The positively charged polysaccharide chitosan is able to increase precorneal residence time of ophthalmic formulations containing active compounds when compared with simple aqueous solutions. The purpose of the study was to evaluate tear concentration of tobramycin and ofloxacin after topical application of chitosan-based formulations containing 0.3% wt/vol of antibiotic and to compare them with 2 commercial solutions: Tobrex® and Floxal®, respectively. The influence of the molecular weight, deacetylation degree, and concentration of 4 different samples of chitosan on pharmacokinetic parameters (area under the curve values [AUCeff] and time of efficacy [teff]) of tobramycin and ofloxacin in tears was investigated over time. It was demonstrated that the 2 chitosan products of high molecular weight (1350 and 1930 kd) and low deacetylation degree (50%) significantly increased antibiotic availability when compared to the controls, with AUCeff showing a 2-to 3-fold improvement. The time of efficacy of ofloxacin was significantly increased from about 25 minutes to 46 minutes by the chitosan of higher Mw (1930 kd) at a concentration of 0.5% wt/vol, whereas a similar performance was achieved by a chitosan of low Mw (580 kd) at a concentration of 1.5% wt/vol in the case of tobramycin.
Chitosan; hydrogel; ophthalmic application; antibiotic; pharmacokinetics
The first objective of this study was to assess the existence of nonresponse bias to a national survey of licensed pharmacists conducted in 2000. Three methods were used to assess nonresponse bias. The second objective of the study was to examine reasons why sampled licensed pharmacists did not respond to the national survey of licensed pharmacists. We used data from 2204 respondents to a national survey of pharmacists and from 521 respondents to a survey of nonrespondents to the national survey. We made comparisons between respondents for 5 variables: employment status, gender, age, highest academic degree, and year of initial licensure. Chi-square tests were used to examine differences in the 5 variables between respondents to the first mailing and second mailing of the survey, early and late respondents to the survey, and respondents to the survey and respondents to the nonrespondent survey. There were no significant differences between first mailing and second mailing respondents, but there were differences in each variable except year of licensure between early and late respondents. These differences likely were due to regional bias possibly related to differences in mailing times. There were differences between respondents and nonrespondents in terms of employment status and year of licensure. The main reasons for not responding to the survey were that it was too long or that it was too intrusive. Overall, the survey methodology resulted in a valid sample of licensed pharmacists. Nonresponse bias should be assessed by surveying nonrespondents. Future surveys of pharmacists should consider the length of the survey and the address where it is sent.
Pharmacy Workforce; Survey Methods; Nonresponse Bias
The development of macromolecules as drugs and drug carriers requires knowledge of their fate in cells. To this end, we studied the internalization and subcellular fate of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers in Hep G2 (human hepatocellular carcinoma) cells. Semiquantitative fluorometry confirmed that galactose was an effective ligand for receptor-mediated endocytosis for Hep G2 cells. The rate of internalization of a galactose-targeted copolymer was almost 2 orders of magnitude larger than that of the nontargeted copolymer. Confocal fluorescent microscopy of both fixed and live cells revealed that the polymer entered the cells by endocytosis. After longer incubation times (typically >8 hours), polymer escaped from small vesicles and distributed throughout the cytoplasm and nuclei of the cells. Polymer that entered the cytoplasm tended to accumulate in the nucleus. Microinjection of the HPMA copolymers into cells' cytoplasm and nuclei indicated that the polymers partitioned to the nucleus. The data from fixed cells was confirmed by microscopy of live, viable cells. To examine the effect of the fluorescent dye on the intracellular fate, polymers with fluorescein, Oregon Green 488, Lissamine rhodamine B, and doxorubicin were tested; no significant differences were observed.
