Pediatric CIDP: Diagnosis and Management. A Single-Center Experience.

Background: Chronic inflammatory demyelinating polyneuropathy (CIDP) is a rare acquired polyneuropathy that especially among youngest children should be differentiated with hereditary neuropathies. Even though upon diagnosis treatment options are similar in children and adults, diagnostic challenges are faced in the pediatric population. Methods: We conducted a retrospective analysis of clinical symptoms, nerve conduction study results, modes of treatment, and final outcome in 37 children aged 3.5-17 years with a final diagnosis of CIDP (18 girls, 19 boys). We established three groups of patients based on age at onset of CIDP: 0-4, 4-13, and 13-18 years. Follow-up ranged from 10 to 222 months. Results: In our analysis, 19/37 patients (51.4%) had an atypical presentation: distal variant of CIDP in 12/37 patients (32.4%) and pure motor variant of CIDP in 5/37 patients (13.5%), and one patient had a pure sensory variant (1/37, 2.7%). Furthermore, 3/37 patients (8.1%) had additional concurring symptoms, including involuntary movements of face muscles (1/37, 2.7%) or hand tremor (2/37, 5.4%). During the follow-up, 23/37 patients (62.2%) received intravenous immunoglobulin (IVIg); 22/37 patients (59.5%) received steroids, 6/37 patients (16.2%) received IVIg and steroids, and 12/37 patients (32.4%) received immunosuppressive drugs, mostly azathioprine, but also methotrexate and rituximab. One patient was treated with plasmapheresis. Complete remission was achieved in 19/37 patients (51.4%) with CIDP in its typical form. Remission with residual symptoms or minimal deficit was observed in 4/37 patients (10.8%), whereas 14/37 patients (37.8%) remain on treatment with gradual improvement. Conclusion: Childhood CIDP may occur in its typical form, but even ~50% of children can present as an atypical variant including distal, pure motor, or pure sensory. Most children have a good prognosis; however, many of them may require long-term treatment. This highlights the importance of an early diagnosis and treatment for childhood CIDP.

Corrigendum: Pediatric CIDP: Diagnosis and Management. A Single-Center Experience.

[This corrects the article DOI: 10.3389/fneur.2021.667378.].

Chemical modification of melphalan as a key to improving treatment of haematological malignancies.

Chemical modification of known, effective drugs is one method to improve chemotherapy. Thus, the object of this study was to generate melphalan derivatives with improved cytotoxic activity in human cancer cells (RPMI8226, HL60 and THP1). Several melphalan derivatives were synthesised, modified in their two important functional groups. Nine analogues were tested, including melphalan compounds modified: only at the amino group, by replacing the amine with an amidine group containing a morpholine ring (MOR-MEL) or with an amidino group and dipropyl chain (DIPR-MEL); only at the carboxyl group to form methyl and ethyl esters of melphalan (EM-MEL, EE-MEL); and in a similar manner at both functional groups (EM-MOR-MEL, EE-MOR-MEL, EM-DIPR-MEL, EE-DIPR-MEL). Melphalan derivatives were evaluated for cytotoxicity (resazurin viability assay), genotoxicity (comet assay) and the ability to induce apoptosis (terminal deoxynucleotidyl transferase dUTP nick end labelling, TUNEL, phosphatidylserine externalisation, chromatin condensation, activity of caspases 3/7, 8 and 9 and intracellular concentration of calcium ions) in comparison with the parent drug. Almost all derivatives, with the exception of MOR-MEL and DIPR-MEL, were found to be more toxic than melphalan in all cell lines evaluated. Treatment of cultures with the derivatives generated a significant higher level of DNA breaks compared to those treated with melphalan, especially after longer incubation times. In addition, all the melphalan derivatives demonstrated a high apoptosis-inducing ability in acute monocytic and promyelocytic leukemia cells. This study showed that the mechanism of action of the tested compounds differed depending on the cell line, and allowed the selection of the most active compounds for further, more detailed investigations.

Antileukemic activity of novel adenosine derivatives.

