Development and validation of a method for the purity determination of (3beta,20R)-4,4-dimethylcholesta-8,14,24-trien-3-ol(FF-MAS) in pharmaceutical products containing recombinant human albumin

A simple and sensitive method has been developed and validated for purity determination of FF-MAS (also known as (3beta,20R)-4,4-dimethylcholesta-8,14,24-trien-3-ol an endogenous substance usually present in the pre-ovulatory follicular fluid) at very low concentrations (200 ng per unit) in pharmaceutical formulations containing RECOMBUMIN (recombinant human albumin) as the matrix. The paper focuses on development of the sample preparation for the product containing recombinant human albumin. After removal of recombinant human albumin by precipitation using a mixture of water and ethanol, the FF-MAS was concentrated by evaporation using a vacuum centrifuge and the prepared sample was analyzed. The purity method was based on a reversed-phase high performance liquid chromatography (RP-HPLC) with ultraviolet absorption detection at 250 nm. The method was validated according to ICH guidelines. The method indicated a significant degree of specificity with good selectivity and no significant effect from the matrix. The limit of detection was found to be 0.3-0.8% (depending on the impurity) corresponding to 1.9-5.1 ng. The limit of quantification was found to be 0.8-2.5% (depending on the impurity) corresponding to 5.2-16 ng. The recovery was found to be between 90 and 101% for the FF-MAS, and 100-129% for the six known impurities. The tested range for FF-MAS was from 320 to 960 ng corresponding to 50-150% of the nominal concentration (640 ng, injection volume is 100 microl). The linearity of each compound (FF-MAS and the six impurities) was investigated. The squared correlation coefficient (r(2)) was 0.999 for FF-MAS (50-150% level) and 0.977-0.998 for the six known impurities (at four levels: 0.20, 0.50, 1.00, 2.00%). The R.S.D. in the repeatability study was found to be 9.2% for the total amount of impurities, and 10.4% for single impurities. The R.S.D. in the intermediate precision study was found to be 10.9% for total impurities, and 12.0% for single impurities. The validation results showed that the method was suitable for the purity analysis. The validated method was then ready for use for samples analysis of phase II clinical studies and the stability investigations of the pharmaceutical product.     

Control Strategy for Small Molecule Impurities in Antibody-Drug Conjugates

Antibody-drug conjugates (ADCs) are an emerging class of biopharmaceuticals. As such, there are no specific guidelines addressing impurity limits and qualification requirements. The current ICH guidelines on impurities, Q3A (Impurities in New Drug Substances), Q3B (Impurities in New Drug Products), and Q6B (Specifications: Test Procedures and Acceptance Criteria for Biotechnological/Biological Products) do not adequately address how to assess small molecule impurities in ADCs. The International Consortium for Innovation and Quality in Pharmaceutical Development (IQ) formed an impurities working group (IWG) to discuss this issue. This white paper presents a strategy for evaluating the impact of small molecule impurities in ADCs. This strategy suggests a science-based approach that can be applied to the design of control systems for ADC therapeutics. The key principles that form the basis for this strategy include the significant difference in molecular weights between small molecule impurities and the ADC, the conjugation potential of the small molecule impurities, and the typical dosing concentrations and dosing schedule. The result is that exposure to small impurities in ADCs is so low as to often pose little or no significant safety risk.     

Development of a Validated LC Method for Separation of Process‐Related Impurities Including the R‐enantiomer of S‐Pramipexole on Polysaccharide Chiral Stationary Phases

Despite the availability of a few methods for individual separation of S‐pramipexole from its process‐related impurities, no common liquid chromatography (LC) method is reported so far in the literature. The present article describes the development of a single‐run LC method for simultaneous determination of S‐pramipexole and its enantiomeric and process‐related impurities on a Chiralpak AD‐H (150 x 4.6 mm, 5μm) column using n‐hexane/ethanol/n‐butylamine (75:25:0.1 v/v/v) as a mobile phase in an isocratic mode of elution at a flow rate of 1.2 ml/min at 30°C. The chromatographic eluents were monitored at a wavelength of 260 nm using a photodiode array detector. Excellent enantioseparation with good resolutions (Rs ≥ 2.88) and peak shapes (A_s ≤ 1.21) for all analytes was achieved. The proposed method was validated according to International Conference Harmonization (ICH) guidelines in terms of accuracy, precision, sensitivity, and linearity. Limits of quantification of impurities (0.25–0.55 μg/ml) indicate the highest sensitivity achievable by the proposed method. The method has an advantage of selectivity and suitability for routine determination of not only chiral impurity but also all possible related substances in active pharmaceutical ingredients of S‐pramipexole. Chirality 27:430–435, 2015. © 2015 Wiley Periodicals, Inc.     

