Investigation of hydrogen bonds in proton transfer complexes derived from the reaction of 2- and 4-(N,N-dimethylamino)pyridines with chloranilic acid.

New complexes of 2-(N,N-dimethylamino)pyridine with chloranilic acid (2-DMAP + CLA) and 4-(N,N-dimethylamino)pyridine with chloranilic acid (4-DMAP + CLA) were synthesized and characterized by single crystal X-ray diffraction, infrared spectroscopy, thermal analysis methods and 1H, 13C and 15N NMR spectroscopy. The NMR spectroscopies were carried out in both, DMSO solution and in the solid state (CPMAS NMR). The 2-DMAP + CLA and 4-DMAP + CLA complexes crystallize in centrosymmetric P-1 and P21/c space group, respectively. In both complexes, the phenomenon of proton transfer is observed, which results in the formation of strong N+-H···O- hydrogen bonds. Thermal decompositions of 2-DMAP + CLA and 4-DMAP + CLA complexes were studied by thermogravimetric analysis. Temperature dependent IR spectra revealed that methyl groups of 4-DMAP + CLA perform fast stochastic reorientational motion at room temperature which is slowed on cooling while in 2-DMAP + CLA reonrientational motion of CH3 groups is much slower due to steric effects.

Clustering of Tadalafil API Samples According to their Manufacturer in the Context of API Falsification Detection.

This paper reports the results of the active pharmaceutical ingredient (API) fingerprint study, organised by the General European Official Medicines Control Laboratory Network (GEON), on tadalafil. A classical market surveillance study, evaluating compliance to the European Pharmacopoeia, was combined with a fingerprint study, the latter to obtain characteristic data for the different manufacturers, allowing the network laboratories to conduct authenticity tests for future samples, as well as to detect substandard and falsified samples. In total, 46 tadalafil API samples from 13 different manufacturers were collected. For all samples fingerprint data was collected through analysis of impurities and residual solvents, mass spectrometric screening, X-ray powder diffraction and proton nuclear magnetic resonance (1H-NMR). Chemometric analysis revealed that all manufacturers could be characterised based on the impurity, residual solvent and 1H-NMR data. Future suspicious samples in the network will therefore be analysed with these techniques in order to attribute the sample to one of the manufacturers. If the sample cannot be attributed, a more profound investigation will be necessary to reveal the origin of the sample. In cases where the suspect sample is claimed to be from one of the manufacturers included in this study, analysis can be limited to the test distinguishing that manufacturer.

Development and application of a liquid chromatography coupled to mass spectrometry method for the simultaneous determination of 23 antineoplastic drugs at trace levels.

The goal of this study was to develop a method for the simultaneous quantification of 23 commonly used antineoplastic drugs in a hospital pharmacy, using ultra-high pressure liquid chromatography separation coupled to tandem mass spectrometry detection (UHPLC-MS/MS). The following drugs were investigated: 5-fluorouracil, cytarabine, ganciclovir, gemcitabine, dacarbazine, methotrexate, pemetrexed, busulfan, topotecan, rentitrexed, ifosfamide, cyclophosphamide, etoposide, irinotecan, doxorubicin/epirubicin, vincristine, docetaxel, paclitaxel, daunorubicin, idarubicin, vinblastine, oxaliplatin and carboplatin. The chromatographic separation was performed on a phenyl-hexyl column (2.1 ×100 mm, 1.7 µm) with a gradient elution of methanol and water containing 10 mM ammonium formate adjusted to pH 4.9. All compounds were analyzed in less than 13 min and detected with a triple quadrupole mass spectrometer operating in MRM mode. Limits of detection (LODs) and limits of quantification (LOQs) were comprised between 0.01 and 5 ng.mL-1, and between 0.5 and 5 ng.mL-1, respectively. Accuracies ranged between 117% and 83% at the LOQ, intermediate and upper LOQ concentrations, with relative standard deviations (RSD) inferior to 8%, for all the antineoplastic drugs. Finally, the UHPLC-MS/MS method was successfully applied to the analysis of surface samples to evaluate the chemical contamination by these highly toxic compounds in a chemotherapy preparation unit in a hospital pharmacy with the purpose of monitoring the exposure of health care professionals.

