Optimizing ophthalmic delivery of a poorly water soluble drug from an aqueous in situ gelling system.

Poorly soluble drugs are often unsuitable to incorporate in ocular in situ gelling systems due to the aqueous based gelling formulations and low volumes administered. For such formulations to be successful, the administered drug must have sufficient solubility to diffuse from the formulation to the eye and should not affect the gelation of the in situ gelling material. Drug salt forms can improve the solubility of poorly soluble drugs, however, as in situ gel forming formulations are often designed to be crosslinked by salts (present the lacrimal fluid) it can make salt forms difficult to formulate. The aim of this study was to develop an in situ gel forming ophthalmic formulation of a poorly soluble drug flurbiprofen (FBP) through cyclodextrin complex formation and to analyse the impact on gelation, release and permeation through the cornea. Hydroxypropyl-beta-cyclodextrin (HßCD) was used as a complexing agent and low acyl gellan gum was added to the FBP- HßCD complex as a water soluble in situ gelling polymer. Measurements were performed using rheo-dissolution, which utilises a rheometer with a modified lower plate that has the unique ability to allow rheological measurement and analysis of drug release simultaneously. An ex-vivo permeation study was also performed using porcine cornea. Rheological measurements in terms of elastic (G') and viscous (G″) modulus showed rapid gelation of the formulation upon contact with simulated lacrimal fluid (SLF). Approximately, 97% FBP was released when 10% HßCD was used and release was decreased to 79% when the amount of HßCD was increased to 20%. The percentage of drug permeation through the cornea was 55% in 300 min whereas the marketed non gelling eye drop formulation containing FBP sodium showed only 37% permeation. The data presented here, revealed that not only could a poorly soluble drug be complexed with cyclodextrin and loaded into an in situ gelling system without interfering with the gelation, but also permeability the of the drug improved.

A survey on IVIVC/IVIVR development in the pharmaceutical industry - Past experience and current perspectives.

The present work aimed to describe the current status of IVIVC/IVIVR development in the pharmaceutical industry, focusing on the use and perception of specific approaches as well as successful and failed case studies. Two questionnaires have been distributed to 13 EFPIA partners of the Oral Biopharmaceutics Tools Initiative and to the Pharmacokinetics Working Party of the European Medicines Agency in order to capture the perspectives and experiences of industry scientists and agency members, respectively. Responses from ten companies and three European Agencies were received between May 21st 2014 and January 19th 2016. The majority of the companies acknowledged the importance of IVIVC/IVIVR throughout the drug development stages and a well-balanced rate of return on investment. However, the IVIVC/IVIVR approach seemed to be underutilized in regulatory submissions. Four of the ten companies stated to have an internal guidance related to IVIVC/IVIVR modelling, whereas three felt that an overall strategy is not necessary. Successful models mainly served to support formulation development and to provide a better mechanistic understanding. There was not yet much experience with safe-space IVIVRs as well as the use of physiologically based modelling in the field of IVIVC. At the same time, the responses from both industry and agencies indicated that there might be a need for a regulatory framework to guide the application of these novel approaches. The relevance of IVIVC/IVIVR for oral IR drug products was recognized by most of the companies. For IR formulations, relationships other than Level A correlation were more common outcomes among the provided case studies, such as multiple Level C correlation or safe-space IVIVR, which could be successfully used for requesting regulatory flexibility. Compared to the responses from industry scientists, there was a trend towards a higher appreciation of the BCS among the regulators, but a less positive attitude towards the utility of non-compendial dissolution methods for establishing a successful IVIVC/IVIVR. The lack of appropriate in vivo data and regulatory uncertainty were considered the major difficulties in IVIVC/IVIVR development. The results of this survey provide unique insights into current IVIVC/IVIVR practices in the pharmaceutical industry. Pursuing an IVIVC/IVIVR should be generally encouraged, considering its high value from both industry and regulators' perspective.

Mathematical Model-Based Accelerated Development of Extended-release Metformin Hydrochloride Tablet Formulation.

