Maria Victoria Silva Elipe

1H NMR structural analysis of human ghrelin and its six truncated analogs.

Human ghrelin, the first recognized natural ligand of growth hormone secretagogue growth hormone secretagogue receptors (GHS-Rs) (M. Kojima, H. Hosada, Y. Date, M. Nakazato, H. Matsuo, and K. Kangawa, Nature, 1999, Vol. 402, pp. 656-660), consists of 28 amino acids of which Ser3 is modified by n-octanoylation. This new peptide hormone has been implicated not only in regulation of the GH secretion but also in regulation of food intake. The discovery of ghrelin opens up more opportunities to study the relationship of ghrelin with metabolic diseases. Until now, only mass spectometry analysis has been reported on the structure of ghrelin. NMR analysis is a suitable way to study if any tertiary structure of unbound ghrelin is present in solution. NMR studies were carried out on human ghrelin and its five truncated analogs. The full-length ghrelin and its fragments exhibited random coil behavior in aqueous solution. Additional studies were carried out on the shortest active segment of human ghrelin, which consists of the first five amino acids of the ghrelin sequence (M. A. Bednarek, S. D. Feighner, S.-S. Pong, K. K. McKee, D. L. Hreniuk, M. V. Silva, V. A. Warrem, A. D. Howard, L. H. Y. Van der Ploeg, and J. V. Heck, Journal of Medical Chemistry, 2000, Vol. 43, pp. 4370-4376), to compare the spectral features with their counterparts in the full-length ghrelin. The NMR data showed behavior similar to ghrelin except for two additional nuclear Overhauser effects (NOEs) between the Phe4 NH and the protons of the beta-methylene of Ser3. CD on human ghrelin and its short active analog in water were indicative of random coil peptides. Molecular modeling based on NMR data was carried out to probe which structural features were similar to growth hormone-releasing peptide-6 (GHRP-6), a hexapeptide that binds to GHS-R releasing GH and stimulating food intake. Modeling suggested some similarities, but they were not of a nature to account for binding properties of these compounds.

Metabolism of MK-0524, a Prostaglandin D2 Receptor 1 Antagonist, in Microsomes and Hepatocytes from Preclinical Species and Humans

(3R)-4-(4-Chlorobenzyl)-7-fluoro-5-(methylsulfonyl)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl acetic acid (MK-0524) is a potent orally active human prostaglandin D2 receptor 1 antagonist that is currently under development for the prevention of niacin-induced flushing. The major in vitro and in vivo metabolite of MK-0524 is the acyl glucuronic acid conjugate of the parent compound, M2. To compare metabolism of MK-0524 across preclinical species and humans, studies were undertaken to determine the in vitro kinetic parameters (Km and Vmax) for the glucuronidation of MK-0524 in Sprague-Dawley rat, beagle dog, cynomolgus monkey, and human liver microsomes, human intestinal microsomes, and in recombinant human UDP glucuronosyltransferases (UGT). A comparison of Km values indicated that UGT1A9 has the potential to catalyze the glucuronidation of MK-0524 in the liver, whereas UGT1A3 and UGT2B7 have the potential to catalyze the glucuronidation in the intestine. MK-0524 also was subject to phase I oxidative metabolism; however, the rate was significantly lower than that of glucuronidation. The rate of phase I metabolism was ranked as follows: rat ∼ monkey > human intestine > dog > human liver with qualitatively similar metabolite profiles across species. In all the cases, the major metabolites were the monohydroxylated epimers (M1 and M4) and the keto-metabolite, M3. Use of inhibitory monoclonal antibodies and recombinant human cytochromes P450 suggested that CYP3A4 was the major isozyme involved in the oxidative metabolism of MK-0524, with a minor contribution from CYP2C9. The major metabolite in hepatocyte preparations was the acyl glucuronide, M2, with minor amounts of M1, M3, M4, and their corresponding glucuronides. Overall, the in vivo metabolism of MK-0524 is expected to proceed via glucuronidation, with minor contributions from oxidative pathways.

Monitoring chemical reactions by low-field benchtop NMR at 45 MHz: pros and cons.