GEONs API fingerprint project: Selection of analytical techniques for clustering of sildenafil citrate API samples.

Through its Active Pharmaceutical Ingredient Working Group (API-WG) the General European Official Medicines Control Laboratory (OMCL) Network (GEON), co-ordinated by the European Directorate for the Quality of Medicines & HealthCare (EDQM), regularly organises market surveillance studies for specific APIs for conformity to their monograph in the European Pharmacopoeia. During the past years some studies were combined with a fingerprint study of the APIs. The idea is to obtain a fingerprint for each manufacturer of the API under investigation, allowing the OMCL network to identify future samples as well as to detect substandard and falsified APIs. This paper reports the results of the latest fingerprint study, organised on sildenafil citrate API samples. Seventy-nine samples from 14 different manufacturers were collected throughout the Network. Fingerprint data was collected through Mid-Infrared spectroscopy, Raman spectroscopy, liquid chromatography for related substances, gas chromatography for residual solvents, X-ray diffraction and Nuclear Magnetic Resonance (NMR) spectroscopy. Chemometrics applied to the collected data showed that all manufacturers could be discriminated based on the data of only three of these tests, i.e. gas chromatography for residual solvents, X-ray diffraction and proton NMR. Suspicious API samples for sildenafil citrate will therefore be analysed in the future with the selected techniques in order to link the sample to a manufacturer or demonstrate the absence of such link. If the sample cannot be attributed to one of the manufacturers, further analysis and research on provenance and identity will be required. Of course, if the suspected sample claims to originate from one of the manufacturers included in the study, analysis can be limited to the test distinguishing this manufacturer.

European fingerprint study on omeprazole drug substances using a multi analytical approach and chemometrics as a tool for the discrimination of manufacturing sources.

Like drug products, Active Pharmaceutical Ingredients (APIs) are subject to substandard and falsification issues, which represent a threat to patient health. In order to monitor the quality of drug substances and prevent the use of non-compliant APIs, Official Medicine Control Laboratories work together in a European network developing coordinated strategies and programmes. The API working group proposed a market surveillance study on omeprazole and omeprazole magnesium with the objectives of controlling the pharmaceutical quality of samples, checking compliance with the monographs of the European Pharmacopoeia, and collecting analytical fingerprints that could be further used to differentiate manufacturing sources for future authenticity investigations. The study described in this article reports the analysis carried out by 7 European laboratories on 28 samples from 11 manufacturers with 5 analytical techniques (related substances with HPLC, residual solvents with GC-MS, near infrared spectroscopy, proton nuclear magnetic resonance spectroscopy and X-ray powder diffractometry). The large amount of resulting analytical data were centralized and treated with two chemometric methods: Principal Component Analysis and Hierarchical Clustering Analysis. Data were analyzed separately and in combination (data fusion), allowing us to conclude that NMR and XRPD were suitable to differentiate samples originating from 9 out of 11 manufacturers. Analytical fingerprints associated with chemometrics were demonstrated to be a valuable methodology to discriminate manufacturers of omeprazole and omeprazole magnesium APIs and detect future substandard and falsified APIs.

Production and characterisation of a candidate hyper-immune serum for the replacement of the Bordetella pertussis mouse antiserum Biological Reference Preparation.

