Molecular evolution of a cytochrome P450 for the synthesis of potential antidepressant (2R,6R)-hydroxynorketamine.

Saturation mutagenesis at seven first-sphere residues of the cytochrome P450 monooxygenase 154E1 (CYP154E1) from Thermobifida fusca YX was applied to construct a variant with only three substitutions that enabled the effective two-step synthesis of the potential antidepressant (2R,6R)-hydroxynorketamine. A recombinant E. coli whole-cell system was essential for GC/MS based medium-throughput screening and at the same time facilitated the oxidation of the substrate (R)-ketamine at a higher scale for product isolation and subsequent NMR analysis.

Performance and toxicity of different absorption enhancers used in the preparation of Poloxamer thermosensitive in situ gels for ketamine nasal administration.

The purpose of this study was to investigate the nasal absorption rate and nasal mucosal toxicity of thermosensitive ketamine in situ gels containing various absorption enhancers. The optimal composition ratio for the gel matrix was determined to be 17.2% Poloxamer 407 and 2% Poloxamer 188, as this combination resulted in solutions with a gelation point within the range found in the nasal cavity. Ketamine gels containing the tested enhancers, namely, ethylenediaminetetraacetic acid disodium salt, hydroxypropyl-ß-cyclodextrin, propylene glycol, or Tween-80, were compared with enhancer-free counterparts to determine the absorption of the drug, in vivo by measuring its plasma levels in rats and in vitro using a Franz diffusion cell. Moreover, the toxicity of each gel type was assessed by microscopic observation of the morphology of rat nasal mucosa as well as by determining the mobility of the mucosal cilia using an established toad model. The results showed that gels containing hydroxypropyl-ß-cyclodextrin could promote the absorption of ketamine without added toxicity compared to enhancer-free gels. Thus, we consider hydroxypropyl-ß-cyclodextrin as the most promising absorption enhancer for the nasal administration of ketamine using in situ gels.

Sustained-release ketamine-loaded nanoparticles fabricated by sequential nanoprecipitation.

Ketamine in sub-anaesthetic doses is an analgesic adjuvant with a morphine-sparing effect. Co-administration of a strong opioid with an analgesic adjuvant such as ketamine is a potential treatment option, especially for patients with cancer-related pain. A limitation of ketamine is its short in vivo elimination half-life. Hence, our aim was to develop biocompatible and biodegradable ketamine-loaded poly(ethylene glycol) (PEG)-block-poly(lactic-co-glycolic acid) (PLGA) nanoparticles for sustained release. Ketamine-encapsulated single polymer PEG-PLGA nanoparticles and double polymer PEG-PLGA/shellac (SH) nanoparticles with a high drug loading of 41.8% (drug weight/the total weight of drug-loaded nanoparticles) were prepared using a new sequential nanoprecipitation method. These drug-loaded nanoparticles exhibited a sustained-release profile for up to 21 days in vitro and for more than 5 days after intravenous injection in mice. Our study demonstrates that high drug loading and a sustained release profile can be achieved with ketamine-loaded PEG-PLGA nanoparticles prepared using this new nanoprecipitation method.

Synthesis and identification of deschloroketamine metabolites in rats' urine and a quantification method for deschloroketamine and metabolites in rats' serum and brain tissue using liquid chromatography tandem mass spectrometry.