The present study investigated the effect of cladribine (CLA) and six of its derivatives containing a formamidine group at position 6 (CLA-FDM, CLA-FPAZ, CLA-FPIR, CLA-FPIP, CLA-FHEX, and CLA-FMOR) on acute promyelocytic, lymphoblastic, and acute monocytic leukemia cells. The role of ATR kinase in deoxycytidine kinase (dCK) activation in response to DNA damage was assessed. The presence of DNA lesions was assessed by measurement phosphorylation of H2AX and by using the alkaline comet assay with proteinase K post-treatment following assessment of the cell cycle. Apoptotic events such as alterations in intracellular calcium concentration, caspase-3/7 activity and increased sub-G1 cell population were measured. CLA derivatives were highly effective against leukemic cells, showing high cytotoxicity, causing DNA fragmentation, and inducing DNA-protein cross-links in leukemic cells. CLA-FMOR showed the highest efficacy. CLA derivatives increased the levels of intracellular calcium ions, caspase-3/7 and the percentage of sub-G1 apoptotic cells and blocked cells in the S phase of the cell cycle to a greater extent than free CLA. The selective ATR inhibitor VE-821 significantly suppressed the increase in dCK activity and decreased basal dCK activity. The present results suggested that ATR kinase controls dCK activity in response to synthetic CLA derivatives.

Lipophilicity profiling of anthracycline antibiotics by microemulsion electrokinetic chromatography-effects on cardiotoxicity and endotheliotoxicity.

Accurate profiling of the lipophilicity of amphoteric compounds might be complex and laborious. In the present work the lipophilicity of 12 anthracycline antibiotics-four parent drugs: doxorubicin, daunorubicin, epidoxorubicin, and epidaunorubicin and eight novel formamidyne derivatives with attached morpholine, hexamethylenoimine or piperidine rings-was determined based on novel approach using MEEKC. In the second stage, lipophilicity was correlated with anthracycline toxicity towards two cell lines. In rat cardiomyoblast cell line (h9c2) a significant correlation between the logP and toxicity was found. The anthracycline lipophilicity was not correlated with toxicity towards the endothelial hybrid cell line (EAhy.926). In conclusion, the lipophilicity of anthracyclines seems to determine their toxicity towards cardiomyoblasts but not on endothelial cells, suggesting a different mechanism of anthracyclines intercellular transport or extrusion in cardiomyoblast and endothelial cells.

Novel oxazolinoanthracyclines as tumor cell growth inhibitors-Contribution of autophagy and apoptosis in solid tumor cells death.

Chemical modification of known, effective drugs are one method to improve the chemotherapy of tumors. We reported ability of oxazoline analogs of doxorubicin (O-DOX) and daunorubicin (O-DAU) to induce apoptosis and autophagy in ovarian and liver cancer cells. Reactive oxygen and nitrogen species (ROS and RNS, respectively), together with intracellular calcium-mediated downstream signaling, are essential for the anticancer effect of these new anthracycline analogs. The changes of mitochondrial membrane potential and induction of the ceramide pathway suggests that these compounds induce cell death by apoptosis. In addition, a significant increase of autophagosome formation was observed by fluorescence assay and acridine orange staining, indicating that the new analogs also induce autophagic cell death. Compared to free DOX- and DAU-treated cells, we observed inhibition of colony formation and migration, a time-dependency between ROS/RNS levels and a greater fall in mitochondrial membrane potential. Altogether, our research broadens the base of molecular oxazolinoanthracyclines targets and reveals that derivatives mediated oxidative stress, ceramide production and increase in intracellular calcium level by mitochondria. Furthermore, our data highlight the importance of mitochondria that simultaneously assume the role of activator of autophagy and apoptosis signals.

Molecular mechanism of action of oxazolinoanthracyclines in cells derived from human solid tumors. Part 2.