For acellular pertussis (aP) vaccines, the current European Pharmacopoeia (Ph. Eur.) monograph Pertussis vaccine (acellular, component, adsorbed) (1356) requires an immunogenicity assay in mice or guinea pigs to assess the potency of each lot of vaccine (Ph. Eur. general method 2.7.16. Assay of pertussis vaccine (acellular)). This biological assay, carried out on the final bulk of the vaccine lot, is based on the measurement of the specific antibody response to the 5 antigenic components (pertussis toxin (PT), Fimbrial haemagglutinin (FHA), pertactin (PRN) and Fimbriae 2 and 3 (FIM2/3)) that are present in the combined aP vaccines. In the mouse assay, serum antibody levels are measured by ELISA. The immunogenicity of a vaccine under test is estimated versus a homologous reference vaccine and a reference antiserum e.g. the first Ph. Eur. Biological Reference Preparation for Bordetella (B.) pertussis mouse anti-serum (BRP1), established in 1998, is used to normalise the titre of antibodies (expressed in ELISA Units (ELU)/mL). In anticipation of the depletion of BRP1 stocks, a project was launched in 2013 by the Biological Standardisation Programme (BSP) of the European Directorate for the Quality of Medicines & HealthCare (EDQM) in order to establish a new standardised reference serum. The project, referred to herein as BSP129, was conducted in 2 phases: 1) the production and characterisation of a mouse serum pool (using a multicomponent aP vaccine marketed in Canada similar to the vaccine used in the BRP1 production as immunogen) and of candidate BRP batches (cBRPs) and 2) an international collaborative study aimed at calibrating the cBRPs in terms of antibody levels against PT, FHA, PRN and FIM2/3. This article presents the design and results of the first phase of the collaborative study to establish the optimal conditions for immunisation and bleeding of mice in order to produce a large pool of hyper-immune serum against the 5 antigens. After the characterisation of this pool, cBRP pilot lots were manufactured by freeze-drying diluted solutions of the hyper-immune serum pool. The pilot lots were then characterised in two Official Medicines Control Laboratories (OMCLs) for their antibody contents against aP vaccine antigens using in-house ELISA (based on methods developed by 2 European vaccine manufacturers) and Multiplex Immunoassay (MIA) methods. The antibody titres recovered demonstrated that a dilution factor of 1/40 could be considered for the scaled-up manufacture of candidate reference preparations (cBRPs). Three batches (15 000 vials) of cBRP were manufactured and fully characterised. In light of the data obtained, and although titration results between the ELISA methods were sometimes discrepant, it was agreed that the establishment study (phase 2) could be launched. Real-time and accelerated stability studies were also included in the first study phase to document the stability of the cBRPs in freeze-dried form and after reconstitution and storage at -20°C±5°C. The results showed that the stability of the freeze-dried cBRPs at usual storage and shipment temperatures is acceptable and that reconstituted cBRP solutions are stable for 12 months at -20°C±5°C. It could therefore be recommended to freeze small aliquots of the 1 mL solution obtained by the reconstitution of one BRP vial in order to store them for use in separate assays. With the application of this strategy, the stocks of the BRP1 replacement batches should cover the needs of OMCLs and manufacturers for at least the next decade.

Low temperature investigations of dynamic properties in l-leucine - chloranilic acid complex.

Inelastic neutron scattering (INS) and infra-red (IR) spectroscopy methods were used for determination of dynamic structure of l-leucine - chloranilic acid complex. A theoretical dynamic pattern calculated by the density functional theory (DFT) method for periodic boundary conditions accompanied the experimental ones. Normal modes in the vibrational spectra were defined and described. The characteristic presence of the Hadzi's trio enriched by numerous submaxima is observed in the wavenumber range 3200-800 cm-1. Bands assigned to CH3 torsion vibrations in the leucine cation were observed at 231 cm-1 and 258 cm-1 in the INS spectrum. Temperature-dependent far-infrared spectra in the temperature range 9 K-290 K were obtained. Vibrational bands were analyzed as a function of temperature. Activation energies for reorientational motion of CH3 and CH2 groups were determined by means of the band shape analysis performed for torsional and twisting vibrations of these groups. The estimated energy is equal to Ea = 2.7 ±â€¯0.2 kJ/mol and Ea = 2.17 ±â€¯0.12 kJ/mol for CH3 and CH2 groups, respectively. A phase transition at about 130 K in the l-leucine - chloranilic acid complex was observed.

