PCT
`WORLD INTELLECTUAL PROPERTY ORGANIZATION
`International Bureau
`INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION 1REATY (PCT)
`WO 95/26325
`
`(11) International Publication Number:
`
`(51) International Patent' Classification 6 :
`C07B 59/00
`
`A2
`
`( 43) International Publication Date:
`
`5 October 1995 (05.10.95)
`
`(21) International Application Number:
`
`PCT/CA95/00154
`
`(22) International Filing Date:
`
`27 March 1995 (27.03.95)
`
`(30) Priority Data:
`08/217,897
`
`25 March 1994 (25.03.94)
`
`us
`
`(81) Designated States: AM, AT, AU, BB, BG,·BR, BY, CA, CH,
`CN, CZ, DE, DK, EE, ES, FI, GB, GE, HU, JP, KE, KG,
`KP, KR, KZ, LK, LR, LT, LU, LV, MD, MG, MN, MW,
`MX, NL, NO, NZ, PL, PT, RO, RU, SD, SE, SI, SK, TJ,
`TT, UA, UZ, VN, European patent (AT, BE, CH, DE, DK,
`ES, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OAPI
`patent (BF, BJ, CF, CG, CI, CM, GA, GN, ML, MR, NE,
`SN, TD, TG), ARIPO patent (KE, MW, SD, SZ, UG).
`
`(71) Applicant: ISOTECHNIKA INC. [CA/CA]; Newton Research
`Building, Room 508A, 11315-87th Avenue, Edmonton, Published
`Without international search report and to be republished
`Alberta T6G 2C2 (CA).
`upon receipt of that report.
`
`(72) Inventors: FOSTER, Robert, R.; 4211 120th Street, Edmonton,
`Alberta T6J 1X9 (CA). LEWANCZUK, Richard; 9136
`81st Avenue, Edmonton, Alberta T6C 0X7 (CA). CAILLE,
`Gilles; 3 A venue Querbes, Outremont, Quebec H2M 2S5
`(CA).
`
`(74) Agents: McKHOOL, Eli, J. et al.; Gowling, Strathy &
`Henderson, Suite 2600, 160 Elgin Street, Ottawa, Ontario
`KlP 1C3 (CA).
`
`(54) Title: ENHANCEMENT OF THE EFFICACY OF DRUGS BY DEUTERATION
`
`(57) Abstract
`
`A method of enhancing the efficiency and increasing the duration of action of drugs (e.g. dihydropyridines) and particularly of
`nifedipine wherein one or more hydrogen atoms are deuterated and wherein the deuterated nifedipine has unexpectedly improved hypotensive
`properties when used in much lower concentrations than nifedipine per se. A method for determining the identity and bioequivalency of a
`new drug is also disclosed wherein the molecular and isotope structure of a new drug is determined by gas chromatography-isotope ratio
`mass spectrometry and compared with the molecular and isotope structure of a known human drug.
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`Apotex Ex. 1019
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`Apotex v. Auspex
`IPR2021-01507
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`

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`FOR THE PURPOSES OF INFORMATION ONLY
`
`Codes used to identify States party to the PCT on the front pages of pamphlets publishing international
`applications under the PCT.
