Impurity assessment, development and validation of an RP-HPLC method for the determination of eleven potential impurities of eltrombopag precursor.

Eltrombopag is an oral non-peptide thrombopoietin receptor (TPO-R) agonist indicated for the treatment of thrombocytopenia in patients with persistent or chronic immune thrombocytopenia (idiopathic thrombocytopenic purpura, ITP) or chronic hepatitis C infection and the treatment of severe aplastic anemia. The purpose of this research was to assess the possible impurities that may carry over to eltrombopag from its precursor Eltro-1 (3'-amino-2'-hydroxy-[1,1'-biphenyl]-3-carboxylic acid) and to develop a specific analytical method for the determination of these impurities. Eltro-1 samples synthesized by two different synthesis routes were investigated during the evaluation and method development studies. Besides the expected process-related impurities (Eltro-1A - Eltro-1J), e.g., starting materials, intermediates, and/or compounds formed from their further reactions, an unknown impurity detected above 0.10% was identified by LC-MS, synthesized and fully characterized by NMR, MS and FTIR (Eltro-1K). Accordingly, an HPLC-RP method for the determination of eleven impurities (Eltro-1A - Eltro-1K) in Eltro-1 was developed and validated according to ICH Q2. The control limits for impurities in Eltro-1 were set at ≤ 0.15% for Eltro-1A - Eltro-1J and ≤ 1.0% for Eltro-1K based on fate, spike-purge and carryover studies and in accordance with the ICH M7 classification for impurities in drug substance. Eltro-1 and eleven impurities at the specification limit were separated from each other and the diluent peaks with sufficient resolution without interference. Separation was performed on a Waters XBridge C18 column (150 × 4.6 mm, 3.5 µm) at 40 °C with a 10 µL injection volume at a detection wavelength of 220 nm and 15 °C sample temperature. The gradient elution is performed at a flow rate of 1.0 mL/min for 40 min with mobile phase A (0.1% orthophosphoric acid in water) and B (acetonitrile) according to the following program: Time (min) / Acetonitrile (%): 0/0, 35/70, 36/0, 40/0. Test and standard solutions were prepared at a concentration of 1.0 mg/mL and 1.0 µg/mL, respectively, using a mixture of mobile phase A and acetonitrile (75/25) as diluent. This is the first specific, selective, sensitive, linear, precise, accurate, and robust HPLC method for the determination of Eltro-1A - Eltro-1K in Eltro-1, which showed no significant degradation under thermal stress, photostability (UV and VIS), and standard accelerated and long-term stability conditions.

Homologous or heterologous COVID-19 vaccine schemes: comparison of immune responses and side effects.

Real-life data are needed regarding the appropriate time and selection of vaccination strategies, homologous or heterologous. We aimed to compare neutralizing antibody levels and side effects in different vaccination schemes. The study included 310 Health Care Workers (HCWs) vaccinated with 5 different schemes. Antispike/RBD IgG levels were measured between 28 and 60 days after the last dose. Side effects in participants were recorded, and pharmacovigilance records were reviewed from the outpatient vaccine clinic. Mean age of the participants was 38 ± 11 years of whom 226 (72.9%) were female, and 84 (27.1%) were male. After booster doses, increasing antibody levels were detected in all groups. Mean antibody levels were detected to be statistically lower in 3 doses of inactivated vaccines group. The side effects were no significant difference between groups. Booster dose administration with mRNA vaccines stands out as the most accurate strategy for those at risk of contracting severe COVID-19 and HCWs caring for this population.

Methylation status, mRNA and protein expression of the SMAD4 gene in patients with non-melanocytic skin cancers.

