Focal segmental glomerulosclerosis (FSGS) is a progressive glomerular disease characterized by podocyte injury, proteinuria, and risk of kidney failure. Until recently, no medicines had been approved by the FDA or European Medicines Agency, with management focused on supportive care and proteinuria reduction. This supplement explores the burden of FSGS from a clinical, humanistic, and economic perspective, informed by the results of 3 systematic literature reviews. FSGS is associated with poorer quality of life and high health care costs, and these outcomes are correlated with disease severity. Kidney Disease: Improving Global Outcomes guidelines recommend monitoring estimated glomerular filtration rate (eGFR) for kidney function; however, it is acknowledged that given the variable decline in FSGS, eGFR is not an ideal trial end point. Consequently, proteinuria reduction has become the preferred surrogate efficacy end point, as supported by the International Society of Glomerular Disease's global Proteinuria and Other Biomarkers as Endpoints for Clinical Trials in Kidney Disease initiative and subsequent FDA endorsement. Available treatments, such as glucocorticoids or calcineurin inhibitors, are limited in efficacy and safety. Among emerging medicines, sparsentan, a dual endothelin and angiotensin receptor antagonist (DEARA), has demonstrated consistent and significant reductions in proteinuria among patients with primary and genetic FSGS resulting in its approval by the FDA to reduce proteinuria in adult and pediatric patients aged 8 years and older with FSGS without nephrotic syndrome. In the phase 2 DUET clinical trial (NCT01613118), those treated with sparsentan showed a urine protein-creatinine ratio reduction of -35.6% (95% CI, -46.3% to -24.6%) vs baseline at 240 weeks. Sparsentan also showed greater reductions in proteinuria vs irbesartan, a standard renin-angiotensin system agent, along with a comparable safety profile in the phase 3 DUPLEX trial (NCT03493685). In model-based projections among real-world registry cohorts, sustained reductions in proteinuria are associated with lower long-term risk of kidney failure. These findings align with the PARASOL-FSGS initiative, supporting the role of proteinuria reduction as a key end point, facilitating pragmatic trial designs to address the persistent treatment gap in FSGS.

A simple, sensitive and efficient ultra performance liquid chromatography-mass spectrometry (UPLC-MS/MS) method for simultaneous quantitative determination of olmesartan and hydrochlorothiazide in human plasma has been developed and validated. Using olmesartan-d4 and hydrochlorothiazide-[13]C-d2 as stable isotope-labeled internal standard (SIL-IS). Plasma samples were processed by protein precipitation. Separated on a ZORBAX Eclipse XDB-Phenyl column (150 mm × 4.6 mm, 5 μm) using a gradient elution with mobile phases methanol and 5 mM ammonium acetate. Detected by electrospray ionization (ESI) in negative ion mode of multiple reaction monitoring (MRM), mass transition ion pairs were m/z 445.1 → 149.0 for olmesartan, m/z 295.9 → 204.9 for hydrochlorothiazide, m/z 449.1 → 149.1 for olmesartan-d4, and m/z 301.0 → 272.0 for hydrochlorothiazide-[13]C-d2. Linear ranges were 10-2000 ng/mL for olmesartan and 1.5-300 ng/mL for hydrochlorothiazide. Mean recoveries of olmesartan, hydrochlorothiazide, olmesartan-d4, and hydrochlorothiazide-[13]C-d2 were 91.31%, 99.34%, 92.62%, and 98.61%, respectively. Our method was well validated in selectivity, carryover, lower limit of quantification (LLOQ), calibration curve, accuracy, precision, dilution effect, matrix effect (normal, hyperlipidemic, and hemolyzed matrices), stability, recovery, and IRS. It was successfully applied in a bioequivalence study of olmesartan medoxomil and hydrochlorothiazide tablets (20/12.5 mg) in healthy Chinese volunteers.