A computational fluid dynamic (CFD) model was developed to predict metformin release from a hydroxypropylmethylcellulose (HPMC) matrix-based extended-release formulation that took into consideration the physical and chemical properties of the drug substance, composition, as well as size and shape of the tablet. New high dose strength (1000 mg) tablet geometry was selected based on the surface area/volume (SA/V) approach advocated by Lapidus/Lordi/Reynold to obtain the desired equivalent metformin release kinetics. Maintaining a similar SA/V ratio across all extended-release metformin hydrochloride (Met XR) tablet strengths that had different geometries provided similar simulations of dissolution behavior. Experimental dissolution profiles of three lots of high-strength tablets agreed with the simulated release kinetics. Additionally, a pharmacokinetic absorption model was developed using GastroPlus™ software and known physicochemical, pharmacokinetic, and in vitro dissolution properties of metformin to predict the clinical exposure of the new high strength (1000 mg) tablet prior to conducting a human clinical bioequivalence study. In vitro metformin release kinetics were utilized in the absorption model to predict exposures in humans for new 1000-mg Met XR tablets, and the absorption model correctly projected equivalent in vivo exposure across all dose strengths. A clinical bioequivalence study was pursued based on the combined modeling results and demonstrated equivalent exposure as predicted by the simulations.

Low incidence of port-site metastasis after robotic assisted surgery for endometrial cancer staging: descriptive analysis.

The purpose of this study was to evaluate the incidence and characteristics of patients with port-site metastasis following robotic assisted surgery for gynecological malignancies. Patients who underwent robotic assisted total laparoscopic hysterectomy and surgical staging at a single institution from November 2006 through November 2011 were retrospectively identified. Medical records were reviewed and the following information was extracted: diagnosis, histology, tumor extension, procedure, complications and post-surgical intervention. Port-site metastases were differentiated between isolated and not isolated. All metastases were confirmed with biopsy and treated with chemotherapy and radiotherapy as indicated. Four hundred forty-six patients with endometrial carcinoma were identified who had undergone robotic assisted hysterectomy and staging. Eight patients were converted to laparotomy and excluded from the study. Of 438 patients, 384 patients were diagnosed with early stages (stages 1 and 2), and 54 were diagnosed with advanced stages (stages 3 and 4). A total of 332 patients underwent pelvic lymphadenectomy regardless of the endometrial cancer stage; of those, 283 with early stage disease underwent pelvic lymphadenectomy, while 49 with advanced stage disease underwent pelvic lymphadenectomy. One hundred seventy-six patients received adjuvant treatment after surgical staging. Four patients were identified with port-site metastases (0.9 %), two patients were reported as isolated metastases. The mean patient age was 63 and mean BMI was 37 kg/m(2). The incidence of port-site metastasis is low after robotic assisted surgery for treatment of endometrial cancer (0.9 %). There is no clear risk factor for development of port-site metastasis or easily identifiable prevention.

Impact of the counterion on the solubility and physicochemical properties of salts of carboxylic acid drugs.

AIM: Salt formation is a widely used approach to improve the physicochemical and solid state properties of an active pharmaceutical ingredient. In order to better understand the relationships between the active drug, the selected counterion and the resultant salt form, crystalline salts were formed using four different carboxylic acid drugs and a closely related series of amine counterions. Thirty-six related crystalline salts were prepared, characterized and the relationship between solubility and dissolution behaviour and other properties of the salt and the counterion studied. METHODS: Salts of four model acid drugs, gemfibrozil, flurbiprofen, ibuprofen and etodolac were prepared using the counterions butylamine, hexylamine, octylamine, benzylamine, cyclohexylamine, tert-butylamine, 2-amino-2-methylpropan-1-ol, 2-amino-2-methylpropan-1,3-diol and tris(hydroxymethyl)aminomethane. Salt formation was confirmed, the salts were characterized and their corresponding solubilities determined and rationalized with respect to the counterions' properties. RESULTS AND CONCLUSION: The properties of the salt highly dependent on the nature of the counterion and, although there is considerable variation, some general conclusion can be drawn. For the alkyl amines series, increasing chain length leads to a reduction in solubility across all the acidic drugs studied and a reduction in melting point, thus contradicting simplistic relationships between solubility and melting point. Small, compact counterions consistently produce crystalline salts with high melting point accompanied with a modest improvement in solubility and the nature of hydrogen bonding between the ions has a major impact on the solubility.