`
`AT
`AU
`BB
`BE
`BF
`BG
`BJ
`BR
`BY
`CA
`CF
`CG
`CH
`CI
`CM
`CN
`cs
`CZ
`DE
`DK
`ES
`Fl
`FR
`GA
`
`Austria
`Australia
`Barbados
`Belgium
`Burkina Faso
`Bulgaria
`Benin
`Brazil
`Belarus
`Canada
`Central African Republic
`Congo
`Switzerland
`COte d'Ivoire
`Cameroon
`China
`Czechoslovakia
`Czech Republic
`Germany
`Denmark
`Spain
`Finland
`France
`Gabon
`
`GB
`GE
`GN
`GR
`HU
`m
`IT
`JP
`KE
`KG
`KP
`
`KR
`KZ
`LI
`LK
`LU
`LV
`MC
`MD
`MG
`ML
`MN
`
`United Kingdom
`Georgia
`Guinea
`Greece
`Hungary
`Ireland
`Italy
`Japan
`Kenya
`Kyrgystan
`Democratic People's Republic
`of Korea
`Republic of Korea
`Kazakhstan
`Liechtenstein
`Sri Lanka
`Luxembourg
`Latvia
`Monaco
`Republic of Moldova
`Madagascar
`Mali
`Mongolia
`
`MR
`MW
`NE
`NL
`NO
`NZ
`PL
`PT
`RO
`RU
`SD
`SE
`SI
`SK
`SN
`TD
`TG
`TJ
`TI'
`UA
`us
`uz
`VN
`
`Mauritania
`Malawi
`Niger
`Netherlands
`Norway
`New Zealand
`Poland
`Portugal
`Romania
`Russian Federation
`Sudan
`Sweden
`Slovenia
`Slovakia
`Senegal
`Chad
`Togo
`Tajikistan
`Trinidad and Tobago
`Ukraine
`United States of America
`Uzbekistan
`Viet Nam
`
`"·
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`(
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`WO95/26325
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`PCT/CA95/00154
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`ENHANCEMENT OF THE EFFICACY OF DRUGS BY DEUTERA TION
`
`REFERENCE TO A REI ATED APPLICATION
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`5
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`This is a continuation-in-part of our copending U.S. Patent Application
`
`...
`
`Serial No. 08/217,897 filed March 25, 1994 which is relied on and incorporated herein
`
`by reference in its entirety.
`
`. BACKGROUND OF THE INVENTION
`
`The present invention relates to a process for enhancing the efficacy of
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`10
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`known pharmaceuticals or drugs, and to the enhanced drugs so produced, by changing
`
`the isotopic form of the molecular structure of the known drug. More particularly, the
`
`present invention relates to the modification of the molecular structure of known drugs
`
`containing one or more hydrogen atoms by deuterating one or more of the hydrogen
`
`atoms to deuterium atoms. The resulting drug is significantly altered and has greatly
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`15
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`improved activity over the known drug. Most particularly this invention relates to a
`
`method of deuterating a dihydropyridine (e.g., nifedipine) whereby the deuterated
`
`nifedipine has an increased hypotensive effect and an increased duration of action on
`
`mammals at lower concentration than does nifedipine.
`
`When pharmaceuticals are synthesized, a carbon back-bone is assembled
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`20
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`having various substituents including carbon, hydrogen, oxygen, nitrogen, etc.
`
`Pharmaceuticals have been designed and synthesized by a number of modes including,
`
`for example, serendipity and molecular modification. These and other methods have
`
`generated a vast number of drugs over the course of time. As such modifications have
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`allowed individual companies to keep a competitive edge in the marketplace, a
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`significant part of the industry's time and resources is spent searching for novel agents
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`within certain pharmacologic classifications, e.g., antihypertensives. Such novel agents
`
`often have different activities from the prototype compounds, thus justifying the monies···
`
`spent for their development.
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`5
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`SUMMARY OF THE INVENTION
`
`It is known that virtually all drugs now marketed include a number of
`
`hydrogen atoms, each of which has a molecular mass of one. It has now been found that
`
`when one or more of the hydrogen atoms on a drug are modified so that their molecular
`
`mass becomes two, the activity of the drug is significantly altered and is even greatly
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`10
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`improved. Thus, for example, isotopic modification of a dihydropyridine, e.g., such as
`
`nifedipine, has resulted in an unexpected change in the hypotensive (blood pressure
`
`lowering) effect in mammals compared to nifedipine per se, and such effects should also
`
`be achieved with humans.
`
`Nifedipine is marketed worldwide as an important drug used in the
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`15
`
`treatment of angina and hypertension. Its structure is as follows:
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`20
`
`By modifying nifedipine by replacing one or more hydrogens of the
`
`methyl groups with deuterium or by replacing one or more of the methyl groups with
`
`CD3, the therapeutic properties of nifedipine can be altered and can even be significantly
`
`improved. For example, by modifying the nifedipine by replacing the two methyl
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`groups at the 2 and 6 positions on the ring with two deuterated groups (CD3), i.e.,
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`replacing 6 hydrogen atoms with six deuterium atoms, the structure of the deuterated
`
`nifedipine is as follows:
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`5
`
`Both of the above molecules are nifedipine and the latter structure is an
`
`isotopic form of the former.