Cardiovascular diseases are the leading causes of deaths worldwide. Complementary therapies, such as medicinal plants, have become increasingly common among treatment options. We propose to investigate the cardioprotective effects of the ethanol-soluble fraction obtained from the aerial parts of Baccharis milleflora in hypertensive rats. Cladodes of B. milleflora were collected, and the aqueous extract was obtained by infusion. The infusion was then treated with ethanol, resulting in the ethanol-soluble fraction of B. milleflora, which was analyzed by LC-DAD-MS. The MS and MS/MS data obtained from the ethanol-soluble fraction of B. milleflora was submitted to the Global Natural Products Social Molecular Networking platform to generate the molecular network, and three main clusters were detected. Wistar-Kyoto and spontaneously hypertensive rats were divided into different experimental groups, including naïve, control (vehicle), hydrochlorothiazide (25 mg/kg), and ethanol-soluble fraction of B. milleflora (30, 100, and 300 mg/kg). The treatment lasted for 28 days. At the end of the treatment, the animals underwent evaluation of renal function, electrocardiographic profile, blood pressure, mesenteric vascular reactivity, serum biochemical parameters, and histopathological analysis. The molecular pathways involved in pharmacological activity were also investigated. After 28 days, animals treated with ethanol-soluble fraction of B. milleflora 30 mg/kg showed a significant antioxidant, diuretic, and antihypertensive response, as well as reversed endothelial dysfunction and left ventricular hypertrophy induced by hypertension. Treatment with a non-selective NO synthase inhibitor, cGMP inhibitor, or non-selective K+ channel blocker suppressed the antihypertensive effects of ethanol-soluble fraction of B. milleflora. Our findings showed that B. milleflora is a species with cardioprotective activity when administered for a prolonged period to hypertensive rats.

Bioanalytical method development and validation are essential for reliable quantification of drugs in biological matrices. This research focuses on developing and validating a UPLC-MS/MS method for the simultaneous determination of bisoprolol and hydrochlorothiazide in human plasma, adhering to established regulatory guidelines for bioanalytical method validation. The development and validation focus on creating a robust and sensitive assay suitable for bioequivalence studies and routine therapeutic drug monitoring. This method utilizes bisoprolol D5 and hydrochlorothiazide C13 D2 as internal standards to enhance accuracy and precision. Chromatographic separation was achieved on a Waters Acquity UPLC BEH C18 column (100 × 2.1 mm, 1.7 μm particle size) with an isocratic mobile phase consisting of 10 mM ammonium formate buffer, methanol, and 0.1% ammonia solution (10:90, v/v). The flow rate was set at 0.3 mL/min, with a retention time of 2.2 min. Multiple reaction monitoring (MRM) was used with positive ESI for bisoprolol (m/z 326.36 → 116.13, internal standard m/z 331.36 → 121.13) and negative ESI for hydrochlorothiazide (m/z 296.11 → 269.00, internal standard m/z 299.11 → 270.00). The analytes and their respective internal standards were co-extracted using a liquid-liquid extraction method with tert-butyl methyl ether as the extraction solvent. Linearity for bisoprolol and hydrochlorothiazide was maintained over a concentration range of 1-100 ng/mL for bisoprolol and 1.0-300 ng/mL for hydrochlorothiazide, respectively, using a weighted least squares linear regression model (1/x). This method achieved a lower limit of quantification (LLOQ) of 1.0 ng/mL, making it highly sensitive for the detection of these analytes. Moreover, the method demonstrated high accuracy, precision, selectivity, and reduced overall analysis time, making it well suited for routine analysis and bioequivalence studies of 10 mg bisoprolol and 25 mg hydrochlorothiazide tablets.

Assessing polymorphic form changes in active pharmaceutical ingredients (APIs) during the early stage is critical for selecting the best polymorphic form required for drug product development. However, this assessment can be constrained by limited API availability. Present methods to assess risk in industry relies on larger volume equipment such as a compaction simulator or texture analyser (TA) that requires larger quantities of API. In the present study, a diamond anvil cell (DAC) was used, that reduces the quantity of material even further to micrograms to investigate the impact of pressure using Hydrochlorothiazide (HCT) as a model API. The powdered API was directly loaded into the DAC sample chamber without a pressure-transmitting medium (PTM), and Raman spectroscopy was used to monitor form changes. A polymorphic transition begins to be observed at 300 MPa pressure in the DAC that is commensurate with the findings in the TA (500 MPa) from Raman and X-ray powder diffraction (XRPD). XRPD analysis revealed that trituration likely causes a transformation back to the original phase as undisturbed samples can be stored for months. In conclusion, the study highlights the effectiveness of the DAC as a material sparing technique for assessing pressure induced polymorphic transition in APIs at the time of compression enabling a real-time monitoring of the process. The DAC successfully detected the polymorphic transition in tabletting compression range requiring significantly less material than the TA. The minimal consumption of API making DAC a preferred method for polymorphic screening particularly at the early stage of development when material availability is limited.