BACKGROUND: SMAD4 is a potent tumor suppressor. SMAD4 loss increases genomic instability and plays a critical role in the DNA damage response that leads to skin cancer development. We aimed to investigate SMAD4 methylation effects on mRNA and protein expression of SMAD4 in cancer and healthy tissues from patients with basal cell carcinoma (BCC), cutaneous squamous cell carcinoma (cSCC), and basosquamous skin cancer (BSC). METHODS AND RESULTS: The study included 17 BCC, 24 cSCC and nine BSC patients. DNA and RNA were isolated from cancerous and healthy tissues following punch biopsy. Methylation-specific polymerase chain reaction (PCR) and real-time quantitative PCR methods were used to examine SMAD4 promoter methylation and SMAD4 mRNA levels, respectively. The percentage and intensity of staining of the SMAD4 protein were determined by immunohistochemistry. The percentage of SMAD4 methylation was increased in the patients with BCC (p = 0.007), cSCC (p = 0.004), and BSC (p = 0.018) compared to the healthy tissue. SMAD4 mRNA expression was decreased in the patients with BCC (p˂0.001), cSCC (p˂0.001), and BSC (p = 0.008). The staining characteristic of SMAD4 protein was negative in the cancer tissues of the patients with cSCC (p = 0.00). Lower SMAD4 mRNA levels were observed in the poorly differentiated cSCC patients (p = 0.001). The staining characteristics of the SMAD4 protein were related to age and chronic sun exposure. CONCLUSIONS: Hypermethylation of SMAD4 and reduced SMAD4 mRNA expression were found to play a role in the pathogenesis of BCC, cSCC, and BSC. A decrease in SMAD4 protein expression level was observed only in cSCC patients. This suggests that epigenetic alterations to the SMAD4 gene are associated with cSCC. TRIAL REGISTRATION: The name of the trial register: SMAD4 Methylation and Expression Levels in Non-melanocytic Skin Cancers; SMAD4 Protein Positivity. The registration number: NCT04759261 ( https://clinicaltrials.gov/ct2/results?term=NCT04759261 ).

Waning immunity to inactive SARS-CoV-2 vaccine in healthcare workers: booster required.

AIMS: Despite high vaccination rates, increasing case numbers continue to be reported with the identification of new variants of concern, and the issue of durability of the vaccine-induced immune response remains hot topic. Real-life data regarding time-dependent immunogenicity of inactivated COVID-19 vaccines are scarce. We aimed to investigate the changes in the antibody at the different times after the second dose of the CoronaVac vaccine. METHODS: The study included 175 HCWs vaccinated with inactive CoronaVac (Sinovac Life Sciences, China) SARS-CoV-2 vaccine in two doses. Anti-spike/RBD IgG levels were measured first, third, and sixth months after the second dose. Chemiluminescent microparticle immunoassay (IgG II Quant test, Abbott, USA), which is 100% compatible with plaque reduction neutralization test, was used. RESULTS: Mean age of the participants was 38 ± 11.23 years (range between 22 and 66) of whom 119 (63.9%) were female, and 56 (32%) were male. Dramatic reductions were demonstrated in median antibody levels particularly in the infection-naïve group, comprising 138 HCWs compared to those with prior history of COVID-19 infection (n = 37) (p < 0.001). There was no difference between the two groups in terms of age, gender, blood groups, BMI, and comorbid diseases. CONCLUSIONS: While antibody positivity remained above 90% in the 6th month after two doses of inactivated vaccine in HCWs, the median titers of neutralizing antibodies decreased rapidly. The decrease was more rapid and significant in those with no history of prior COVID-19 infection. In this critical phase of the pandemic, where we are facing the dominance of the Omicron variant after Delta, booster doses have become vital.

Heterologous booster COVID-19 vaccination elicited potent immune responses in HCWs.

