Advances in the Specificity of Mass Spectrometry and Nuclear Magnetic Resonance Spectroscopy Based Structural Characterisation Methods for Synthetic Oligonucleotides.

Identity testing is a critical part in the development of a therapeutic synthetic oligonucleotide. Tandem Mass Spectrometry (MS/MS) is commonly used for the analysis of oligonucleotides to obtain structural and sequence information, however there are challenges resulting from chemical modifications introduced to improve their pharmacokinetics and stability. For these structurally complex oligonucleotides, Nuclear Magnetic Resonance (NMR) Spectroscopy has found limited use for characterisation and identity testing, as only partial NMR resonance assignment for oligonucleotides is achieved without isotopic labelling methodologies. Regardless of the choice of method used for oligonucleotide analysis, the specificity is of critical importance. In this work, in-source dissociation mass spectrometry and proton (1H) and carbon (13C) NMR at high temperature were used to analyse danvatirsen, a 16 nucleotide phosphorothioate antisense oligonucleotide, and its closely related switch sequences. Both approaches have shown specificity to distinguish danvatirsen from these similar sequences.

Monitoring apoptosis in intact cells by high-resolution magic angle spinning 1 H NMR spectroscopy.

Apoptosis maintains an equilibrium between cell proliferation and cell death. Many diseases, including cancer, develop because of defects in apoptosis. A known metabolic marker of apoptosis is a notable increase in 1 H NMR-observable resonances associated with lipids stored in lipid droplets. However, standard one-dimensional NMR experiments allow the quantification of lipid concentration only, without providing information about physical characteristics such as the size of lipid droplets, viscosity of the cytosol, or cytoskeletal rigidity. This additional information can improve monitoring of apoptosis-based cancer treatments in intact cells and provide us with mechanistic insight into why these changes occur. In this paper, we use high-resolution magic angle spinning (HRMAS) 1 H NMR spectroscopy to monitor lipid concentrations and apparent diffusion coefficients of mobile lipid in intact cells treated with the apoptotic agents cisplatin or etoposide. We also use solution-state NMR spectroscopy to study changes in lipid profiles of organic solvent cell extracts. Both NMR techniques show an increase in the concentration of lipids but the relative changes are 10 times larger by HRMAS 1 H NMR spectroscopy. Moreover, the apparent diffusion rates of lipids in apoptotic cells measured by HRMAS 1 H NMR spectroscopy decrease significantly as compared with control cells. Slower diffusion rates of mobile lipids in apoptotic cells correlate well with the formation of larger lipid droplets as observed by microscopy. We also compared the mean lipid droplet displacement values calculated from the two methods. Both methods showed shorter displacements of lipid droplets in apoptotic cells. Our results demonstrate that the NMR-based diffusion experiments on intact cells discriminate between control and apoptotic cells. Apparent diffusion measurements in conjunction with 1 H NMR spectroscopy-derived lipid signals provide a novel means of following apoptosis in intact cells. This method could have potential application in enhancing drug discovery by monitoring drug treatments in vitro, particularly for agents that cause portioning of lipids such as apoptosis.

NMR and Thermal Studies for the Characterization of Mass Transport and Phase Separation in Paracetamol/Copovidone Hot-Melt Extrusion Formulations.

The formulation of drug/polymer amorphous solid dispersions (ASDs) is one of the most successful strategies for improving the oral bioavailability of poorly soluble active pharmaceutical ingredients (APIs). Hot-melt extrusion (HME) is one method for preparing ASDs that is growing in importance in the pharmaceutical industry, but there are still substantial gaps in our understanding regarding the dynamics of drug dissolution and dispersion in viscous polymers and the physical stability of the final formulations. Furthermore, computational models have been built to predict optimal processing conditions, but they are limited by the lack of experimental data for key mass transport parameters, such as the diffusion coefficient. The work presented here reports direct measurements of API diffusion in pharmaceutical polymer melts, using high-temperature pulsed-field gradient NMR. The diffusion coefficient of a model drug/polymer system (paracetamol/copovidone) was determined for different drug loadings and at temperatures relevant to the HME process. The mechanisms of the diffusion process are also explored with the Stokes-Einstein and Arrhenius models. The results show that diffusivity is linked exponentially to temperature. Furthermore, this study includes rheological characterization, differential scanning calorimetry (DSC), and 1H ssNMR T1 and T1ρ measurements to give additional insights into the physical state, phase separation, and API/polymer interactions in paracetamol/copovidone ASD formulations.