`
`BRIEF DESCRIPTION OF THE DRAWING
`
`The present invention will be further understood with reference to the
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`10
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`drawings, wherein:
`
`Figure 1 shows the hypotensive effect of the various concentrations of the
`
`deuterated nifedipines on the treated rats as compared with nifedipine per se;
`
`Figures 2 and 3 show use dependent inhibition of control nifedipine
`
`(Nifedipine B) and deuterated nifedipine (Nifedipine D) on T type calcium channels;
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`15
`
`Figure 4 shows the effect of control nifedipine and deuterated nifedipine
`
`on calcium current inhibition as a function of pulse frequency;
`
`Figure 5 shows the effect of control nifedipine and deuterated nifedipine
`
`on use dependency;
`
`Figures 6(a) and (b) show the effect of control and deuterated nifedipine
`
`20
`
`on mean arterial pressure;
`
`Figures 7(a) and (b) show concentration-effect relationships for control
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`(Figure 7(a)) and deuterated (Figure 7(b)) nifedipine fitted using an asymmetric
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`sigmoidal model;
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`Figures 8(a) and (b) show concentration-effect relationships for control
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`(Figure S(a)) and deuterated (Figure S(b)) nifedipine fitted using logistic dose response
`
`model;
`
`Figures 9a and 9b show the dose-response effect for the control and
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`5
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`deuterated (test) nicardipines;
`
`Figure · 10 shows the duration of effect was greater for deuterated
`
`nicardipine compared to control nicardipine;
`
`Figure 11 shows the hypotensive effect of the various concentrations of
`
`the deuterated verapamil on the treated rats as compared with verapamil per se;
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`10
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`Figure 12 shows the duration of effect was greater for deuterated
`
`verapamil compared to control verapamil;
`
`Figure 13 shows a three dimensional "fingerprint" of seven nifedipine
`
`..
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`preparations;
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`15
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`preparations;
`
`Figure 14 shows a two dimensional "fingerprint" of nine nifedipine
`
`Figure 15 shows a two dimensional (oxygen vs. carbon) "fingerprint" of
`
`solatol crude tablets;
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`Figure 16 shows "fingerprints" of three different hair samples from three
`
`different people; and
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`20
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`Figure 17 shows a two dimensional carbon-oxygen "fingerprint" of three
`
`liquors.
`
`DETAILED DFSCRIPTION OF THE INVENTION
`
`Such modification of nifedipine (and other dihydropyridines such as, for
`
`example, nicardipine, nimodipine, niludipine, nisoldipine, nitrendipine, felodipine,
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`ifradipine and arnlodipine) can be achieved by dissolving the nifedipine in a deuteration
`
`tube in a mixture of deuterochloroform and deuterium oxide, and then adding a minor
`
`amount of trifluoroacetic anhydride and deuteroacetone thereto and mixing ·therewith.·
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`The solution is then frozen within the tube, preferably by immersing the tube in liquid
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`1oo
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`5
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`nitrogen and then sealing the tube. The sealed tube is then heated at a temperature with
`
`the range of from about 50° to about 65°C, and preferably within the range of about 55°
`
`to about 60 ° C and maintained at that temperature for a period of time sufficient to
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`deuterate the methyl group at the 2 and 6 positions on the nifedipine to CD3 • A time of
`
`from about 150 to about 180 hours is effective to complete the reaction, with a time of
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`10
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`about 160 to 170 hours being preferred.
`
`Deuterated nifedipine was synthesized in the following manner:
`
`EXAMPLE I
`
`80 mg of nifedipine in powder form was placed into a special deuteration
`
`· tube and dissolved therein in a mixture of 2 ml of deuterochloroform and 0.5 ml of
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`15
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`deuterium oxide after which 0.2 ml of trifluoroacetic anhydride and 2 ml of
`
`deuteroacetone were added and mixed therewith. The solution was frozen in liquid
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`nitrogen and the tube flame sealed under nitrogen. The tube was then heated at 57°C
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`for 168 hours after which it was cooled and opened. The contents of the tube were
`
`transferred to a round-bottom flask and the solvent was removed in vacuo on a rotovap.