The objective of our study was to evaluate the antibody responses of health care workers (HCWs) who were vaccinated with booster dose BNT162b2 6 months after 2 doses of the CoronoVac vaccine. The study included 318 HCWs vaccinated with inactive CoronaVac SARS-CoV-2 vaccine in 2 doses. Anti-spike/RBD IgG levels were measured immediately before and 1 month after the booster dose. In the sixth month after CoronaVac vaccination, the median of antibody levels of 1212.02 AU/ML, while it was 9283 AU/mL after BNT162b2 vaccination. IgG antibody titers of over 1050 AU/mL (which is equivalent to 1:80 dilution in the plaque reduction neutralization test) were detected in HCWs 15.09% and 97.8%, respectively. Our results showed that antibody titers increased 8-fold after the booster dose. We believe that the administration of the mRNA vaccine as a booster dose can provide more effective protection against COVID-19 infection, especially in individuals with risk factors.

The Effect of MMP-1 on Wound Healing and Scar Formation.

BACKGROUND: In this study, we investigated the effect of matrix metalloproteinase-1 (MMP-1) on wound healing on skin in a model produced in rats. METHODS: Sixteen Sprague-Dawley male rats were included in the study. The four full-thickness skin wound was created on the dorsal area of each rat with 4.4 mm punch. The rats were randomly divided into two groups. MMP-1 and saline were administered intraperitoneally once daily for 7 days. The biopsies were taken from the separate wounds on the 4th, 7th, 14th and 21st days of the experiment. The lymphocytic response, vascular proliferation, fibroblast proliferation, epithelial hyperplasia, foreign body reaction, ulcer formation, acute inflammation, keloid scar formation and hypertrophic scar formation were compared in each group in histopathologically. RESULTS: In our study, epithelial hyperplasia on 14th day was significantly higher in the MMP-1 group compared to the control group (p < 0.05). The lymphocytic response on 4th and 21th days, the vascular proliferation on 4th day, the fibroblast proliferation on 4th and 7th days, the acute inflammation on 4th day and the hypertrophic scar formation on 7th, 14th, 21st days were significantly lower in the MMP-1 group compared to the control group (p < 0.05). No statistically significant difference was found in comparison with other parameters (p > 0.05). CONCLUSIONS: MMP-1 improves the wound-healing process of skin with higher epithelial hyperplasia and reduces scar formation in the animal model. Therefore, MMP-1 can potentially be used as an effective anti-fibrogenic agent for preventing or treating the hypertrophic scar. NO LEVEL ASSIGNED: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Synthesis of New Di- and Triamides as Potential Organocatalysts for Asymmetric Aldol Reaction in Water.

New di- or triamide organocatalysts derived from (L)-proline were synthesized and successfully used in the direct asymmetric aldol reaction of aliphatic ketones and aromatic aldehydes in water at 0oC in the presence of benzoic acid as co-catalyst. (S)-methyl-2-((S)-3-hydroxy-2-((S)-3-pyrrolidine-2-carboxamido)propanamido)-3-phenylpropanoate (7c) as organocatalyst showed best results under these reaction conditions, and good diastereoselectivities (up to 99%), enantioselectivities (up to 98%) and yields (up to 91%) were observed.

CD56, CD57, HBME1, CK19, Galectin-3 and p63 immunohistochemical stains in differentiating diagnosis of thyroid benign/malign lesions and NIFTP.

Detection of thyroid carcinoma has been steadily increased in the past few decades. After the recognition of NIFTP, also gain importance to differentiate benign tumors (follicular adenoma) from follicular patterned variants of papillary thyroid carcinoma (invasive and infiltrative follicular variant papillary thyroid carcinoma), and low-risk lesions of thyroid (NIFTP). Follicular patterned proliferations of thyroid still persists as a battle for pathologists. In this study, we aimed to analyze the most commonly used immunohistochemical stains "HBME1, CK19, Galectin-3", adding the new ones "CD56, CD57, and p63". Study groups were; nodular hyperplasia, follicular adenoma, NIFTP, infiltrative follicular variant PTC, classical variant PTC (CVPTC) and follicular carcinoma. Each group consisted of twenty cases. The sections were stained with CD56, CD57, p63, CK19, HBME1 (Mesotel cell), Galectin-3 antibody. Although the expression of CD56 was high in benign follicular lesions, FC could not be excluded in this group. CD57 was high in malignant follicular group and NIFTP. Interestingly, p63 was found highly expressed in FVPTC, which might be promising to predict invasiveness in follicular group of lesions. CK19, Galectin-3 and HBME1 were found quietly prominent in CVPTC in concordance with the previous reports.