Online monitoring of a photocatalytic reaction by real-time high resolution FlowNMR spectroscopy.

We demonstrate how FlowNMR spectroscopy can readily be applied to investigate photochemical reactions that require sustained input of light and air to yield mechanistic insight under realistic conditions. The Eosin Y mediated photo-oxidation of N-allylbenzylamine is shown to produce imines as primary reaction products from which undesired aldehydes form after longer reaction times. Facile variation of reaction conditions during the reaction in flow allows for probe experiments that give information about the mode of action of the photocatalyst.

Asymmetric substitutions of 2-lithiated N-boc-piperidine and N-Boc-azepine by dynamic resolution.

Proton abstraction of N-tert-butoxycarbonyl-piperidine (N-Boc-piperidine) with sBuLi and TMEDA provides a racemic organolithium that can be resolved using a chiral ligand. The enantiomeric organolithiums can interconvert so that a dynamic resolution occurs. Two mechanisms for promoting enantioselectivity in the products are possible. Slow addition of an electrophile such as trimethylsilyl chloride allows dynamic resolution under kinetic control (DKR). This process occurs with high enantioselectivity and is successful by catalysis with substoichiometric chiral ligand (catalytic dynamic kinetic resolution). Alternatively, the two enantiomers of this organolithium can be resolved under thermodynamic control with good enantioselectivity (dynamic thermodynamic resolution, DTR). The best ligands found are based on chiral diamino-alkoxides. Using DTR, a variety of electrophiles can be used to provide an asymmetric synthesis of enantiomerically enriched 2-substituted piperidines, including (after Boc deprotection) the alkaloid (+)-beta-conhydrine. The chemistry was extended, albeit with lower yields, to the corresponding 2-substituted seven-membered azepine ring derivatives.

Dynamic resolution of N-Boc-2-lithiopiperidine.

Dynamic thermodynamic resolution of N-Boc-2-lithiopiperidine is possible using a chiral ligand; the two enantiomers of this organolithium can be resolved with selectivities of up to 85 : 15 from a selection of 26 chiral diamino-alkoxide ligands screened.

Asymmetric lithiation-substitution of amines involving rearrangement of borates.

Asymmetric lithiation of substituted benzylamines, N-Boc-pyrrolidine, or N-Boc-indoline using Beak's methodology was followed by electrophilic quench with trialkylboranes. The resulting borate intermediates rearrange with concomitant C-N bond breakage to give, after oxidation, chiral secondary alcohols with high enantioselectivity.

Implications of a congenitally abnormal valve: a study of 1025 consecutively excised aortic valves.

BACKGROUND: An increasing proportion of patients with congenitally abnormal aortic valves (AV) present for AV replacement. AIMS: To review morphological changes in a large contemporary patient population undergoing AV replacement. METHODS: A detailed review was conducted for all 1025 patients who underwent AV replacement from 2002 to 2005, including the clinical indication for surgery, the type of native AV disease, the pathological changes observed in each valve and the need for related surgery. RESULTS: Tricuspid (TAV), bicuspid (BAV) and unicuspid (UAV) aortic valves were observed in 64.5%, 31.9% and 3.0% of all patients respectively. A decreased number of cusps was associated with increasing predilection for male gender (83.9%, 73.4%, 59.2% for UAV, BAV, TAV respectively), a younger patient age at surgery (41.6 (14.3), 61.3 (12.8), 67.5 (12.9) years), and an increased occurrence of pathological changes in the cusps, including calcification of both the cusp and the base, ossification and ulceration. UAV and BAV were also associated with increasing replacement of the ascending aorta due to dilatation and aneurysm formation (54.8, 38.8%, 16.6%). The incidence of infective endocarditis and rheumatic heart disease was 3.8% and 11.2% of all excised valves respectively. CONCLUSION: UAV and BAV were increasingly likely to affect men, fail at an earlier age, and show an increasing incidence of pathological changes in the cusps and ascending aorta than TAV. These results suggest that TAV, BAV and UAV may represent a phenotypic continuum of a similar disease process.

Dynamic kinetic and kinetic resolution of N-Boc-2-lithiopiperidine.

