrospective scrutiny of both interferon and nucleos(t)ide analogues (NUC) studies failed to provide robust evidence for HCC chemoprevention, due to a number of confoundings in the studies that were originally designed to assess the antiviral activity of interferon therapy. However, the reanalysis of outcomes following patients stratification for risk factors of HCC, provided a clue to find an association between NUC therapy and a reduced risk of liver cancer in non cirrhotic patients, only. In the hepatitis C scenario, a meta analysis of 30 observational studies of patients treated with interferon demonstrated a more than 70% reduction of HCC risk occurring independently of severity of underlying liver fibrosis which was less pronounced in aged patients and those with more advanced liver fibrosis. While the reasons for the residual risk of HCC in virological responders remain largely unexplained, international societies recommend surveillance for HCC of both HBV and HCV responders to antiviral therapy.

Administration of deoxyribonucleosides or inhibition of their catabolism as a pharmacological approach for mitochondrial DNA depletion syndrome.

Mitochondrial DNA (mtDNA) depletion syndrome (MDS) is characterized by a reduction in mtDNA copy number and consequent mitochondrial dysfunction in affected tissues. A subgroup of MDS is caused by mutations in genes that disrupt deoxyribonucleotide metabolism, which ultimately leads to limited availability of one or several deoxyribonucleoside triphosphates (dNTPs), and subsequent mtDNA depletion. Here, using in vitro experimental approaches (primary cell culture of deoxyguanosine kinase-deficient cells and thymidine-induced mtDNA depletion in culture as a model of mitochondrial neurogastrointestinal encephalomyopathy, MNGIE), we show that supplements of those deoxyribonucleosides (dNs) involved in each biochemical defect (deoxyguanosine or deoxycytidine, dCtd) prevents mtDNA copy number reduction. Similar effects can be obtained by specific inhibition of dN catabolism using tetrahydrouridine (THU; inhibitor of cytidine deaminase) or immucillin H (inhibitor of purine nucleoside phosphorylase). In addition, using an MNGIE animal model, we provide evidence that mitochondrial dNTP content can be modulated in vivo by systemic administration of dCtd or THU. In spite of the severity associated with diseases due to defects in mtDNA replication, there are currently no effective therapeutic options available. Only in the case of MNGIE, allogeneic hematopoietic stem cell transplantation has proven efficient as a long-term therapeutic strategy. We propose increasing cellular availability of the deficient dNTP precursor by direct administration of the dN or inhibition of its catabolism, as a potential treatment for mtDNA depletion syndrome caused by defects in dNTP metabolism.

Acute intravenous infusion of an adenosine regulating agent improves left ventricular function in dogs with advanced heart failure.

PURPOSE: GP531 is a second generation adenosine regulating agent (ARA) that increases concentrations of endogenous adenosine, a natural cardioprotective agent, in ischemic/hypoxic tissue. This study examined the effects of acute intravenous infusions of GP531 on left ventricular (LV) systolic and diastolic function in dogs with advanced chronic heart failure (HF) (LV ejection fraction, EF <30 %). METHODS: Six dogs with intracoronary microembolization-induced HF received a constant intravenous infusion of GP531 (10 µg/kg/min) or vehicle (normal saline) for 6 h in random order 1 week apart. Hemodynamic measurements were made at baseline and at 1, 2, 3, 4, 5 and 6 h after initiating drug infusion. Myocardial oxygen consumption (MVO2) was measured at baseline and 4 and 6 h. LV pressure-volume relationship (PVR) was measured at baseline and 6 h. RESULTS: Vehicle infusions had no effect on indexes of LV systolic and diastolic function. GP531 infusion had no effect on heart rate or mean aortic pressure but significantly decreased LV end-diastolic pressure, end-diastolic volume, end-systolic volume and end-diastolic wall stress. GP531 significantly increased LV EF (27 ± 1 at baseline to 34 ± 1 after 6 h of drug infusion, p < 0.05), deceleration time of early mitral inflow velocity and the slope of end-systolic PVR without increasing MVO2. CONCLUSIONS: Results of the study indicate that approaches which increase the local release of adenosine in failing LV myocardium, such as ARAs, have a favorable impact on LV performance. These observations support the continued development of ARA's for the treatment of acute HF syndromes.