Assessment of QT liabilities in drug development

Since the publication, in 1997, of the CPMP (Committee for Proprietary Medicinal Products) Points to Consider document on "The assessment of potential for QT prolongation by non-cardiovascular medicinal products," both regulatory bodies and the pharmaceutical industry have paid increasing attention to the conduct of careful preclinical studies on the subject. Regulatory attention has focused on the drafting of Safety Pharmacology guidelines through the ICH (International Conference on Harmonization) process, which resulted in approval by the ICH and acceptance by the three main regions (USA, Europe, and Japan) of the ICH S7A guideline. The guideline does not deal only with cardiovascular studies and does not provide guidance on QT investigations. This part has been deferred to a second guideline (ICH S7B). Nevertheless, pharmaceutical companies have implemented screening strategies aimed at selecting compounds that do not present QT liabilities. These strategies can differ according to the pharmaceutical class, while experimental models differ according to the stage of development of the compound. Several in vitro models are employed in discovery (radioligand binding, high-throughput patch clamp, efflux, and fluorescence assays). These models, coupled with in silico methods, allow companies to screen a high number of compounds. Other in vitro models, applied later in the R&D process (action potential duration, APD, in Purkinje fibers or papillary muscle and the isolated heart) are useful in better describing the activity of compounds on cardiac ion channels. The most robust and accepted in vivo test is represented by telemetry studies in conscious non-rodents.     

Normal phase and reverse phase HPLC-UV-MS analysis of process impurities for rapamycin analog ABT-578: application to active pharmaceutical ingredient process development

ABT-578, an active pharmaceutical ingredient (API), is a semi-synthetic tetrazole derivative of the fermented polyene macrolide rapamycin. Reverse phase (RP)-HPLC-UV-MS and normal phase (NP)-HPLC-UV-MS methods employing an LC/MSD trap with electrospray ionization (ESI) have been developed to track and map all significant impurities from the synthetic process. Trace-level tracking of key impurities occurring at various process points was achieved using complimentary methodologies, including a stability indicating reverse phase HPLC method capable of separating at least 25 starting materials and process-related impurities from the API (YMC-Pack Phenyl column, UV-MS, 210 nm) and a targeted reverse phase HPLC method capable of separating very polar compounds from crude reaction mixtures (Phenomenex Synergi Polar RP column, UV, 265 nm). In addition, a normal phase HPLC method condition with post-column modifier infusion is described for the separation of epimeric impurities, and analysis of aqueous-sensitive reactive species (YMC-Pack SIL column, UV-MS, 278 nm). Process control strategies were established with these combinations of analytical technologies for impurities analyses to enable a rich understanding of the ABT-578 process.     

晚期早产儿颅内出血的相关因素分析

目的：探讨晚期早产儿颅内出血的相关因素,指导晚期早产儿颅内出血的防治。方法：2011年9月至2012年8月我院收治晚期早产儿253例,其中有210例行头颅MRI检查,以经头颅MRI检查确诊颅内出血30例为ICH组,同时随机抽取同期住院的经头颅MRI证实无颅内出血晚期早产儿60例作为对照组。应用SPSS 17.0进行统计学分析。结果：1.ICH组产前激素应用率显著低于对照组（P〈0.05）。2.ICH组经阴分娩、胎膜早破、代谢性酸中毒发生率显著高于对照组（P〈0.05）。3.Logistic回归分析显示产前激素是颅内出血的保护因素（P〈0.05）,而经阴分娩、胎膜早破（P〈0.01）、代谢性酸中毒（P〈0.05）是颅内出血的危险因素。结论：产前应用激素是晚期早产儿颅内出血的保护因素,经阴分娩、胎膜早破、代谢性酸中毒是晚期早产儿颅内出血高危因素。     

