Enzymatic synthesis of isotopomers of tyramine labeled with deuterium and tritium

The combined chemical and enzymatic methods of synthesis of five isotopomers of L ‐tyrosine, L ‐Tyr, and their derivatives, i.e. corresponding isotopomers of tyramine (TA), labeled with deuterium and tritium have been reported. Two‐step synthesis consists with introduction of deuterium or tritium label into intermediate L ‐Tyr using isotope exchange followed by enzymatic decarboxylation using enzyme tyrosine decarboxylase (EC 4.1.1.25). This way five isotopomers of L ‐tyrosine, i.e. [2‐²H]‐L‐, [2‐³H]‐L‐,[2‐²H/³H]‐L‐, [3′,5′‐²H₂]‐L‐,[3′,5′‐³H₂]‐L ‐Tyr, and six isotopomers of tyramine i.e. [1S‐²H]‐, [1S‐³H]‐, [1S‐²H/³H]‐, [3′,5′‐²H₂]‐, [3′,5′‐²H₂]‐, [2′,6′‐²H₂]‐TA were obtained. Copyright © 2007 John Wiley & Sons, Ltd.     

Synthesis of isotopomers of dopamine labeled with deuterium or tritium

The synthesis of four isotopomers of dopamine labeled with deuterium or tritium is reported. The ring labeled [2′,5′,6′‐²H₃]‐, and [2′,5′,6′‐³H₃]‐dopamine were obtained using acid catalyzed isotopic exchange between dopamine and heavy or tritiated water respectively. Two selectively labeled isotopomers, i.e. [1R‐²H]‐, and [1R‐³H]‐dopamine were synthesized by enzymatic decarboxylation of L ‐DOPA using the enzyme tyrosine decarboxylase (EC 4.1.1.25) from Streptococcus faecalis. Copyright © 2006 John Wiley & Sons, Ltd.     

布洛芬HPMC骨架片药物释放因素研究

目的 考察影响布洛芬亲水性骨架片体外释药的各种因素。方法 以羟丙基甲基纤维素（HPMC）为骨架材料，用湿法制粒和粉末直接压片法制备缓释骨架片，并考察HPMC用量，粒度，制备方法，片子大小及其它辅料对布洛芬HPMC骨架片的体外释药的影响。结果 布洛芬HPMC骨架片的体外释药均符合Higuchi方程。HPMC的用量，粒度和制法，片子大小对布洛芬的释放速率均有显著影响。湿法制片的释药速率比干法慢。布洛共姝释药速率随HPMC粒度的减小和片子的增大而减慢。淀粉，PVP、MCC、EC的加入（每片HPMC的含量不变）均减慢布洛芬释药速率。结论 HPMC用量、粒度、制备方法、片子大小及其它辅料为布洛芬骨架片释放速率的主要因素。     

Enzymatic synthesis of tritium‐labelled isotopomers of histamine

Three tritium‐labelled isotopomers of histamine (HA) have been synthesized using combined chemical and enzymatic methods. In the first step the tritium‐labelled isotopomers of histidine have been obtained by catalysed exchange with tritiated water. These intermediates have been converted into [1S‐³H]‐HA, [1R‐³H]‐HA and [2′,4′,1S,‐³H₃]‐HA using the enzyme histidine decarboxylase (HDC, EC 4.1.1.22) from Lactobacillus 30a. Copyright © 2004 John Wiley & Sons, Ltd.     

Synthesis of tritium labeled isotopomers of L‐tyrosine

The synthesis of four selectively labeled isotopomers of L ‐tyrosine, (L ‐Tyr), using chemical and enzymatic methods is reported. Four tritium labeled isotopomers of L ‐phenylalanine (L ‐Phe) – [2‐³H]‐, [2′,6′‐³H₂]‐, [3R‐³H]‐ and [3S‐³H]‐ have been synthesized using a combination of chemical and enzymatic methods. The labeled isotopomers of L ‐Phe have been converted into [2 ‐³H]‐, [2′,6′‐³H₂]‐, [3R‐³H]‐, and [3S‐³H]‐L ‐Tyr by using the enzyme L ‐phenyl‐alanine 4′‐monooxygenase. Copyright © 2002 John Wiley & Sons, Ltd.     