Novel Synthetic Approaches for Bisnaphthalimidopropyl (BNIP) Derivatives as Potential Anti-Parasitic Agents for the Treatment of Leishmaniasis.

Leishmaniasis is a neglected parasitic disease that is widely seen in more than 60 countries worldwide, including Turkey and its subcontinental region. There are several chemotherapy agents for the treatment of leishmaniasis, including pentavalent antimonials-i.e., sodium stibogluconate (Pentostan) and meglumine antimoniate (Glucantim), pentamidine, conventional amphotericin B deoxycholate, miltefosine, paramomycin (aminosidine), and liposomal amphotericin B. However, these therapies are usually unsatisfactory due to dose-limiting toxicity issues and limited efficacy. Furthermore, resistance gained by parasites endangers future success of these therapies. Addressing these issues, the development of novel drugs with high efficacy has a vital importance. Latest studies have shown that bisnaphthalimidopropyl (BNIP) derivatives display high activity against Leishmaniasis parasites by selectively targeting parasitic sirtuin proteins and interacting with DNA. Despite the promising anti-parasitic activity, the low solubility and toxicity on human macrophages are the limitations to overcome. This study describes the new synthesis strategies for existing-i.e., BNIPDaoct and BNIPDanon-and novel BNIP derivatives differing in respect of their alkyl linker chain lengths. The new synthesis approach provides certain advantages compared to its existing alternatives reported in the literature. The proposed methodology does not only decrease the number of synthesis steps and production time of BNIPDaoct and BNIPDanon, but also provides higher yields, thereby making the synthesis highly cost-effective.

Conservative Surgical Treatment of a Case of Placenta Accreta.

Placenta accreta syndromes are associated with increased maternal mortality and morbidity. Cesarean hysterectomy is usually performed in cases of placenta accreta syndrome. Fertility sparing methods can be applied. In the present study, we report a successful segmental uterine resection method for placenta accreta in the anterior uterine wall in a cesarean section case. A 39-year-old woman underwent an elective cesarean section at 38 + 2 weeks. A placental tissue with an area of 10 cm was observed extending from the anterior uterine wall to the serosa, 2 cm above the uterine incision line. The placental tissue was removed with the help of monopolar electrocautery. The uterine incision was continuously sutured. The patient was discharged on the second postoperative day. The placental pathology was reported as placenta accreta. The American College of Obstetricians and Gynecologists (ACOG) generally recommends cesarean section hysterectomy in cases of placenta accreta because removal of placenta associated with significant hemorrhage. Conservative and fertility sparing methods include placenta left in situ, cervical inversion technique and triple-P procedure. There are several studies reporting that segmental uterine resection is performed with and without balloon placement or artery ligation. Segmental uterine resection may be an alternative to cesarean hysterectomy to preserve fertility or to protect the uterus in cases of placenta accreta when there is no placenta previa.

Conservative surgical treatment of a case of placenta accreta

Abstract Placenta accreta syndromes are associated with increased maternal mortality and morbidity. Cesarean hysterectomy is usually performed in cases of placenta accreta syndrome. Fertility sparing methods can be applied. In the present study, we report a successful segmental uterine resection method for placenta accreta in the anterior uterine wall in a cesarean section case. A 39-year-old woman underwent an elective cesarean section at 38 + 2 weeks. A placental tissue with an area of 10 cm was observed extending fromthe anterior uterine wall to the serosa, 2 cm above the uterine incision line. The placental tissue was removed with the help of monopolar electrocautery. The uterine incision was continuously sutured. The patient was discharged on the second postoperative day. The placental pathology was reported as placenta accreta. The American College of Obstetricians and Gynecologists (ACOG) generally recommends cesarean section hysterectomy in cases of placenta accreta because removal of placenta associated with significant hemorrhage. Conservative and fertility sparing methods include placenta left in situ, cervical inversion technique and triple-P procedure. There are several studies reporting that segmental uterine resection is performed with and without balloon placement or artery ligation. Segmental uterine resection may be an alternative to cesarean hysterectomy to preserve fertility or to protect the uterus in cases of placenta accreta when there is no placenta previa. received