HPMA copolymer; subcellular trafficking; endocytosis; microinjection; confocal fluorescent microscopy
The gene encoding the human muscarinic receptor, type 1 (CHRM1), was genotyped from 245 samples of the Coriell Collection (Coriell Institute for Medical Research, Camden, NJ). Fifteen single nucleotide polymorphisms (SNPs) were discovered, 9 of which are located in the coding region of the receptor. Of these, 8 represent synonymous SNPs, indicating that CHRM1 is highly conserved in humans. Only a single allele was found to contain a nonsynonymous SNP, which encodes an amino acid change of Cys to Arg at position 417. This may have functional consequences because a C417S point mutation in rat M1 was previously shown to affect receptor binding and coupling. Furthermore, 0 of 4 SNPs within CHRM1 previously deduced from sequencing of the human genome were found in this study despite a prediction that a majority of such inferred SNPs are accurate. The consensus sequence of CHRM1 obtained in our study differs from the deposited reference sequence (AC NM_000738) in 2 adjacent nucleotides, leading to a V173M change, suggesting a sequencing error in the reference sequence. The extraordinary sequence conservation of the CHRM1 gene-coding region was unexpected as M1-knockout mice show only minimal functional impairments.
pharmacogenetics; muscarinic acetylcholine; receptor; single nucleotide polymorphism; G protein coupled receptor; CHRM1
Receptor binding studies were performed on 24 soft anticholinergic agents and 5 conventional anticholinergic agents using 4 cloned human muscarinic receptor subtypes. The measured pKi values of the soft anticholinergic agents ranged from 6.5 to 9.5, with the majority being in the range of 7.5 to 8.5. Strong correlation was observed between the pKis determined here and the pA2 values measured earlier in guinea pig ileum contraction assays. The corresponding correlation coefficients (r2) were 0.80, 0.73, 0.81, and 0.78 for pKi(m1), pKi(m2), pKi(m3), and pKi(m4), respectively. Quantitative structure-activity relationship (QSAR) studies were also performed, and good characterization could be obtained for the soft anticholinergics containing at least 1 tropine moiety in their structure. For these compounds, the potency as measured by the pKi values was found to be related to geometric, electronic, and lipophilicity descriptors. A linear regression equation using ovality (Oe), dipole moment (D), and a calculated log octanol-water partition coefficient (QLogP) gave reasonably good descriptions (r=0.88) for the pKi(m3) values.
drug design; soft drugs; receptor binding; metabolism; drug evaluation; muscarinic antagonists
Statistical analysis and Monte Carlo simulation were used to characterize uncertainty in the allometric exponent (b) of xenobiotic clearance (CL). CL values for 115 xenobiotics were from published studies in which at least 3 species were used for the purpose of interspecies comparison of pharmacokinetics. The b value for each xenobiotic was calculated along with its confidence interval (CI). For 24 xenobiotics (21%), there was no correlation between log CL and log body weight. For the other 91 cases, the mean±standard deviation of the b values was 0.74±0.16; range: 0.29 to 1.2. Most (81%) of these individual b values did not differ from either 0.67 or 0.75 at P=0.05. When CL values for the subset of 91 substances were normalized to a common body weight coefficient (a), the b value for the 460 adjusted CL values was 0.74; the 99% CI was 0.71 to 0.76, which excluded 0.67. Monte Carlo simulation indicated that the wide range of observed b values could have resulted from random variability in CL values determined in a limited number of species, even though the underlying b value was 0.75. From the normalized CL values, 4 xenobiotic subgroups were examined: those that were (i) protein, and those that were (ii) eliminated mainly by renal excretion, (iii) by metabolism, or (iv) by renal excretion and metabolism combined. All subgroups except (ii) showed a b value not different from 0.75. The b value for the renal excretion subgroup (21 xenobiotics, 105 CL values) was 0.65, which differed from 0.75 but not from 0.67.
allometric scaling; body-weight exponent; clearance; metabolism; metabolic rate; pharmacokinetics; Monte Carlo simulation; power law
The solubility of 4 analogues of efavirenz was studied as a function of pH. The study evaluated the ionization behavior and determined the relative contribution of electronegative substituents versus resonance effects on the pKa value of the cyclic carbamate. The most profound lowering effect on the pKa was due to the presence of multiple electronegative substituents and in particular the trifluoromethyl and acetylene groups. The presence of chlorine on the benzoxazinone ring was found to have a slight impact on the pKa, although to a lesser extent. In the absence of any functional groups on the benzoxazinone ring system, the pKa shifted to a value of 13.2, which is 3 pH units above that of efavirenz and more closely correlates with typical literature values for cyclic carbamates.