BACKGROUND/AIM: Oxazolinodoxorubicin (O-DOX) and oxazolinodaunorubicin (O-DAU) are derivatives of anthracyclines (DOX and DAU) with a modified daunosamine moiety. We aimed to clarify their mechanisms of action by investigating intracellular accumulation and effects on the cell cycle, phosphatidylserine externalization, and proteasome 20S activity. MATERIALS AND METHODS: Experimental model consisted of SKOV-3, A549 and HepG2 cells. Compounds were used at the concentration of 80nM. Intracellular accumulation, drug uptake, and proteasome 20S activity were evaluated by fluorimetric methods. The effects on the cell cycle and phosphatidylserine externalization were measured by flow cytometry. RESULTS: O-DOX was equivalent to DOX in terms of inducing G2/M arrest, but O-DAU was less potent in SKOV-3, HepG2, and A549 cells. O-DOX had the greatest effect on initiating apoptosis in all tested cells. Externalization of phosphatidylserine was significantly higher following O-DOX treatment compared with control cells and cells incubated with DOX. The intracellular accumulation and uptake of the derivatives were similar to those of the reference drugs. Tested compounds are able to activate proteasome 20S activity. CONCLUSION: Our results extended the understanding of the toxicity, mechanism of action, and biochemical properties of oxazoline derivatives of doxorubicin and daunorubicin, including their effects on cell cycle, apoptosis and DNA degradation.

In Vitro Antileukemic Activity of Formamidine Epidoxorubicin Analogs.

BACKGROUND/AIM: Epidoxorubicin is an anthracycline agent. The present study was undertaken to compare the antileukemic potential of epidoxorubicin and its two formamidine analogs containing either a morpholine moiety (EPIFmor) or a hexamethyleneimine moiety (EPIFhex) in the amidine group. MATERIALS AND METHODS: The experiments were performed in vitro on MOLT-4 cells using spectrophotometry, Coulter electrical impedance, flow cytometry, and light microscopy methods. RESULTS: The leukemia cell responses to the action of the anthracyclines were manifested in their different viability, count and volume, degree of apoptosis and necrosis, activity of caspases -8, -9, and -3/7, mitochondrial membrane potential, and in the cell-cycle distribution. In general, epidoxorubicin appeared to be the most active, and EPIFmor was more active than EPIFhex against MOLT-4 cells. CONCLUSION: The structural modifications of epidoxorubicin in the amidine group were responsible for the varied action of its formamidine analogs on human acute lymphoblastic leukemia cells.

Effects of structural modification of the daunosamine moiety of anthracycline antibiotics on pKa values determined by capillary zone electrophoresis.

The thermodynamic acid dissociation constants (pKa1 and pKa2) of 16 anthracycline antibiotics, including doxorubicin (DOX) and daunorubicin (DAU), their epimers, epidoxorubicin (EDOX) and epidaunorubicin (EDAU), as well as novel anthracycline derivatives containing piperidine (FPIP), morpholine (FMOR) and hexamethylenoimine (FHEX) rings in the formamidine group of the daunosamine moiety were determined by analysis of the dependence between measured electrophoretic mobility and the pH of the buffer using the capillary zone electrophoresis method. The results obtained confirmed the ampholytic character of anthracyclines with at least two ionization states. The determined values were in the range of 8.36-9.28 and 9.38-11.48 for pKa1 and pKa2 arising from ionization of amino and phenolic groups, respectively. Structural modifications in the daunosamine moiety of the studied anthracyclines affected their pharmacological properties, such as antiproliferative activity.

Dopa-responsive dystonia or early-onset Parkinson disease - Genotype-phenotype correlation.

OBJECTIVE: Dopa-responsive dystonia (DRD) is a rare form of hereditary movement disorder with onset in childhood, characterized by gait difficulties due to postural dystonia with marked improvement after low doses of levodopa. Mutations in the GCH1 gene are the most common cause of DRD, however, in some cases when the disease is associated with parkinsonism mutations in the PARK2 gene may be identified. The aim of this study was to analyze and compare genotype-phenotype correlation. MATERIAL/PARTICIPANTS: Four families with inter- and intrafamilial variability of progressive gait dysfunction due to lower limb dystonia occurring in childhood or adolescence were included in the analysis. METHODS: General and neurological examination was performed for all affected family members and asymptomatic mutation carriers. The molecular analysis encompassed GCH1 and PARK2 genes. RESULTS: All probands were clinically diagnosed with DRD. The molecular analysis revealed, however, that the dopa-responsive dystonia phenotype was caused by a mutation in the GCH1 gene in three families and in the PARK2 gene in one family. Obtained results allowed to establish the final diagnosis for all families as DYT5a or early-onset Parkinson disease (EO-PD). CONCLUSIONS: Reported cases confirm that the DRD phenotype may have heterogeneous genetic background and may be caused by point mutations or rearrangements in the GCH1 gene as well as in the PARK2 gene. Differential diagnosis and genetic tests covering the analysis of genes causative for DRD and EO-PD should be obligatory in both disorders diagnostics as DRD, mainly adolescent onset dystonia, may be associated with parkinsonism.

