Molecular Mechanisms Involved in the Chemical Instability of ONC201 and Methods to Counter Its Degradation in Solution.

Glioblastoma is one of the most common and aggressive forms of brain tumor, a rare disease for which there is a great need for innovative therapies. ONC201, a new drug substance, has been used in a compassionate treatment program where the choice of dosage form and regimen have yet to be justified. The prior knowledge needed to anticipate ONC201 stability problems has recently been partially addressed, by (i) showing that ONC201 is sensitive to light and oxidation and (ii) identifying the molecular structures of the main degradation products formed. The aim of the work presented here was to improve our understanding of the degradation pathways of ONC201 using data from ab initio calculations and experimental work to supplement the structural information we already published. The C-H bonds located αto the amine of the tetrahydropyridine group and those located alpha to the imine function of the dihydroimidazole group exhibit the lowest bond dissociation energies (BDEs) within the ONC201 molecule. Moreover, these values drop well below 90 kcal.mol-1 when ONC201 is in an excited state (S1; T1). The structures of the photoproducts we had previously identified are consistent with these data, showing that they would have resulted from radical processes following the abstraction of alpha hydrogens. Concerning ONC201's sensitivity to oxidation, the structures of the oxidation products matched the critical points revealed through mapped electrostatic potential (MEP) and average local ionization energy (ALIE). The data obtained from ab initio calculations and experimental work showed that the reactivity of ONC201 to light and oxidation conditions is highly dependent on pH. While an acidic environment (pH < 6) contributes to making ONC201 quantitatively more stable in solution in the face of oxidation and photo-oxidation, it nevertheless seems that certain chemical groups in the molecule are more exposed to nucleophilic attacks, which explains the variation observed in the profile of degradation products formed in the presence of certain antioxidants tested. This information is crucial to better understand the stability results in the presence of antioxidant agents and to determine the right conditions for them to act.

Development of carbon monoxide-releasing molecules conjugated to polysaccharides (glyco-CORMs) for delivering CO during obesity.

Metal carbonyls have been developed as carbon monoxide-releasing molecules (CO-RMs) to deliver CO for therapeutic purposes. The manganese-based CORM-401 has been recently reported to exert beneficial effects in obese animals by reducing body weight gain, improving glucose metabolism and reprogramming adipose tissue towards a healthy phenotype. Here, we report on the synthesis and characterization of glyco-CORMs, obtained by grafting manganese carbonyls on dextrans (70 and 40 kDa), based on the fact that polysaccharides facilitate the targeting of drugs to adipose tissue. We found that glyco-CORMs efficiently deliver CO to cells in vitro with higher CO accumulation in adipocytes compared to other cell types. Oral administration of two selected glyco-CORMs (5b and 6b) resulted in CO accumulation in various organs, including adipose tissue. In addition, glyco-CORM 6b administered for eight weeks elicited anti-obesity and positive metabolic effects in mice fed a high fat diet. Our study highlights the feasibility of creating carriers with multiple functionalized CO-RMs.

3D-Printed, Liquid-Filled Capsules of Concentrated and Stabilized Polyphenol Epigallocatechin Gallate, Developed in a Clinical Trial.

In vitro studies have shown that epigallocatechin gallate (EGCG), the most potent antioxidant of the green tea polyphenol catechins, is able to effectively prevent the formation of amyloid plaques and induce their clearance. However, its high chemical reactivity promotes high chemical instability, which represents a major obstacle for the development of pharmaceutical forms containing solubilized EGCG, an essential condition for a better systemic passage via the oral route. After discovering that EGCG forms a deep eutectic with choline chloride, we exploited this property to formulate and patent liquid-filled capsules containing 200-800 mg of soluble EGCG in easy-to-administer sizes. The gelatin envelopes used are of the conventional type and their filling has been achieved using 3D printing technology. Not only did the EGCG-choline complex allow the formulation of hydrophilic solutions with a high concentration of active substance but it also contributed significantly to its chemical stability, since after at least 18 months of storage at 25 °C/60% RH and one year at 40 °C/75% RH, the capsules show unchanged hardness, chromatographic profiles and antioxidant activity compared to T0. Preclinical studies in monkeys showed that bioavailability was increased by a factor of 10 compared to marketed capsules comprising EGCG powder. This pharmaceutical development was conducted in the context of upcoming clinical trials to evaluate EGCG alone or in combination when treating transthyretin and light-chain cardiac amyloidosis.

Identification of the Major Degradation Pathways of Selumetinib.