Asymmetric substitution of 2-lithiopiperidines can be achieved by dynamic resolution; the organolithium is formed as a racemic mixture by proton abstraction (or tin-lithium exchange) and is resolved in the presence of a chiral ligand.

Hemodilution during cardiopulmonary bypass is an independent risk factor for acute renal failure in adult cardiac surgery.

BACKGROUND: This observational study sought to determine whether the degree of hemodilution during cardiopulmonary bypass is independently related to perioperative acute renal failure necessitating dialysis support. METHODS: Data were prospectively collected on consecutive patients undergoing cardiac operations with cardiopulmonary bypass from 1999 to 2003 at a tertiary care hospital. The independent relationship was assessed between the degree of hemodilution during cardiopulmonary bypass, as measured by nadir hematocrit concentration, and acute renal failure necessitating dialysis support. Multivariate logistic regression was used to control for variables known to be associated with perioperative renal failure and anemia. RESULTS: Of the 9080 patients included in the analysis, 1.5% (n = 134) had acute renal failure necessitating dialysis support. There was an independent, nonlinear relationship between nadir hematocrit concentration during cardiopulmonary bypass and acute renal failure necessitating dialysis support. Moderate hemodilution (nadir hematocrit concentration, 21%-25%) was associated with the lowest risk of acute renal failure necessitating dialysis support; the risk increased as nadir hematocrit concentration deviated from this range in either direction (P = .005). Compared with moderate hemodilution, the adjusted odds ratio for acute renal failure necessitating dialysis support with severe hemodilution (nadir hematocrit concentration <21%) was 2.34 (95% confidence interval, 1.47-3.71), and for mild hemodilution (nadir hematocrit concentration >25%) it was 1.88 (95% confidence interval, 1.02-3.46). CONCLUSIONS: Given that there is an independent association between the degree of hemodilution during cardiopulmonary bypass and perioperative acute renal failure necessitating dialysis support, patient outcomes may be improved if the nadir hematocrit concentration during cardiopulmonary bypass is kept within the identified optimal range. Randomized clinical trials, however, are needed to determine whether this is a cause-effect relationship or simply an association.

Long-term results of bioprosthetic mitral valve replacement: the pericardial perspective.

Pericardial valve bioprostheses were introduced in early 1970s and were widely used in the 1980s. The longterm results with the Ionescu-Shiley valve, the first commercially available pericardial valve, were disappointing because of high rate cusp tears during the first decade after implantation. The enthusiasm for this type of bioprosthetic valve was further hampered by the premature failure of the Hancock pericardial valve. The long-term results of aortic valve replacement with the Carpentier-Edwards pericardial valve, which was introduced in 1981, indicated that that valve was durable and the issue of cusp tears had been resolved by an appropriate design. This knowledge prompted surgeons to revisit the merits of pericardial valves for mitral valve replacement and several other pericardial valves are now commercially available. The largest data on long-term results are with the Carpentier-Edwards pericardial mitral valve. The reported freedom from structure valve failure ranged from 69% to 85% at 10 years in patient population with mean age of 60 to 70 years. Young age is a major determinant of valve failure, which is largely due to calcification. There are also long-term data, albeit more limited on the Sorin Pericarbon and Mitroflow valves used for mitral valve replacement. This paper review the published experience with various pericardial bioprosthetic valves used for mitral valve replacement during the past 3 decades.

Reoperations on the aortic valve combined with replacement of the ascending aorta.

OBJECTIVE: To review the experience with reoperations on the aortic valve combined with replacement of the ascending aorta. PATIENTS AND METHODS: From 1991 to 2000, 237 patients underwent reoperations on the aortic valve combined with replacement of the ascending aorta. The study consisted of 188 men and 49 women, with a mean age of 51 years. The operation was urgent or emergent in 44% of cases. Many patients (42%) were in New York Heart Association Class IV, and 24 had active infective endocarditis. The ascending aorta was replaced previously in 46 patients, while the remaining patients had aneurismal dilation. An aortic valve sparing operation was performed in 14 patients and aortic valve replacement in 223. The ascending aorta was replaced in all patients as follows: as a composite graft in 166 and supracoronary in 71. Mechanical valves were used in 145 (61%) patients. RESULTS: The operative mortality was 9%. Postoperative complications were common and 30% of patients suffered an adverse event (death or complication). No independent predictor of operative mortality could be identified but urgent/emergent surgery, advanced functional class, infective endocarditis, coronary artery disease, and replacement of the transverse aortic arch were associated with higher operative mortality by chi-square analysis. The survival at 5 years was 74%+/-4% for patients who had composite replacement of the aortic valve and ascending aorta. CONCLUSIONS: Reoperations on the aortic valve combined with replacement of the ascending aorta can be performed with acceptable operative risk and good mid-term survival.