Characterization of 5-Fluorouracil Release from Hydroxypropylmethylcellulose Compression-Coated Tablets

Hydrogel compression-coated tablets are able to release the core drug after a period of lag time and have potential for colon-specific drug delivery based on gastrointestinal transit time concept. This study investigated the factors influencing in vitro release characteristics of a model drug 5-fluorouracil from hydroxypropylmethycellulose (HPMC) compression-coated tablets. The core tablet, prepared by a wet granulation compression method, was designed to disintegrate and dissolute quickly. To prepare the compression-coated tablets, 50% of the HPMC/lactose coat powder was precompressed first, followed by centering the core tablet and compressing with the other 50% of the coat powder. Release characteristics were evaluated in distilled water by using a Chinese Pharmacopoeia rotatable basket method. Effect of HPMC viscosity, lactose content in outer shell, and overall coating weight of outer shell on release lag time (T_lag), and zero-order release rate (k) were studied. Release of drug from compression-coated tablets began after a time delay as a result of hydrogel swelling/retarding effect, followed by zero-order release for most of the formulations studied. HPMC of higher viscosity (K4M and K15M) provided better protection of the drug-containing core, showing increased release lag time and slower release rate. Incorporating lactose in outer shell led to decrease of T_lag and increase of k. T_lag and k are exponentially and linearly correlated to lactose content, expressed as weight percentage of the outer shell. Larger coating weight (W) of outer shell produced larger coating thickness (D) around core tablet, which resulted in increase in T_lag and decrease in k. There was good fitting of a linear model for each of the four variables W, D, T_lag, and k. Hardness of the compression-coated tablets and pHs of the release media had little effect on drug release profile. It is concluded that the release lag time and release rate are able to be tailored through adjusting the formulation variables to achieve colon-specific drug delivery of 5-fluorouracil.     

Enzymatic synthesis of tritium‐labelled isotopomers of L‐DOPA

The synthesis of two isotopomers of L ‐DOPA labelled selectively with tritium is reported. In the intermediate step [3S‐³H]‐, and [3′,5′‐³H₂]‐L ‐tyrosine, have been obtained using a combination of chemical and enzymatic methods. The labelled isotopomers of L ‐tyrosine, L ‐Tyr, have been converted into, [3S‐³H]‐, and [5′‐³H]‐L ‐DOPA using the enzyme mushroom tyrosinase (monophenol oxidase, EC 1.14.18.1) from Neurospora crassa. Copyright © 2005 John Wiley & Sons, Ltd.     

Synthesis of deuterium labelled 2‐bromobenzylamine by directed ortho‐metalation chemistry

Directed ortho‐metalation (DoM) strategy has been applied for the development of a short procedure for the regiospecific synthesis of [phenyl‐²H₄]‐2‐bromo‐benzylamine 6 starting from commercially available [phenyl‐²H₅]‐benzoyl chloride 1 . A strong isotope effect was observed during the ortho‐substitution. Copyright © 2005 John Wiley & Sons, Ltd.     

Formulation development and optimization of bilayer tablets of aceclofenac

Objective: The objective of the present study was to develop bilayer tablets of aceclofenac that are characterized by initial burst drug release followed by sustained release of drug.

Methods: The fast-release layer of the bilayer tablet was formulated using microcrystaline cellulose (MCC) and HPMC K4M. The amount of HPMC E4M (X₁) and MCC (X₂) was used as independent variables for optimization of sustained release formulation applying 3² factorial design. Three dependent variables were considered: percentage of aceclofenac release at 1 h, percentage of aceclofenac release at 12 h, and time to release 50% of drug (t_50%). The composition of optimum formulation of sustained release tablets were employed to formulate double layer tablets.

Results: The results indicate that X₁ and X₂ significantly affected the release properties of aceclofenac from sustained release formulation. The double layer tablets containing fast-release layer showed an initial burst drug release of more than 30% of its drug content during first 1 h followed by sustained release of the drug for a period of 24 h.

Conclusion: The double layer tablets for aceclofenac can be successfully employed as once-a-day oral-controlled release drug delivery system characterized by initial burst release of aceclofenac for providing the loading dose of drug.     