Reactive Impurities in Excipients: Profiling,Identification and Mitigation of Drug–Excipient Incompatibility

Reactive impurities in pharmaceutical excipients could cause drug product instability, leading to decreased product performance, loss in potency, and/or formation of potentially toxic degradants. The levels of reactive impurities in excipients may vary between lots and vendors. Screening of excipients for these impurities and a thorough understanding of their potential interaction with drug candidates during early formulation development ensure robust drug product development. In this review paper, excipient impurities are categorized into six major classes, including reducing sugars, aldehydes, peroxides, metals, nitrate/nitrite, and organic acids. The sources of generation, the analytical method for detection, the stability of impurities upon storage and processing, and the potential reactions with drug candidates of these impurities are reviewed. Specific examples of drug–excipient impurity interaction from internal research and literature are provided. Mitigation strategies and corrective measures are also discussed.     

Amount of Bleeding and Hematoma Size in the Collagenase-Induced Intracerebral Hemorrhage Rat Model

The aggravated risk on intracerebral hemorrhage (ICH) with drugs used for stroke patients should be estimated carefully. We therefore established sensitive quantification methods and provided a rat ICH model for detection of ICH deterioration. In ICH intrastriatally induced by 0.014-unit, 0.070-unit, and 0.350-unit collagenase, the amount of bleeding was measured using a hemoglobin assay developed in the present study and was compared with the morphologically determined hematoma volume. The blood amounts and hematoma volumes were significantly correlated, and the hematoma induced by 0.014-unit collagenase was adequate to detect ICH deterioration. In ICH induction using 0.014-unit collagenase, heparin enhanced the hematoma volume 3.4-fold over that seen in control ICH animals and the bleeding 7.6-fold. Data suggest that this sensitive hemoglobin assay is useful for ICH detection, and that a model with a small ICH induced with a low-dose collagenase should be used for evaluation of drugs that may affect ICH.     

Impact of ciprofloxacin impurities on bacterial growth,antibiotic resistance development and content assays

In addition to active pharmaceutical ingredient (API), antibiotics may contain small amounts of excipients and impurities and be prone to accumulation of degradation products. There has been limited work characterizing how these substances impact bacterial growth and antibiotic resistance development. We investigated how two ciprofloxacin (CIP) impurities, fluoroquinolonic acid (FQA) and ciprofloxacin ethylenediamine analogue (CEA), impact growth and antibiotic resistance in Escherichia coli. Additionally, we investigated how these impurities impact a frequently used API content assay. Both impurities displayed modest antimicrobial activity compared to the CIP API. The effective antimicrobial activity of a medicine containing increased impurity levels may permit bacterial growth and resistance development. Our results also suggest that increasing exposure concentration and duration to CEA and FQA, independent of CIP, can promote antibiotic resistance development. However, at concentrations of 100% and below the MIC of the API, impurities had limited contributions to resistance development compared to the CIP API. From a methodological standpoint, we found that UV spectrophotometry may be inadequate to account for antibiotic impurities or degradation products. This can lead to incorrect estimations of API content and we propose additional multi-wavelength measures when using UV spectrophotometry to help identify impurities or degradation.     

Aspects of melatonin manufacturing and requirements for a reliable active component

Commercially available melatonin was found to contain impurities associated with eosinophilia-myalgia syndrome (EMS). From sample analysis, remarkable differences in impurity profiles between the active ingredient from various suppliers could be found. An industrial process was developed which guarantees a high purity melatonin active ingredient. All potential impurities have been characterized and synthetized for analytical conformity with pharmaceutical regulations. To avoid any side effects from impurities, only high-purity melatonin should be utilized from the laboratory through to commercialization.     

