Pore networks and polymer rearrangement on a drug-eluting stent as revealed by correlated confocal Raman and atomic force microscopy.

Drug release from and coating morphology on a CYPHER sirolimus-eluting coronary stent (SES) during in vitro elution were studied by correlated confocal Raman and atomic force microscopy (CRM and AFM, respectively). Chemical surface and subsurface maps of the SES were generated in the same region of interest by CRM and were correlated with surface topography measured by AFM at different elution times. For the first time, a direct correlation between drug-rich regions and the coating morphology was made on a drug-eluting medical device, linking drug release with pore formation, pore throats, and pore networks. Drug release was studied on a drug-eluting stent (DES) system with a multicomponent carrier matrix (poly(n-butyl methacrylate) [PBMA] and poly(ethylene-co-vinyl acetate) [PEVA]). The polymer was found to rearrange postelution because confluence of the carrier polymer matrix reconstituted the voids created by drug release.

Forced degradation studies of rapamycin: identification of autoxidation products.

The immunosuppressant drug rapamycin, also known as Sirolimus, underwent autoxidation under mild conditions to give numerous monomeric and oligomeric compounds, which were generally characterized by size-exclusion chromatography and NP-HPLC with UV and MS detection. Some of the more predominant products, epoxides and ketones, were isolated and identified. Two epoxides and 10S-epimer of rapamycin were described for the first time. Observed rapamycin isomers were also addressed. Computational chemistry was used to provide mechanistic insights. Formation of the majority of the rapamycin products could be rationalized with free radical-mediated autoxidation reactions involving alkene and alcohol sites. Methodological aspects of oxidative stress testing are discussed.

Mass balance in rapamycin autoxidation.

The immunosuppressant drug rapamycin is a complex polyene-containing natural product which undergoes autoxidation. The resulting product mixtures contained numerous monomeric and oligomeric compounds, which represented challenges for addressing mass balance in forced degradation studies and in analysis of aged developmental drug-eluting stents. A combination of SEC with ultraviolet and refractive index detection and RP-HPLC was used to account for drug loss and product formation. The mass balance methodology was subsequently validated for the determination of rapamycin and composite rapamycin autoxidation product material in developmental stent samples. This mass balance approach may find general applicability in other situations where drugs degrade to a plethora of products, which cannot be determined individually and summed.

Investigation of mass-balance issue in e-beam sterilized paclitaxel eluting coronary stents by SPME/GC-MS.

Paclitaxel eluting coronary stents were sterilized by e-beam in a closed system, to investigate sterilization related mass-balance issues and evaluate potential volatile paclitaxel degradation products. A solid-phase microextraction (SPME) method utilizing a polydimethyl-siloxane/divinyl-benzene (PDMS/DVB) fiber was optimized for extracting the volatiles from the head-space of the sterilized stents. GC-MS was used for separation, identification, and quantitation of the components. Benzaldehyde and benzoic acid were identified as paclitaxel related volatile degradation products. Three groups of stents were included in the study, a control group (not exposed to e-beam), a group sterilized at 25 kGy, and a final group sterilized at 75 kGy. The stents sterilized by e-beam at 75 kGy contained significantly higher levels of benzoic acid relative to the controls and the stents at 25 kGy contained intermediate levels of benzoic acid. The benzaldehyde levels increased in the 25 kGy e-beam sterilized stents relative to the control but remained fairly constant in the 75 kGy e-beam sterilized stents relative to the 25 kGy e-beam results. Mechanism for the formation of benzoic acid and benzaldehyde from paclitaxel was proposed. The levels of benzoic acid and benzaldehyde observed on the stents did not resolve the original mass-balance issue, but most likely contribute to the lack of mass balance observed for paclitaxel.

Thrombogenic collagen-mimetic peptides: Self-assembly of triple helix-based fibrils driven by hydrophobic interactions.