Deschloroketamine (2-(methylamino)-2-phenyl-cyclohexanone) is a ketamine analog belonging to a group of dissociative anesthetics, which have been distributed within the illicit market since 2015. However, it was also being sold as 'ketamine' misleading people to believe that they were getting genuine ketamine. Dissociative anesthetics have also come to the attention of the psychiatric field due to their potential properties in the treatment of depression. At present, there is a dearth of information on deschloroketamine related to its metabolism, biodistribution, and its mechanism of action. We have therefore carried out a metabolomics study for deschloroketamine via non-targeted screening of urine samples employing liquid chromatography combined with high-resolution mass spectrometry. We developed and validated a multiple reaction monitoring method using a triple quadrupole instrument to track metabolites of deschloroketamine. Furthermore, significant metabolites of deschloroketamine, (trans-dihydrodeschloroketamine, cis- and trans-dihydronordeschloroketamine, and nordeschloroketamine), were synthesized in-house. The prepared standards were utilized in the developed multiple reaction monitoring method. The quantification method for serum samples provided intra-day accuracy ranging from 86% to 112% with precision of 3% on average. The concentrations of cis/trans-dihydronordeschloroketamines and trans-dihydrodeschloroketamine were lower than 10 ng/mL, nordeschloroketamine and deschloroketamine ranged from 0.5 to 860 ng/mL in real samples. The quantification method for brain tissue provided intra-day accuracy ranging from 80% to 125% with precision of 7% on average. The concentrations of cis/trans-dihydronordeschloroketamines and trans-dihydrodeschloroketamine ranged from 0.5 to 70 ng/g, nordeschloroketamine and deschloroketamine varied from 0.5 to 4700 ng/g in real samples.

Synthesis of carbon-14 labeled ketamine and norketamine.

Ketamine is a well-known general anesthetic that inhibits cerebral NMDA receptors. Norketamine is a major circulating metabolite of this drug. A nasal spray formulation of esketamine, the S enantiomer of ketamine, is under development for the management of treatment-resistant depression. To assess the pharmacokinetic properties, C-14 labeled ketamine and norketamine were prepared separately from commercially available [14 C]CuCN through a five-step sequence with the C-14 label at the quaternary carbon of the cyclohexyl ring. Chiral resolution of [14 C]ketamine and chiral column separation of [14 C]norketamine resolved/separated the (S)-enantiomers from (R)-enantiomers.

Simple Enantioselective Syntheses of (2R,6R)-Hydroxynorketamine and Related Potential Rapid-Onset Antidepressants.

A novel strategy for accessing cyclic α-amino ketones enantioselectively has opened a simple synthetic route to the antidepressant (2R,6R)-hydroxynorketamine and numerous analogues. Mechanistically guided catalyst selection was essential in an initial olefin epoxidation step. In a second crucial step, the epoxide was subjected to a novel O → N displacement that occurred with retention of configuration through the use of Al- or Ti-based azides, which promote epoxide activation and internal cis delivery of N3 to carbon.

Direct asymmetric amination of α-branched cyclic ketones catalyzed by a chiral phosphoric acid.

Here we report the direct asymmetric amination of α-substituted cyclic ketones catalyzed by a chiral phosphoric acid, yielding products with a N-containing quaternary stereocenter in high yields and excellent enantioselectivities. Kinetic resolution of the starting ketone was also found to occur on some of the substrates under milder conditions, providing enantioenriched α-branched ketones, another important building block in organic synthesis. The utility of this methodology was demonstrated in the short synthesis of (S)-ketamine, the more active enantiomer of this versatile pharmaceutical.

Structure-activity relationships for ketamine esters as short-acting anaesthetics.

A series of aliphatic esters of the non-opioid anaesthetic/analgesic ketamine were prepared and their properties as shorter-acting analogues of ketamine itself were explored in an infused rat model, measuring the time after infusion to recover from both the anaesthetic (righting reflex) and analgesic (response to stimulus) effects. The potency of the esters as sedatives was not significantly related to chain length, but Me, Et and i-Pr esters were the more dose potent (up to twofold less than ketamine), whereas n-Pr esters were less potent (from 2- to 6-fold less than ketamine). For the Me, Et and i-Pr esters recovery from anaesthesia was 10-15-fold faster than from ketamine itself, and for the n-Pr esters it was 20-25-fold faster than from ketamine. A new dimethylamino ketamine derivative (homoketamine) had ketamine-like sedative effects but was slightly less potent than, but ester analogues of homoketamine had very weak sedative effects.

Improved methods for thermal rearrangement of alicyclic α-hydroxyimines to α-aminoketones: synthesis of ketamine analogues as antisepsis candidates.