Control of the heparosan <Emphasis Type="Italic">N</Emphasis>-deacetylation leads to an improved bioengineered heparin

The production of the anticoagulant drug heparin from non-animal sources has a number of advantages over the current commercial production of heparin. These advantages include better source material availability, improved quality control, and reduced concerns about animal virus or prion impurities. A bioengineered heparin would have to be chemically and biologically equivalent to be substituted for animal-sourced heparin as a pharmaceutical. In an effort to produce bioengineered heparin that more closely resembles pharmaceutical heparin, we have investigated a key step in the process involving the N-deacetylation of heparosan. The extent of N-deacetylation directly affects the N-acetyl/N-sulfo ratio in bioengineered heparin and also impacts its molecular weight. Previous studies have demonstrated that the presence and quantity of N-acetylglucosamine in the nascent glycosaminoglycan chain, serving as the substrate for the subsequent enzymatic modifications (C5 epimerization and O-sulfonation), can impact the action of these enzymes and, thus, the content and distribution of iduronic acid and O-sulfo groups. In this study, we control the N-deacetylation of heparosan to produce a bioengineered heparin with an N-acetyl/N-sulfo ratio and molecular weight that is similar to animal-sourced pharmaceutical heparin. The structural composition and anticoagulant activity of the resultant bioengineered heparin was extensively characterized and compared to pharmaceutical heparin obtained from porcine intestinal mucosa.     

A review on biocide reduced susceptibility due to plasmid-borne antiseptic-resistant genes—special notes on pharmaceutical environmental isolates

Biocides (antiseptics and disinfectants) are widely used in hospitals and pharmaceutical industries for contamination control. The emergence of reduced susceptibility to biocides is the major concern and this is caused by various factors, among which plasmid-mediated resistance is common. Many publications describe the antibiotic resistance and mechanisms in a clinical setting. However, there are only limited studies available worldwide addressing the molecular mechanisms of biocide resistance in the pharmaceutical sector. In addition, there is a considerable lack of scientific reports regarding minimum inhibitory concentration (MIC) values of typical biocides against pharmaceutical cleanroom environmental isolates. This review analyses the plasmid-mediated resistance in typical pharmaceutical micro-organisms and prevalence of biocide-resistant genes among common clinical and pharmaceutical isolates. This review discusses the MIC values of biocides in pharmaceutical environmental isolates, indicating the importance of the correlation between the presence or absence of biocide-resistant genes and reduced susceptibility of MIC values. This review recommends that pharmaceutical organizations adopt policies and test methodologies to examine the MICs of common cleanroom biocides against the most common types of cleanroom environmental isolates.     

A New Role Discovered for IGTP: The Protective Effect of IGTP in ICH-Induced Neuronal Apoptosis

Interferon gamma-induced GTPase (IGTP), which is also named Irgm3, has been widely described in regulating host resistance against intracellular pathogens. Previous researches have demonstrated that IGTP exerts beneficial function during coxsackievirus B3 (CVB3) infection. However, little information is available regarding the role of IGTP in central nervous system. Here, our study revealed that IGTP may have an essential role during ICH-induced neuronal apoptosis. We found the expression level of IGTP adjacent to hematoma was strongly increased after ICH, accompanied with the up-regulation of proliferating cell nuclear antigen (PCNA), active-caspase-3, p-GSK-3β, and Bax. IGTP was also observed to be co-localized with PCNA in astrocytes and active-caspase-3 in neurons, indicating its association with astrocyte proliferation and neuronal apoptosis after ICH. Finally, in vitro study, knocking down IGTP with IGTP-specific siRNA promoted active-caspase-3, p-GSK-3β, and Bax expression, and led to more severe neuronal apoptosis after ICH. All these results above suggested that IGTP might play a critical role in protecting neurons from apoptosis after ICH.     

Photostability Issues in Pharmaceutical Dosage Forms and Photostabilization

Photodegradation is one of the major pathways of the degradation of drugs. Some therapeutic agents and excipients are highly sensitive to light and undergo significant degradation, challenging the quality and the stability of the final product. The adequate knowledge of photodegradation mechanisms and kinetics of photosensitive therapeutic entities or excipients is a pivotal aspect in the product development phase. Hence, various pharmaceutical regulatory agencies, across the world, mandated the industries to assess the photodegradation of pharmaceutical products from manufacturing stage till storage, as per the guidelines given in the International Conference on Harmonization (ICH). Recently, numerous formulation and/or manufacturing strategies has been investigated for preventing the photodegradation and enhancing the photostability of photolabile components in the pharmaceutical dosage forms. The primary focus of this review is to discuss various photodegradation mechanisms, rate kinetics, and the factors that influence the rate of photodegradation. We also discuss light-induced degradation of photosensitive lipids and polymers. We conclude with a brief note on different approaches to improve the photostability of photosensitive products.     