Selumetinib is administered orally in capsule form and is indicated for the treatment of neurofibromatosis. To facilitate dosage adjustments, liquid preparations, such as solutions or suspensions, are to be developed. This led, first, to determine the stability profile of soluble or dispersed selumetinib and, secondly, to look for ways to stabilize the active substance. The degradation kinetics of selumetinib as a function of stress conditions were determined and compared. The degradation products were detected and identified by LC-HRMSn. In solution, selumetinib is sensitive to oxidation and degrades by photooxidation. In both cases, the side chain represented by the oxoamide group is concerned, leading to the formation of an amide derivative for the first case and an ester derivative for the second. The identification of such degradation mechanisms allowed us to study, in a targeted way, processes aiming at stabilizing the active molecule.

Unequal Behaviour between Hydrolysable Functions of Nirmatrelvir under Stress Conditions: Structural and Theoretical Approaches in Support of Preformulation Studies.

Nirmatrelvir is an antiviral drug approved for the treatment of COVID-19. The available dosage form consists of tablets marketed under the brand name PAXLOVID®. Although knowledge of nirmatrelvir's intrinsic stability may be useful for any potential development of other pharmaceutical forms, no data regarding this matter is available to date. Preliminary forced degradation studies have shown that the molecule is stable under oxidative and photolytic conditions, while hydrolytic conditions, both acidic and basic, have proven deleterious. Indeed, the molecule presents a priori several functions that can undergo hydrolysis, i.e., three amide moieties and a nitrile function. However, considering the degradation products formed under forced conditions and which were detected and identified by LC-UV-HRMSn, the hydrolysis process leading to their formation is selective since it involved only 2 of the 4 hydrolysable functions of the molecule. Ab initio studies based on density functional theory (DFT) have helped better understand these reactivity differences in aqueous media. Some hydrolyzable functions of nirmatrelvir differ from others in terms of electrostatic potential and Fukui functions, and this seems to correlate with the forced degradation outcomes.

Mixed Polymeric Micelles for Rapamycin Skin Delivery.

Facial angiofibromas (FA) are one of the most obvious cutaneous manifestations of tuberous sclerosis complex. Topical rapamycin for angiofibromas has been reported as a promising treatment. Several types of vehicles have been used hitherto, but polymeric micelles and especially those made of d-α-tocopherol polyethylene glycol 1000 succinate (TPGS) seem to have shown better skin bioavailability of rapamycin than the so far commonly used ointments. To better understand the influence of polymeric micelles on the behavior of rapamycin, we explored it through mixed polymeric micelles combining TPGS and poloxamer, evaluating stability and skin bioavailability to define an optimized formulation to effectively treat FA. Our studies have shown that TPGS improves the physicochemical behavior of rapamycin, i.e., its solubility and stability, due to a strong inclusion in micelles, while poloxamer P123 has a more significant influence on skin bioavailability. Accordingly, we formulated mixed-micelle hydrogels containing 0.1% rapamycin, and the optimized formulation was found to be stable for up to 3 months at 2-8 °C. In addition, compared to hydroalcoholic gel formulations, the studied system allows for better biodistribution on human skin.

Simple Approach to Enhance Green Tea Epigallocatechin Gallate Stability in Aqueous Solutions and Bioavailability: Experimental and Theoretical Characterizations.

Because of its antioxidant, antimutagenic, and anti-infectious properties, epigallocatechin gallate (EGCG) is the most interesting compound among the green tea catechins polyphenols. However, its health effects are inconclusive due to its very low bioavailability, largely due to a particular instability that does not allow EGCG to reach the potency required for clinical developments. Over the last decade, many efforts have been made to improve the stability and bioavailability of EGCG using complex delivery systems such as nanotechnology, but these efforts have not been successful and easy to translate to industrial use. To meet the needs of a large-scale clinical trial requiring EGCG in a concentrated solution to anticipate swallowing impairments, we developed an EGCG-based aqueous solution in the simplest way while trying to circumvent EGCG instability. The solution was thoroughly characterized to sort out the unexpected stability outcome by combining experimental (HPLC-UV-mass spectrometry and infrared spectroscopy) and computational (density functional theory) studies. Against all odds, the EGCG-sucrose complex under certain conditions may have prevented EGCG from degradation in aqueous media. Indeed, in agreement with the ICH guidelines, the formulated solution was shown to be stable up to at least 24 months under 2-8 °C and at ambient temperature. Furthermore, considerable improvement in bioavailability in rats, against EGCG powder formulated in hard-gel capsules, was shown after gavage. Thus, the proposed formulation may provide an easily implementable platform to administer EGCG in the context of clinical development.

Ruxolitinib photodegradation mechanisms by theoretical and experimental chemistry.