Investigation of gammaE-crystallin target protein binding to bovine lens alpha-crystallin by small-angle neutron scattering.

alpha-Crystallin, one of the main constituent proteins in the crystalline lens, is an important molecular chaperone both within and outside the lens. Presently, the structural relationship between alpha-crystallin and its target proteins during chaperone action is poorly understood. It has been hypothesised that target proteins bind within a central cavity. Small-angle neutron-scattering (SANS) experiments in conjunction with isotopic substitution were undertaken to investigate the interaction of a target lens protein (gammaE-crystallin) with alpha-crystallin (alpha(H)) and to measure the radius of gyration (Rg) of the proteins and their binary complexes in solution under thermal stress. The size of the alpha(H) in D(2)O incubated at 65 degrees C increased from 69+/-3 to 81+/-5 A over 40 min, in good agreement with previously published small-angle X-ray scattering (SAXS) and SANS measurements. Deuterated gammaE-crystallin in H(2)O buffer (gammaE(D)/H(2)O) and hydrogenous gammaE-crystallin in D(2)O buffer (gammaE(H)/D(2)O) free in solution were of insufficient size and/or too dilute to provide any measurable scattering over the angular range used, which was selected primarily to investigate gammaE:alpha(H) complexes. The evolution of the aggregation size/shape as an indicator of alpha(H) chaperone action was monitored by recording the neutron scattering in different H:D solvent contrasts under thermally stressed conditions (65 degrees C) for binary mixtures of alpha(H), gammaE(H), and gammaE(D). It was found that Rg(alpha(H):gammaE(D)/D(2)O)>Rg(alpha(H):gammaE(H)/D(2)O)>Rg(alpha(H)/D(2)O) and that Rg(alpha(H):gammaE(H)/D(2)O) approximately Rg(alpha(H)/D(2)O). The relative sizes observed for the complexes weighted by the respective scattering powers of the various components imply that gammaE-crystallin binds in a central cavity of the alpha-crystallin oligomer, during chaperone action.

Complementing structural information of modular proteins with small angle neutron scattering and contrast variation.

Many macromolecules in the cell function by forming multi-component assemblies. We have applied the technique of small angle neutron scattering to study a nucleic acid-protein complex and a multi-protein complex. The results illustrate the versatility and applicability of the method to study macromolecular assemblies. The neutron scattering experiments, complementing X-ray solution scattering data, reveal that the conserved catalytic domain of RNase E, an essential ribonuclease in Escherichia coli (E. coli), undergoes a marked conformational change upon binding a 5'monophosphate-RNA substrate analogue. This provides the first evidence in support of an allosteric mechanism that brings about RNA substrate cleavage. Neutron contrast variation of the multi-protein TIM10 complex, a mitochondrial chaperone assembly comprising the subunits Tim9 and Tim10, has been used to determine a low-resolution shape reconstruction of the complex, highlighting the integral subunit organization. It shows characteristic features involving protrusions that could be assigned to the six subunits forming the complex.

Interaction of cationic lipid vesicles with model cell membranes--as determined by neutron reflectivity.

Transfection of cells by DNA (for the purposes of gene therapy) can be effectively engineered through the use of cationic lipid/DNA "lipoplexes", although the transfection efficiency of these lipoplexes is sensitive to the neutral "helper" lipid included. Here, neutron reflectivity has been used to investigate the role of the helper lipid present during the interaction of cationic lipid vesicles with model cell membranes. Dimethyldioctadecylammonium bromide (DDAB) vesicles were formed with two different helper lipids, 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) and cholesterol, and the interaction of these vesicles with a supported phospholipid bilayer was determined. DOPE-containing vesicles were found to interact faster with the membrane than those containing cholesterol, and vesicles containing either of the neutral helper lipids were found to interact faster than when DDAB alone was present. The interaction between the vesicles and the membrane was characterized by an exchange of lipid between the membrane and the lipid aggregates in solution; the deposition of vesicle bilayers on the surface of the membrane was not apparent.

Molecular and mesoscale structures in hydrophobically driven aqueous solutions.