pKa; Ionization; Benzoxazinone; Carbamate; Solubility; Efavirenz Analogs
This study evaluated the effect of parathyroid hormone (PTH) infusion alone or in combination with salmon calcitonin (sCT) in ovariectomized (OVX) rats and compared it with daily PTH injections alone or in combination with sCT infusion. Female Sprague-Dawley rats were divided randomly into 6 groups and were either bilaterally ovariectomized or underwent a sham operation; they were then treated for 4 weeks, beginning the day after surgery. Each group of OVX rats received either PTH infusion (group 1), PTH+sCT infusion (group 2), sCT infusion+daily PTH injection (group 3), or daily PTH injection (group 4). One group each of OVX (group 5) and sham-operated rats (group 6) received daily injections of vehicle alone. PTH was injected at 80 μg/kg/day and infused at 40 μg/kg/day, whereas sCT was infused at 10 μg/kg/day. The animals were sacrificed 28 days after treatment, and cancellous bone volume was measured in the tibial metaphysis. Similar to daily PTH injections, continuous infusion of PTH alone increased cancellous bone volume significantly over that seen in vehicle-treated OVX and sham-operated rats. Although cancellous bone volume after continuous infusion of PTH+sCT was also significantly higher than that seen in vehicle-treated OVX and sham-operated rats, the increase was significantly lower than with the other 3 nonvehicle treatments. The increase in cancellous bone volume after administration of sCT infusion along with daily PTH injections was not different from that with daily PTH injections alone. Thus, at the doses tested, the beneficial effects of PTH injection were not apparently improved by PTH infusion or by combination with sCT.
Salmon calcitonin; human parathyroid hormone (1–34); infusion; ovariectomized rats; cancellous bone volume
Objective. The complex composition-activity relationship of botanicals such as St John's Wort (SJW) presents a major challenge to product development, manufacture, and establishment of appropriate quality and performance standards for the formulated products. As part of a larger study aimed at addressing that challenge, the goals of the present study are to (1) determine and compare the phytochemical profiles of 3 commercial SJW extracts; (2) assess the possible impact of humidity, temperature, and light on their stability; and (3) evaluate several physical properties important to the development of solid dosage forms for these extracts. Methods. An adapted analytical method was developed and validated to determine phytochemical profiles and assess their stability. The extract physical properties measured were particle size (Malvern Mastersizer), flow (Carr's compressibility index; minimum orifice diameter), hygroscopicity (method of Callahan et al), and low-pressure compression physics (method of Heda et al). Results. The phytochemical properties differed greatly among the extracts and were extremely sensitive to changes in storage conditions, with marked instability under conditions of elevated humidity. All extracts exhibited moderate to free-flow properties and were very hygroscopic. Compression properties varied among the extracts and differed from a common use excipient, microcrystalline cellulose. Conclusions. Three commercial sources of SJW extracts exhibited different physical and chemical properties. Standardization to 1 or 2 marker compounds does not ensure chemical equivalence nor necessarily equivalent pharmacological activity. Flow and compression properties appear suitable for automatic capsule-filling machines, but hydroscopicity and the moisture sensitivity of the phytochemical profile are concerns.
Hypericum perforatum; St John's Wort; Nutraceuticals
This study was undertaken to determine whether the gravimetric method provided an accurate measure of water flux correction and to compare the gravimetric method with methods that employ nonabsorbed markers (eg, phenol red and 14C-PEG-3350). Phenol red, 14C-PEG-3350, and 4-[2-[[2-(6-amino-3-pyridinyl)-2-hydroxyethyl]amino]ethoxy]-methyl ester, (R)-benzene acetic acid (Compound I) were co-perfused in situ through the jejunum of 9 anesthetized rats (single-pass intestinal perfusion [SPIP]). Water absorption was determined from the phenol red. 14C-PEG-3350, and gravimetric methods. The absorption rate constant (ka) for Compound I was calculated. Both phenol red and 14C-PEG-3350 were appreciably absorbed, underestimating the extent of water flux in the SPIP model. The average ±SD water flux (μg/h/cm) for the 3 methods were 68.9±28.2 (gravimetric), 26.8±49.2 (phenol red), and 34.9±21.9 (14C-PEG-3350). The (average±SD) ka for Compound I (uncorrected for water flux) was 0.024±0.005 min−1. For the corrected, gravimetric method, the average±SD was 0.031±0.001 min−1. The gravimetric method for correcting water flux was as accurate as the 2 “nonabsorbed” marker methods.