Dilation of the sinotubular junction causes aortic insufficiency after aortic valve replacement with the Toronto SPV bioprosthesis.

OBJECTIVE: This study was undertaken to examine the causes of late aortic insufficiency in patients who had aortic valve replacement with the Toronto SPV bioprosthesis (St Jude Medical, Inc, St Paul, Minn). METHODS: From 1991 to 1996, 174 patients with a mean age of 63 +/- 11 years underwent aortic valve replacement with the Toronto SPV bioprosthesis and were evaluated annually by Doppler echocardiographic studies to assess valve function. The diameters of the aortic root were retrospectively measured in all patients who had aortic insufficiency and also in a random sample of 23 patients without aortic insufficiency. The mean follow-up was 5.8 years (range 4 to 9 years). RESULTS: Aortic insufficiency greater than 1+ developed in 19 patients. The diameter of the sinotubular junction increased in these patients and did not change in those without aortic insufficiency. The ratio between the diameter of the sinotubular junction and the size of the Toronto SPV bioprosthesis increased in patients who had aortic insufficiency and did not change in those without aortic insufficiency. Both 2-way analysis of covariance and analysis by a mixed linear model demonstrated a significant difference in slopes between the patients with aortic insufficiency greater than 1+ and in those without insufficiency for the ratio of the diameter of the sinotubular junction/diameter of the Toronto SPV relationships over time (aortic insufficiency. Year; P <.001). Structural valve deterioration was observed in 5 valves, and in 4 of them the sinotubular junction of the aortic root had dilated. The freedom from structural valve deterioration was 99% +/- 1% for patients without aortic insufficiency and 82% +/- 12% for those with aortic insufficiency of more than 1+ at 8 years (P =.004). One patient had moderate aortic insufficiency without structural valve deterioration and dilation of the sinotubular junction. CONCLUSIONS: Dilation of the sinotubular junction causes aortic insufficiency after aortic valve replacement with the Toronto SPV bioprosthesis and increases the risk of structural valve deterioration. Banding the sinotubular junction may prevent dilation and enhance the durability of this valve.

Aortic valve-sparing operations for aortic root aneurysm.

The object of this report is to review the clinical outcomes of aortic valve-sparing operations in patients with aortic root aneurysm. From May 1988 to June 2000, 120 patients with aortic root aneurysm with or without aortic insufficiency underwent aortic valve-sparing operations. There were 83 men and 37 women whose mean age was 46 years (range 16 to 72 years). Forty-eight patients had the Marfan syndrome, and 22 had either acute or chronic type A aortic dissections. Reconstruction of the aortic root was performed using the technique of reimplanation of the aortic valve in 64 patients and the remodeling of the aortic root in 56. The mean follow-up was 35 months (range 2 to 145 months), and it was complete. Aortic valve function was assessed by echocardiography. There were 2 operative and 5 late deaths. The 10-year survival for patients with aortic root aneurysm was 88% +/- 4%. Two patients have required aortic root replacement: one on the first postoperative day because of severe aortic insufficiency and one paraplegic patient 12 years after surgery because of infective endocarditis with aortic root abscess. Both patients survived reoperation. The 10-year freedom from aortic root reoperation was 99% +/- 1%. Three patients suffered transient ischemic attacks. The 10-year freedom from thromboembolic events was 89% +/- 5%. Seven patients had moderate aortic insufficiency at the latest echocardiographic study. The 10-year freedom from severe or moderate aortic insufficiency was 83% +/- 8%. Aortic valve-sparing operations have provided excellent clinical outcomes and few valve-related complications. The function of the reconstructed aortic root remains unchanged in most patients during the first decade of follow-up.

Results of aortic valve-sparing operations.