Monitoring chemical reactions is the key to controlling chemical processes where NMR can provide support. High‐field NMR gives detailed structural information on chemical compounds and reactions; however, it is expensive and complex to operate. Conversely, low‐field NMR instruments are simple and relatively inexpensive alternatives. While low‐field NMR does not provide the detailed information as the high‐field instruments as a result of their smaller chemical shift dispersion and the complex secondary coupling, it remains of practical value as a process analytical technology (PAT) tool and is complimentary to other established methods, such as ReactIR and Raman spectroscopy. We have tested a picoSpin‐45 (currently under ThermoFisher Scientific) benchtop NMR instrument to monitor three types of reactions by 1D 1H NMR: a Fischer esterification, a Suzuki cross‐coupling, and the formation of an oxime. The Fischer esterification is a relatively simple reaction run at high concentration and served as proof of concept. The Suzuki coupling is an example of a more complex, commonly used reaction involving overlapping signals. Finally, the oxime formation involved a reaction in two phases that cannot be monitored by other PAT tools. Here, we discuss the pros and cons of monitoring these reactions at a low‐field of 45 MHz by 1D 1H NMR. Copyright

An Efficient, Regioselective Amination of 3,5-Disubstituted Pyridine N-Oxides Using Saccharin as an Ammonium Surrogate

A process for the regioselective amination of unsymmetrical 3,5-substituted pyridine N-oxides has been developed utilizing cheap, readily available saccharin as an ammonium surrogate. High conversions of the corresponding saccharin adducts have been achieved under mild reaction conditions. In situ deprotection under acidic conditions allows for a one-pot process to substituted aminopyridines. High regioselectivities were obtained from a variety of 3,5-disubstituted pyridine N-oxides.

Application of LC–NMR for the study of the volatile metabolite of MK-0869, a substance P receptor antagonist

LC-NMR was applied to identify the polar volatile metabolite of MK-0869. MK-0869, a morpholine-based compound containing a triazolone ring, is a very potent NK(1) receptor antagonist. Currently, it is in development as an anti-emesis agent in chemotherapy treatments. The primary metabolites of MK-0869, M1 and M2, are non-polar and lack the triazolone ring. Incubation of [14C]M1 with liver microsomes from male rats produced a very polar and volatile metabolite, M3. Analysis was not possible by LC-MS or by conventional NMR because of poor ionization, small molecular weight and volatility, leaving chemical derivatization and LC-NMR as alternative methods. Reduction of M3 with NaBH(4) resulted in a derivative that had the same retention time as p-fluorophenylethylene glycol on HPLC. A small aliquot of the solution containing M3 was passed through the LC of the LC-NMR system, which was connected on-line with a radioactivity detector. The simultaneous UV and radioactivity chromatograms thus identified the chromatographic UV peak that was associated with the metabolite. Analysis was carried out by stop-flow on another portion of this fraction. From the chemical derivatization and the analysis by LC-NMR, M3 is shown to be p-fluoro-alpha-hydroxyacetophenone. Further studies using LC-NMR showed that M3 could be generated from both M1 and M2 in NADPH-dependant reactions catalyzed by microsomes containing recombinant human CYP2C19, CYP1A2 or CYP3A4.

Application of 19 F time‐domain NMR to measure content in fluorine‐containing drug products

It is necessary to show that the active content in the dosage form of drugs is within a certain narrow range of the label claim. In case of fluorinated drugs, the active content can be measured by high field solid state NMR because the excipients lack fluorine. To make NMR reachable to any laboratory, simple to use, and at a low cost, measurement of 19 F nucleus using a 23 MHz (for 1H) low field benchtop time‐domain (TD) NMR was investigated. Three fluorinated drug products, cinacalcet, lansoprazole, and ciprofloxacin, were chosen for this study. The doses for these drug products range from 15 to 500 mg. The average drug content measured using 19 F TD‐NMR compares well with the reported label claims for the three drugs tested. 19 F TD‐NMR is a simple and non‐destructive technique to measure drug content in tablets. In addition, the accessibility and simplicity of the technique makes it an excellent process analytical technology tool for development and manufacturing in the pharmaceutical industry. Copyright

Performance of new 400-MHz HTS power-driven magnet NMR technology on typical pharmaceutical API, cinacalcet HCl