intestinal perfusion model; permeability; rat; water flux; multiple linear regression
This study was designed to theoretically investigate the influence of drug release properties, characterized by the disintegration of a solid dosage form and dissolution of drug particles, on the systemic exposure of highly soluble drugs in immediate release products. An absorption model was developed by considering disintegration of a solid dosage form, dissolution of drug particles, gastrointestinal transit flow, and intestinal absorption processes. The absorption model was linked to a conventional pharmacokinetic model to evaluate the effect of disintegration and dissolution on the peak exposure (Cmax) and total exposure of area under the curve (AUC). Numerical methods were used to solve the model equations. The simulations show that the effect of disintegration of a dosage form and dissolution of drug particles depend on the permeability of a drug, with a low-permeability drug having a greater effect. To provide similar exposure to an oral solution formulation, a solid dosage form containing a low-permeability drug would need to dissolve more rapidly than a solid dosage form containing a high-permeability drug. It was shown theoretically for poorly permeable drugs that the disintegration rate constant has to be greater than 9 hour−1 (equivalent to approximately 90% in 30 minutes) to make both AUC and Cmax ratios higher than .9, ensuring the confidence interval of .80 to 1.25. The rapid in vitro release requirement of at least 85% dissolved in 30 minutes is sufficient for highly soluble and highly permeable drugs. However, for highly soluble and poorly permeable drugs, the appropriate in vitro release requirement seems to be 90% dissolved in 30 minutes.
Small intestinal transit; dissolution; disintegration; absorption modeling; bioequivalence
The objective of this study was to demonstrate the use of transmission Fourier transform near-infrared (FT-NIR) spectroscopy for quantitative analysis of an active ingredient in a translucent gel formulation. Gels were prepared using Carbopol 980 with 0%, 1%, 2%, 4%, 6%, and 8% ketoprofen and analyzed with an FT-NIR spectrophotometer operated in the transmission mode. The correlation coefficient of the calibration was 0.9996, and the root mean squared error of calibration was 0.0775%. The percent relative standard deviation for multiple measurements was 0.10%. The results prove that FT-NIR can be a good alternative to other, more time-consuming means of analysis for these types of formulations.
FT-NIR; Transmission Spectroscopy; Carbopol; Topical Formulations; Ketoprofen
Numerous genes encode G protein-coupled receptors (GPCRs)-a main molecular target for drug therapy. Estimates indicate that the human genome contains approximately 600 GPCR genes. This article addresses therapeutic implications of sequence variations in GPCR genes. A number of inactivating and activating receptor mutations have been shown to cause a variety of (mostly rare) genetic disorders. However, pharmacogenetic and pharmacogenomic studies on GPCRs are scarce, and therapeutic relevance of variant receptor alleles often remains unclear. Confounding factors in assessing the therapeutic relevance of variant GPCR alleles include 1) interaction of a single drug with multiple closely related receptors, 2) poorly defined binding pockets that can accommodate drug ligands in different orientations or at alternative receptor domains, 3) possibility of multiple receptor conformations with distinct functions, and 4) multiple signaling pathways engaged by a single receptor. For example, antischizophrenic drugs bind to numerous receptors, several of which might be relevant to therapeutic outcome. Without knowing accurately what role a given receptor subtype plays in clinical outcome and how a sequence variation affects drug-induced signal transduction, we cannot predict the therapeutic relevance of a receptor variant. Genome-wide association studies with single nucleotide polymorphisms could identify critical target receptors for disease susceptibility and drug efficacy or toxicity.