Gut microbiota analysis reveals a marked shift to bifidobacteria by a starter infant formula containing a synbiotic of bovine milk-derived oligosaccharides and Bifidobacterium animalis subsp. lactis†CNCM I-3446.

Non-digestible milk oligosaccharides were proposed as receptor decoys for pathogens and as nutrients for beneficial gut commensals like bifidobacteria. Bovine milk contains oligosaccharides, some of which are structurally identical or similar to those found in human milk. In a controlled, randomized double-blinded clinical trial we tested the effect of feeding a formula supplemented with a mixture of bovine milk-derived oligosaccharides (BMOS) generated from whey permeate, containing galacto-oligosaccharides and 3'- and 6'-sialyllactose, and the probiotic Bifidobacterium animalis subsp. lactis (B. lactis) strain CNCM I-3446. Breastfed infants served as reference group. Compared with a non-supplemented control formula, the test formula showed a similar tolerability and supported a similar growth in healthy newborns followed for 12 weeks. The control, but not the test group, differed from the breast-fed reference group by a higher faecal pH and a significantly higher diversity of the faecal microbiota. In the test group the probiotic B. lactis increased by 100-fold in the stool and was detected in all supplemented infants. BMOS stimulated a marked shift to a bifidobacterium-dominated faecal microbiota via increases in endogenous bifidobacteria (B. longum, B. breve, B. bifidum, B. pseudocatenulatum).

Conformations and intermolecular interactions pattern in solid chloroxylenol and triclosan (API of anti-infective agents and drugs). A (35)Cl NQR, (1)H-(14)N NQDR, X-ray and DFT/QTAIM study.

Two antibacterial and antifungal agents, chloroxylenol (4-chloro-3,5-dimethyl-phenol) and triclosan (5-chloro-2-(2',4'-dichlorophenoxy)-phenol), were studied experimentally in solid state with an X-ray, (35)Cl-nuclear quadrupole resonance (NQR) and (17)O-nuclear quadrupole double resonance (NQDR) spectroscopies and, theoretically, with the density functional theory/quantum theory of atoms in molecules (DFT/QTAIM). The crystallographic structure of triclosan, which crystallises in space group P31 with one molecule in the asymmetric unit [a = 12.64100(10), b = 12.64100(10), c = 6.71630(10) Å], was solved with an X-ray and refined to a final R-factor of 2.81% at room temperature. The NQR frequencies of (35)Cl and (17)O were detected with the help of the density functional theory (DFT) assigned to particular chlorine and oxygen sites in the molecules of both compounds. The NQR frequencies at (35)Cl sites in chloroxylenol and triclosan were found to be more differentiated than frequencies at the (17)O site. The former better describes the substituent withdrawing effects connected to π-electron delocalization within the benzene rings and the influence of temperature; whereas, those at the (17)O site provide more information on O-H bond and intermolecular interactions pattern. The conformation adopted by diphenyl ether of triclosan in solid state was found to be typical of diphenyl ethers, but the opposite to those adopted when it was bound to different inhibitors. According to an X-ray study, temperature had no effect on the conformation of the diphenyl ring of triclosan, which was the same at 90 K and at room temperature (RT). The scattering of NQR frequencies reproduced by the DFT under assumption of the X-ray data at 90 K and RT is found to be a good indicator of the quality of resolution of the crystallographic structure.

Nature of isomerism of solid isothiourea salts, inhibitors of nitric oxide synthases, as studied by 1H-14N nuclear quadrupole double resonance, X-ray, and density functional theory/quantum theory of atoms in molecules.