Ruxolitinib is a Janus Kinase inhibitor currently approved for the treatment of myelofibrosis. It is also a promising drug for the treatment of skin and infectious diseases. In terms of pharmaceutical stability, although ruxolitinib has been established as being sensitive to light, no data on photodegradation processes are available to date, while these may be useful for quality risk management and any potential development of other pharmaceutical forms for other routes of administration. One way to partially fill this gap was to carry out a study that combines a consistent determination of the most sensitive sites of the molecule to photolysis through theoretical calculations based on functional density, with the identification of the main photodegradation products obtained after forced degradation. This integrated approach has shown converging results describing the mechanisms based on photo-oxidation that can lead to the opening of the pyrrole ring. Having access to the structure of the degradation products and intermediates then made it possible to carry out an in silico evaluation of their potential mutagenicity and it appears that some of them feature alert structures.

Intrinsic stability of the antiviral drug umifenovir by stress testing and DFT studies.

Umifenovir is an antiviral drug approved in China and Russia for the treatment of influenza. The available dosage form consists of capsules marketed under the brand name Arbidol®. Due to its broad spectrum, umifenovir may also be used in other viral contexts, alone or combined with other antiviral drugs. Although knowledge of umifenovir intrinsic stability may be useful for any potential development of other pharmaceutical forms for other routes of administration and for quality risk management, no data regarding this matter is available to date. In this study, the exploration of the molecule's behaviour under hydrolytic, oxidative and photolytic conditions was carried out experimentally and supported by density functional theory (DFT) studies. It comes out that umifenovir is sensitive to these stress conditions giving rise to 6 structurally characterized degradation products. The one-electron oxidation process produced on the sulphur atom is probably the main cause of umifenovir degradation with reference to the structures of the degradation products formed and the DFT data.

Comparison of the In Vitro and Ex Vivo Permeation of Existing Topical Formulations Used in the Treatment of Facial Angiofibroma and Characterization of the Variations Observed.

Rapamycin has been used topically to treat facial angiofibromas associated with tuberous sclerosis for more than a decade. In the absence of a commercial form, a large number of formulations have been clinically tested. However, given the great heterogeneity of these studies, particularly with regard to the response criteria, it was difficult to know the impact and thus to compare the relevance of the formulations used. The objective of this work was therefore to evaluate the link between the diffusion of rapamycin and the physico-chemical characteristics of these different formulations on Strat-M® membranes as well as on human skin using Franz cells. Our results underline the importance of the type of vehicle used (hydrogel > cream > lipophilic ointment), the soluble state of rapamycin and its concentration close to saturation to ensure maximum thermodynamic activity. Thus, this is the first time that a comparative study of the different rapamycin formulations identified in the literature for the management of facial angiofibromas has been carried out using a pharmaceutical and biopharmaceutical approach. It highlights the important parameters to be considered in the development and optimization of topical rapamycin formulations with regard to cutaneous absorption for clinical efficacy.

Post hoc study to investigate the potential causes of poor quality of cardiovascular medicines collected in sub-Saharan countries.

OBJECTIVES: The incidence of cardiovascular diseases is increasing and there is a growing need to provide access to quality cardio drugs in Africa. In the SEVEN study, we analysed 1530 cardiovascular drug samples randomly collected from 10 African countries. By that time, of the seven drugs products analysed, only those containing amlodipine and captopril had very low assay values with active substance contents that could be less than 75% of those expected. In this article we investigate complementary aspects of the amlodipine and captopril samples so to explain the previously observed low assays for these two drugs. DESIGN: Post hoc analysis of the captopril and amlodipine drugs samples and their packages collected in the context of the SEVEN study. SETTING: 10 countries were concerned: Benin, Burkina Faso, Congo, Democratic Republic of the Congo, Guinea, Côte d'Ivoire, Mauritania, Niger, Senegal and Togo. PARTICIPANTS: Local scientists and hospital practitioners collected the drug samples in the 10 African countries. OUTCOME MEASURES: The drug amount and the relative amounts of drug impurities, as well as the main compounds of the drugs packaging, were analysed. RESULTS: Identification of the blister packaging of the samples led to separate both amlodipine and captopril drug samples in two groups. Mann Whitney's bilateral test showed a significant difference (p<0.0001) between the median value of the captopril dosage when tablets are packaged in blisters providing higher protection to humidity (n=105) as opposed to the tablets packaged in blisters providing lower humidity protection (n=130). CONCLUSION: Based on these results, particular attention should be paid to the materials and types of packaging used in order to minimise the lack of control over the exposures and drug circuits present in these different countries.