OBJECTIVE: To review the late results of valve-sparing operations in patients with aortic root aneurysm and in those with ascending aortic aneurysm and aortic insufficiency. METHODS: From May 1988 to June 2000, 120 patients with aortic root aneurysm and 68 with ascending aortic aneurysm and aortic insufficiency underwent aortic valve-sparing operations. Patients with aortic root aneurysm were younger, were predominantly male, and had less severe aortic insufficiency than patients with ascending aortic aneurysm, who were older and often had aneurysm of the transverse arch. Forty-eight patients with aortic root aneurysm had the Marfan syndrome. The prevalence of aortic dissection was similar in both groups. Reconstruction of the aortic root was performed by reimplanation of the aortic valve in 64 patients and by remodeling of the aortic root in 56. Patients with ascending aortic aneurysm and aortic insufficiency were treated by replacement of the ascending aorta with reduction in the diameter of the sinotubular junction. Approximately two thirds of the latter patients also required replacement of the transverse aortic arch. The mean follow-up was 35 +/- 31 months for patients with aortic root aneurysm and 26 +/- 23 months for those with ascending aortic aneurysm. RESULTS: There were 2 operative and 5 late deaths in patients with aortic root aneurysm and 1 operative and 9 late deaths in patients with ascending aortic aneurysm. The 5-year survival for patients with aortic root aneurysm was 88% +/- 4% and for patients with ascending aortic aneurysm, 68% +/- 12% (P =.01). Severe aortic insufficiency developed in 2 patients, and they required aortic valve reoperation. The 5-year freedom from aortic valve reoperation was 99% +/- 1% for patients with aortic root aneurysm and 97% +/- 4% for those with ascending aortic aneurysm. Seven patients had moderate aortic insufficiency at the latest echocardiographic study. The 5-year freedom from severe or moderate aortic insufficiency was 90% +/- 4% in patients who had aortic root aneurysm and 98% +/- 2% in those who had ascending aortic aneurysm. CONCLUSIONS: Aortic valve-sparing operations have provided excellent clinical outcomes and few valve-related complications. The function of the reconstructed aortic root remains unchanged in most patients during the first 5 years of follow-up.

Redo aortic root replacement: experience with 31 patients.

BACKGROUND: Aortic root re-replacement is being performed with increased frequency. Limited information is available regarding the surgical approaches and clinical outcomes of this reoperation. METHODS: Between May 1980 and May 1999, 31 patients (mean age, 45 +/- 15 years) underwent redo composite replacement of the aortic valve and ascending aorta. Indications for reoperation were prosthetic valve endocarditis in 12 patients (39%), failed biological valve in 17 (55%), and false aneurysm in 2 (6%). At reoperation, mechanical valves were implanted in 24 patients and biologic valves in 7. All patients with endocarditis had annular abscess and required reconstruction of the left ventricular outflow tract before implantation of a new valved conduit. Mechanical valves were used in 24 patients, aortic homograft in 4, and bioprosthetic valves in 3. The coronary button technique was used to reimplant the coronary arteries whenever possible. Extension of one or both coronary arteries with a short segment of saphenous vein or a synthetic graft was used in 16 patients (52%). The aortic arch was replaced in 7 patients (23%). RESULTS: There was one operative death (3%) because of rupture of an abdominal aortic aneurysm. The mean follow-up was 47 +/- 46 months and was 100% complete. There were five late deaths (16%), three of which were cardiac related. The actuarial survival was 71% +/- 12% at 5 years. Three patients experienced recurrent prosthetic valve endocarditis 4 months to 8 years after operation. The 8-year freedom from endocarditis for patients operated on for endocarditis was 82% +/- 11% compared with 100% for those operated on for other reasons (p = 0.1). At the last follow-up, 21 of 25 survivors (84%) were in New York Heart Association functional classes I or II, and 4 were in class III. CONCLUSIONS: Redo aortic root replacement can be performed with good early and late results. Patients operated on for prosthetic root endocarditis may have an increased risk of recurrent late endocarditis.

Fifteen-year experience with the mitral Carpentier-Edwards PERIMOUNT pericardial bioprosthesis.