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`20
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`All operations were conducted under reduced intensity of light. Using conventional 1H
`
`nuclear magnetic resonance (NMR) the deuterium substitution was calculated to be 95 %
`
`of the C-2 and C-6 methyl groups shown above.
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`The effect of the deuterated nifedipine on the blood pressure in rats was
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`then determined as follows:
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`EXAMPLE II
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`Spontaneously hypertensive rats
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`(SHR) were anesthetized with
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`pentobarbital (65 mg/kg, intraperitoneally) and a carotid artery and jugular vein:
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`cannulated. Blood pressure was continuously monitored via the carotid artery cannula.
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`5
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`Nifedipine samples, both deuterated and non-deuterated, dissolved in dimethylsulfoxide
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`(DMSO) were diluted in saline so that the final injected concentration of DMSO was less
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`than 0.025% by volume. Aliquots of appropriate dilutions were then injected
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`intravenously in the SHR and blood pressure effects monitored for at least two hours
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`following injection. Doses used were 0.CXXXH, 0.00002, 0.000025, and 0.00005
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`10
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`millimoles per rat in the control group and in the test group. All rats were within 25
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`grams of body weight of each other.
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`The results are shown in Figure 1. At the three lower concentrations the
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`hypotensive effect of deuterated nifedipine was greater than that of regular nifedipine
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`(p=0.08 by Wilcoxon rank-sum test). Effective doses of 50% of the rats (ED50's) were
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`15
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`calculated on the basis of the results from the above doses and results were: (1) Log
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`ED50 deuterated nifedipine: -4.48 (-4.53 to-4.43, 95% confidence interval); and (2) Log
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`ED50 regular nifedipine:
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`-4.36 (-4.40 to -4.31, 95% confidence interval). As the
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`confidence intervals do not overlap, there is a statistical difference in the potency of the
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`two nifedipine products, with the deuterated nifedipine unexpectedly having the greatest
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`20
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`potency.
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`The effect of deuterated nifedipine on calcium channel blocking activity
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`was studied and the studies were carried out using the whole cell version of the patch
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`clamp method, as follows:
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`EXAMPLEm
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`NIE-115 cells (neuroblastoma cell line) were used and these cells were
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`cultured using conventional tissu~ culture techniques. For study, cells were used six to ·
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`eight hours after trypsinization and replating. In this state the predominant calcium
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`channel expressed was the T-type channel, which was used for the current studies.
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`..
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`Patch clamping was carried out using the following external and internal
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`solutions: External solution (in mM): BaC12 20, Tris 105, KCl 5, CsCl 5, HEPES 20,
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`glucose 20 and tetrodotoxin 0.0005. Internal solutions (in mM): CsCl 130, ATP-Na2
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`2, HEPES 20, glucose 5, MGC12 5, cAMP 0.25, and EGTA 10. Osmolarity of all
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`10
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`solutions was adjusted to 310-320 mOsm and the pH adjusted to 7.4 using HCl, NaOH,
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`CsOH or Ba(OH)2 as required. The Petri dish containing the cells was mounted on the
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`stage of an inverted phase contrast microscope. Pipettes fabricated from thin-walled
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`borosilicate glass, and containing the internal solution, were advanced to the selected cell
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`surface using a micromanipulator. Suction facilitated the formation of a membrane patch
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`15
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`with resistance in the range of 20-30 gigaohms. Test pulses in increments of 10 mV
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`were applied for 200 msec with at least 5 sec allowed for channel recovery between
`
`pulses. Basal channel activity was measured based on the peak inward currents. After
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`addition of the nifedipine solutions to the appropriate concentration, 3 minutes were
`
`allowed for the drug to reach equilibrium concentrations. After this time current-voltage
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`20
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`relationships were re-tested and the results expressed as the percentage of control current
`
`obtained (i.e., 100% indicates no channel blocking activity).
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`Preliminary results at concentrations of 1 x 1 o-6 and 1 x 10-5 showed no
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`statistical difference between channel conductance for deuterated and non-deuterated
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`nifedipine (70% vs. 77%, and 35% and 43%, respectively, p-not significant). During
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`these studies, however, it was noted that a difference did exist between the channel
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`activation dependency of the two nifedipine compounds. As usual, normal nifedipine
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`showed blocking activity which is dependent on the state of activation of the · channels
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`(normally blocking is facilitated when channels are more "active"). This effect was not
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`5
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`seen for deuterated nifedipine; rather, it seemed to show continual maximal effect
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`..