Ketamine is an analgesic/anesthetic drug, which, in combination with other drugs, has been used as anesthetic for over 40 years. Ketamine induces its analgesic activities by blocking the N-methyl-D-aspartate (NMDA) receptor in the central nervous system (CNS). We have reported that low doses of ketamine administrated to patients before incision significantly reduced post-operative inflammation as reflected by reduced interleukin-6 (IL-6) sera-levels. Our data demonstrated in a rat model of Gram-negative bacterial-sepsis that if we inject a low dose of ketamine following bacterial inoculation we reduce mortality from approximately 75% to 25%. Similar to what we have observed in operated patients, the levels of TNF-α and IL-6 in ketamine-treated rats were significantly lower than in septic animals not treated with ketamine. On the base of these results, we have designed and synthesized series of new analogues of ketamine applying a thermal rearrangement of alicyclic α-hydroxyimines to a-aminoketones in parallel arrays. One of the analogues (compound 6e) displayed high activity in down-regulating the levels of IL-6 and TNF-α in vivo as compared to ketamine.

Synthesis of deuterium labeled ketamine metabolite dehydronorketamine-d4.

A convenient synthesis of ketamine metabolite dehydronorketamine-d(4), starting from commercially available deuterium labeled bromochlorobenzene, was achieved. Key steps include Grignard reaction, regioselective hydroxybromination, Staudinger reduction, and dehydrohalogenation.

Efecto de dosis bajas de ketamina en la analgesia postoperatoria y consumo de morfina tras cirugía de revascularización miocárdica / Efficacy of low doses of ketamine in postoperative analgesia and the use of morphine after myocardial revascularisation surgery

Objetivos. Valorar la eficacia de la ketamina administrada intraoperatoriamente a dosis bajas en infusión continua (8mg/kg/min) en la analgesia postoperatoria y en reducir el consumo de morfina en las primeras 24h del postoperatorio de cirugía de derivación coronaria tras anestesia basada en remifentanilo. Material y método. Estudio prospectivo, aleatorio y doble ciego en 60 pacientes, ASA III–IV, programados para ser sometidos a cirugía de revascularización miocárdica sin circulación extracorporea. Técnica anestésica estándar con propofol y remifentanilo entre 0,5–1mg/kg/min, tras la inducción anestésica, el grupo K (n=30) recibió una infusión de ketamina constante de 8mg/kg/min y el grupo P (n=30) suero salino 0,9% al mismo ritmo de infusión por vía intravenosa. Antes de finalizar la cirugía se administró 0,15mg/kg de morfina vía intravenosa. La analgesia postoperatoria se realizó con morfina 3mg cada 5min cuando la escala verbal simple (1). Valoramos el consumo de remifentanilo propofol intraoperatorio, tiempo hasta administrarse el primer analgésico, consumo de morfina en las primeras 24h, intensidad del dolor mediante escala verbal simple y escala visual analógica, parámetros hemodinámicos, respiratorios, nivel de sedación y efectos adversos. Resultados. No hemos encontrado diferencias estadísticamente significativas en las características demográficas, así como en el consumo e infusiones medias de remifentanilo y propofol de ambos grupos. Los pacientes del grupo K retrasaron significativamente el momento de solicitar la primera dosis de analgésico con respecto al grupo P (p<0,03), el consumo de morfina en la reanimación y el consumo total en las primeras 24h no mostró diferencias significativas entre ambos grupos de estudio. Resultados. Los parámetros hemodinámicos, respiratorios y nivel de sedación permanecieron estables durante todo el periodo de estudio sin diferencias estadísticamente significativas entre ambos grupos. El control del dolor postoperatorio fue adecuado en ambos grupos (escala visual analógica <30, escala verbal simple <2) sin diferencias significativas durante las 24h de estudio. En la incidencia de efectos adversos no hubo diferencias significativas entre ambos grupos, la incidencia de nauseas y vómitos postoperatorios fue del 20% en el grupo K y del 30% en el grupo P. Ningún paciente presentó alucinaciones o disforia. Conclusiones. Dosis bajas de ketamina en infusión continua durante el periodo intraoperatorio de cirugía coronaria prolongan el tiempo de demanda de la primera dosis de morfina, sin reducir el consumo de remifentanilo intraoperatorio, ni el consumo total de morfina durante en las primeras 24h de postoperatorio (AU)