G Protein-Coupled; Receptors; Drug Therapy; Pharmacogenomics; Pharmacogenetics
Receptor-binding ligands have been incorporated into DNA/polyethylenimine (PEI) complexes to enhance cell binding and cellular internalization. This study characterizes receptor-mediated uptake of DNA/PEI complexes on a cellular basis. A novel assay based on flow cytometry was applied, discriminating between total cell-associated and extracellularly bound DNA complexes. Receptor-mediated uptake of ligand-containing DNA/PEI (molecular weight, 800 kd) complexes was found to occur quickly (within 1 hour), whereas unspecific uptake through adsorptive endocytosis is less efficient or requires extended periods to reach the same degree of internalization. Rapid, receptor-mediated internalization requires a small complex size; however, large, aggregated complexes show higher gene expression. Using PEI 25 kd conjugated to large proteins such as transferrin or antibodies, improper condensation with DNA leads to suboptimal uptake and gene expression, whereas partial replacement of ligand-PEI with unconjugated PEI increases both uptake and transfection. In contrast, the 8 kd protein epidermal growth factor conjugated to PEI 25 kd properly condenses DNA and mediates specific uptake into human adenocarcinoma (KB) cells. Modification of the complex surface with appropriate amounts of poly(ethylene glycol) (PEG) does not block ligand-mediated internalization. A higher degree of PEGylation reduces the internalization of transferrin or antibody-containing complexes to a level similar to that of ligand-free complexes. In contrast, epidermal growth factor-mediated uptake is less effected by excessive PEGylation.
gene transfer; receptor mediated endocytosis; polyethylenimine; flow cytometry; poly(ethylene glycol)
This study evaluated tableting compression by using internal and external lubricant addition. The effect of lubricant addition on the enzymatic activity of trypsin, which was used as a model drug during the tableting compression process, was also investigated. The powder mixture (2% crystalline trypsin, 58% crystalline lactose, and 40% microcrystalline cellulose) was kneaded with 5% hydroxypropyl cellulose aqueous solution and then granulated using an extruding granulator equipped with a 0.5-mm mesh screen at 20 rpm. After drying, the sample granules were passed through a 10-mesh screen (1680 μm). A 200-mg sample was compressed by using 8-mm punches and dies at 49, 98, 196, or 388 MPa (Mega Pascal) at a speed of 25 mm/min. The external lubricant compression was performed using granules without lubricant in the punches and dies. The granules were already dry coated by the lubricant. In contrast, the internal lubricant compression was performed using sample granules (without dry coating) containing 0.5% lubricant. At 98 MPa, for example, the compression level using the external lubricant addition method was about 13% higher than that for internal addition. The significantly higher compressing energy was also observed at other MPas. By comparison, the friction energy for the external addition method calculated based on upper and lower compression forces was only slightly larger. The hardness of tablets prepared using the internal addition method was 34% to 48% lower than that for the external addition method. The total pore volume of the tablet prepared using the external addition method was significantly higher. The maximum ejection pressure using the no-addition method (ie, the tablet was prepared using neither dry-coated granules nor added lubricant) was significantly higher than that of other addition methods. The order was as follows: no addition, external addition, and then internal addition. The ejection energy (EE) for internal addition was the lowest; for no addition, EE was the highest. In the dissolution test, the tablets obtained using external addition immediately disintegrated and showed faster drug release than those prepared using internal addition. This result occurred because the water penetration rate of the tablet using the external addition was much higher. The trypsin activity in tablets prepared using the external addition method was significantly higher than that produced using the internal addition method at the same pressure. All these results suggest that the external addition method might produce a fast-dissolution tablet. Because the drug will be compressed using low pressure only, an unstable bulk drug may be tableted without losing potency.