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`regardless of channel status. This suggests that the binding of deuterated nifedipine to
`
`the calcium channel is enhanced even though there may be no difference in potency.
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`Clinically, this means that deuterated nifedipine may have a longer half-life of the
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`receptor and/or that it may have a constant effect across blood pressure ranges (normally
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`1 O
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`the higher the blood pressure, the greater the hypotensive effect of calcium channel
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`blockers). It is expected that the deuterated nifedipine would act in a similar manner in
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`humans having high blood pressure.
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`While it has been noted that deuterizing one or more hydrogen atoms in
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`known pharmaceutical compounds will enhance or alter the activity of such compounds,
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`15
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`it is believed that the activity of such compounds may also be ~tered by substituting a
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`different isotope for one or more of the other atoms in the compound. It is known that
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`several varieties of other atoms, such as carbon, nitrogen, oxygen, tin, etc., exist which
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`differ in their atomic mass. These differing species of atoms are referred to as isotopes
`
`and differ only in the number of neutrons in the nucleus.
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`20
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`EXAMPLE IV
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`Use dependency of calcium channel inhibition-Repetitive activation of
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`calcium channels progressively reduces the peak inward current. Such a decrease in
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`current occurs more markedly the greater the frequency of stimulation of the channels.
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`In the presence of a "use dependent" antagonist, the decrease in current with repetitive
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`stimulation is enhanced. Use dependency implies that the antagonist binds cumulatively
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`in small increments during subse.quent channel activations. After 2 or 3 channel
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`activations in the case of calcium channels, a steady state of inhibition is reached. . The
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`underlying implication of use dependence is that a given antagonist ( drug) binds more
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`5
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`strongly to the active channel. A drug which shows greater use dependent channel
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`inhibition is presumed to have a greater affinity for the active calcium channel.
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`In the following set of examples, calcium channels were depolarized with
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`repetitive pulses of current at the intervals indicated. Such studies were carried out
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`using the whole cell version of the patch-clamp. The reduction of inward current as a
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`10
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`function of the interval between pulses in the absence of any drug was taken as the
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`control. Control (i.e., non-deuterated) and deuterated nifedipine were then applied to
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`the cells at a concentration of 5 micromolar. The decrease in inward current as a
`
`function of frequency of stimulation (i.e., use dependence) was then compared between
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`the two nifedipine preparations.
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`15
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`Figure 2 shows use dependent inhibition of control nifedipine (Nifedipine
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`B) and deuterated nifedipine (Nifedipine D) on T type calcium channels in NlE 115
`
`cells. This figure represents inward current flow and its inhibition by the nifedipines.
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`As frequency of stimulation increases, inward current upon repetitive stimulation
`
`decreases (use dependence). Nifedipine is seen to decrease inward current, but at
`
`20
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`ihcreased stimulation frequency deuterated nifedipine is more effective than control
`
`nifedipine (panel C). These differences are further shown in Figures 3-5.
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`Figure 3 represents the inhibitory effect of the two nifedipines on inward
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`calcium current for repetitive 1 second pulses (depolarizations). At this pulse frequency
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`deuterated nifedipine is more effective at blocking calcium current than control current
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`(p < .05 by repeated measures ANOV A). Concentrations of nifedipine were 5
`
`micromolar in both cases.
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`Figure 4 shows the effect of the two nifedipines · on calcium current'
`
`inhibition as a function of pulse frequency. At 1 and O. 3 second intervals deuterated
`
`5
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`nifedipine was more effective than control nifedipine in blocking the calcium channels.
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`Figure 5 shows the effect of the two nifedipines on use dependency. At
`
`all frequencies deuterated nifedipine showed greater use dependent calcium channel
`
`inhibition. Such inhibition was even more marked as frequency of stimulation increased.