After years towards higher field strength magnets, nuclear magnetic resonance (NMR) technology in commercial instruments in the past decade has expanded at low and high magnetic fields to take advantage of new opportunities. At lower field strengths, permanent magnets are well established, whereas for midrange and high field, developments utilize superconducting magnets cooled with cryogenic liquids. Recently, the desire to locate NMR spectrometers in nontypical NMR laboratories has created interest in the development of cryogen‐free magnets. These magnets require no cryogenic maintenance, eliminating routine filling and large cryogen dewars in the facility. Risks of spontaneous quenches and safety concerns when working with cryogenic liquids are eliminated. The highest field commercially available cryogen‐free NMR magnet previously reported was at 4.7 T in 2013. Here we tested a prototype cryogen‐free 9.4‐T power‐driven high‐temperature‐superconducting (HTS) magnet mated to commercial NMR spectrometer electronics. We chose cinacalcet HCl, a typical active pharmaceutical ingredient, to evaluate its performance towards structure elucidation. Satisfactory standard 1D and 2D homonuclear and heteronuclear NMR results were obtained and compared with those from a standard 9.4‐T cryogenically cooled superconducting NMR instrument. The results were similar between both systems with minor differences. Further comparison with different shims and probes in the HTS magnet system confirmed that the magnet homogeneity profile could be matched with commercially available NMR equipment for optimal results. We conclude that HTS magnet technology works well providing results comparable with those of standard instruments, leading us to investigate additional applications for this magnet technology outside a traditional NMR facility.

Application of hyphenated NMR in industry

This chapter reviews the applications of hyphenated NMR in industry from 2010 to middle of 2015. The chapter is divided into sections focused on applications on LC-NMR, NMR hyphenated to other analytical techniques such as CD, SEC, LCCC, SPE, and SPE-MS, and applications of hyphenated NMR to other non-analytical techniques such as reactor vessels to monitor reactions by NMR with high field and low field NMR instruments.

A Multidisciplinary Investigation to Determine the Structure and Source of Dimeric Impurities in AMG 517 Drug Substance

In the initial scale-up batches of the experimental drug substance AMG 517, a pair of unexpected impurities was observed by HPLC. Analysis of data from initial LC-MS experiments indicated the presence of two dimer-like molecules. One impurity had an additional sulfur atom incorporated into its structure relative to the other impurity. Isolation of the impurities was performed, and further structural elucidation experiments were conducted with high-resolution LC-MS and 2D NMR. The dimeric structures were confirmed, with one of the impurities having an unexpected C-S-C linkage. Based on the synthetic route of AMG 517, it was unlikely that these impurities were generated during the last two steps of the process. Stress studies on the enriched impurities were carried out to further confirm the existence of the C-S-C linkage in the benzothiazole portion of AMG 517. Further investigation revealed that these two dimeric impurities originated from existing impurities in the AMG 517 starting material, N-acetyl benzothiazole. The characterization of these two dimeric impurities allowed for better quality control of new batches of the N-acetyl benzothiazole starting material. As a result, subsequent batches of AMG 517 contained no reportable levels of these two impurities

Comparative disposition and metabolism of paraherquamide in sheep, gerbils and dogs

The disposition and metabolism of paraherquamide (PHQ), a potent and broad-spectrum anthelminthic, were examined in sheep, dogs, and gerbils. The metabolism of PHQ in these species was extensive and marked by significant species differences both in vitro and in vivo. In sheep and gerbils, PHQ metabolism occurs mainly at the pyrrolidine moiety, generating several metabolites that, for the most part, retained nematodicidal activity in vitro. In dogs, the dioxepene group was also extensively metabolized, ultimately resulting in formation of a catechol and loss of pharmacological activity. After oral administration of [3H]PHQ to intact sheep, gerbils, and dogs, the majority of the administered radioactivity was recovered in feces. Intact PHQ accounted for 0% (dogs) to ∼30% (sheep and gerbils) of drug-related material in feces. A detailed investigation of the composition of the intestinal content of sheep indicated that a significant amount of the dose was still present in the rumen 24 h after dose and that PHQ underwent significant dehydration in the cecum. The oral pharmacokinetic parameters of PHQ in sheep and dogs suggest that its absorption is rapid in both species but that its apparent elimination rate is significantly higher in the dog (t1/2 ∼ 1.5 h) than it is in sheep (t1/2 ∼ 8.5 h). The short elimination half-life and the absence of PHQ or other active components in the dog gastrointestinal tract provide a potential explanation of the lack of efficacy of PHQ in this species.