The diuretic drug hydrochlorothiazide (HCT) is associated with an increased risk of non-melanoma skin cancer upon UV exposure. The underlying cellular and molecular mechanisms behind this association remain elusive. Herein, a human skin model to assess the photocarcinogenic effects of HCT is established. Skin biopsies collected from human body donors are treated with HCT and irradiated with 300 mJ/cm[2] low dose UVA or UVB or with 5 J/cm[2] high dose UVA. In HCT-treated biopsies but not in control, low dose UVA irradiation results in activation and nuclear translocation of the tumor-suppressor protein p53 accompanied by an upregulated gene expression of p53-negative regulator MDM2. High dose UVA additionally provokes DNA damage and initiation of pro-inflammatory gene expression. In contrast, UVB induces pronounced DNA damage, p53 protein activation, gene expression of MDM2 and inflammatory marker genes in both HCT-treated biopsies and untreated control. In summary, in HCT-treated skin biopsies, activation of the p53-MDM2 axis, induction of DNA damage, and inflammatory response depends on UVA-dosage and may influence skin carcinogenesis over time. This human model eliminates the need for animal testing and mitigates species difference, offering a valuable tool for future drug development and safety testing.

The pathogenesis of cutaneous tumors has been known for decades yet remains largely unexplained or incompletely understood. The reason for this mystery lies in the concepts of photosensitivity and phototoxicity: how do they arise or what actually causes them? Recently published data in the medical literature link certain nitrosamines such as nitrosomorpholine, for example, to gene and phototoxicity in humans. A number of other nitrosamines analogous in action and structure are found as contaminants in about 300 of the most widely distributed pharmaceuticals worldwide: NDEA, NDMA, NMBA and many others. These contaminated drugs include beta blockers/ bisoprolol/, thiazide diuretics/ hydrochlorothiazide/, antiarrhythmics/ propafenone/, ACE inhibitors/ lisinopril/, but also a number of other drugs which are, according to the FDA, found to have contaminants with a certain carcinogenic potency ranging between 1 and 5. The phototoxicity and genotoxicity of these contaminants, attributed to the pathogenesis of skin tumors, still remain a mystery. The problems of the intake of the above-mentioned groups of drugs arise mainly on the basis of the official bulletins of the regulatory bodies, namely that: in practice, the intake of polymedication could in many cases also be considered as regular, permanent, long-term intake of contaminants/carcinogens/mutagens of heterogeneous type, also known as nitrosamines or NDSRIs. Nitrosamines are genome modifiers in humans and cause acquired mutations. Their concomitant administration in the context of standard, but currently not yet officially declared as contaminated polymedication, would be able to block certain tumor suppressor genes (p53) as well as activate RAS oncogenes. Or in practice- daily administration of a particular combination of drugs could activate the cascades of carcinogenesis regulating the genesis of skin cancer. Precisely because of this fact, it should not be surprising to anyone that the concurrent intake of the aforementioned drugs could also be associated with the clinical manifestation of multiple keratinocytic tumors. We describe a consecutive case of a patient who developed 4 keratinocytic tumors: 2 basal cell carcinomas, 1 keratoacanthoma, and 1 squamous cell carcinoma on a background of potentially contaminated polymedication with propafenone, lisinopril, hydrochlorothiazide, and bisoprolol. Recently published innovative international data on the topic are discussed in the context of concepts such as drug-mediated nitrosogenesis, photonitrosо-carcinogenesis and metabolic programming/ reprogramming of the tumor cell.

