IE51585B1 - Method for the quantitative determination of encainide - Google Patents

Description

This invention relates to a process for the quantitative determination of ennainide by non-ionic bonding.

According to the invention there is provided a process for the quantitative determination of encainide and the 05 femethyl and 3-nethoxy-O-demethyl metabolites thereof in a biological fluid involving extraction thereof from said fluid and separation thereof by high pressure liquid chromotography cn a silicic acid colum using an organic solvent as mobile phase, wherein said raobiLe phase is the mixture ethanol:water: BEthanesulfomc acid 500:30:0.1.

Encainide hydrochloride is an antiarrhythric compound which is also referred to in the literature as Ml 9067 (USAN And Ihe USP Dictionary of Drug Names 1980, page 122, United States Harmacopeial Convention, Inc. 12601 Twinhroak Parkway, Rockville, ® 20852, Library of Congress Catalog Card No. 72-88571).

Encainide has the following structural formula. lbs following publications describe the chemical synthesis of encainide, a number of analogs thereof, and the antiarrhythmic properties of these compounds in animals.

Dykstra, et al., J. Med. Chem., 16 1015-1020 (1973).

Dykstra and Minielli, U.S. Patent No. 3,931,195 patented January 6, 1976.

Byrne, et al., J. Pharmacology and Bperimental Therapeutics, 5 200, 147-154 (1977).

Assay methods for encainide and its metabolites are disclosed in the following references.

Mayol and Gaitans, Therap. Drug Monitoring, _1, 507-S24 (1979).

Mayol, U.S. Patent Application Serial No. 155,338 filed June 2, 1980.

In Patent Specification No. ΐ there is described and claimed 4-Hydroxy-3-nethoxy-N-[2-[2-(1methyl-2-piperidinyl )-ethyl j phenyl) benzamide.

Hie structures of encainide and a nurrber of analogs thereof which are described in the foregoing Dykstra, et al. publication and patent are shown in the following table as well as the conpound of Patent Specification No. . The ccnpound nunbers used in the table are the sane as the example nutters enployed in the Dykstra, et al. patent. Ccnpound No. A is the oonpcund cf Patent Specification No. · Compound No. Bi R3 89 (O-denethylencainide, or CEE) HO- H- ch3- 107 (encainide, or E) CH30- H- ®3- 108 H- CH3O- ch3- 109 CH3O- CH3O- ch3- 139 (N-demethylencainide, or NDE) CH3O- H- H- A (3-methoxy-O-denettol-encainide, or 3-nethoxy ODE) HO- CH3O- CK3- Caipound Nos. 8S and 139 have been identified by Mayol 10 and Garmans (op. cit.) as metabolites of encainide (Caipound No. 107) in which publication tbs forner is referred to as ODE anc the latter of NDE. The former (ODE) has been identified as a principal netabolite which is sonEtimss present in plas.iB in amounts several tines the concentration of encainide following treatment with the latter. As a result of an inprovement of the high pressure liquid chromatography assay method described in the iteyol and Ganmans publication, which inprovement constitutes the present invention, it has been found that caipound A is also a principal metabolite of encainide in man following oral administration of the latter, and that it is present in concentrations in the blood plasne substantially greater than that of Caipound Na 89 two or more tours following tha aanir.istration of a 50 mg dose of encainide hydrochloride orally. The HPLC assay method described in the Mayol and Gamnens publication has now been found to have been incapable of distinguishing between Caipound Nos. 89 and Caipound A. It is thought that Caipound A is produced in the body from Caipound No. 89 by a biological transformation process.

For comparative purposes the biological activity of the compounds whose structures are shown in the above table are presented in the following table in which comparative antiarrhythmic activity is given as determined by the laboratory screening test referred to in the Dykstra, et al. paper above.

This is referred to in the table as Test I. Test XI is an approximation of the oral toxicity of these oonpounds in mice, and Tests III and IV refer to the human metabolism results observed following oral administration of an antiarrhythmic oral dose of 75 mg total dose of encainide hydrochloride to patients.

Biological Activity I Antiarrhythmic activity for ventricular arrhythmia in mice produced by chloroform inhalation; ED50 expressed as mg/kg body weight, intraperitoneal administration, J.K. Lawson J. Pharmacol. Expt. Ther. 160, 22 (1968).