Deoxycytidine kinase is overexpressed in poor outcome breast cancer and determines responsiveness to nucleoside analogs.

Only a minority of breast cancer patients responds to chemotherapy and we lack predictive biomarkers that help to select a patient-tailored therapy that takes into consideration the molecular heterogeneity of the cancer type. Responsiveness to the clinically important nucleoside analogs gemcitabine and decitabine may be critically determined by Deoxycytidine kinase (DCK) expression as this enzyme is required to convert the inactive prodrugs into their pharmacologically active forms. Here, we examined whether DCK is differentially expressed in breast cancer and evaluated whether DCK expression levels control responsiveness to these nucleoside analogs in vitro by experimentally modulating DCK expression levels. We examined DCK expression in gene expression data sets of breast tumors including the series of 295 consecutive patients that have been classified into low or high risk for recurrence using the MammaPrint 70 gene profile. We found that DCK is expressed at higher levels in patients having poor clinical outcome as judged by the MammaPrint assay. As such, patients that have a poor prognosis may thus be susceptible to treatment with nucleoside analogs. In support of this, we found a causal relationship between DCK levels and sensitivity to these nucleoside analogs in breast cancer cell lines. The data indicate that breast cancers that are at high risk of recurrence express higher levels of DCK, which we find to be strongly correlated to a favorable response to nucleoside analogs. The data suggest that DCK expression in breast cancer could be exploited to select patients that are likely to respond to treatment with nucleoside analogs.

Prevalence of the mutational pattern E44D/A and/or V118I in the reverse transcriptase (RT) gene of HIV-1 in relation to treatment with nucleoside analogue RT inhibitors.

It has been reported that a new pattern of mutations, E44D/A and/or V118I, in the reverse transcriptase (RT) gene of HIV-1 confers a moderate level of resistance to lamivudine in the absence of the M184V mutation. The prevalence of this mutational pattern was studied in HIV-1 isolates obtained from 280 patients. These mutations were not identified in the RT sequences from 23 antiretroviral-naive patients but were detected in 82 (31.9%) of the 257 RT sequences obtained from nucleoside reverse transcriptase inhibitors (NRTI)-experienced patients. Mutation at codon 44 was identified in 41 patients (7 mutations E44A and 34 mutations E44D), mutation V118I was identified in 73 patients and a combination of mutations at codons 44 and 118 was found in 32 patients. Multivariate analysis showed an association between the E44D/A and/or V118I mutational pattern and the RT mutations D67N, T69D, L210W, and T215Y/F. No relationship was observed between this mutational pattern and the lamivudine-specific resistance mutation M184V. The prevalence of these mutations increased significantly with the number of drug regimens experienced and a prevalence of 42.4% was observed in patients who had received >or= 4 antiretroviral regimens. A relationship was found between the E44D/A and/or V118I mutational pattern and experience with didanosine or stavudine but not with lamivudine. The results suggest that the development of the E44D/A and/or V118I mutational pattern is frequent in patients treated with NRTIs. Thymidine analogues and didanosine, but not lamivudine, could promote the development of these mutations.

Ototoxicity associated with use of nucleoside analog reverse transcriptase inhibitors: a report of 3 possible cases and review of the literature.

Although a variety of adverse effects have been attributed to treatment with nucleoside analog reverse transcriptase inhibitors (NRTIs) for human immunodeficiency virus type 1 (HIV-1) infection, only 5 cases of ototoxicity have been reported in the literature. We describe 3 additional cases of possible NRTI-associated ototoxicity in HIV-1-infected patients, all of whom were aged >45 years, had a history of noise-induced hearing loss, and reported tinnitus and deterioration in hearing in the setting of antiretroviral therapy. Reductions in mitochondrial DNA content induced by NRTIs, as well as mitochondrial DNA mutations associated with aging and HIV-1 infection, all may contribute to auditory dysfunction in older patients with HIV-1 infection. Prospective studies are necessary to determine the incidence of tinnitus and hearing loss among HIV-1-infected patients and their relationship to the use of NRTIs.