An innovative and efficient synthesis of stable isotope labelled 1‐methyl‐5‐(4,4,5,5‐tetramethyl‐1,3,2‐dioxaborolan‐2‐yl)‐1H‐pyrazole via [13C42H3] N‐methylpyrazole

In support of a programme to develop a treatment for cancer, a stable isotope labelled version of the drug candidate was required. The key labelled intermediate was [¹³C₄²H₃] N‐methylpyrazole prepared by a novel bisacetal cyclisation. This was prepared from commercially available diethyl [¹³C₃] malonate and [¹³C²H₃] iodomethane. Copyright © 2012 John Wiley & Sons, Ltd.     

Synthesis of [2H3]‐labelled sulfamethoxazole and its main urinary metabolites

Sulfamethoxazole was labelled at positions 3, 5, and 4′ by H/D exchange in a mixture of 5% ²H₂SO₄ and 95% ²H₂O (v/v) under reflux for 72h with good isotope incorporation and acceptable yield. Subsequently, [²H₃]‐sulfamethoxazole‐N₁‐glucuronide and [²H₃]‐N₄‐acetyl‐sulfamethoxazole were synthesized. Copyright © 2007 John Wiley & Sons, Ltd.     

Preparation of human drug metabolites using fungal peroxygenases

The synthesis of hydroxylated and O- or N-dealkylated human drug metabolites (HDMs) via selective monooxygenation remains a challenging task for synthetic organic chemists. Here we report that aromatic peroxygenases (APOs; EC 1.11.2.1) secreted by the agaric fungi Agrocybe aegerita and Coprinellus radians catalyzed the H₂O₂-dependent selective monooxygenation of diverse drugs, including acetanilide, dextrorphan, ibuprofen, naproxen, phenacetin, sildenafil and tolbutamide. Reactions included the hydroxylation of aromatic rings and aliphatic side chains, as well as O- and N-dealkylations and exhibited different regioselectivities depending on the particular APO used. At best, desired HDMs were obtained in yields greater than 80% and with isomeric purities up to 99%. Oxidations of tolbutamide, acetanilide and carbamazepine in the presence of H₂¹⁸O₂ resulted in almost complete incorporation of ¹⁸O into the corresponding products, thus establishing that these reactions are peroxygenations. The deethylation of phenacetin-d₁ showed an observed intramolecular deuterium isotope effect [(k_H/k_D)_obs] of 3.1 ± 0.2, which is consistent with the existence of a cytochrome P450-like intermediate in the reaction cycle of APOs. Our results indicate that fungal peroxygenases may be useful biocatalytic tools to prepare pharmacologically relevant drug metabolites.     

曲尼司特缓释片的制备及其释药影响因素研究

目的 制备了曲尼司特凝胶骨架片。方法 采用HPMC K4M、K15M为凝胶骨架材料，进行了处方研究；通过测定制剂体外释放度，评价了该缓释片处方。结果 曲尼司特缓释片体外释药符合Higuchi方程，其释药速率常数Kr为0．193h^1/2。影响缓释片体外释药的因素有骨架材料的种类、用量、粘合剂的种类和释药介质的pH等。结论 缓释片具有明显的缓释作用，可缓慢释药12h。     

Synthesis of carbon‐14 and stable isotope labelled NN414: a potent potassium channel opener

Currently, NN414, a potent β‐cell selective potassium channel opener, is undergoing clinical trials for the treatment of type 2 diabetes. Here, we report the synthesis of carbon‐14 and stable isotope labelled NN414 for use in metabolic studies and as an internal standard in pharmacokinetic assays, respectively. The carbon‐14 labelling was performed in two steps starting from an advanced intermediate. This provided [¹⁴C]NN414 in 60% overall radiochemical yield with a specific activity of 58mCi/mmol. The stable isotope labelling was accomplished from benzyl tert‐butyl malonate in eight steps using [¹³C,²H₃]iodomethane and [²H₂]dibromomethane as the source of carbon‐13/deuterium. The synthetic sequence, which included a Mannich reaction followed by deamination, a Simmons–Smith‐type cyclopropanation and a modified Curtius reaction, provided [¹³C,²H₅]NN414 in 8.6% overall yield. Copyright © 2004 John Wiley & Sons, Ltd.     