`
`Based on the above data, deuterated nifedipine is seen to have greater use
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`10
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`dependent inhibition of calcium channels. This means that deuterated nifedipine is more
`
`effective than regular nifedipine at blocking the calcium channels as the frequency of
`
`stimulation increases. Because channel activation is greater in pathological conditions
`
`such as hypertension or angina, deuterated nifedipine would be expected to be more
`
`efficacious/potent in . these disorders than regular nifedipine.
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`In non-pathological
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`15
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`conditions where channel activation is not as great, however, the activity of deuterated
`
`nifedipine would approach that of regular nifedipine. Such a characteristic is extremely
`
`appealing as it means that the relative potency of the drug (e.g., deuterated nifedipine)
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`would vary directly with the severity of the condition. Thus, for example, the drug
`
`would seemingly "know" how much to reduce the blood pressure in order to achieve a
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`20
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`particular blood pressure goal. Currently, as the severity of a condition (e.g.,
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`hypertension, angina) increases, the dose of nifedipine necessary to treat such a condition
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`also increases. Such may not be the case for deuterated nifedipine.
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`EXAMPLEV
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`Effect of deuterated and control nifedipines on blood pressure in
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`, normotensive Sprague-Dawley rats. Control and deuterated nifedipines were dissolved ·
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`in minimal volumes of ethanol and diluted until the final concentration of ethanol was
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`5
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`less than 0.04%. One milliliter doses of the two nifedipines at the indicated
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`concentrations were then injected into pen~barbi~-anesthetized Sprague-Dawley rats
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`weighing between 300 and 350 grams. The maximum change in mean arterial pressure,
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`as well as the duration of hypotensive response, were measured directly by means of an
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`intra-arterial catheter. Only one drug dose was given to each rat and a minimum of 60
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`10
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`minutes was allowed for blood pressure to ·return to baseline. Results are shown in the
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`figures discussed below. At each time period the duration of effect was greater for
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`deuterated nifedipine compared to control nifedipine. However, because the duration
`
`of response might be dependent on the magnitude of decrease in blood pressure,
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`equipotent doses of the two formulations were compared. In this comparison, control
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`15
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`nifedipine at a concentration of 2 x 10-3 molar and deuterated nifedipine at a
`
`concentration of 1 x 10-3 molar were compared (relative potencies -45.8 vs. -40.3 mmHg
`
`control vs. deuterated, p=NS). At these doses, despite an equivalent blood pressure
`
`effect, the duration of action of control nifedipine was 46.5 min and the duration of
`
`action of deuterated nifedipine was 62.2 min (p=.02). Thus, the duration of action of
`
`20
`
`deuterated nifedipine is greater than that of control nifedipine independent of blood
`
`pressure lowering effectiveness (i.e., both potency and duration of action differ).
`
`Figure 6 shows the effect of control and deuterated nifedipine on mean
`
`arterial pressure;: Figure 6(a) shows actual values plotted, Figure 6(b) shows curves
`
`fitted to data by logistic dose-response regression. A total of six .rats were tested at each
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`dose. As can be seen, at all doses deuterated nifedipine was more potent than control
`
`nifedipine (p < .05 by repeated measures ANOVA). MAP = mean arterial pressure
`
`Figure 7 shows concentration-effect relationships for control (Figure 7(a)) ·
`
`and deuterated (Figure 7(b)) nifedipine fitted using an asymmetric sigmoidal model.
`
`5
`
`Fitted values plus 95 % confidence intervals are shown. From these two graphs, based
`
`,I
`
`on a lack of overlap between the confidence inteivals, it is evident that the
`
`concentration-effect relationships for the two nifedipines differ. To be more precise,
`
`curve fitting was carried out using the equation: y=a+b(l-(1 +exp((x+dln(211e-l)-c)/d))·
`
`e). On this basis the parameters a, b and e all differed statistically at p < . 05.
`
`10
`
`Figure 8 shows concentration-effect relationships for control (Figure 8(a))
`
`and deuterated (Figure 8(b)) nifedipine fitted using logistic dose response model. Fitted
`
`values plus 95 % confidence inteivals are shown. From these two graphs, based on a
`
`lack of overlap between
`
`the confidence
`
`intervals,
`
`it
`
`is evident
`
`that
`
`the
`
`concentration-effect relationships for the two nifedipines differ. To be more precise,
`
`15
`
`curve fitting was carried out using the equation: y=a+b/(l+(x/c)"). On this basis the
`
`parameters a and ball differed statistically at p < .05.