Collagens are integral structural proteins in animal tissues and play key functional roles in cellular modulation. We sought to discover collagen model peptides (CMPs) that would form triple helices and self-assemble into supramolecular fibrils exhibiting collagen-like biological activity without preorganizing the peptide chains by covalent linkages. This challenging objective was accomplished by placing aromatic groups on the ends of a representative 30-mer CMP, (GPO)(10), as with l-phenylalanine and l-pentafluorophenylalanine in 32-mer 1a. Computational studies on homologous 29-mers 1a'-d' (one less GPO), as pairs of triple helices interacting head-to-tail, yielded stabilization energies in the order 1a' > 1b' > 1c' > 1d', supporting the hypothesis that hydrophobic aromatic groups can drive CMP self-assembly. Peptides 1a-d were studied comparatively relative to structural properties and ability to stimulate human platelets. Although each 32-mer formed stable triple helices (CD) spectroscopy, only 1a and 1b self-assembled into micrometer-scale fibrils. Light microscopy images for 1a depicted long collagen-like fibrils, whereas images for 1d did not. Atomic force microscopy topographical images indicated that 1a and 1b self-organize into microfibrillar species, whereas 1c and 1d do not. Peptides 1a and 1b induced the aggregation of human blood platelets with a potency similar to type I collagen, whereas 1c was much less effective, and 1d was inactive (EC(50) potency: 1a/1b >> 1c > 1d). Thus, 1a and 1b spontaneously self-assemble into thrombogenic collagen-mimetic materials because of hydrophobic aromatic interactions provided by the special end-groups. These findings have important implications for the design of biofunctional CMPs.

Multivariate analysis applied to the study of spatial distributions found in drug-eluting stent coatings by confocal Raman microscopy.

Multivariate data analysis was applied to confocal Raman measurements on stents coated with the polymers and drug used in the CYPHER Sirolimus-eluting Coronary Stents. Partial least-squares (PLS) regression was used to establish three independent calibration curves for the coating constituents: sirolimus, poly(n-butyl methacrylate) [PBMA], and poly(ethylene-co-vinyl acetate) [PEVA]. The PLS calibrations were based on average spectra generated from each spatial location profiled. The PLS models were tested on six unknown stent samples to assess accuracy and precision. The wt % difference between PLS predictions and laboratory assay values for sirolimus was less than 1 wt % for the composite of the six unknowns, while the polymer models were estimated to be less than 0.5 wt % difference for the combined samples. The linearity and specificity of the three PLS models were also demonstrated with the three PLS models. In contrast to earlier univariate models, the PLS models achieved mass balance with better accuracy. This analysis was extended to evaluate the spatial distribution of the three constituents. Quantitative bitmap images of drug-eluting stent coatings are presented for the first time to assess the local distribution of components.

An investigation of adhesion in drug-eluting stent layers.

An atomic force microscopy (AFM) method was developed to quantify the adhesion forces between and cohesive forces within the layers of a drug-eluting stent (DES). Surface pairs representing both the individual components and the complete chemistry of each layer within the DES were prepared. As a model, the CYPHER Sirolimus-eluting Coronary Stent was studied. This DES consists of a stainless steel stent substrate, a parylene C primer layer, and a drug-eluting layer that contains poly(ethylene-co-vinyl acetate), poly(n-butyl methacrylate), and sirolimus (rapamycin). Coated AFM tips and two-dimensional substrates or coupons, which act as surrogates to the CYPHER Stent, were prepared and characterized. The force-displacement measurements were conducted to evaluate the adhesion between the middle parylene C layer and the 316L stainless steel substrate, the adhesion between the parylene C layer and the outer drug-eluting layer, and the cohesion between the three constituents of the drug-eluting layer. The average adhesion forces between the parylene C to drug layer varied from 88 to 167 nN, and the drug layer-to-drug layer interactions were between 194 and 486 nN within the model CYPHER Stent coating. All the adhesion forces measured were larger than those observed for gold-gold interactions, which yielded a pull of force of 19 nN (Zong et al., J Appl Phys 2006;100:104313-104323).