Identification of the key pathway of oxazolinoanthracyclines mechanism of action in cells derived from human solid tumors.

Oxazolinodoxorubicin (O-DOX) and oxazolinodaunorubicin (O-DAU) are novel anthracycline derivatives with a modified daunosamine moiety. In the present study, we evaluated the cytotoxicities, genotoxicities and abilities of O-DOX and O-DAU to induce apoptosis in cancer cell lines (SKOV-3; A549; HepG2), and compared the results with their parent drugs. We assessed antiproliferative activity by MTT assay. We evaluated apoptosis-inducing ability by double-staining with fluorescent probes (Hoechst 33258/propidium iodide), and by determining expression levels of genes involved in programmed cell death by reverse transcription-polymerase chain reaction. Genotoxicities of the compounds were tested by comet assays. Oxazolinoanthracyclines demonstrated high anti-tumor activity. O-DOX had significantly higher cytotoxicity, apoptosis-inducing ability, and genotoxicity compared with parental doxorubicin (DOX) in all tested conditions, while O-DAU activity differed among cell lines. The mechanism of oxazoline analog action appeared to involve the mitochondrial pathway of programmed cell death. These results provide further information about oxazoline derivatives of commonly used anthracycline chemotherapy agents. O-DOX and O-DAU have the ability to induce apoptosis in tumor cells.

Spectroscopic studies of anthracyclines: Structural characterization and in vitro tracking.

A broad spectroscopic characterization, using ultraviolet-visible (UV-vis) and Fourier transform infrared absorption as well as Raman scattering, of two commonly used anthracyclines antibiotics (DOX) daunorubicin (DNR), their epimers (EDOX, EDNR) and ten selected analogs is presented. The paper serves as a comprehensive spectral library of UV-vis, IR and Raman spectra of anthracyclines in the solid state and in solution. The particular advantage of Raman spectroscopy for the measurement and analysis of individual antibiotics is demonstrated. Raman spectroscopy can be used to monitor the in vitro uptake and distribution of the drug in cells, using both 488nm and 785nm as source wavelengths, with submicrometer spatial resolution, although the cellular accumulation of the drug is different in each case. The high information content of Raman spectra allows studies of the drug-cell interactions, and so the method seems very suitable for monitoring drug uptake and mechanisms of interaction with cellular compartments at the subcellular level.

Subcellular localization of anthracyclines in cultured rat cardiomyoblasts as possible predictors of cardiotoxicity.

In this study, we compared the cellular uptake, intracellular localization and cytotoxicity of two groups of anthracycline derivatives in cultured H9c2(2-1) rat cardiomyoblasts. The first group consisted of doxorubicin (DOX) and two of its derivatives containing a formamidino group (-N = CH-N<) at the C-3' position with a morpholine (DOXM) or a hexamethyleneimine (DOXH) ring. The second group consisted of daunorubicin (DRB) and its derivatives containing a morpholine (DRBM) or a hexamethyleneimine (DRBH) ring. DOXH and DRBH were taken up by cardiomyoblasts more efficiently than estimated for other tested anthracyclines. The cellular uptakes of DOXM and DRBM were reduced compared to those of the parent compounds. Applied structural modifications of DOX and DRB influenced the subcellular localization of the tested derivatives. DOX and DOXH were localized primarily in nuclei, whereas the other anthracyclines were found in the nuclei and cytoplasm. The percentages of the compounds that accumulated in the nuclei were 80.2 and 54.2 % for DOX and DOXH, respectively. The lowest nuclear accumulation values were observed for DRBM (19.9 %), DRBH (21.9 %) and DOXM (23.7 %). The ability of anthracyclines to accumulate in the nuclei correlated with their DNA binding constants (r = 0.858, P = 0.029). A correlation was found between the accumulation of the tested anthracyclines in the nuclei of cardiomyoblasts and their cardiotoxicity in vivo, which was observed in our previous study. We suggest that cytotoxicity and the anthracycline accumulation level in the nuclei of cultured cardiomyoblasts could be used for early prediction of their cardiotoxicity.