Tableting; Trypsin; Preparation; Compression; Dissolution
Heparin employed in cardiovascular surgeries often leads to a high incidence of bleeding complications. Protamine employed in heparin reversal, however, can cause severe adverse reactions. In an attempt to address this clinical problem, we developed low molecular weight protamine (LMWP) as a potentially effective and less toxic heparin antagonist. A homogeneous 1880-d peptide fragment, termed LMWP-TDSP5 and containing the amino acid sequence of VSRRRRRRGGRRRR was derived directly from protamine by enzymatic digestion of protamine with thermolysin. In vitro studies demonstrated that TDSP5 was capable of neutralizing various anticoagulant functions of both heparin and commercial low molecular weight heparin preparations. In addition, TDSP5 exhibited significantly reduced crossreactivity toward mouse sera containing antiprotamine antibodies. TDSP5 showed a decrease in its potential in activating the complement system. All of these findings suggested the possibility of markedly reduced protamine toxicity for TDSP5.
In this article, we conducted preliminary in vivo studies to further demonstrate the feasibility and utility of using LMWP as a nontoxic clinical protamine substitute. Dogs were chosen as test animals because they were known to magnify the typical human response to protamine. By using a full spectra of biological and clinical assays for heparin, including the anti-IIa and anti-Xa chromogenic assays and the activated partial, thromboplastin time and TCT clotting assays, TDSP5 showed that it could completely neutralize all these different anticoagulant functions of heparin in dogs. Although administration of protamine in dogs produced a significant reduction in mean arterial blood pressure (−14.9 mm Hg) and elevation in pulmonary artery systolic pressure (+5.0 mm Hg), the use of TDSP5 in dogs did not elicit any statistically significant change in any of the variables measured. Furthermore, the use of LMWP also significantly reduced the protamine-induced transient thrombocytopenic and granulocytopenic responses. The white blood cell counts and platelet counts decreased to 82.1% and 60.0% of baseline, respectively, in dogs given intravenous protamine compared to 97.8% and 88.6% of baseline in dogs receiving TDSP5. These preliminary findings indicated that LMWP could potentially provide an effective and safe means to control both heparin- and protamine-induced complications.
Heparin Neutralization; Protamine Toxicity; aPTT/TCT Heparin Clotting Assays; Anti-IIa Anti-Xa Chromogenic Assays; Hemodynamic/Hematologic Responses
Patients undergoing anticoagulation with heparin or low molecular weight heparin (LMWH) require a superior antidote that possesses more selective biological actions and a better safety profile than protamine. We had previously developed 2 low molecular weight protamine (LMWP) fractions (TDSP4 and TDSP5) from thermolysin-digested protamine as potential nontoxie, heparin-neutralizing agents. In this, the second article in this series, studies focused on in vitro evaluation of heparin/LMWH-neutralizing efficacy and putative toxicity. These LMWP fractions, particularly TDSP5, were effective and fully capable of neutralizing a broad spectrum of heparin-induced anticoagulant activities (ie, aPTT, anti-Xa, and anti-IIa activities). Additionally, these LMWP fractions could neutralize the activities of commercial LMWH. As assessed by the anti-Xa assay, TDSP5 was as effective as, although less potent than, protamine in reversing the activity of Mono-Embolex (molecular weight 5000–7000) and 2 other different sizes (molecular weight of 3000 and 5000 d) of LMWH preparations. Furthermore, compared with protamine, TDSP5 exhibited a much-reduced toxicity and thus an improved safety profile, as reflected by its reduced ability to activate the complement system and cross-react with the antiprotamine antibodies, which are 2 primary indices of protamine toxicity.