Objectives. To assess the efficacy of ketamine in low doses by continuous infusion (8mg/kg/min) given during surgery and the reduction in morphine use in the first 24h after coronary bypass surgery with remifentanil based anaesthesia. Material and method. Randomised, prospective, double blind study on 60 patients, ASA III–IV, scheduled to have off-pump myocardial revascularisation surgery. A standard anaesthetic technique with propofol and remifentanil between 0.5−1mg/kg/min, after induction of anaesthesia, the ketamine (K) group (n=30) received a continuous infusion of 8mg/kg/min and the propofol and remifentanil (P) group (n=30) received 0.9% physiological saline intravenously at the same infusion rate. Before the end of the surgery 0.15mg/kg of morphine was given intravenously. Post-operative analgesia consisted of 3mg of morphine every 5min when the simple verbal scale was <1 (SVS<1). The intra-operative use of remifentanil–propofol was determined, as well as, the time to giving the first analgesic, pain intensity using the SVS and visual analogue scale (VAS), haemodynamic and respiratory parameters, level of sedation and adverse effects. Results. No statistically significant differences were found between the demographic parameters, or in the mean use and infusions of remifentanil and propofol by both groups. The patients from the K group significantly delayed the time of requesting the first analgesic dose compared to the P group (P<0.03). There were no significant differences between both study groups in the use of morphine in recovery and the total use in the first 24h. Results. The haemodynamic and respiratory parameters, and the sedation level remained stable during the whole period of the study, with no statistically significant differences between both groups. Post-operative pain control was adequate in both groups VAS<30, SVS<2), with no significant differences during the 24h of the study. There no significant differences between groups, in the incidence of adverse effects. The incidence of nausea and post-operative vomiting was 20% in group K and 30% in group P. None of the patients had hallucinations or dysphoria. Conclusions. Low doses of ketamine in continuous infusion during the intra-operative period of coronary surgery prolongs the time of demanding the first morphine dose, without reducing the use of intra-operative remifentanil, or the total use of morphine in the first 24h after surgery (AU)

Comparative pharmacology in the rat of ketamine and its two principal metabolites, norketamine and (Z)-6-hydroxynorketamine.

(Z)-6-Hydroxynorketamine (3), a secondary metabolite of the dissociative anesthetic agent ketamine (1), was synthesized, and its central nervous system (CNS) properties were compared to those of the parent drug and the primary metabolite, norketamine (2). Administration of compounds 1 and 2 to rats (40 mg/kg iv) produced general anesthesia and also led to marked increases in spontaneous locomotor activity during the postanesthetic recovery phase. These effects were of significantly longer duration with 1 than with 2. In contrast, the same dose of 3 produced neither general anesthesia nor CNS excitation, despite the fact that 3 entered brain tissue readily from the systemic circulation. It is concluded that the CNS effects of 1 are attenuated by metabolism to 2 and are abolished by subsequent hydroxylation to produce 3. Moreover, the results suggest that the desirable anesthetic properties of 1 and related arylcyclohexylamines may be inseparable from their ability to produce adverse postanesthetic emergence reactions.

Stereochemical studies of demethylated ketamine enantiomers.

The enantiomorphs of norketamine, 2-(o-chlorophenyl)-2-aminocyclohexanone, were synthesized and screened for biological activity. Resolution was achieved by fractional crystallization of the tartrate salts. Stereochemical purity was determined using standard GC or GC-MS analysis. Preliminary pharmacological evaluations revealed that intraperitoneally injected dextrorotatory norketamine caused a greater duration of loss of righting reflex in mice than the levorotatory isomer.