Formamidinodoxorubicins are more potent than doxorubicin as apoptosis inducers in human breast cancer cells.

BACKGROUND/AIM: The ability of five formamidinodoxorubicins to induce apoptosis of MCF-7 breast cancer cells was tested. All these compounds were modified at C-3' and contain a formamidine group (-N=CH-NRR), with the rest of the cyclic secondary amine (HNRR) of a gradually increasing ring size. MATERIALS AND METHODS: Cytotoxicity was assessed using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. To analyze apoptosis, double staining using fluorescence probes Hoechst 33258/propidium iodide (PI) and annexin V- Fluorescein isothiocyanate/PI was carried-out. Additionally, the TdT-mediated dUTP nick-end labelling test and activity of caspase 3 were determined. RESULTS: The four tested derivatives displayed a significant increase in antiproliferative activity in comparison to doxorubicin. All of the tested derivatives induced caspase-dependent apoptosis of MCF-7 cells. CONCLUSION: DOX-F MOR and DOX-F PAZ analogs are more potent apoptosis inducers than doxorubicin.

Cytotoxicity and induction of apoptosis by formamidinodoxorubicins in comparison to doxorubicin in human ovarian adenocarcinoma cells.

BACKGROUND/AIM: In this study we investigated the effect of DOX and five of its derivatives containing a formamidine group (NCHNRR) at the 3' position with pyrrolidine (DOX-F PYR), piperidine (DOX-F PIP), morpholine (DOX-F MOR), N-methylpiperazine (DOX-F PAZ) and hexamethyleneimine (DOX-F HEX) ring on SKOV-3 ovarian cancer cells. We have focused on the anti-proliferative activity and the value of apoptosis induced by tested analogues. MATERIALS AND METHODS: The following methods were used: spectrophotometric assay with MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide); fluorimetric assays - double staining with Hoechst 33258 and propidium iodide (PI), measurement of caspase-3 activity; flow cytometry methods - phosphatidylserine (PS) externalization using Annexin V-FITC and PI fluorochromes, and TUNEL assay. RESULTS: All of the investigated derivatives were considerably more cytotoxic to the SKOV-3 cell line than DOX. The predominant type of cell death induced by the anthracycline analogues was apoptosis. Necrotic cells represented only a small percentage (<5%) of all cells. The number of apoptotic cells was dependent on the compound and the incubation time. Moreover, a significant increase in caspase-3 activity, DNA fragmentation, and morphological changes in ovarian cells were observed predominantly in new DOX analogues. CONCLUSIONS: All new formamidine derivatives of DOX were effective against ovarian cancer cells. They induced mainly the apoptotic pathway of cell death mediated by caspase-3. The most promising results were obtained for DOX-F MOR and DOX-F PAZ. The least potent was DOX-F HEX.

Cell-cycle disturbance and induction of programmed death by new formamidine analogs of daunorubicin.

BACKGROUND/AIM: Structural modifications of daunorubicin are an important way to change its anticancer activity. For this reason, formamidinodaunorubicins have been synthesized. The present study was undertaken to determine and compare the in vitro effects of daunorubicin and its new formamidine derivatives on human acute leukemia MOLT-4 and ML-1 cells. MATERIALS AND METHODS: The experiments were performed on human acute lymphoblastic leukemia MOLT-4 cells and human acute myeloblastic leukemia ML-1 cells. The study was conducted using flow cytometry and light microscopy methods. RESULTS: The various patterns of temporary changes in the cell cycle and DNA fragmentation, as well as the extent of mitotic catastrophe, apoptosis, and necrosis, were determined. The anti-leukemic activities of the new daunorubicin analogs were weaker than that of daunorubicin. CONCLUSION: The influence of these anthracyclines on cell-cycle progression, DNA damage, and induction of mitotic catastrophe and cell death depended on the agent and its concentration, the time interval after application, and the cell line used. The structural modifications of daunorubicin were responsible for the different cytotoxic effects of the two formamidinodaunorubicins.