Isothioureas, inhibitors of nitric oxide synthases, have been studied experimentally in solid state by nuclear quadrupole double resonance (NQDR) and X-ray methods and theoretically by the quantum theory of atoms in molecules/density functional theory. Resonance frequencies on (14)N have been detected and assigned to particular nitrogen sites in each molecule. The crystal packings of (S)-3,4-dichlorobenzyl-N-methylisothiouronium chloride with the disordered chlorine positions in benzene ring and (S)-butyloisothiouronium bromide have been resolved in X-ray diffraction studies. (14)N NQDR spectra have been found good indicators of isomer type and strength of intra- or intermolecular N-H···X (X = Cl, Br) interactions. From among all salts studied, only for (S)-2,3,4,5,6-pentabromobenzylisothiouronium chloride are both nitrogen sites equivalent, which has been explained by the slow exchange. This unique structural feature can be a key factor in the high biological activity of (S)-2,3,4,5,6-pentabromobenzylisothiouronium salts.

X-ray powder diffractometry and liquid chromatography studies of sibutramine and its analogues content in herbal dietary supplements.

The contemporary societies of the developed countries are prone to use traditional far-east medicines as remedies for all diseases. Some of them, such as obesity, might be classified as civilization diseases. Combating the problem, people try not only several miraculous diets but also herbal infusions (teas) and variety of "herbal" preparations. All these believing that such treatment is healthy and harmless as far as it is "natural". Leaving out of the way the question if herbal medicines can be taken safely without doctors' control the query arises if the common preparations are strictly natural and herbal. Here we report examples of quality studies of such medicines using both X-ray powder diffraction (XRPD) and liquid chromatography (LC) with various types of detection: ultraviolet (UV), coulometric electrode array (CEAD) and time-of-flight mass spectrometry (TOF-MS). Especially the XRPD assisted with an optical microscopy seems to be useful as a fast screening method of general sample composition of such preparations. First of all it can discriminate between capsules containing pure herbal materials and those with some chemical.

The identification of rimonabant polymorphs, sibutramine and analogues of both in counterfeit Acomplia bought on the internet.

Acomplia was ordered over the internet resulting in the delivery of counterfeit Acomplia and imitation products. The tablets were analyzed for the presence of rimonabant. Using LC-DAD-MSn the presence of effective quantities of rimonabant was confirmed in samples A-D. Samples A and D also contained traces of the rimonabant analogue NIDA-41020. Furthermore, NIR spectroscopy on the tablets indicated the presence of an unapproved rimonabant polymorph in samples C and D which was confirmed by Raman spectroscopy and X-ray diffraction (XRD). In sample E a low dose of sibutramine was found as well traces of N-desmethylsibutramine and bis-N-desmethylsibutramine. Rimonabant was withdrawn from the market because of serious adverse events and lack of efficacy. The availability of poor quality products with rimonabant, impurities and unapproved polymorphs is worrying. Suspect weight-loss medicines should be screened for the presence of novel analogues.

Structural study of selected polyhalogenated benzimidazoles (protein kinase CK2 inhibitors) by nuclear quadrupole double resonance, X-ray, and density functional theory.

Protein kinase CK2 inhibitors, polyhalogenated benzimidazoles, have been studied experimentally in solid state by NMR-NQR double resonance and X-ray and theoretically by the density functional theory (DFT). Six resonance frequencies on (14)N have been detected and assigned to particular nitrogen sites in each polyhalogenated benzimidazole molecule. The effects of prototropic annular tautomerism and polymorphism related to stable cluster formation due to intermolecular hydrogen bonding interactions on the (14)N NQR parameters have been analyzed within the DFT and AIM (atoms in molecules) formalism. The studies suggest that all polyhalogenobenzimidazoles are isostructural and can exhibit polymorphism and that (14)N NQR is very sensitive to hydrogen bondings but less sensitive to the specific arrangement of the hydrogen bonded molecules. NQDR and DFT results suggest the presence of the prototropic annular tautomerism 50:50, which is in a good agreement with the X-ray and (1)H NMR data.