Synthesis of the NK3 receptor antagonist AZD2624 in C‐14‐, H‐3‐ and C‐13‐labeled forms

In support of a program to develop an antipsychotic treatment for schizophrenia, three labeled forms of the NK3 receptor antagonist AZD2624 have been prepared. [³H₂]AZD2624 was synthesized by tritiodehalogenation for use in receptor occupancy and autoradiographic studies. [¹³C₆]AZD2624 was prepared for use as an internal standard through the intermediacy of [¹³C₆]isatin, and two C‐14 isotopomers of AZD2624 were prepared from [¹⁴C]benzoic acid and [¹⁴C]isatin for a variety of DMPK studies. Copyright © 2011 John Wiley & Sons, Ltd.     

Endothelin receptors in the human coronary artery,ventricle and atrium

Summary In the present experiments we investigated endothelin (ET) receptors in the human coronary artery, and in ventricular and atrial muscle using quantitative receptor autoradiography. Displacement of [¹²⁵I]Sf6b (Sarafotoxin S6b) (30 pM)- and [¹²⁵I]ET-1 (30 pM)-labeled binding sites was studied using ET-1, the ET_A receptor selective ligand BQ-123 (cyclo[D-Asp-L-Pro-D Val-L-Leu-D-Trp-]), and the ETB receptor selective ligand [Ala^1,3,11,15]ET-1.Specific binding was more dense in atrium and coronary artery (relative optical density (r.o.d.): 0.14±0.01 and 0.16±0.01, respectively) than in ventricular muscle (r.o.d.: 0.10±0.01). In the coronary artery, binding was especially dense in the media. ET-1 displaced [¹²⁵I]ET-1 and [¹²⁵I]Sf6b monophasically in atrium, ventricle and coronary artery. [Ala^1,3,11,15]ET 1 and BQ-123 displaced [¹²⁵I]ET-1 and [¹²⁵I]Sf6b-labeled sites biphasically in the ventricle and in the atrium. In the human coronary artery, [Ala^1,3,11,15]ET-1 and BQ-123 displaced [¹²⁵I]ET-1-labeled sites monophasically (pIC₅₀): ET-1 (9.72±0.12) > BQ-123 (6.84±0.08) > [Ala^1,3,11,15]ET-1 (6.40±0.12). In contrast, [Ala^1,3,11,15]ET-1 and BQ-123 displaced [¹²⁵I] Sf6b-labeled coronary artery sites biphasically (high affinity pIC₅₀: BQ-123, 9.03±0.25;[Ala^1,3,11,15]ET-1, 8.40±0.14; low affinity pIC₅₀: BQ-123, 7.24±0.14; [Ala^1,3,11,15]ET-1, 6.99±0.09).These data indicate that both [¹²⁵I]ET-1 and [¹²⁵I] Sf6b-labeled ET_A and ET_B binding sites in human ventricular and atrial muscle. In the human coronary artery, both radioligands labeled ET_A binding sites, but [¹²⁵I] Sf6b also labeled a non-ET_A, non-ET_B binding site with relatively high affinity for both BQ-123 and [Ala^1,3,11,15] ET-1. Correspondence to W. A. Bax, at the above address     

Disposition and clinical pharmacokinetics of theophylline after administration of a new sustained release tablet

Summary The systemic disposition of theophylline after taking a new, sustained release tablet (Theolair Retard^® 250 mg, Theolair S. R.^®, Riker Laboratories) has been studied in 8 hospitalized patients. Absolute bioavailability was determined from the ratios of the areas under the serum concentration-time curves after intake of the tablet and after intravenous infusion of aminophylline in the same patient. The absolute bioavailability of Theolair Retard^® 250 mg was 110.9±20.8% (mean ± SD). Maximal serum concentrations were reached after 7.3±3.5 h, the large intersubject variation being due to differences in gastric emptying time. The tablets appear to release theophylline slowly in acid conditions, but more rapidly in an alkaline medium. Invasion was found to be either monophasic with a rate constant of about 0.8 h^–1 (intestine), or biphasic with rate constants of 0.2 h^–1 (stomach) and 0.8 h^–1 (intestine). The peak levels accounted for 7.9±2.2 mg · 1^–1. The profiles of the serum concentration-time curves were such that the concentrations remained above 80% of c^max for 6.5±3.3 h. The relevant pharmacokinetic parameters (half-life of elimination, total body clearance and volume of distribution) were determined and were used to calculate the individual dosage regimens required to obtain therapeutic serum concentrations. The optimal dosing interval to obtain an average steady state serum concentration of 12.5 mg · l^–1 was 9.8±3.1 h.     