Boron substituted deoxyribonucleosides as cytotoxic agents.

Base substituted boronated nucleosides and phosphate modified nucleotides were examined for their cytotoxic activity in both murine and human tissue cultured cancer cells. These derivatives demonstrated better activity against the growth of single cell suspensions than solid cell tumor cell growth. A detailed mode of action study showed that 2'deoxyriboadenosine-N7-cyanoborane 6 suppressed Tmolt3 DNA synthesis preferentially with the major target of the agent being the purine de novo pathway. The activities of one of the regulatory enzymes of the pathway were reduced by the agents, i.e. PRPP-amido transferase. Other sites in the cell which were moderately affected by the agent were nucleoside kinase activities. DNA polymerase alpha and dihydrofolate reductase activities. The DNA molecule itself did not appear to be a target of the compound.

Purine and 1-deazapurine ribonucleosides and deoxyribonucleosides: synthesis and biological activity.

A series of 6-(hydroxylamino)purine and -1-deazapurine nucleosides were synthesized and tested for their antitumor and adenosine deaminase inhibitory activity. All the examined molecules displayed an in vitro activity comparable to that of the reference compounds 6-(hydroxylamino)-9-beta-D-ribofuranosylpurine (HAPR) and ara-A, their ID50 ranging from 0.9 microM to approximately 100 microM. The 6-hydroxylamino derivatives of 1-deazapurine 9, 12, and 17 and also the blocked compound 13 are inhibitors of ADA whereas the purine derivatives 4 and 6 and the nitro compounds 11 and 16 are resistant to the enzyme. 7-(Hydroxylamino)-3-(2-deoxy-beta-D-erythro-pentofuranosyl)-3H-imi dazo[4,5- b]pyridine, the less cytotoxic but the most active ADA inhibitor in the series (Ki = 2.7 x 10(-7)), greatly potentiates the antitumor activity of ara-A in vitro.

[The modification of radiation effect by nucleoside related compounds on a murine tumor given with local radiotherapy and on survival following whole body irradiation].

We have evaluated the effect of nucleoside related compounds on a murine tumor given with local radiotherapy and on the survival rate following whole body irradiation. C3H/He mouse transplanted FM3A tumor and ddY mice were used. The enhancement ratio (ER) for the tumor treated by radiation and nucleoside related compounds was 1.02-1.67. For the normal tissue, Ara-A and BVAU given with radiation had no effect on the survival rate. In the combination of 50-800 mg/kg of ACV and whole body irradiation, the 50% survival day after 7 Gy was shorter than that of radiation alone. When 25-400 mg/kg of 3'-dG was given with radiation, the 50% survival day was longer. When combined with radiation, LD50/30 for ACV and 3'-dG were 3.9 and 6.3 Gy, respectively. ACV showed radiosensitizing effect with Dose modifying factor (DMF) at 1.1, on the other hand, 3'-dG showed radioprotective effect with DMF at 1.47. Our results suggested that some nucleoside analogues might be useful as radiosensitizer.

Preparation and antileukemic screening of some new 6'-deoxyhexopyranosyladenine nucleosides.

9-beta-D-Fucopyranosyladenine (1) has weak antileukemic activity against L1210 cells grown in culture. Several new 6'-deoxyhexopyranosyladenine nucleosides were synthesized by standard procedures and assayed for activity. The new nucleosides were 9-(6-deoxy-beta-D-glucopyranosyl)adenine (2), 9-(6-deoxy-beta-D-allopyranosyl)adenine (3), 9-(6-deoxy-alpha-L-talopyranosyl)adenine (4), 9-alpha-D-rhamnopyranosyladenine (5), and 9-(6-deoxy-alpha-L-idopyranosyl)adenine (6). In addition, 9-(6-deoxy-alpha-L-sorbofuranosyl)adenine (7) was isolated from the same preparation as 6. None of the new nucleosides 2-7 had activity against L1210 cells in culture. A number of other known nucleosides related in structure to 1 were also tested for activity. One of these, 9-alpha-L-arabinopyranosyladenine, had activity, but was significantly weaker than 1.