Special attention is given to the pharmacological treatment of combined medication of Carvedilol and hydrochlorothiazide which is the most effective and the most beneficial therapy for hypertensive patients with diabetes and various metabolic comorbidities. This work represents spectrophotometric platform scenarios based on factorized spectrum (FS) using interpoint data difference resolution scenarios (IDDRS) coupled with spectrum subtraction method (SS) for the concurrent quantification of carvedilol (CAR) and hydrochlorothiazide (HCT) when present together in a combination without the need for any initial physical separation steps. This IDD resolution scenario based on manipulating the zero-order spectra (D[0]) of both drugs in the mixture with various spectral features at different wavelength regions (200-400 nm), region I (220-250 nm), region II (240-300 nm) and region III (270-320 nm) via absorbance resolution (AR) and induced absorbance resolution (IAR) methods coupled with corresponding spectrum subtraction (SS). The calibration curves were established across the linearity ranges of 2.0-12.0 µg/mL at 242.50 nm and 4.0-40.0 µg/mL at 285.5 nm for CAR and 1.0-11.0 µg/mL at 226.10 nm and 2.0-20.0 µg/mL at 270.5 nm for HCT. Moreover, methods' validation was confirmed via ICH guidelines. A Multicenter comparison between sensitivity, specificity in respect resolution sequence were applied using different wavelength regions with various concentration ranges was applied and finally spectral resolution recommendation is issued and cumulative validation score (CVS) is calculated as an indicator in the risk analysis. In quality control laboratories, the studied approaches are applicable for conducting analysis on the mentioned drugs. In addition, the selection of spectrophotometry aligns with the principles of green analytical chemistry, an approach that resonates with the overarching theme of minimizing environmental impact. Via four metric tools named: analytical greenness (AGREE), green analytical procedure index (GAPI), analytical eco-scale, and national environmental method index (NEMI), methods' greenness profile was guaranteed.

Nephrogenic diabetes insipidus (NDI) is a rare genetic disorder primarily associated with mutations in the arginine vasopressin receptor 2 (AVPR2) gene or the aquaporin 2 (AQP2) gene, resulting in impaired water reabsorption in the renal tubules. This report describes a case of a young male patient with NDI from China with a history of polydipsia and polyuria for over 15 years. Laboratory examinations of the proband indicated low urine-specific gravity and osmolality. Urologic ultrasound revealed severe bilateral hydronephrosis in both kidneys, bilateral dilatation of the ureters, roughness of the bladder wall, and the formation of muscle trabeculae. The diagnosis of diabetes insipidus was confirmed by water deprivation tests. The administration of posterior pituitary hormone did not alter urine-specific gravity, and osmolality remained at a low level (<300 mOsm/kg). Based on these findings, and the genetic tests of the proband and his parents were performed. A missense mutation (c.616 G>C) in exon 3 of the AVPR2 gene of the proband was found, caused by the substitution of amino acid valine to leucine at position 206 [p.Val206Leu], which was a hemizygous mutation and consistent with X-chromosome recessive inheritance. The administration of oral hydrochlorothiazide improves the symptoms of polydipsia and polyuria in the proband. This novel AVPR2 gene mutation may be the main cause of NDI in this family, which induces a functional defect in AVPR2, and leads to reduced tubular reabsorption of water.

A novel and highly sensitive high-performance thin-layer chromatographic (HPTLC) method was developed and validated to quantify a combination of five pharmaceutical mixtures spiked to human plasma. The compounds comprised Amlodipine (AML) along with five angiotensin II receptor antagonist drugs (AIIRAs), namely Olmesartan (OLM), Telmisartan (TLM), Candesartan (CAN), Losartan (LOS), and Irbesartan (IRB). HPTLC was performed on silica gel 60 F254 plates using a mobile phase of Toluene: ethyl acetate: methanol: acetone: acetic acid (6:1.5:1:0.5:1, v/v/v/v/v). In a pioneering move, a reflectance/fluorescence detection mode was employed to identify two concurrently administered drugs at different pH levels for the first time. This method utilized the same chromatographic system, incorporating a specific measurement for AML at a neutral medium to achieve its maximum fluorescence at a 360 nm excitation wavelength, and measuring emission using a 540 nm optical filter. The process involved obtaining a very low fluorescence response from AIIRA. Subsequently, to enhance AIIRA's fluorescence, the plate was sprayed with perchloric acid to transition to a strong acidic medium, ultimately attaining the maximum fluorescence of AIIRA using various excitation wavelengths and a 400 nm emission filter. Through this strategic process, we could optimize the fluorescence signals of both drugs, thereby elevating the sensitivity of detection for this drug combination. AML demonstrated a linear range of 18-300 ng/band, while AIIRAs drugs exhibited a linear range of 6-150 ng/band. The method satisfied the International Conference on Harmonization (ICH) criteria for recovery, precision, repeatability, and robustness, showcasing exceptional sensitivity. The approach was successfully applied to quantify AML and AIIRAs drugs in both bulk drug and plasma samples, achieving high recovery percentages and minimal standard deviations.