The Views of Healthcare Professionals,Drug Developers and Regulators on Information about Older People Needed for Rational Drug Prescription

Background

The ICH E7 guideline intends to improve the knowledge about medicines in geriatric patients. As a legislative document, it might not reflect the needs of healthcare professionals. This study investigated what information healthcare professionals, regulatory agencies and pharmaceutical industries consider necessary for rational drug prescribing to older individuals.

Methods and Findings

A 29-item-questionnaire was composed, considering the representation in trials, pharmacokinetics, efficacy, safety, and convenience of use in older individuals, with space for additions. Forty-three European professionals with an interest in medication for older individuals were included. In order to investigate their relevance, five items were included in a second questionnaire, with 11 control items. Median scores, differences between clinical and non-clinical respondents and response consistency were analysed. Consistency was present in 10 control items. Therefore, all items of the first questionnaire and the five additional items were analysed. Thirty-seven (86%) respondents returned the first questionnaire; 31/37 (84%) the second. Information about age-related differences in adverse events, locomotor effects, drug-disease interactions, dosing instructions, and information about the proportion of included 65+ patients was considered necessary by most respondents. Clinicians considered information significantly more important than the non-clinical respondents about the inclusion of 75+, time-until-benefit in older people, anticholinergic effects, drug-disease interactions, and convenience of use. Main study limitations are the focus on information for daily practice, while the ICH E7 guideline is a legislative document focused on market approval of a new medicine. Also, a questionnaire with a Likert scale has its limitations; this was addressed by providing space for comments.

Conclusions

This study reveals that items considered necessary are currently not included in the ICH E7 guideline. Also, clinicians’ and non-clinicians’ opinions differed significantly in 15% of the items. Therefore, all stakeholders should collaborate to improve the availability of information for the rational prescribing to older individuals.     

Optimization of a recombinant human growth hormone purification process using quality by design

This work describes a strategy to optimize a downstream processing of a recombinant human growth hormone (rhGH) by incorporating a quality by design approach toward meeting higher quality specifications. The optimized process minimized the presence of impurities and degradation by-products during manufacturing by the establishment of in-process controls. Capillary zone electrophoresis, reverse phase, and size-exclusion chromatographies were used as analytical techniques to establish new critical process parameters for the solubilization, capture, and intermediate purification steps aiming to maintain rhGH quality by complying with pharmacopeial specifications. The results indicated that the implemented improvements in the process allowed the optimization of the specific recovery and purification of rhGH without compromising its quality. In addition, this optimization facilitated the stringent removal of the remaining impurities in further polishing stages, as demonstrated by the analysis of the obtained active pharmaceutical ingredient.     

The role and therapeutic potential of heat shock proteins in haemorrhagic stroke

Heat shock proteins (HSPs) are induced after haemorrhagic stroke, which includes subarachnoid haemorrhage (SAH) and intracerebral haemorrhage (ICH). Most of these proteins function as neuroprotective molecules to protect cerebral neurons from haemorrhagic stroke and as markers to indicate cellular stress or damage. The most widely studied HSPs in SAH are HSP70, haeme oxygenase‐1 (HO‐1), HSP20 and HSP27. The subsequent pathophysiological changes following SAH can be divided into two stages: early brain injury and delayed cerebral ischaemia, both of which determine the outcome for patients. Because the mechanisms of HSPs in SAH are being revealed and experimental models in animals are continually maturing, new agents targeting HSPs with limited side effects have been suggested to provide therapeutic potential. For instance, some pharmaceutical agents can block neuronal apoptosis signals or dilate cerebral vessels by modulating HSPs. HO‐1 and HSP70 are also critical topics for ICH research, which can be attributed to their involvement in pathophysiological mechanisms and therapeutic potential. However, the process of HO‐1 metabolism can be toxic owing to iron overload and the activation of succedent pathways, for example, the Fenton reaction and oxidative damage; the overall effect of HO‐1 in SAH and ICH tends to be protective and harmful, respectively, given the different pathophysiological changes in these two types of haemorrhagic stroke. In the present study, we focus on the current understanding of the role and therapeutic potential of HSPs involved in haemorrhagic stroke. Therefore, HSPs may be potential therapeutic targets, and new agents targeting HSPs are warranted.     