`
`EXAMPLE VI
`
`Effect of deuterated control nicardipines on blood pressure
`
`in
`
`normotensive Sprague-Dawley rats. Control and deuterated nicardipines (prepared, as
`
`20
`
`described below, in a manner analogous to the preparation of deuterated nifedipine) were
`
`dissolved in minimal volumes of ethanol and diluted until the final concentration of
`
`ethanol was less than 0.04%. Doses of the two nicardipines as indicated were then
`
`injected into pentobarbital-anesthetized Sprague-Dawley rats weighing between 300 and
`
`350 grams. The maximum change in mean arterial pressure, as well as the duration of
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`hypotensive response, were measured directly by means of an intra-arterial catheter.
`
`Only one drug dose was given to each rat and a minimum of 60 minutes was allowed
`
`for blood pressure to return to baseline
`
`Potency-Figures 9a and 9b show the dose-response effect for the control
`
`5
`
`and deuterated (test) nicardipines. Although the confidence intervals of the curves
`
`overlap in areas, statistical comparison of ED16, ED50, and ED84 showed the following
`
`differences:
`
`ED16 control
`
`6. llxlo-6 mmoles
`
`95% CI: 2.2-16.Sxlo-6
`
`deuterated
`
`46.2xlo-6 mmoles
`
`37.3-57.2x1Q-6
`
`EDso control
`
`2.54x10-3 mmoles
`
`95 % CI: 1.53-4.20x.10-3
`
`deuterated
`
`2.llxl0·3 mmoles
`
`1.48-3.00xl0-3
`
`ED34 control
`
`1.06 mmoles
`
`95% CI: 0.38-2.91
`
`deuterated
`
`0.09 mmoles
`
`0.077-0.119
`
`,:, % Cl = ~, % connaence mterval
`
`15
`
`From the above table, it can be seen that the confidence intervals are
`
`exclusive for both ED16 and ED84• This indicates that the nature of the dose response
`
`relationships differs. This is confirmed by differing slope functions of 416 for control
`
`nicardipine and 46 for deuterated nicardipine (as calculated by Litchfield-Wilcoxon
`
`20
`
`method). Thus, the potencies of the two formulations differ.
`
`Two-way analysis of variance using dose and formulation as independent
`
`variables and reduction in mean arterial pressure as the dependent variable revealed the
`
`following ANOV A table:
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`factor.
`value
`
`d.f.
`
`sum of
`squares
`
`mean
`square
`
`PCT/CA95/00154
`
`F
`
`p
`
`5
`
`drug
`dose
`drug X dose
`
`1
`17
`17
`
`323
`5531
`6464
`
`323
`3255
`380
`
`3.11
`30.8
`3.6
`
`.. 082
`.0001
`.0001
`
`Although there is no difference at a p < . 05 level for drug differences
`
`10
`
`(there is a difference at the.! level, however), the lack of difference is likely due to the
`
`many doses clustered around the BD50 where the curves do not differ. There is a
`
`marked drug x dose interaction, however, which does implies a difference in the nature
`
`of the dose-response curves
`
`Duration of action-At most time periods the duration of effect was greater
`
`15
`
`for deuterated nicardipine compared to control nicardipine (Figure 10). Because the
`
`duration of response might be dependent on the magnitude of decrease in blood pressure,
`
`however, equipotent doses of the two formulations were compared. In this comparison,
`
`control nicardipine at a dose of 1 x 10-9 moles and deuterated nicardipine at a
`
`concentration of 3 x 10-9 moles were compared (relative potenci~ - 13. 8 vs -13. 8 mmHg
`control vs deuterated, p = NS). At these doses, despite an equivalent blood pressure
`
`effect, the duration of action of control nicardipine was 5.4 + 3.8 (SD) min and the
`
`duration of action of deuterated nicardipine was 15.0 + 6.4 (SD) min (p=.049 by
`
`Mann-Whitney U-test). Thus, the duration of action of deuterated nicardipine is greater
`
`than that of control nicardipine independent of blood pressure lowering effectiveness.