BACKGROUND: This multicenter study concerning the mitral PERIMOUNT valve previously reported clinical results at 12 years; this report updates the performance to 15 years postoperatively. METHODS: The 435 patients (mean age 60.7+/-11.6 years; 41.1% male) underwent implantation with the PERIMOUNT valve between 1984 and 1989 at seven institutions. Follow-up was complete for 96.1% of the cohort. The mean follow-up was 8.1+/-4.4 years (range 0 to 15.4 years) for a total of 3492 patient-years. RESULTS: There were 34 (7.8%) operative deaths, one (0.2%) valve related. The late mortality rate was 5.3%/patient-year (2.2%/patient-year valve related). At 14 years, the overall actuarial survival rate was 37.1%+/-3.3% (63.1%+/-4.4% valve related). Actuarial freedom from complications at 14 years was as follows: thromboembolism, 83.8%+/-3.2% (1.1%/patient-year); hemorrhage, 86.6%+/-3.2% (1.1%/patient-year); and explant due to structural valve deterioration (SVD), 68.8%+/-4.7%. Actual freedom from explant due to SVD was 83.4%+/-2.3%. Rates of structural failure decreased with increasing age at implant. CONCLUSIONS: The Carpentier-Edwards PERIMOUNT Pericardial Bioprosthesis is a reliable choice for a tissue valve in the mitral position, especially in patients more than 60 years of age.

Clinical outcomes after aortic valve replacement with the Toronto stentless porcine valve.

BACKGROUND: Hemodynamic benefits of the Toronto stentless porcine valve have been documented. Clinical well-being and freedom from major valve-related events have been less well defined. METHODS: A total of 447 patients were prospectively followed for up to 8 years (1,745.2 valve years total, 3.9 valve years/patient). The patient demographics included 66% men, mean age 65 years, New York Heart Association functional class III-IV 55%, concomitant coronary artery bypass grafting 41%. RESULTS: We found that 83.7% of patients were in New York Heart Association functional class I and 80.8% had 0 to 1+ aortic insufficiency. Mean gradient at 6 years (n = 75) was 4.4 mm Hg and mean effective orifice area (EOA) 2.4 cm2. Late adverse event rates per patient per year were: embolism 1.0%, endocarditis 0.4%, thrombosis 0%, structural deterioration 0.2%, explant 0.3%, and valve-related death 0.6%. Freedom from valve-related death at 6 years was 95.8%; from cardiac death 96.3%. Freedom from endocarditis was 98.4%, from embolism 93.9%, from structural deterioration 97.4%, and freedom from explant 98.1%. For patients older than 60 years, freedom from structural deterioration was 100%. CONCLUSIONS: These results confirm satisfactory clinical outcomes after aortic valve replacement with the Toronto stentless porcine valve, with a low incidence of valve-related adverse events as long as 96 months after valve replacement.

The efficacy of the Cox/maze procedure combined with mitral valve surgery: a matched control study.

OBJECTIVE: To evaluate the results of the maze procedure combined with mitral valve (MV) surgery in patients with chronic atrial fibrillation (AF). METHODS: From 1994--1999, 47 patients with chronic AF underwent the maze procedure combined with MV surgery (maze group). They were compared to 47 patients matched for age, sex, left ventricular function and type of MV surgery (non-maze group). The maze group had less severe symptoms but larger left atrium, and AF of longer duration than the non-maze group. One surgeon performed all operations in both groups of patients. RESULTS: There were two early deaths in the maze group (4.5%) and one (2.2%) in the non-maze group. The duration of cardiopulmonary bypass (P=0.0001) and aortic crossclamping (P=0.0001) were greater in the maze group. Mean follow-up was 26+/-3 months in the maze group and 32+/-4 months in the non-maze group, and was 100% complete. Three-year survival was 96+/-3% for the maze group compared to 85+/-7% for the non-maze group (P=0.16). At the latest follow-up, 75% of the maze patients were in sinus rhythm compared to 36% of the non-maze patients (P=0.0004); 38% of the maze group were on coumadin postoperatively, compared to 69% in the non-maze group (P=0.003); and patients in the maze group were on fewer antiarrhythmic medications (P=0.0002). Three-year freedom from thromboembolic complications was 100% for the maze group compared to 83+/-7% for the non-maze group (P=0.03). CONCLUSIONS: In this retrospective study the maze procedure did not seem to increase operative mortality of MV surgery, was effective in eliminating atrial fibrillation, and reduced the risk of thromboembolic complications and the need for long-term anticoagulation after mitral valve repair or replacement with a bioprosthesis.