[Evaluation of the Ocular Response Analyzer in ocular hypertension, glaucoma, and normal populations. Prospective study on 329 eyes]. / Evaluation des facteurs biomécaniques cornéens mesurés à l'Ocular Response Analyzer dans l'hypertension intraoculaire, le glaucome primitif à angle ouvert et chez le sujet normal. Etude prospective sur 329 yeux.

OBJECTIVES: (1) Evaluate and analyze the Ocular Response Analyzer (ORA) in three groups of patients: glaucoma, intraocular hypertension, and normal; (2) study corneal hysteresis values in the three groups according to age; and (3) compare intraocular pressure values measured with the ORA with intraocular pressure measured with Goldmann (IOPGoldmann) and pulsed air (IOPair) applanations. MATERIAL AND METHODS: This prospective, single-center study included 329 eyes divided into three groups: normal (n=207), intraocular hypertension (n=55), and primary angle glaucoma (n=67). Corneal hysteresis (CH), IOP corneal-compensated (IOPcc), and Goldmann correlated IOP (IOPg) measurements were provided by the ORA device for all patients. Ultrasonic central corneal thickness (CCT US), and intraocular pressure measured with Goldmann and pulsed air tonometry were also assessed in each eye. RESULTS: The mean values were: IOPGoldmann 14.4+/-3.4 mmHg, IOPair 15.5+/-3.6 mmHg, CCT 542.1+/-36.6 microm, CH 10+/-1.7 mmHg, IOPcc 16.6+/-4.1 mmHg, and IOPg 15.7+/-3.9 mmHg. All the IOP measurements (IOPGoldmann, IOPair, IOPg, IOPcc) were strongly correlated into the three age groups. The mean CH in the glaucoma (9.8 mmHg) and intraocular hypertension (9.6 mmHg) groups was lower than in the normal group (10.3 mmHg), but there was no difference between the three groups for the CH values, with an age-related analysis of the three groups. The CH was correlated with CCT US in the three groups. IOP measures were not strongly correlated with CH except for IOPcc. There was a negative correlation (-0.79) between CH and IOPcc. DISCUSSION/CONCLUSION: In our study, the mean ORA, CCT US, and IOP values in the normal group were similar to those found in the literature. These are the first CH values reported for a normal group according to age. We confirm the good correlation between all the IOP measurements. The mean corneal hysteresis value was low in glaucoma and intraocular hypertension, but there was no difference between the three groups for the CH values, when the three groups were analyzed according to age. There was a correlation between corneal hysteresis and central corneal thickness. Moreover, IOPcc seems to be the best evaluation of IOP with no influence from corneal biomechanical factors. The Ocular Response Analyzer and corneal hysteresis should be considered a useful parameter for patients with intraocular hypertension and/or glaucoma.

Technical quality of root fillings performed by dental students at the dental teaching centre in Reims, France.

AIM: To evaluate the technical quality of root fillings performed by undergraduate students at a dental teaching centre in France. METHODOLOGY: A random sample of 419 records of patients who received dental treatment at the dental service of the teaching Hospital, in Reims, France between 2005 and 2006 was investigated. Evaluation of root filled was based on radiographical criteria defined by the French National Health Service. The length of root fillings, the radiodensity and the presence of voids in the root filling or between root filling and root canal walls were recorded and scored. Chi-square analysis was used to determine statistically significant differences between the technical quality of root fillings and tooth type. RESULTS: Of the 304 teeth included in the study, 69% had an adequate length of root filling and 42.7% had a dense root filling without voids; only 30.3% of teeth fulfilled these criteria at the same time. The relationship between the technical quality of root fillings and tooth type was statistically significant (P < 0.001), the highest percentage of adequate root fillings occurred in single-rooted teeth (36.1%). The highest percentage of inadequate root fillings according to the criteria of root filling length and lateral adaptation was found in molar teeth (71.9%). CONCLUSION: Overall, the technical quality of root fillings performed by undergraduate students was poor.

[Practical assessment. Ocular hypertension]. / Bilan en pratique. L'hypertonie oculaire isolée.