`
`Comparison of duration of action by two-way ANOV A using dose and
`
`formulation as independent variables and duration of action as the dependent variable
`
`revealed the following ANOV A table:
`
`20
`
`25
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`factor
`value
`
`d.f.
`
`sum of
`squares
`
`mean
`square
`
`PCT/CA95/00154
`
`F
`
`p
`
`5
`
`drug
`dose
`drug x dose
`
`1
`14
`14
`
`5697
`.. 68292
`10678
`
`5697
`4878
`237
`
`24. 0
`20. 6
`3. 2
`
`• 0001
`.0001
`.0002
`
`These results show significant differences in the duration of action of the
`
`10
`
`two drugs as well as implying a difference in the nature of the dose-duration of action
`
`relationship.
`
`Based on the above data, deuterated nicardipine differs from control
`
`nicardipine both in the nature of the blood pressure-lowering dose-response effect as well
`
`as in duration of action.
`
`15
`
`EXAMPLE VII
`
`Preparation of deuterated nicardipine. Such modification of nicardipine
`
`(and other dihydropyridines) can be achieved by dissolving the. nicardipine in a
`
`deuteration tube in a mixture of deuterochloroform and deuterium oxide, and then adding
`
`a minor amount of trifluoroacetic anhydride and deuterocacetone thereto and mixing
`
`20
`
`therewith. The solution is then frozen within the tube, preferably by immersing the tube
`
`in liquid nitrogen and then sealing the tube. The sealed tube is then heated at a
`
`temperature within the range of from about 50° to about 65°C, and preferably within
`
`the range of about 55° to about 60°C, and maintained at that temperature for a period
`
`of time sufficient to deuterate the methyl group at 2 3:Ild 6 positions on the nicardipine
`
`25
`
`to CD3• A time of from about 150 to about 180 hours is effective to complete the
`
`reaction, with a time of about 160 to 170 hours being preferred.
`
`Deuterated nicardipine was synthesized in the following manner: 80 mg
`
`of nicardipine in powder form was placed into a special deuteration tube and dissolved
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`therein in a mixture of 2 ml of deuterochloroform and 0.5 ml of deuterium oxide after
`
`which 0.2 ml of trifluoroacetic anhydride and 2 ml of deuteroacetone were added and
`
`.mixed therewith. The solution was frozen in liquid nitrogen and the tube flarried sealed
`under nitrogen. The tube was then heated at src for 168 hours after which it was
`
`5
`
`cooled and opened. The contents of the tube were transferred to a round-bottom flask
`
`and the solvent was removed in vacuo on a_ rotov~. All operations were conducted
`
`under reduced intensity of light. Using conventional 1H nuclear magnetic resonance
`
`(NMR) the deuterium substitution was calculated to be 95 % of the C-2 and C-6 methyl
`
`groups shown above.
`
`10
`
`Given the profound effects of substitution of deuterium for hydrogen in
`
`the methyl groups attached to positions 2 and 6 of the dihydropyridine ring, and given
`
`the fact that all dihydropyridine calcium channel blockers have at least one methyl group
`
`in these positions (in fact, all dihydropyridines have 2 methyls except amlodipine which
`
`only has a 6 methyl, substitution of the 2 methyl with CH20CH2CH2NH2 interestingly
`
`15
`
`drastically increases the duration of action of this dihydropyridine), deuteration of these
`
`groups in any other dihydropyridine would be expected to have the same effect as in
`
`nifedipine.
`
`EXAMPLE VIII
`
`Effect of Deuterated and Control Verapamils on Blood Pressure in
`
`20
`
`Normotensive Sprague-Dawley Rats.
`
`Deuteration ofveJ'i\Pil1)il-verapamil hydrochloride was added to a solution
`
`of 25 % deuterated sulfuric acid in deuterated water (v/v) and deuterated methanol. The
`
`solution was stirred for 140 hours at 90°C. The pH was adjusted to 12.0 and the
`
`mixture extracted with ethyl acetate. The combined ethyl acetate extracts were washed
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`with water, dried over magnesium sulfate and evaporated to yield a viscous oil. This
`
`oil was dissolved in ether and ethereal hydrochloride was added to precipitate the
`
`hydrochloride salt. The salt was collected by .