Investigation of the effect of hyperglycemia on intracerebral hemorrhage by proteomic approaches

Intracerebral hemorrhage (ICH) is associated with high mortality and disability, and hyperglycemia worsens the clinical and neurological outcomes of patients with ICH. In this study, we utilized proteomic approaches to investigate the role of hyperglycemia in ICH. Hyperglycemia was induced by intraperitoneal injection of streptozotocin (STZ) in adult Sprague-Dawley male rats; ICH was induced by stereotaxic infusion of collagenase/heparin into the right striatum. It was observed that the size of induced hemorrhage was significantly larger in the hyperglycemic group (n=6 in each group). On the first day after ICH, an apparent decrease in the bilateral grasp was also observed for the lesioned hyperglycemic rats compared with normoglycemic ones. When employing 2-DE and MS to examine the proteomes of perihematomal and control regions in individual hyperglycemic and normoglycemic rats, eight differentially expressed protein targets were identified. Most noteworthy, in response to ICH significant increase of albumin was ubiquitously observed in the brains of normoglycemic rats but not in the brains of hyperglycemic rats. Coincidentally, more significant neuronal apoptosis were found in the perihematomal regions of hyperglycemic rats. These observations described suggest the protection role of albumin in acute stage of ICH, which may be dependent on different blood sugar levels.     

Acetazolamide alleviates sequelae of hyperglycaemic intracerebral haemorrhage by suppressing astrocytic reactive oxygen species

Abstract

Hyperglycaemia is associated with the poor outcome after intracerebral haemorrhage (ICH). Acetazolamide (AZA), a kind of carbonic anhydrogenase (CA) inhibitor, its effectiveness in ICH had been reported. However, the connections between AZA and ICH, especially in hyperglycaemia condition had never been defined. In this study, adult Sprague–Dawley rats were administered with vehicle or streptozotocin (STZ) to render them into normoglycaemic (NG) or hyperglycaemic (HG), respectively. Collagenase was then injected into the striatum. The NG or HG ICH rats treated with vehicle control or 5?mg/kg AZA (oral gavage) underwent haemorrhagic area assessments on the 1st, 4th, and 7th day after ICH. The coverage of pericytes was examined by immunohistochemistry. Reactive oxygen species (ROS) levels were assessed in mouse astrocyte cell line treated with vehicle or 20?μmol/L of AZA in culture media according to two different glucose concentrations. AZA reduced the haematoma size, improved neurobehavioral functions, suppressed astrocytic ROS production in vitro, and preserved cerebral pericytes coverage, which are even more remarkable in HG conditions. The present study indicates that AZA may alleviate some sequelae after ICH, especially in poorer prognostic HG rats through the suppression of astrocytic ROS production.     

New approaches with two cyano columns to the separation of acetaminophen, phenylephrine, chlorpheniramine and related compounds

The development of new pharmaceutical forms with classical active compounds generates new analytical problems. That is the case of sugar-free sachets of cough-cold products containing acetaminophen, phenylephrine hydrochloride and chlorpheniramine maleate. Two cyanopropyl stationary phases have been employed to tackle the problem. The Discovery cyanopropyl (SUPELCO) column permitted the separation of the three actives, maleate and excipients (mainly saccharine and orange flavour) with a constant proportion of aqueous/ organic solvent (95:5, v/v) and a pH gradient from 7.5 to 2. The run lasted 14 min. This technique avoids many problems related to baseline shifts with classical organic solvent gradients and opens great possibilities to modify selectivity not generally used in reversed phase HPLC. On the other hand, the Agilent Zorbax SB-CN column with a different retention profile permitted us to separate not only the three actives and the excipients but also the three known related compounds: 4-aminophenol, 4-chloracetanilide and 4-nitrophenol in an isocratic method with a run time under 30 min. This method was validated following ICH guidelines and validation parameters showed that it could be employed as stability-indicating method for this pharmaceutical form.