Physical and chemical stability of a generic etoposide formulation as an alternative to etoposide phosphate.

The generic Mylan® etoposide (ETP) has been investigated as an alternative for Etopophos®, in part due to a global shortage of the latter. The generic alternative is different both in its formulation and in its very limited stability (6 h at 25 °C against 4 days for Etopophos®) once reconstituted in ready-to-use chloride or glucose solutions. Its intrinsic stability has been thoroughly studied under various conditions. Two degradation products resulting from hydrolysis were characterized by LC-HR-MSn and supported by density functional theory calculations of the frontier molecular orbitals energies, molecular electrostatic potential mapping, and Mulliken charge analysis. Chemical degradation increases with temperature and can be fitted to a zero order kinetic model with a half-life of 119 days and a kinetic constant of 0.0028 mM day-1. Precipitation was only observed in solutions at 5 °C and -20 °C indicating that at these temperatures the reconstituted solutions are thermodynamically metastable. In conclusion, ETP at concentrations of 0.68 and 1 mM prepared and stored at 25 °C under good manufacturing practices remained unchanged over a period of 21 days irrespective of the nature of the solvents or the type of container.

Pemetrexed degradation by photocatalytic process: Kinetics, identification of transformation products and estimation of toxicity.

This study employed a UV-A/visible/TiO2 system to investigate the degradation of pemetrexed, an antifolate agent used in chemotherapy. The laboratory-scale method employed a photostability chamber that could be used to study multiple samples. Reversed-phase HPLC coupled with high-resolution ESI-LTQ-Orbitrap mass spectrometry was used to determine the transformation products (TPs) of PEME. Based on the identified TPs and existing chemical knowledge, the mechanism of degradation of the target compound was proposed. Concentrations were monitored as a function of time, and the degradation kinetics were compared. The structures of seven TPs, four of which have not been described to date, were proposed. Most of the TPs stemmed from OH radical additions to the dihydropyrrole moiety and oxidative decarboxylation of the glutamate residue. Based on the elucidated structures, a computational toxicity assessment was performed, showing that the TPs with higher log D values than the parent compound are more toxic than the PEME itself. To support these findings, the toxicities of irradiated samples on Vibrio fischeri were monitored over time. The experimental results corresponded well with the results of previous computational studies.

Substandard drugs among five common antihypertensive generic medications: an analysis from 10 African countries.

OBJECTIVE: Hypertension results in more deaths than any other risk factor and has been on the rise in sub-Saharan Africa over the past few decades. Generic drugs have helped improve accessibility and affordability of antihypertensive therapy in developing countries. However, assessment of quality standards of these products is important. We performed a quality assessment of five commonly used antihypertensive generic drugs in 10 sub-Saharan African countries and studied the impact of price on quality. METHODS: Drug samples were prospectively collected using standardized methods between 2012 and 2014. We developed a validated reversed-phase liquid chromatography with tandem mass spectrometry method to accurately quantify the active ingredient in a certified public laboratory. Quality was defined based on the percentage ratio of measured to expected dosage of active ingredient. RESULTS: A total of 1185 samples were assessed, of which 70.0% were generic (n = 830). Among the generic drugs, the percentage of poor-quality drugs was 24.3% (n = 202/830). The percentage ratio of measured to expected dosage of active ingredient ranged from 49.2 to 111.3%; the majority (81.7%) of the poor-quality samples had insufficient quantity of the active ingredient. Moreover, poor quality was not associated with purchase price of the drug. CONCLUSION: In this study from 10 sub-Saharan African countries, nearly one-quarter of the available generic antihypertensive drugs were found to be of poor quality. Concerted measures to improve the quality of antihypertensive drugs could lead to major improvements in hypertension control with attendant reduction of its deleterious consequences in low-income and middle-income countries.

Restructuration kinetics of amphiphilic intraocular lenses during aging.

Photooxidation and hydrolysis are the two primary aging factors of intraocular lenses. Opacifications, dislocations, glistening and yellowing of the implanted acrylic lenses, which are due to chain scissions and depolymerization, are the consequences of aging from the clinical perspective. The purpose of this study was to examine the consequence of the aging of intraocular lenses on chemical and surface properties. Acrylic lenses made of poly acrylic-co-polystyrene polymer were artificially aged by photooxidation and hydrolysis from 2 to 20 years. Degradation products were observed by Reverse-phase High-Performance Liquid Chromatography RP-HPLC and thermogravimetric analysis (TGA). The surface, which was analyzed by atomic force microscopy (AFM) and fibronectin adhesion kinetics, was chosen as an indicator of intraocular biocompatibility. Low-molecular-weight degradation products (LMWP) result from chain scission under both hydrolysis and photooxidation. The osmotic effects of water enable degradation products to migrate through the polymer. A portion of the degradation products exudate in the surrounding center, whereas a portion link with lateral chains of the polymer. At the same time, the surface roughness evolves to externalize the most hydrophilic chains. As a result, the fibronectin adhesion level decrease with time, which indicates the existence of a biocompatible kinetic for implanted intraocular lenses.