Since Kauzmann's seminal 1959 paper, the hydrophobic interaction has dominated thinking on the forces that control protein folding and stability. Despite its wide importance in chemistry and biology, our understanding of this interaction at the molecular level remains poor, with little experimental evidence to support the idea of water ordering close to a non-polar group that is at the centre of the standard model for the source of the entropic driving force. Developments over recent years in neutron techniques now enable us to see directly how a non-polar group actually affects the molecular structure of the water in its immediate neighbourhood. On the basis of such work on aqueous solutions of small alcohols, the generally accepted standard model is found to be wanting, and alternative sources of the entropic driving force are suggested. Moreover, the fact that we can now follow changes in hydrogen bonding as the alcohol concentration is varied gives us the possibility of explaining the concentration dependence of the enthalpy of mixing. Complementary studies of solute association on the mesoscopic scale show a rich concentration and temperature behaviour, which reflects a complex balance of polar and non-polar interactions. Unravelling the detailed nature of this balance in simple aqueous amphiphiles may lead to a better understanding of the forces that control biomolecular structural stability and interactions.

Amphipols: polymeric surfactants for membrane biology research.

Membrane proteins classically are handled in aqueous solutions as complexes with detergents. The dissociating character of detergents, combined with the need to maintain an excess of them, frequently results in more or less rapid inactivation of the protein under study. Over the past few years, we have endeavored to develop a novel family of surfactants, dubbed amphipols (APs). APs are amphiphilic polymers that bind to the transmembrane surface of the protein in a noncovalent but, in the absence of a competing surfactant, quasi-irreversible manner. Membrane proteins complexed by APs are in their native state, stable, and they remain water-soluble in the absence of detergent or free APs. An update is presented of the current knowledge about these compounds and their demonstrated or putative uses in membrane biology.

Detergent organisation in crystals of monomeric outer membrane phospholipase A.

The structure of the detergent in crystals of outer membrane phospholipase A (OMPLA) has been determined using neutron diffraction contrast variation. Large crystals were soaked in stabilising solutions, each containing a different H(2)O/D(2)O contrast. From the neutron diffraction at five contrasts, the 12 A resolution structure of the detergent micelle around the protein molecule was determined. The hydrophobic beta-barrel surfaces of the protein molecules are covered by rings of detergent. These detergent belts are fused to neighbouring detergent rings forming a continuous three-dimensional network throughout the crystal. The thickness of the detergent layer around the protein varies from 7-20 A. The enzyme's active site is positioned just outside the hydrophobic detergent zone and is thus in a proper location to catalyse the hydrolysis of phospholipids in a natural membrane. Although the dimerisation face of OMPLA is covered with detergent, the detergent density is weak near the exposed polar patch, suggesting that burying this patch in the enzyme's dimer interface may be energetically favourable. Furthermore, these results indicate a crucial role for detergent coalescence during crystal formation and contribute to the understanding of membrane protein crystallisation.

Neutron and X-ray scattering by ox corneal stroma differentially loaded with bound anions.

Ox corneas at near physiological hydration were subjected to two variables: the amount of chloride ions bound to them and exposure of various mixtures of H(2)O/D(2)O as solvent. The preparations were then exposed to a neutron beam and the contrast match points, at which the collagen fibrils of the corneal stroma most nearly matched the scattering density of the various H(2)O/D(2)O mixtures, were measured. In both cases of high and low bound chloride, the contrast match points of the collagen fibril were equal, indicating that there were no significant changes in the water of electrostriction at the fibril surface when chloride ions bind to the stroma. The data suggest that the ligands which bind anions to corneal stroma are not located at the collagen fibril surface. When the chloride binding ligands were extracted from the corneal stroma there were significant changes in the structure of the fibrils. We suggest that the chloride binding ligands may be located within the collagen fibril.

Concomitant cisplatin and extended field radiation therapy in patients with cervical and endometrial cancer.