Melanotic Schwannomas Are Rarely Seen Pigmented Tumors with Unpredictable Prognosis and Challenging Diagnosis.

Melanotic Schwannoma (MS) is rarely seen and potentially malignant neoplasm that is categorized as a variant of Schwannoma. MS most frequently involves intracranial structures followed by posterior nerve roots in the spinal canal. Approximately 50% of the cases with MS have psammomatous calcifications and this type of MS is related to Carney complex with autosomal dominant inheritance. Most cases of MS are benign, though 10% of them are malignant with metastatic potential. MS mimics melanoma and the differential diagnosis should be made excluding other melanin producing neoplasms especially melanoma. Case 1. A 42-year-old hypertensive male presented for checkup. He had a well-defined extraspinal oval lesion measuring 3.5 × 2.5 cm near right adrenal. Case 2. A 22-year-old female presented with neurofibromatosis-2, bilateral acoustic schwannomas and café au lait lesions on sacrococcygeal region. She had an intradural extramedullary lesion measuring 6.1 × 2.0 cm at L1-2 level. MS is a rare neoplasm composed of Schwann cells and melanin pigment. These tumors are usually benign but they may become aggressive. The biologic behavior of MS is difficult to predict; the patients have to be followed up for a longer period due to its malignant potential.

Parotid Duct Repair with Intubation Tube: Technical Note.

The parotid duct can be damaged in traumatic injuries and surgical interventions. Early diagnosis and treatment of a duct injury is of great importance because complications such as sialocele and salivary gland fistula may develop if the duct is not surgically repaired. We think the cuff of an intubation tube is an ideal material in parotid duct repair, because of its technical characteristics, easiness of availability, and low-cost. In this paper, we described the use of the cuff cannula of an intubation tube for the diagnosis and treatment of parotid duct laceration, as a low-cost and easy to access material readily available in every operating room.

Investigating KRAS/BRAF mutation in oropharyngeal squamous cell carcinomas: a preliminary study.

OBJECTIVES: This study aims to investigate the role of KRAS/BRAF gene mutation in the pathogenesis of oropharyngeal squamous cell carcinoma (OSCC). PATIENTS AND METHODS: A total of 26 OSCC patients (23 males, 3 females; mean age 60 years; range 41 to 77 years) diagnosed between January 2003 and November 2013 were included in the study. The methods used in our study were quantitative fluorescence polymerase chain reaction for KRAS/BRAF mutation analysis. RESULTS: Ten of the tumors were located at the tongue base, 12 in the tonsil and four at the floor of mouth. The mean tumor size was 3.8 cm. Six of the tumors were well differentiated, 18 were moderately differentiated and two were poorly differentiated. All cases were analyzed for KRAS and BRAF gene mutations and none of them showed gene mutations. CONCLUSION: We could not find any relation between OSCC and KRAS/BRAF gene mutations in our short case file. The role of mutations should be analyzed in larger series in OSCC to predict new targeted therapy modalities.

Determination of HER2 and p53 Mutations by Sequence Analysis Method and EGFR/Chromosome 7 Gene Status by Fluorescence in Situ Hybridization for the Predilection of Targeted Therapy Modalities in Immunohistochemically Triple Negative Breast Carcinomas in Turkish Population.