Heparin/LMWH neutralization; protamine toxicity; aPTT clotting assay; anti-Xa assay; complement Activation; immunogenicity; cross-reactivity
Low molecular weight protamine (LMWP) appears to be a promising solution for heparin neutralization without the protamine-associated catastrophic toxic effects. The feasibility of this hypothesis was proven previously by using a peptide mixture produced from proteolytic digestion of protamine. To further examine the utility of this compound as an ultimate nontoxic protamine substitute, detailed studies on the purification and characterization of LMWP including the precise amino acid sequence, structure-function relationship, and possible mechanism were conducted. A number of LWMP fragments, composed of highly cationic peptides with molecular weights ranging from 700 to 1900 d, were prepared by digestion of native protamine with the protease thermolysin. These fragments were fractionated using a heparin affinity chromatography, and their relative binding strengths toward heparin were elucidated. Five distinct fractions were eluted at NaCl concentration ranging from 0.4 to 1.0 M and were denoted as TDSP1 to TDSP5, in increasing order of eluting ionic strength. Among these 5 fractions, TDSP4 and TDSP5 contained 3 LMWP peptide fragments, and they were found to retain the complete heparin-neutralizing function of protamine. By using a peptide mass spectrometry (MS) fingerprint mapping technique, the amino acid sequences of the microheterogeneous LMWP fragments in all these 5 elution fractions were readily identified. A typical structural scaffold made by arginine clusters in the middle and nonarginine residues at the N-terminal of the peptide sequence was observed for all these LMWP fragments. By aligning the sequences with the potency in heparin neutralization of these LMWP fragments, it was found that retention of potency similar to that of protamine required the presence of at least 2 arginine clusters in the LMWP fragments; such as the sequence of VSRRRRRRGGRRRR seen in the most potent LMWP fraction-TDSP5. The above finding was further validated by using a synthetic LMWP analogue-CRRRRRRR-and it was found that its heparin-neutralizing ability was increased by changing from a monomeric to a dimeric structure of this analogue peptide. Based on these results, the structural requirement for a compound to function as an effective heparin antidote and the possible mechanism involved in heparin neutralization were established.
Heparin LMWH neutralization; Protamine toxicity; LMWP peptide sequences; MS fingerprint mapping; Mechanism of heparin neutralization
The fibrinolytic enzyme from southern copperhead snake venom, fibrolase, contains 1 mole of zine per mole of protein, belongs to the major family of metalloproteinases known as the metzincins, and has been shown to degrade fibrin clots in vitro and in vivo. The purpose of this study was to develop a 3-dimensional model of fibrolase to investigate the geometry of conserved and variable sequences between members of the snake venom metalloproteinases. When compared to atrolysin C (form D) or adamalysin II (metzincins with completely different substrate specificity), fibrolase has approximately 60% overall sequence identity and nearly 100% sequence similarity in the active site. We used the crystal structure of adamalysin II to build a 3-dimensional homology model of fibrolase. Three disulfide bonds were constructed (the highly conserved disulfide bond [118–198] was maintained from the adamalysin II structure and 2 new disulfide bonds were introduced between residues 158–182 and 160–165). We used Sculpt 2.5 and HyperChem 5.0 to “dock” a substrate fragment octapeptide (HTEKLVTS), and a water molecule into the active site cleft. We calculated the differential average homology profile for fibrolase compared to 8 hemorrhagic and 5 nonhemorrhagic metzincins. We then determined the sequence regions that might be responsible for their substrate specificity. Our 3-dimensional homology model shows that the variable sequences lie on the periphery of the identified active site region containing the His triangle; this indicates that substrate specificity may depend on surface residues that are not directly associated with the active site.