The purpose of this study is to evaluate the toxicity and safety of concomitant cisplatin (CDDP) and extended field radiation therapy (EFRT) in patients with cervical cancer (CxCA) and endometrial cancer (EnCA). Twenty-five patients were analyzed retrospectively for treatment-related morbidity from 1989 to 1998. Fourteen patients had CxCA and 11 patients had EnCA. Eighteen patients (72%) had surgery prior to radiotherapy and chemotherapy. EFRT was delivered by a four-field technique to the pelvis and para-aortic regions. CDDP at 100 mg/m2 was given over 5 days during 1st and 4th week of EFRT. EFRT dose for EnCA and CxCA was 45 Gy. Toxicity was analyzed using the RTOG toxicity criteria. Twenty-four (96%) of the 25 patients completed the prescribed therapy. Of the 14 patients with CxCA, three (21%) had no toxicity, three (21%) had grade 1-2, and eight (58%) had grade 3-4 hematologic toxicities. Overall six (24%) had grade 3-4 acute gastrointestinal toxicities, three (21%) of these patients were treated for cervix cancer and three (27%) patients were treated for endometrial cancer. The worst (Grade 3-4) toxicities in 15 patients occurred after the 4th week of radiotherapy. In six of 25 (24%) patients radiation treatments had to be delayed due to toxicities. The median delay of treatment was 10.5 days (range 7-31 days). Of the six patients who had grade 3-4 acute gastrointestinal toxicities, four (66%) had undergone exploratory laparotomy and lymph node sampling prior to start of chemoradiation. We conclude that concomitant EFRT and CDDP appears to be safe with moderate but manageable toxicity. Toxicity is most severe after the 4th week of treatment. Morbidity may be worse in patients with prior laparotomy.

In vitro-in vivo correlation (IVIVC) models for metformin after administration of modified-release (MR) oral dosage forms to healthy human volunteers.

The objective of the current study was to develop and evaluate the internal predictability for level C and A in vitro-in vivo correlation (IVIVC) models for prototype modified-release (MR) dosage forms of metformin. In vitro dissolution data for metformin were collected for 22 h using a USP II (paddle) method. In vivo plasma concentration data were obtained from 8 healthy volunteers after administration of immediate-release (IR) and MR dosage forms of metformin. Linear level C IVIVC models were developed using dissolution data at 2.0 and 4.0 h and in vitro mean dissolution time (MDT). A deconvolution-based level A model was attempted through a correlation of percent in vivo input obtained through deconvolution and percent in vitro dissolution obtained experimentally. Further, basic and extended convolution level A IVIVC models were attempted for metformin. Internal predictability for the IVIVC models was assessed by comparing observed and predicted values for C(max) and AUC(INF). The results suggest that highly predictive level C models with prediction errors (%PE) of <5% could be developed. Mean percent in vivo input for metformin was incomplete from all formulations and did not exceed 35% of dose. The deconvolution-based level A models for all MR formulations were curvilinear. However, a unique IVIVC model applicable to all MR formulations could not be developed using the deconvolution approach. The basic convolution level A model, which used in vitro dissolution as the in vivo input, had %PE values as high as 103%. Using an extended convolution approach, which modeled the absorption of metformin using a Hill function, a level A IVIVC model with %PE as low as 11% was developed. In conclusion, the current work indicates that level C and A IVIVC models with good internal predictability may be developed for a permeability- and absorption window-limited drug such as metformin.

Hierarchical assembly of the Alu domain of the mammalian signal recognition particle.

The mammalian signal recognition particle (SRP) catalytically promotes cotranslational translocation of signal sequence containing proteins across the endoplasmic reticulum membrane. While the S-domain of SRP binds the N-terminal signal sequence on the nascent polypeptide, the Alu domain of SRP temporarily interferes with the ribosomal elongation cycle until the translocation pore in the membrane is correctly engaged. Here we present biochemical and biophysical evidence for a hierarchical assembly pathway of the SRP Alu domain. The proteins SRP9 and SRP14 first heterodimerize and then initially bind to the Alu RNA 5' domain. This creates the binding site for the Alu RNA 3' domain. Alu RNA then undergoes a large conformational change with the flexibly linked 3' domain folding back by 180 degrees onto the 5' domain complex to form the final compact Alu ribonucleoprotein particle (Alu RNP). We discuss the possible mechanistic consequences of the likely reversibility of this final step with reference to translational regulation by the SRP Alu domain and with reference to the structurally similar Alu RNP retroposition intermediates derived from Alu elements in genomic DNA.

Small angle neutron scattering and gel filtration analyses of neutrophil NADPH oxidase cytosolic factors highlight the role of the C-terminal end of p47phox in the association with p40phox.

