Application of Near-Infrared Spectroscopy for Screening of Chlorothiazide Sodium Vials.

Chlorothiazide sodium for injection, USP, is a diuretic and antihypertensive medication in the form of a white or practically white, sterile, lyophilized powder. Each vial contains 500 mg of chlorothiazide sodium, equivalent to 500 mg of chlorothiazide, and 250 mg of mannitol as an inactive ingredient. The pH is adjusted with sodium hydroxide. Chlorothiazide sodium has a molecular weight of 317.71 amu. Since 2020 there have been multiple national shortages of chlorothiazide. Recent studies target chlorothiazide's low bioavailability, aiming to enhance it through nanoparticle production via a supercritical method. The drug's solubility in supercritical carbon dioxide (scCO2) is vital, with measurements ranging from 0.417×10-5 to 1.012×10-5 mole fraction under specific conditions. Adding co-solvents, like ethanol, DMSO, and acetone, to scCO2 boosts solubility, with ethanol proving most effective, enhancing solubility by 2.02-11.75 times. Intra-lot variability was discovered in a sample of a lot of chlorothiazide sodium by the University of Kentucky Drug Quality Task Force. Two vials of six screened in one lot were displaced from the center of the lot by 4.0 and 4.2 SDs, respectively. Inter-lot variability was confirmed in the near-IR spectra of 204 vials obtained from 28 different lots of chlorothiazide sodium. Using full spectrum BEST analysis 13 vials (6.4%) were outliers.

Screening for quality with process analytical technology in a health-system pharmacy: A primer.

PURPOSE: The University of Kentucky Drug Quality Study team briefly reviews the growing concerns over pharmaceutical manufacturing quality in the globalized environment, reviews the historical approach by the US Food and Drug Administration (FDA) that prioritizes process over product in enforcing quality with manufacturers, reviews the science of process analytical technology (PAT) such as near-infrared (NIR) spectroscopy, illustrates the use of PAT methods for assessing uniformity and quality in injectable pharmaceuticals, and demonstrates the application of NIR spectroscopy in a health-system pharmacy setting while maintaining current good practice quality guidelines and regulations (cGxP). SUMMARY: Given that the current approach to monitoring quality in pharmaceutical manufacturing was developed in the late 1960s at a time when manufacturing was mostly domestic, the current approach prioritizes process over product, and the global footprint of manufacturing is straining federal resources to fulfill their task of monitoring quality, an approach to augment the quality monitoring process has been developed. PAT methodologies are supported by FDA for monitoring quality and offer a fast, low-cost, nondestructive solution. Given that the Accreditation Council for Pharmacy Education has not required qualitative/quantitative analysis and drug assaying in the pharmacy curriculum for several decades, the authors spend time explaining the science behind one of these PAT methodologies, NIR spectroscopy. This primer reviews the application of this technology in the health-system pharmacy setting and the relevant clinical applications. CONCLUSION: Utilizing PAT methodologies such as NIR spectroscopy, health-system pharmacies can gain insights about whether process controls are in place or lacking in FDA-approved formulations.

Variability in Content of Hydrocortisone Sodium Succinate.

SOLU-CORTEFⓇ Sterile Powder is a type of anti-inflammatory glucocorticoid that contains hydrocortisone sodium succinate as its active ingredient. It can be administered intravenously or intramuscularly, and comes in several packages including 100 mg plain vials without diluent. The diluent, which is part of the ACT-O-VIAL system, contains only Water for Injection and no preservatives. The pH of each formula is adjusted with sodium hydroxide to ensure it falls within the specified range of 7 to 8 after reconstitution. Intralot variability was detected in lot GA6092. Measuring in the PC subspace using just PCs 4, 5 and 6, vial 12 plots 4.2 BEST SDs from the center of the cluster, and vial 7 is 3.7 SDs from the center. Vial 18 appears 3.1 SDS from the center of the cluster (3/18, 17%). Interlot variability was also found in the spectral library (lots GA6092, GK7048, GM6839, GR8925, FL8062, FN6860, FR1914, and FR5098) containing the spectra of 126 hydrocortisone sodium succinate vials.