Developmental therapeutics and the acquired immunodeficiency syndrome.

Patients with the acquired immunodeficiency syndrome (AIDS) die of overwhelming infections as a consequence of the destruction of the T4 subset of lymphocytes. Approaches to the treatment of AIDS have involved attempts to reestablish immune competence as well as treat opportunistic infections. The discovery of the human T-lymphotropic virus type III, which causes AIDS, has provided a specific target for screening antiviral drugs. There are many potential screening targets, from surface-binding proteins to viral integration and assembly, but most of the recent efforts have been aimed at developing drugs to inhibit the unique viral DNA polymerase (reverse transcriptase). The early studies with 3'-azido-3'-deoxythymidine (AZT) have provided encouraging results.

Strategies for antiviral therapy in AIDS.

The retrovirus that causes AIDS has revealed enough of its life history for a variety of therapeutic strategies to be apparent. Some of these are suitable for immediate application in clinical trials or have already yielded positive results in some patients.

Synthesis, cell growth inhibition, and antitumor screening of 2-(p-n-butylanilino)purines and their nucleoside analogues.

Derivatives of N2-(p-n-butylphenyl)guanine (BuPG) and 2-(p-n-butylanilino)adenine (BuAA) were synthesized and tested as inhibitors of mammalian DNA polymerase alpha, cell growth, and macromolecule synthesis. 2-(p-n-Butylanilino)-6-chloropurine (BuACl) served as a useful intermediate to prepare a series of 6-substituted analogues. BuACl, as its sodium salt, reacted with 2-deoxy-3,5-di-p-toluoyl-beta-D-ribofuranosyl chloride in acetonitrile to give 64% of the corresponding 9-beta nucleoside (blocked BuAdCl) and only 14% of the 7-beta isomer. Deblocking and substitution of chlorine in BuAdCl generated a series of 2-(p-n-butylanilino)-9-(2-deoxy-beta-D-ribofuranosyl)purine derivatives. Reaction of the sodium salt of BuACl with (2-acetoxyethoxy)methyl bromide also afforded, after deblocking and substitution of the 6-chloro group, a series of 2-(p-n-butylanilino)-9-[(2-hydroxyethoxy)methyl]purines. The bases synthesized were inhibitors of DNA polymerase alpha isolated from Chinese hamster ovary cells, the most potent compounds being 6-methoxy and 6-methylthio derivatives of 2-(p-n-butylanilino)purine. When tested for their ability to inhibit [3H]thymidine incorporation into DNA in HeLa cell cultures and the growth of exponentially growing HeLa cells, 9-(2-deoxy-beta-D-ribofuranosyl) derivatives had greater potency than their base counterparts, but "adenine" analogues, such as 2-(p-n-butylanilino)-2'-deoxyadenosine (BuAdA, IC50 = 1 microM), were considerably more potent than N2-(p-n-butylphenyl)-2'-deoxyguanosine (BuPdG, IC50 = 25 microM). Derivatives bearing the 9-[(2-hydroxyethoxy)methyl] group were nearly as potent inhibitors of [3H]thymidine incorporation in these experiments as the corresponding deoxyribonucleosides. Base and deoxynucleoside derivatives also inhibited cellular RNA synthesis, and several compounds, at high concentrations, inhibited protein synthesis. BuPG, BuAA, and four deoxyribonucleoside derivatives of 2-(p-n-butylanilino)purines were tested against P-388 lymphocytic leukemia in mice. None of the compounds increased the survival time of test animals, but two of them, BuAdA and its 6-desamino derivative BuAdP, were lethal at the highest concentration used (400 mg/kg).

Therapeutic effects of 9-beta-D-arabinofuranosyladenine and 2'-deoxycoformycin combinations on intracerebral leukemia.

Drug combinations of 9-beta-D-arabinofuranosyladenine and 2'-deoxycoformycin were active in the therapy of mice with intracerebral implants of the L1210 tumor. In in vivo mouse brain adenosine deaminase studies, inhibition of 9-beta-D-arabinofuranosyladenine deamination for periods of up to 24 hr was found after a single i.p. dose of 0.002 mmole/kg.