The NADPH oxidase of phagocytic cells is regulated by the cytosolic factors p47(phox), p67(phox), and p40(phox) as well as by the Rac1-Rho-GDI heterodimer. The regulation is a consequence of protein-protein interactions involving a variety of protein domains that are well characterized in signal transduction. We have studied the behavior of the NADPH oxidase cytosolic factors in solution using small angle neutron scattering and gel filtration. p47(phox), two truncated forms of p47(phox), namely, p47(phox) without its C-terminal end (residues 1-358) and p47(phox) without its N-terminal end (residues 147-390), and p40(phox) were found to be monomeric in solution. The dimeric form of p67(phox) previously observed by gel filtration experiments was confirmed. Our small angle neutron scattering experiments show that p40(phox) binds to the full-length p47(phox) in solution in the absence of phosphorylation. We demonstrated that the C-terminal end of p47(phox) is essential in this interaction. From the comparison of the presence or absence of interaction with various truncated forms of the proteins, we confirmed that the SH3 domain of p40(phox) interacts with the C-terminal proline rich region of p47(phox). The radii of gyration observed for p47(phox) and the truncated forms of p47(phox) (without the C-terminal end or without the N-terminal end) show that all these molecules are elongated and that the N-terminal end of p47(phox) is globular. These results suggest that the role of amphiphiles such as SDS or arachidonic acid or of p47(phox) phosphorylation in the elicitation of NADPH oxidase activation could be to disrupt the p40(phox)-p47(phox) complex rather than to break an intramolecular interaction in p47(phox).

Effects of ligand binding on the association properties and conformation in solution of retinoic acid receptors RXR and RAR.

In higher eukaryotes, vitamin A derived metabolites such as 9-cis and all-trans retinoic acid (RA), are involved in the regulation of several essential physiological processes. Their pleiotropic physiological effects are mediated through direct binding to cognate nuclear receptors RXRs and RARs that act as regulated transcription factors belonging to the superfamily of nuclear hormone receptors. Hormone binding to the structurally conserved ligand-binding domain (LBD) of these receptors triggers a conformational change that principally affects the conserved C-terminal transactivation helix H12 involved in transcriptional activation. We report an extensive biophysical solution study of RAR alpha, RXR alpha LBDs and their corresponding RXR alpha/RAR alpha LBD heterodimers combining analytical ultracentrifugation (AUC), small-angle X-ray and neutron scattering (SAXS and SANS) and ab initio three-dimensional shape reconstruction at low resolution. We show that the crystal structures of RXRs and RARs LBDs correlate well with the average conformations observed in solution. Furthermore we demonstrate the effects of 9-cisRA and all-transRA binding on the association properties and conformations of RXR alpha and RAR alpha LBDs in solution. The present study shows that in solution RAR alpha LBD behaves as a monomer in both unliganded and liganded forms. It confirms the existence in solution of a ligand-induced conformational change towards a more compact form of the LBD. It also confirms the stability of the predicted RXR alpha/RAR alpha LBD heterodimers in solution. SAS measurements performed on three different types of RXR alpha/RAR alpha LBD heterodimers (apo/apo, apo/holo and holo/holo) with respect to their ligand-binding site occupancy show the existence of three conformational states depending on the progressive binding of RA stereoisomers on RAR alpha and RXR alpha LBD subunits in the heterodimeric context. These results suggest that the subunits are structurally independent within the heterodimers. Our study also underlines the particular behaviour of RXR alpha LBD. In solution unliganded RXR alpha LBD is observed as two species that are unambiguously identified as homotetramers and homodimers. Molecular modelling combined with SAS data analysis allows us to propose a structural model for this autorepressed apo-tetramer. In contrast to the monomeric state observed in the crystal structure, our data show that in solution active holo-RXR alpha LBD bound to 9-cisRA is a homodimer regardless of the protein concentration. This study demonstrates the crucial role of ligands in the regulation of homodimeric versus heterodimeric association state of RXR in the NR signalling pathways.

Combined results from solution studies on intact influenza virus M1 protein and from a new crystal form of its N-terminal domain show that M1 is an elongated monomer.