Quality Variations in Thyrotropin Alfa.

Thyrotropin alfa is a heterodimeric glycoprotein containing human thyroid stimulating hormone (TSH). It is used as an adjunctive diagnostic tool for serum thyroglobulin (Tg) testing with or without radioiodine imaging in the follow-up of patients with well-differentiated thyroid cancer who have previously undergone thyroidectomy. Inter-lot variability in the Fourier transform near-infrared spectra of 30 samples obtained from four separate lots of Thyrogen® was detected in the Drug Quality Study (DQS). The vials fell into two distinct groups (rtst = 0.90, rlim= 0.98, p=0.02). In addition, one vial of the 30 (3%) appeared 4.7 multidimensional SDs from all of the other vials, suggesting that it also represents a different material.

Spectrometric Analysis of Dantrolene Sodium.

Dantrolene sodium is a direct-acting skeletal muscle relaxant. Dantrolene sodium for injection is indicated, along with suitable supportive measures, for the management of sudden, severe hypermetabolism of skeletal muscle typical of malignant hyperthermia crises in patients of any age. The formulation scanned in this work was designed to be injected intravenously. Intra-lot and inter-lot variability in the spectra of REVONTO™ (dantrolene sodium) was measured in the Drug Quality Study (DQS) using Fourier transform near-infrared spectrometry (FTNIR). Spectra of 69 vials from lot 20REV01A contained two groups (n1=56 vials, n2=13 vials) when scanned with an FTNIR. The two groups of spectra in lot 20REV01A were found to be 66.7 SDs apart using a subcluster detection test, suggesting that the two groups were manufactured differently. As a result, all available samples of dantrolene were examined. A library of spectra of 141 vials of dantrolene from 4 lots were found to contain 3 separate groups, also suggesting that different vials contain different materials.

Spectrometric Assessment of Generic and Brand Drug Quality for a Sentinel Screening Network.

The U.S. Food and Drug Administration (FDA) is a worldwide leader among analogous regulatory organizations in other countries. The FDA uses current good manufacturing practices to regulate the processes that produce drugs. Nevertheless, investigative journalists have pointed out problems in the drug supply, and pharmacies are not required to test the drugs they receive. The University of Kentucky Drug Quality Study does perform screening on the sterile injectable drugs that it receives and regularly reports new findings to FDA, practitioners, and the public. A Sentinel Screening Network of academic health systems could provide independent data on drug quality to FDA not available through manufacturers.

Spectral Intra-Lot and Inter-Lot Variability in Carfilzomib.

Carfilzomib is a prescription injectable drug approved for use by the FDA as an antineoplastic agent, part of a drug class of medications known as proteasome inhibitors, and used to stop and slow the growth and progression of cancer cells within the body. The drug is approved as an agent to treat multiple myeloma. It is provided as a single-use vial that contains 60 mg of carfilzomib as a sterile, white to off-white lyophilized cake or powder. Intra-lot and inter-lot variability in the spectra of carfilzomib vials was detected in the Drug Quality Study (DQS) using Fourier transform near-infrared spectrometry (FTNIR). One of 12 vials of lot 1143966 manufactured for Onyx Pharmaceuticals, Inc. appeared 4.7 multidimensional standard deviations (SDs) from the other 11 vials in a 3-D space formed by the first 3 principal components, which captured 81% of the total spectral variation. Spectra of 168 vials from 18 lots in the spectral library formed two groups in the 3-D space formed by the first 3 principal components. One group contained 155 vials and the other group contained 13 vials. The 2 groups had different locations and scales using a subcluster detection test at p=0.02.

Assessment of Vecuronium Quality Using Near-Infrared Spectrometry.