Determination of relative response factors of impurities in paclitaxel with high performance liquid chromatography equipped with ultraviolet and charged aerosol detectors.

A case study was conducted to determine the relative response factors (RRFs) of paclitaxel-related impurities by high performance liquid chromatography (HPLC) equipped with an ultraviolet (UV) detector and charged aerosol detector (CAD) in tandem. The peak response using CAD was independent of analyte structure in an isocratic analysis for this application. After a sample containing known and unknown impurities was analyzed with HPLC-UV-CAD, an empirical approach was developed to calculate the RRFs for all impurities. The RRFs of known impurities were also determined by linear calibration curves. For known impurities, the RRFs values determined with two approaches are comparable. The new approach is effective yet simpler to determine the RRFs for unknown impurities or degradation products since the need for obtaining authentic pure materials was eliminated.

Methodology for the preparation of 2-argininylbenzothiazole.

An efficient process to produce kilogram quantities of a key argininylbenzo[d]thiazole intermediate was developed for the preparation of the tryptase inhibitor RWJ-56423. A variety of activated arginine esters and benzo[d]thiazole nucleophiles were evaluated as coupling partners. Our work led to the selection and optimization of an argininyl imidazolide ester and benzothiazol-2-yl MgCl nucleophile. This paper focuses on the preparation, use, and stability of the benzothiazol-2-yl Grignard reagents.

A new procedure for preparation of carboxylic acid hydrazides.

The standard method for preparing carboxylic acid hydrazides is hydrazinolysis of esters in alcoholic solutions. However, when applied to alpha,beta-unsaturated esters, the main product typically is the pyrazolidinone resulting from an undesired Michael-type cyclization. Other alternative methodologies reported for direct preparation of hydrazides from acids are inefficient. We developed an efficient and general process, involving preforming activated esters and/or amides followed by reaction with hydrazine, for the preparation of hydrazides including those of alpha,beta-unsaturated acids. This process gives the desired hydrazides in excellent yield and purity under mild conditions.

Determination of the absolute configuration of a key tricyclic component of a novel vasopressin receptor antagonist by use of vibrational circular dichroism.

We present the results of a study using vibrational circular dichroism (VCD) for (+)-1, which furnished an unambiguous determination of its absolute configuration as S. The most abundant conformation of (+)-1 in CDCl(3) solution was also established.

Reductive Amination of Aldehydes and Ketones with Sodium Triacetoxyborohydride. Studies on Direct and Indirect Reductive Amination Procedures(1).

Sodium triacetoxyborohydride is presented as a general reducing agent for the reductive amination of aldehydes and ketones. Procedures for using this mild and selective reagent have been developed for a wide variety of substrates. The scope of the reaction includes aliphatic acyclic and cyclic ketones, aliphatic and aromatic aldehydes, and primary and secondary amines including a variety of weakly basic and nonbasic amines. Limitations include reactions with aromatic and unsaturated ketones and some sterically hindered ketones and amines. 1,2-Dichloroethane (DCE) is the preferred reaction solvent, but reactions can also be carried out in tetrahydrofuran (THF) and occasionally in acetonitrile. Acetic acid may be used as catalyst with ketone reactions, but it is generally not needed with aldehydes. The procedure is carried out effectively in the presence of acid sensitive functional groups such as acetals and ketals; it can also be carried out in the presence of reducible functional groups such as C-C multiple bonds and cyano and nitro groups. Reactions are generally faster in DCE than in THF, and in both solvents, reactions are faster in the presence of AcOH. In comparison with other reductive amination procedures such as NaBH(3)CN/MeOH, borane-pyridine, and catalytic hydrogenation, NaBH(OAc)(3) gave consistently higher yields and fewer side products. In the reductive amination of some aldehydes with primary amines where dialkylation is a problem we adopted a stepwise procedure involving imine formation in MeOH followed by reduction with NaBH(4).