Triple negative breast cancer (TNBC), an agressive subtype accounts nearly 15 % of all breast carcinomas. Conventional chemotherapy is the only treatment modality thus new, effective targeted therapy methods have been investigated. Epidermal growth factor receptor (EGFR) inhibitors give hope according to the recent studies results. Also therapeutic agents have been tried against aberrant p53 signal activity as TNBC show high p53 mutation rates. Our aim was to detect the incidence of mutations/amplifications identified in TNBC in our population. Here we used sequence analysis to detect HER2 (exon 18-23), p53 (exon 5-8) mutations; fluorescence in situ hybridization (FISH) method to analyse EGFR/chromosome 7 centromere gene status in 82 immunohistochemically TNBC. Basaloid phenotype was identified in 49 (59.8 %) patients. EGFR amplification was noted in 5 cases (6.1 %). All EGFR amplified cases showed EGFR overexpression by immunohistochemistry (IHC). p53 mutations were identified in 33 (40.2 %) cases. Almost 60 % of the basal like breast cancer cases showed p53 mutation. Only one case showed HER2 mutation (exon 20:g.36830_3). Our results showed that gene amplification is not the unique mechanism in EGFR overexpression. IHC might be used in the decision of anti-EGFR therapy in routine practice. p53 mutation rate was lower than the rates reported in the literature probably due to ethnic differences and low sensitivity of sanger sequences in general mutation screening. We also established the rarity of HER2 mutation in TNBC. In conclusion EGFR and p53 are the major targets in TNBC also for our population.

A case of isolated abducens nerve paralysis in maxillofacial trauma.

Nervus abducens is a pure motor nerve located in the pons. It retracts the eyeball laterally by stimulating rectus lateralis muscle. In case of their paralysis, diplopia and restriction in the eye movements while looking sideways, are seen. Since the same signs are seen due to the muscle entrapment in blowout fractures, its differential diagnosis has importance in terms of the treatment protocol and avoiding unnecessary operations. In this article, we present a 22-year-old male patient who was referred to our department due to the prediagnosis of blowout fracture following maxillofacial trauma. However, he was diagnosed with abducens nerve paralysis after the consultations and analysis and his restriction of movement was resolved via systemic steroid treatment instead of unnecessary operation.

[Immunohistochemical characteristics of triple negative/basal-like breast cancer]. / Triple-Negatif/Bazal Benzeri Meme Kanserinin Immünohistokimyasal Özellikleri.

OBJECTIVE: Triple-negative-breast-cancer that accounts for 10-20% of all breast carcinomas is defined by the lack of estrogen receptor, progesterone receptor, HER2 expression, and agressive clinical behavior. Triple-negative-breast-cancer is categorized into basal like and other types. The basal-like subtype is characterized by the expression of myoepithelial/basal markers. MATERIAL AND METHOD: We studied 41 immunohistochemically triplenegative- breast-cancer patients to determine EGFR, Cytokeratine 5/6, p53, Ki67, GCDFP-15 expression profiles, HER2 and Chromosome 17 centromere gene status by fluorescence-in-situ-hybridization method. RESULTS: Histological type was invasive ductal carcinoma in 90.2% of the tumors. p53, Ki67, GCDFP-15 mean positivity rates were 55.6%, 51.7%, and 3.2%, respectively. GCDFP-15 positivity was noted in 8 cases of which 6 were Cytokeratine 5/6 negative. The cut-off value for Cytokeratine 5/6 positivity was 5%. EGFR immunoreactivity was grouped into 0, 1+ as negative; 2+, 3+ as positive categories. Cytokeratine 5/6 was positive in 56,1%, EGFR was positive in 51.2% of the patients. The relation between Cytokeratine 5/6 and EGFR expression was statistically significant (p < 0.01). None of the cases showed HER2 amplification by fluorescence-in-situ-hybridization method. CONCLUSION: GCDFP-15 alone is not a useful marker to detect the metastasis of basaloid type breast cancers. Cytokeratine 5/6 and EGFR expressions showed correlation so these markers are reliable to diagnose basaloid type tumors with a 5% cut-off value.