Fighting fake medicines: First quality evaluation of cardiac drugs in Africa.

BACKGROUND: The growing menace of poor quality and falsified drugs constitutes a major hazard, compromising healthcare and patient outcomes. Efforts to assess drug standards worldwide have almost exclusively focused on anti-microbial drugs; with no study to date on cardiovascular drugs. Our study aims to assess quality of seven routinely used cardiovascular medications (anticoagulants, antihypertensives and statins) in ten Sub-Saharan African countries. METHODS: Drugs were prospectively collected using standardized methods between 2012 and 2014 from licensed (random pharmacies) and unlicensed (street-markets) places of sale in Africa. We developed a validated reversed-phase liquid chromatography with tandem mass spectrometry method to accurately quantify the active ingredient in a certified public laboratory. Three quality categories were defined based on the ratio of the measured to the expected dosage of the active ingredient: A (good quality): 95% to 105%, B (low quality): 85 to 94.99% or 105.01 to 115%, C (very low quality): <85% or >115%. RESULTS: All expected medicines (n=3468 samples) were collected in Benin, Burkina-Faso, Congo-Brazzaville, the Democratic Republic of Congo, Guinea, Côte d'Ivoire, Mauritania, Niger, Togo and Senegal. Out of the 1530 samples randomly tested, poor quality (types B and C) was identified in 249 (16.3%) samples. The prevalence of poor quality was significantly increased in certain specific drugs (amlodipine 29% and captopril 26%), in generic versions (23%) and in drugs produced in Asia (35%). The proportion of poor quality reached 50% when drugs produced in Asia were sold in street-markets. CONCLUSION: In this first study assessing the quality of cardiovascular drugs in Africa, we found a significant proportion of poor quality drugs. This requires continued monitoring strategies.

Investigating therapeutic usage of combined Ticagrelor and Aspirin through solid-state and analytical studies.

The mainstay treatment for patients with acute coronary syndrome is an oral route dual antiplatelet therapy with a P2Y12-receptor antagonist and Aspirin (ASA). To improve patient adherence to such treatments, combination therapies (polypill) are envisioned. Physicochemical solid-state studies have been carried out to develop a preformulation strategy of ASA with the P2Y12-receptor antagonist Ticagrelor (TIC). The investigations were carried out using differential scanning calorimetry, liquid chromatography-high resolution-multistage mass spectrometry (LC-HR-MSn) and as complementary techniques Fourier transform infrared measurements and thermogravimetric analysis. A simple eutectic transition at 98°C with a mole fraction for the eutectic liquid of 0.457 has been observed and the mixing of ASA and TIC molecules in each other's crystal structures appears to be limited. No cocrystals of TIC and ASA have been found. The appearance of the eutectic liquid was linked with a clear onset of chemical instability of the two pharmaceuticals. The decomposition mechanism in the liquid phase involves prior decomposition of ASA, whose residues react with well-identified TIC interaction sites. Seven interaction products were observed by LC-HR-MSn linked to corresponding degradation products. The most important degradation pathway is N-dealkylation. In conclusion, polypills of ASA and TIC are a viable approach, but the decomposition of ASA should be avoided by eliminating high temperatures and high humidity.

RP-HPLC detection and dosage method for acrylic monomers and degradation products released from implanted medical devices.

Acrylic copolymers are useful in medical therapeutics. As in dental implants or intraocular lenses, acrylics are present in many medical devices or drug adjuvants. Industrial using of acrylics is still important in painting or textile manufacturing. Scientific research background has proved that acrylic suffer for depolymerized and cross-linking mechanisms under heating and photo-oxidative conditions. Those aging processes could lead to release of unreacted monomers and degradation products. We developed a new RP-HPLC method with good resolution, recovery, linearity, detection and quantification limits that is efficient for acrylic monomers quantification in in vitro and in vivo saline solution matrices. This method allows the detection of copolymer and medical devices degradation products too. Both the limit of quantification and the limit of detection for monomers and degradation products are above cytotoxic concentrations for human epithelial cells. Those biological results confirm the interest of the method for dosage of unreacted acrylics after polymerization and for the research of degradation products in body fluids as aqueous humor.