This study employed Fourier Transform near-infrared spectrometry to assess the quality of vecuronium bromide, a neuromuscular blocking agent. Spectral data from two lots of vecuronium were collected and analyzed using the BEST metric, principal component analysis (PCA) and other statistical techniques. The results showed that there was variability between the two lots and within each lot. Several outliers in the spectral data suggested potential differences in the chemical composition or sample condition of the vials. The outliers were identified and their spectral features were examined. A total of eight unique outliers were found in the PC space from PCs 1 to 9, so 22% of the total vials were outliers. The study findings suggest that the manufacturing process of vecuronium bromide may have been operating outside of a state of process control. Further investigation is needed to determine the source of these variations and their impact on the safety and efficacy of the drug product.

Spectrometric Analysis of Process Variations in Remifentanil.

ULTIVA® (remifentanil hydrochloride) is a sterile, nonpyrogenic, preservative-free, white to off-white lyophilized powder for intravenous (IV) administration after reconstitution and dilution. Each vial contains 1, 2, or 5 mg of remifentanil base; 15 mg glycine; and hydrochloric acid to buffer the solutions to a nominal pH of 3 after reconstitution. ULTIVA® is a µ-opioid agonist with rapid onset and peak effect, and short duration of action. Intra-lot and inter-lot variability in the spectra of ULTIVA® was measured in the Drug Quality Study (DQS) using Fourier transform near-infrared spectrometry (FTNIR). In 6 vials sampled, 1 came from lot 220453F while 5 came from lot 30020BF. The 1 vial sampled from lot 220453F appeared 122 multidimensional SDs from the other vials from lot 30020BF, suggesting that it represents a different formulation or material. Consequently, additional spectra from other lots were analyzed. Spectra of 90 vials from 9 lots in the spectral library contained vials that were outside the main group (50.3 SDs using a subcluster detection test), suggesting that the 35 library vials (39% of the total) contain different materials from the other 55 vials.

Potential Process Control Issues with Pemetrexed.

Pemetrexed is a folate analog metabolic inhibitor used in treatment of locally advanced or metastatic nonsquamous non-small cell lung cancer and for mesothelioma. Intra-lot and inter-lot variability in the spectra of ALIMTA® was detected in the Drug Quality Study (DQS) using Fourier transform near-infrared spectrometry (FTNIR). One vial of 12 (8%) sampled from lot S20I013A appeared 3.0 multidimensional SDs from the other vials, suggesting that it represents a different material. Consequently, additional spectra from other lots were analyzed. Spectra of 147 vials from 23 lots in the spectral library contained 14 vials that were outside the main group (26.4 SDs using a subcluster detection test), suggesting that the 14 library vials (9.5% of the total) also contain differing materials.

Variability in Content of Piperacillin and Tazobactam Injection.

Piperacillin and Tazobactam Injection is a combination product consisting of a penicillin-class antibacterial, piperacillin, and a beta-lactamase inhibitor, tazobactam, indicated for the treatment of patients with moderate to severe infections caused by susceptible isolates of bacteria. In the past decade some quality problems have been noted by the US Food and Drug Administration (FDA) with the Apotex Corp. manufacturing. Intra-lot and inter-lot variability in the spectra of Piperacillin and Tazobactam Injection 3.375 g was detected in the Drug Quality Study (DQS) using Fourier transform near-infrared spectrometry (FTNIR). One vial of 6 (17%) sampled from lot AD103008F3 appeared 14.4 multidimensional SDs from the other vials, suggesting that it represents a different material. Spectra of 132 vials from 19 lots in the spectral library contained 4 vials that were outside the group (21.0 SDs using a subcluster detection test), suggesting that the 4 library vials (3%) also contain differing materials.

FTNIR Spectrometry of Micafungin Sodium Quality.

Intra-lot and inter-lot variability in the spectra of micafungin was detected in the Drug Quality Study (DQS) using Fourier transform near-infrared spectrometry (FTNIR). Two vials of 6 vials sampled from Fresenius Kabi lot ACP106 appeared 7.9 and 14.0 standard deviations (SDs) from the center of the rest of the vials on the DQS FTNIR screening assay. Spectra of 48 vials from 7 lots in the library showed 2 outliers at 8.3 and 9.8 SDs from the center of the rest of the library, suggesting they represent different material.