Stereoselective excretion of (3-methoxy-4-sulphooxyphenyl)ethylene glycol (MHPG sulphate) in the dog

Summary 3-Methoxy-4-hydroxyphenylethylene glycol (MHPG) labelled with six deuterium atoms ([²H₆]MHPG) was infused into two female greyhound dogs. Plasma and urine were analyzed for endogenous MHPG and [²H₆]MHPG and their conjugates by the technique of selected ion monitoring gas chromatography-mass spectrometry. This analysis showed that the major forms of MHPG and [²H₆]MHPG in the plasma and urine of the greyhounds were the sulphate conjugates. However, the plasma concentration of [²H₆]MHPG sulphate increased continuously during the infusion period and the renal clearance of this compound was found to be much less than that of the endogenous sulphate. The explanation that this was due either to saturation of a renal transport mechanism or to a deuterium isotope effect was eliminated. Stereochemical analysis showed that, while the [²H₆]MHPG used for infusion was a racemic mixture of two stereoisomers, much more of the natural(–)stereoisomer of [²H₆]MHPG sulphate was excreted in the dog urine. It was also shown that both the (+) and (–)stereoisomers of [²H₆]MHPG sulphate were sulphoconjugated at the 4-position of the aromatic ring.     

Zero-Order Release Formulation of Oxprenolol Hydrochloride with Swelling and Erosion Control

Zero-order release of oxprenolol hydrochloride was obtained by controlling the swelling and erosion of the matrix. This formulation involves only mixing of drug, hydroxypropylmethylcellulose (HPMC), and sodium carboxymethylcellulose (Na CMC) at the ratio of 1:0.4:1.6, respectively, and compressing the mixture directly into tablets. The in vitro release pattern from this optimized matrix tablet was reproducible. Accelerated stability studies revealed that the optimized formulation remains stable for an approximately 2-year shelf life. This sustained-release (SR) tablet was evaluated in dogs, and for comparison a conventional (CV) formulation was also given at the same dose level. Plasma oxprenolol levels were monitored by a sensitive and specific high-performance liquid chromatographic (HPLC) method. Significant differences in the pharmacokinetic parameters, i.e., lower C _max, higher values of t _max, MRT, AUC, and plasma concentration at 24 hr, and nearly constant plasma levels over 12 hr, indicated that the SR matrix tablet is superior to the CV rapid-releasing formulation. The in vitro release parameters and in vivo pharmacokinetics correlated well.     

Deuterium isotope effect on the metabolism of N-nitrosodimethylamine and related compounds by cytochrome P4502E1

1. Deuteration of N-nitrosodimethylamine (NDMA) decreases its carcinogenicity, and produces an isotope effect on its metabolism in vivo. Consistent with these results are the observations that deuteration caused a 5-fold increase in the apparent K_m, but not the V_max the demethylation and denitrozation of NDMA in acetone-induced rat liver microsomes. These microsomes are a good source of cytochrome P4502E1.

2. For demethylation of Z-[²H₃]NDMA and E-[²H₃)NDMA, the K_m values were indistinguishable, and were between the values for those of NDMA and [²H₆]NDMA. Almost all the formaldehyde formed was derived from the non-deuterated methyl group. indicating a lack of stereoselectivity in the demethylation of NDMA.

3. NDMA and [²H₆]NDMA displayed apparent K_i values of 59 and 441 μM, respectively, for N-nitrosodiethylamine deethylase, showing an apparent isotope effect of 0.13, and displayed an isotope effect of 0.21 in the K_i values for p-nitrophenol hydroxylase.

4. With acetone and deuterated acetone as inhibitors for p-nitrophenol hydroxylase, the isotope effect on the K_i was 0.11. Similar deuterium isotope effects were also observed with acetone and dimethylformamide as competitive inhibitors for NDMA demethylase.

5. In the microsomal oxidation of ethanol, a deuterium isotope effect of about five was observed in the V_max/K_m when carbon-1 was deuterated, but was not observed in the V_max.

6. Results illustrate a unique deuterium isotope effect on the K_m values of reactions catalysed by P4502E1.