In vitro induction of apoptosis and necrosis by new derivatives of daunorubicin.

BACKGROUND/AIM: The comparative effects of daunorubicin, and its new formamidine derivatives containing either a morpholine moiety (DAUFmor) or a hexamethyleneimine moiety (DAUFhex) in the amidine group, on induction of programmed cell death were determined. MATERIALS AND METHODS: The experiments were performed on human acute lymphoblastic leukemia MOLT-4 cells and human acute myeloblastic leukemia ML-1 cells. The research was conducted using the flow cytometry annexin V-fluorescein (FITC)/propidium iodide (PI) method and tetramethylrhodamine ethyl ester (TMRE) assay. RESULTS: The various patterns of temporary changes of early apoptotic cells, late apoptotic and necrotic cells, and in the frequency of the acute leukemia cells with high values of mitochondrial membrane potential (MMP) were found. Phosphatidylserine externalization, plasma membrane disruption, and changes in MMP occurring in the leukemia cells were dependent on the agent tested, its concentration, the time intervals after daunorubicin, DAUFmor, and DAUFhex application, and on the leukemia cell line used. CONCLUSION: The structural modifications of daunorubicin producing two new analogs, DAUFmor and DAUFhex, induced the different responses of MOLT-4 and ML-1 cells to triggering of programmed death.

Effects of structural modifications of daunorubicin on in vitro antileukemic activity.

BACKGROUND/AIM: In the search for new derivatives of anthracycline antibiotics, formamidinodaunorubicins containing in the amidine group either a morpholine moiety (DAUFmor) or a hexamethyleneimine moiety (DAUFhex) were synthesized. The biological effects of daunorubicin (DAU), DAUFmor and DAUFhex were compared. MATERIALS AND METHODS: The experiments were performed on human acute lymphoblastic leukemia MOLT-4 cells and human acute myeloblastic leukemia ML-1 cells. The research was conducted using the spectrophotometric 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay and the electronic Beckman-Coulter method. RESULTS: Temporary changes in the leukemia cell viability, size and count were found. The antileukemic activities of the new DAU analogs were weaker than that of daunorubicin. MOLT-4 cells were more sensitive than ML-1 cells to the action of all agents. Among the formamidinodaunorubicins, DAUFmor appeared to be more active in ML-1 cells than DAUFhex, but there were not differences between the analyzed values in MOLT-4 cells. CONCLUSION: The structural modifications of daunorubicin were responsible for the different antileukemic potentials of the two formamidinodaunorubicins.

Oxazolinodoxorubicin - a promising new anthracycline.

Oxazolinodoxorubicin, a doxorubicin analog with a modified daunosamine moiety was synthesized. The properties of this compound and the parent doxorubicin were compared. The cytotoxicity in vitro studies against several human tumor cell lines (PC-3, MCF-7, SW707, HL-60, RPMI 8226, ACHN) showed higher antiproliferative potency for this new compound. Moreover, its ability to completely overcome the drug resistance of cancer cells in vitro was revealed (LoVo, LoVo/DX, MES-SA, MES-SA/DX5, HL-60, HL-60/Vinc, HL-60/MX2 cell lines). Cellular uptake analyzed on HL-60 and HL-60/MX2 cells, demonstrated higher penetration levels of oxazolinodoxorubicin compared to that of doxorubicin. In animal experiments, general toxicity of oxazolinodoxorubicin was lower than that observed for doxorubicin. Furthermore, similar antitumor effects was observed in NOD/SCID mice bearing resistant HL-60/Vinc leukemia tumor and in mice treated with the new or parent compounds. The presented results suggest that oxazolinodoxorubicin is a new anthracycline with an advantageous biological activity profile.