In cases of high intraocular pressure, the patient's hypertension must first be confirmed and its characteristics delineated with a diurnal IOP curve or at least several different measures. Central corneal thickness should be measured, since a thin cornea is a risk factor of progression toward glaucomatous neuropathy. Later, the optic nerve head and retinal nerve fibers should be tested (HRT, GDx, and OCT), to determine whether the ocular hypertension is associated with other findings, and visual field analysis should be carried out (standard automated perimetry, blue-yellow perimetry, FDT), completed by a search for associated risk factors. Therapeutic decisions will be based on this workup, keeping in mind that in the majority of cases, monitoring and information are sufficient but necessary; treatment should, however, be initiated in cases of ocular hypertension with a high risk of progression.

[Clinical consequences of dioxins exposure during tooth development]. / Conséquences cliniques des effets des dioxines sur le développement dentaire.

Commonly designed by the term "dioxins", polychlorinated aromatic hydrocarbons are environmental pollutants leading to several toxic effects during development and growth in embryo and child. The general consequences of dioxin exposure are particularly well-documented whereas only few data are mentioned by the experts concerning tooth development. However, studies performed in rodents have shown many disruptions during odontogenesis and enamel mineralisation. Moreover, recent epidemiological studies have demonstrated in human the incidence of dioxin exposure on enamel hypomineralisation and hypodontia. The aim of this review is to report recent data about consequences of dioxin exposure on tooth development, tooth being considered as a biological marker of potential dioxin poisoning.

[Diagnostic laser in glaucoma: scanning laser polarimetry (GDx VCC) and confocal scanning laser tomography (HRT)]. / Les lasers diagnostiques dans le glaucome: la polarimétrie à balayage laser (GDx VCC) et la tomographie confocale par balayage laser (HRT).

Automated structural measurements of retinal nerve fibers and optic nerve head are possible with new lasers providing objective and reproductible data for analysis. Scanning laser polarimetry (GDx VCC), based on retardation of polarized light, assesses peripapillary nerve fiber layer thickness. Confocal scanning laser tomography yields precise topographic maps of the optic disc and peripapillary retina. The advantages, applications for glaucoma detection, both in a screening setting as well as for monitoring progression, limitations and pitfalls need to be well known and results should be analyzed with clinical data.

[Corneal burns and matrix metalloproteinases (MMP-2 and -9): the effects of human amniotic membrane transplantation]. / Brûlures cornéennes et métalloprotéases: influence des greffes de membranes amniotiques.

PURPOSE: Human amniotic membranes have recently been used in ophthalmology to restore deleted ocular surface after burns. Matrix metalloproteinases-2 and -9 have been implicated in the development of neovascularization. In this study, MMP-2 and MMP-9 expression was analyzed by in situ zymography on rabbit corneal chemical burns with and without human amniotic membrane graft. METHOD: Corneal neovascularization was induced in 10 Fauve de Bourgogne rabbits by means of a heptanol chemical burn on controlled deep keratotomy using a Chiron ALK-E corneal shaper. Half of rabbits received acute amniotic membrane transplantation 30mn after chemical burn; the remaining five rabbits received medical treatment. In situ zymography is a recent nondestructive technique which preserved the fine morphological details of the cornea and showed the active enzyme location in different corneal layers. The MMP-2 and -9 substrate was gelatin, which was detected by fluorescent microscopy. RESULTS: There was an overexpression of MMP-2 and -9 in corneal burns versus control corneas. Expression of MMP-2 and -9 was low in corneal burn without amniotic membrane graft. Following amniotic membrane transplantation, MMP-2 and -9 were strongly expressed and clinical neovascularization and inflammation decreased. Active enzymes were located in epithelium layers in the uncovered group. In the covered group, the active enzymes were located in the anterior and posterior stromal layers. CONCLUSION: The results support a role for MMP-2 and MMP-9 in corneal burn neovascularization. Amniotic membrane transplantation can play a protective role by up-regulation of their biological expression.