The amino-terminal domain of influenza A virus matrix protein (residues 1-164) was crystallized at pH 7 into a new crystal form in space group P1. This packing of the protein implies that M1(1-164) was monomeric in solution when it crystallized. Otherwise, the structure of the M1 fragment in the pH 7 crystals was the same as the monomers in crystals formed at pH 4 where crystal packing resulted in dimer formation [B. Sha and M. Luo, 1997, Nature Struct. Biol. 4, 239-244]. Analysis of intact M1 protein, the N-terminal domain, and the remaining C-terminal fragment (residues 165-252) in solution also showed that the N-terminal domain was monomeric with the same dimensions as determined from the crystal structure. Intact M1 protein was also monomeric but with an elongated shape due to the presence of the C-terminal part. Circular dichroism showed that the C-terminal part of M1 contained helical structure. A model for soluble M1 is presented, based on the assumption that the C-terminal domain is spherical, in which the N- and C-terminal domains are connected by a linker sequence which is available for proteolytic attack.

In-vitro in-vivo correlation models for glibenclamide after administration of metformin/glibenclamide tablets to healthy human volunteers.

In this study, level C and A in-vitro in-vivo correlation (IVIVC) models were developed for glibenclamide. In-vitro dissolution data were collected for the glibenclamide component of three metformin/glibenclamide tablets using a USP Type II apparatus. In-vivo plasma concentration data were obtained after administration of the prototype formulations to 24 healthy volunteers and subject to deconvolution analysis to obtain percentage in-vivo absorbed profiles. Multiple linear level C models were developed for CMAX and AUC(0-48) using percentage in-vitro dissolved data at 10, 45 and 120 min. Initially, the level A model was constructed for the first 2 h only, based on availability of in-vitro data. Another level A model was attempted using a time-scaled approach, with percentage in-vivo absorbed at time t and percentage in-vitro dissolved at time t/I as the correlating data. Internal predictability was evaluated for the level C and time-scaled level A models. For all level C approaches, linear regression models with r2 > 0.99 were determined. The prediction errors (% PE) for Cmax and AUC(0-48) were less than 1% for all formulations at all three chosen time points. The deconvolution analysis indicated biphasic absorption for glibenclamide, with one phase occurring at 2-3h and another at 6-12h after dose administration. The level A model using 2-h data was not unique for all formulations and was therefore not developed. The time-scaling factor I correlated highly (r2 = 0.99) with in vitro mean dissolution time (MDT). A linear regression time scaled model (r2 = 0.97) was successfully developed using in-vitro and in-vivo data from all 3 formulations. However, the internal predictability of the time-scaled model was poor, with % PE values for Cmax and AUC(0-48) being as much as 30.5% and 18.7%, respectively. The results indicate that level C models have good internal predictability. Though a time-scaled level A IVIVC model was successfully developed, the model was found to have poor internal predictability.

Critical role of micelles in pancreatic lipase activation revealed by small angle neutron scattering.

In the duodenum, pancreatic lipase (PL) develops its activity on triglycerides by binding to the bile-emulsified oil droplets in the presence of its protein cofactor pancreatic colipase (PC). The neutron crystal structure of a PC-PL-micelle complex (Hermoso, J., Pignol, D., Penel, S., Roth, M., Chapus, C., and Fontecilla-Camps, J. C. (1997) EMBO J. 16, 5531-5536) has suggested that the stabilization of the enzyme in its active conformation and its adsorption to the emulsified oil droplets are mediated by a preformed lipase-colipase-micelle complex. Here, we correlate the ability of different amphypathic compounds to activate PL, with their association with PC-PL in solution. The method of small angle neutron scattering with D(2)O/H(2)O contrast variation was used to characterize a solution containing PC-PL complex and taurodeoxycholate micelles. The resulting radius of gyration (56 A) and the match point of the solution indicate the formation of a ternary complex that is similar to the one observed in the neutron crystal structure. In addition, we show that either bile salts, lysophospholipids, or nonionic detergents that form micelles with radii of gyration ranging from 13 to 26 A are able to bind to the PC-PL complex, whereas smaller micelles or nonmicellar compounds are not. This further supports the notion of a micelle size-dependent affinity process for lipase activation in vivo.