Lack of Content Uniformity in MMR Vaccine.

The measles, mumps, and rubella (MMR) vaccine is a vaccine used to prevent measles, mumps, and rubella (German measles). The vaccine is mandated for children to attend public school in nearly all US states. However, measles cases have been increasing in the past decade, and quality problems have recently been noted by the US Food and Drug Administration (FDA) with the vaccine manufacturer. Intra-lot and inter-lot variability in the spectra of MMR (measles, mumps, and rubella) vaccine was detected in the Drug Quality Study (DQS) using Fourier transform near-infrared spectrometry (FTNIR). Six vials of 12 (50%) sampled from Merck lot U006488 appeared 14.5 SDs from the other vials on a subcluster detection test, suggesting that they represent different material. Spectra of 198 vials from 12 lots in the spectral library contained 140 vials in one tight ellipsoidal group, and 58 vials (30%) were outside that group (39.7 SDs using a subcluster detection test), suggesting that the library vials also contain differing materials.

Levothyroxine Variations by Process Analytical Technology.

Intra-lot and inter-lot variability in the spectra of levothyroxine was detected in the Drug Quality Study (DQS) using Fourier transform near-infrared spectrometry (FTNIR). Two vials of 12 vials sampled from Athenex lot AFN102 appeared 10.1 and 9.1 SDs from the center of the rest of the vials on the DQS FTNIR screening assay. Spectra of 108 vials from six lots in the library clustered in two groups (p=0.02), suggesting they represent different material.

Spectrometric Results of Process Variations in Dacarbazine.

Intra-lot and inter-lot variability in dacarbazine was detected in the Drug Quality Study (DQS) using Fourier transform near-infrared spectrometry (FTNIR). One vial of six vials sampled from Fresenius Kabi Lot 6125612 appeared 7.8 SDs from the center of the rest of the vials on the DQS FTNIR screening assay. Spectra of 54 vials from six lots in the library clustered in two groups (p=0.02), suggesting they represent different material.

Bridging integrator 1 (BIN1) protein expression increases in the Alzheimer's disease brain and correlates with neurofibrillary tangle pathology.

Recent genome wide association studies have implicated bridging integrator 1 (BIN1) as a late-onset Alzheimer's disease (AD) susceptibility gene. There are at least 15 different known isoforms of BIN1, with many being expressed in the brain including the longest isoform (iso1), which is brain-specific and localizes to axon initial segments and nodes of Ranvier. It is currently unknown what role BIN1 plays in AD. We analyzed BIN1 protein expression from a large number (n = 71) of AD cases and controls from five different brain regions (hippocampus, inferior parietal cortex, inferior temporal cortex, frontal cortex (BA9), and superior and middle temporal gyri). We found that the amount of the largest isoform of BIN1 was significantly reduced in the AD brain compared to age-matched controls, and smaller BIN1 isoforms were significantly increased. Further, BIN1 was significantly correlated with the amount of neurofibrillary tangle (NFT) pathology but not with either diffuse or neuritic plaques, or with the amount of amyloid-ß peptide. BIN1 is known to be abnormally expressed in another human disease, myotonic dystrophy, which also features prominent NFT pathology. These data suggest that BIN1 is likely involved in AD as a modulator of NFT pathology, and that this role may extend to other human diseases that feature tau pathology.

Obesity and diabetes cause cognitive dysfunction in the absence of accelerated ß-amyloid deposition in a novel murine model of mixed or vascular dementia.