Fibrolase; Metzincin; Homology Model; Substrate Specificity; Docking; Differential Average Homology
This study investigates the crystallization of the endogenous surfactant nonoxynol 100 in Eudragit NE30D-free films during storage and the influences of nonoxynol 100 on the dissolution of diphenhydramine hydrochloric acid (HCL) pellets coated with Eudragit NE30D before and after aging at ambient conditions. Polarizing light microscopy showed that when Eudragit NE30D-free films were stored at ambient conditions, off-white, flower-shaped crystals formed and increased in the polymer film as storage time increased. Also, x-ray diffraction showed polymer crystals in the aged free film. Thermogravimetric analysis showed no evidence of combined volatile molecules with the polymer molecules, and Fourier transformed infrared spectroscopy (FTIR) data suggested the same chemical composition of the polymer before and after phase separation. Further, from normal light microscopy, the appearance of the melting droplets in the polymer film indicated that the polymer molecules did not form the crystals. After the extraction of nonoxynol 100 by water, the free film formed by the water-extracted Eudragit NE30D was found free of the crystals after aging at the same conditions. The combination of the thermogravimetric analysis, FTIR, and microscopy showed that the origin of the crystals in dry Eudragit NE30D-free films came from nonoxynol 100, and not from the polymer molecules themselves. Monitoring by differential scanning calorimeter, it was found that the rates of crystallization of nonoxynol 100 were faster when the films were stored at 30°C and 40°C than when stored at ambient conditions and 45°C. When stored at −5°C, the crystallization rate was nearly zero. As the temperature got closer to melting temperature, the crystallization rate was very low because the system was in a thermodynamically disfavored state. The rate gradually increased and finally passed through a maximum as the crystallization temperature decreased. As the temperature kept decreasing, the crystallization rate became small again and eventually stopped because the system turned into a kinetically disfavored state. Because the phase transition of nonoxynol 100 in Eudragit NE30D occurred at ambient conditions, its influence on the dissolution of diphenhydramine HCL pellets coated with Eudragit NE30D was studied. Three different levels of nonoxynol 100 were used in Eudragit NE30D dispersions to make 3 different batches of Eudragit NE30D film-coated, controlled-release diphenhydramine HCL pellets. The results showed the dissolution rate increased as the level of nonoxynol 100 increased in the coating formula. Compared to the commonly used water-soluble additive human peripheral mononuclear cell, nonoxynol 100 was more effective in enhancing the dissolution of diphenhydramine HCL from pellets coated with Eudragit NE30D. Further study showed that the phase separation of the surfactant during aging tends to stabilize or slightly increase dissolution rates at higher surfactant levels.
Eudragit NE30D; Endogenous Surfactant; Free Film; Diphenhydramine HCL Pellets
The penetration of paclitaxel into multilayered solid tumors is time- and concentration-dependent, a result of the drug-induced apoptosis and changes in tissue composition. This study evaluates whether this tissue penetration property applies to other highly protein-bound drugs capable of inducing apoptosis. The penetration of doxorubicin was studied in histocultures of prostate xenograft tumors and tumor specimens obtained from patients who underwent radical prostatectomy. The kinetics of drug uptake and efflux in whole tumor histocultures were studied by analyzing the average tumor drug concentration using high-pressure liquid chromatography. Spatial drug distribution in tumors and the drug concentration gradient across the tumors were studied using fluorescence microscopy. The results indicate that drug penetration was limited to the periphery for 12 hours in patient tumors and to 24 hours in the more densely packed xenograft tumors. Subsequently, the rate of drug penetration to the deeper tumor tissue increased abruptly in tumors treated with higher drug concentrations capable of inducing apoptosis (i.e., >5 μm), but not in tumors treated with lower concentrations. These findings indicate a time- and concentration-dependent penetration of doxorubicin in solid tumors, similar to that of paclitaxel. We conclude that doxorubicin penetration in solid tumors is time- and concentration-dependent and is enhanced by drug-induced cell death.
Doxorubicin; Delivery; Apoptosis; Solid Tumor
Detailed models of solute transport through the stratum corneum (SC) require an interpretation of apparent bulk diffusion coefficients in terms of microscopic transport properties. Modern microscopy techniques provide a tool for evaluating one key property—lipid pathway tortuosity—in more detail than previously possible. Microscopic lipid pathway measurements on alkali expanded human SC stained with the lipid-soluble dyes methylene blue, Nile red, and oil red O are described. Brightfield, differential interference contrast, fluorescence, and laser scanning confocal optics were employed to obtain 2-dimensional (2-D) and 3-dimensional (3-D) images. The 2-D techniques clearly outlined the corneocytes. Confocal microscopy using Nile red yielded a well-delineated 3-D structure of expanded SC. Quantitative assessment of the 2-D images from a small number of expanded SC samples led to an average value of 3.7 for the ratio of the shortest lipid-continuous pathway to the width of the membrane. This was corrected for the effect of alkaline expansion to arrive at an average value of 12.7 for the same ratio prior to swelling.
Stratum Corneum; Alkaline Expansion; Microscopy; Lipid Pathlength; Tortuosity