II ALD50/ATD5O mouse treated orally; ALDjq is the ^proximate lethal dose of half the animals,· ATD50 is the approximate lowest dose where signs of physiologic or neurologic deficit appear in half the animals, expressed as mg/kg body weight.

III Time following oral dosing of humans with 75 mg. of encainide hydrochloride for clearance (plasma concentration <20 ng./ml.) from blood plasma,· hcurs.

TV Approximate half life in hunan blood plasma following 2o oral dosing with encainide hydrochloride; hcurs.

Conpound No. I II III IV 89 1.7-2.8* 25-50/5-10 >16 4-6 107 (encainide) 5.3-15* 50-100/5-10 6 2.5-3 108 10 100/10-25 ♦* 109 10 100/25 «· 139 28 147/15.7 + ♦ A 9.5 250/31.3-62.5 >16 4.6 * Range of various determinations.

" Conpounds have not been administered to man nor observed as metabolites of encainidp in nan or animals.

+ Occurrence in plasma following oral administration of encainide to nan has been rare, and value has not been determinable.

The foregoing results reflect approximately equivalent antiarrhythmic activity for each of these substances except for Conpound No. 89 which is somewhat more potent but also somewhat more toxic in mice. Conpound A differs from and enjoys the advantages over each of Conpound Nos. 89 and 107 of reduced toxicity and of a more prolonged presence in the blood.

EXAMPLE Assay Procedure A. Extraction of Biological Fluid.- To 1 ml of plasma cr urine in a screw-capped tube there is added 0.2 ml of 0.5 H.Tris-HCl buffer (2-amino-2-hydroxynethyl-l,3-propanediol), pH 8.5, and 10 ml of n-butyl chloride containing 5% by volume of isopropanol. The sanple is then shaken on an oscillating go muer followed by centrifugation to separate the phases. A 9 ml aliquot of the organic layer is rentjved and evaporated to dryness under a stream of nitrogen and the residue is redissolved in 100 ml of ethanol. A 50 nil aliquot of this solution is injected onto the column for high pressure liquid chronBtography.

B. High Pressure Liquid Chronatography (HPLC) Conditions.A normal phase silicic acid column having dimensions 3.9 nm by 30 cm in length eg lipped with a variable wavelength UV detector set at 254 nm is used. The mobile phase employed consists cf 500 ml of ethanol, 30 ml of water, and 0.1 ml of oethanesulfonic acid at a flow rate of 1 ml per minute. A series of standards containing 0, 25, 100 and 500 ng/ml of each conpound to be assayed was prepared using pooled human plasma. These stand* nte were treated as described above. Quantification of the detector 5158S response was achieved by digital integration or measurement of peak heights. The standard curve for each component was «instructed by linear regression of the detector response versus concentration from the plasma standards. The concentration of coepound in the sanple was then interpolated from these curves C. Results.- The relative order of elution of encainide and its O-demethjl and 3-rethoxy O-demethyl metabolites from the ociunr anc their retention times is as fellows: Conpound to. 89, 8.3 minutes,- Conpound A, 9.7 minutes; Conpound No. 107, 11·* minutes. The last endogenous plasma oomponent eluted from the colixnn has a retention tire of 3.6 minutes and the baseline is very stable in the region where the metabolites elute. Thus, no interference from endogenous plasma components occurs. Due to differences in extraction efficiencies, specific molar absorptivities, and retention tines for the individual components, the sensitivity of the assay for each of these exponents is as follows: Compound No. 89, 10 ng/ml,· Conpound A, 20 ng/ml; Conpound No. 107, 15 ng/ml.

Claims (2)

CLAIMS:

1. A process for the quantitative determination of encainide and the O-demsthji and 3-methoxy-O-daiEthyl metabolites thereof in a biological fluid involving extraction thereof from said 5 fluid and separation thereof by high pressure liquid chraratography on a silicic acid column using an organic solvent as mobile phase, wherein said mobile phase is the mixture ethanol: water: nethanesulfonic acid 500:30:0.1 by volume.

2. A process according to claim 1, substantially as herein10 before described.