Mid-life obesity and type 2 diabetes mellitus (T2DM) confer a modest, increased risk for Alzheimer's disease (AD), though the underlying mechanisms are unknown. We have created a novel mouse model that recapitulates features of T2DM and AD by crossing morbidly obese and diabetic db/db mice with APPΔNL/ΔNLx PS1P264L/P264L knock-in mice. These mice (db/AD) retain many features of the parental lines (e.g. extreme obesity, diabetes, and parenchymal deposition of ß-amyloid (Aß)). The combination of the two diseases led to additional pathologies-perhaps most striking of which was the presence of severe cerebrovascular pathology, including aneurysms and small strokes. Cortical Aß deposition was not significantly increased in the diabetic mice, though overall expression of presenilin was elevated. Surprisingly, Aß was not deposited in the vasculature or removed to the plasma, and there was no stimulation of activity or expression of major Aß-clearing enzymes (neprilysin, insulin degrading enzyme, or endothelin-converting enzyme). The db/AD mice displayed marked cognitive impairment in the Morris Water Maze, compared to either db/db or APPΔNLx PS1P264L mice. We conclude that the diabetes and/or obesity in these mice leads to a destabilization of the vasculature, leading to strokes and that this, in turn, leads to a profound cognitive impairment and that this is unlikely to be directly dependent on Aß deposition. This model of mixed or vascular dementia provides an exciting new avenue of research into the mechanisms underlying the obesity-related risk for age-related dementia, and will provide a useful tool for the future development of therapeutics.

Antisense oligonucleotide against GSK-3ß in brain of SAMP8 mice improves learning and memory and decreases oxidative stress: Involvement of transcription factor Nrf2 and implications for Alzheimer disease.

Glycogen synthase kinase (GSK)-3ß is a multifunctional protein that has been implicated in the pathological characteristics of Alzheimer's disease (AD), including the heightened levels of neurofibrillary tangles, amyloid-beta (Aß), and neurodegeneration. In this study we used 12-month-old SAMP8 mice, an AD model, to examine the effects GSK-3ß may cause regarding the cognitive impairment and oxidative stress associated with AD. To suppress the level of GSK-3ß, SAMP8 mice were treated with an antisense oligonucleotide (GAO) directed at this kinase. We measured a decreased level of GSK-3ß in the cortex of the mice, indicating the success of the antisense treatment. Learning and memory assessments of the SAMP8 mice were tested post-antisense treatment using an aversive T-maze and object recognition test, both of which observably improved. In cortex samples of the SAMP8 mice, decreased levels of protein carbonyl and protein-bound HNE were measured, indicating decreased oxidative stress. Nuclear factor erythroid-2-related factor 2 (Nrf2) is a transcription factor known to increase the level of many antioxidants, including glutathione-S transferase (GST), and is negatively regulated by the activity of GSK-3ß. Our results indicated the increased nuclear localization of Nrf2 and level of GST, suggesting the increased activity of the transcription factor as a result of GSK-3ß suppression, consistent with the decreased oxidative stress observed. Consistent with the improved learning and memory, and consistent with GSK-3b being a tau kinase, we observed decreased tau phosphorylation in brain of GAO-treated SAMP8 mice compared to that of RAO-treated SAMP8 mice. Lastly, we examined the ability of GAO to cross the blood-brain barrier and determined it to be possible. The results presented in this study demonstrate that reducing GSK-3 with a phosphorothionated antisense against GSK-3 improves learning and memory, reduces oxidative stress, possibly coincident with increased levels of the antioxidant transcriptional activity of Nrf2, and decreases tau phosphorylation. Our study supports the notion of GAO as a possible treatment for AD.

Transgenic models of Alzheimer's disease: better utilization of existing models through viral transgenesis.

Animal models have been used for decades in the Alzheimer's disease (AD) research field and have been crucial for the advancement of our understanding of the disease. Most models are based on familial AD mutations of genes involved in the amyloidogenic process, such as the amyloid precursor protein (APP) and presenilin 1 (PS1). Some models also incorporate mutations in tau (MAPT) known to cause frontotemporal dementia, a neurodegenerative disease that shares some elements of neuropathology with AD. While these models are complex, they fail to display pathology that perfectly recapitulates that of the human disease. Unfortunately, this level of pre-existing complexity creates a barrier to the further modification and improvement of these models. However, as the efficacy and safety of viral vectors improves, their use as an alternative to germline genetic modification is becoming a widely used research tool. In this review we discuss how this approach can be used to better utilize common mouse models in AD research. This article is part of a Special Issue entitled: Animal Models of Disease.