United States Patent
`Uda et al.
`
`119]
`
`[11] Patent Number:
`
`4,504,470
`
`[45] Date of Patent:
`
`Mar. 12, 1985
`
`[54] PHARMACEUTICAL PREPARATIONS
`CONTAINING TRH OR ITS ANALOGUE
`
`[56]
`
`References Cited
`U.S. PATENT DOCUMENTS
`
`[75]
`
`Inventors: Yoshiaki Uda; Takatsuka Yashiki,
`both of Takarazuka, Japan
`
`[73] Assignee:
`
`Takeda Chemical Industries, Ltd.,
`Osaka, Japan
`
`[21] App1.No.: 377,592
`
`[22] Filed:
`
`May 12, 1982
`
`Foreign Application Priority Data
`[30]
`May 14, 1981
`[JP]
`Japan ................................ .. 56/73293
`
`Int. c1.3 ................... .. A61K 37/00; co7c 103/52
`[51]
`[52] U.S. C1. ........................ .. 514/19; 260/112.5 TR
`[58] Field of Search ............................... .. 424/177, 14;
`260/112.5 TR
`
`............ .. 260/112.5 TR
`5/1976 Fujino et al.
`3,959,247
`4,059,692 11/1977 Takahashi et al.
`.
`....... .. 424/177
`
`4,211,769
`7/1980 Okada et al.
`..... ..
`..
`4,250,163
`2/1981 Nagai et a1.
`......................... .. 424/14
`
`Primary Exczminer—De1bert R. Phillips
`Attorney, Agent, or Firm—Wenderoth, Lind & Ponack
`
`[57]
`
`ABSTRACT
`
`A non-parenteral pharmaceutical preparation contain-
`ing TRH, its salt, or its analogue is produced by em-
`ploying the drug and a hydroxycarboxylic acid or poly-
`carboxylic acid of 2 to 8 carbon atoms, and having the
`pH of the preparation adjusted to 2 to 6.
`
`11 Claims, No Drawings
`
`   
`


`
`Lannett Holdings, Inc. LAN 1012
`
`

`
`1
`
`4,504,470
`
`(I1)
`
`R-NH—CH'—CO-N
`
`CO—NH—A
`
`[A is H, alkyl, aralkyl, alkoxyalkyl, hydroxyalkyl or
`alkoxy; R is
`
`co— or -0
`
`0
`T ]—co—
`
`HN
`
`s
`
`0*
`
`and when A _is other than H, may also be
`
`¢ CO—; X is "'CH2'—, —CH2CH2— or "'S"';
`
`NH
`
`0
`
`R and each of the other constituent amino acid residues
`may have L- or D-configuration or be racemic] and
`salts thereof (Japanese Patent Application Laid-open
`No. 116465/1977; U.S. Pat. No. 4,100,152).
`A
`In said formula (II), the alkyl represented by A pref-
`erably contains 1
`to 10 carbon atoms and to straight-
`chained or branched, and may for example be methyl,
`ethyl, propyl, i-propyl, butyl, sec-butyl,
`tert-butyl,
`i-
`butyl, amyl, hexyl, octyl, nonyl or decyl. The aralkyl
`represented by A is preferably one having the formula:
`
`_.R'
`
`wherein R’ is alkylene. The alkylene may for example
`be methylene, ethylene, 1,3-trimethylene (—CH2CH2C-
`I-I2—), propylene
`
`CH3
`1
`(‘CH-CH2—),
`
`tetramethylene (——CH2CH2CH2CH2—) or 2-methyl-
`trimethylene
`
`as
`(— CI-I2CH2CH2—).
`
`The alkoxyalkyl represented by A preferably contains
`up to 9 carbon atoms and is straight-chained or
`branched and may for example be methoxymethyl,
`methoxyethyl, propoxypropyl, butoxybutyl or methox-
`yoctyl. The alkoxy represented by A preferably con-
`tains up to 9 carbon atoms and is straight-chained or
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`PHARMACEUTICAL PREPARATIONS
`CONTAINING TRH OR ITS ANALOGUE
`
`This invention relates to a non-parenteral pharmaceu-
`tical preparation containing L-pyroglutamyl-L—histidyl-
`L-prolinamide (thyrotropin releasing hormone, herein-
`after referred to briefly as TRH) or its analogue.
`TRH is known to stimulate the release of thyroid
`stimulating hormone from the pituitary gland and, also,
`to act on the central nervous system, and there are also
`several known analogues thereof which have activities
`similar to those of TRH.
`TRH and its analogues are all peptides, and because
`peptides generally are only sparingly lipophilic and are
`susceptible to enzymatic degradation in the gastrointes-
`tinal tract, these compounds are used almost exclusively
`as parenteral preparations. However, injections require
`the skilled hands of specialists and cause pain in recipi-
`ents. Therefore, especially for repeated-dose adminis-
`tration, more convenient, easy-to-use dosage forms are
`desirable.
`To overcome the above problem, the present inven-
`tors explored the possibility of developing non-parent-
`eral dosage forms for TRH or its analogue. As a result,
`it was discovered that if a hydroxycarboxylic acid or
`polycarboxylic acid is incorporated in a preparation of
`TRH or its analogue, there can be expected an efficient
`absorption of the active compound in vivo even when it
`is administered by a non-parenteral route. This inven-
`tion has been accomplished on the basis of the above
`finding.
`This invention is therefore concerned with a non-par-
`enteral pharmaceutical preparation containing TRH, its
`salt or its analogue and a hydroxycarboxylic acid or
`polycarboxylic acid of 2 to 8 carbon atoms, or tropic
`acid and having its pH adjusted to 2 to 6, and with a
`method for producing a non-parenterally administered
`drug form which comprises using a therapeutically
`effective dosage amount of L-pyroglutamyl-L-histidyl-
`L-prolinamide, its salt or its analogue capable of being
`absorbed into the blood stream and an adjuvant of hy-
`droxycarboxylic acid or polycarboxylic acid of 2 to 8
`carbon atoms, or tropic acid, said adjuvant being pres-
`ent in said drug form in a sufficient amount to be effec-
`tive in enhancing said absorption ratio, and wherein the
`pH of said drug form is adjusted to 2 to 6.
`TRH, which is employed in accordance with this
`invention, has the following structure:
`
`(I)
`
`CONH2
`
`CO--NH-CH—CO—N
`
`1C
`
`H2
`
`%\ N
`
`I-IN-J
`
`O
`
`¢: ;\N
`
`H
`
`Salts of TRH include those with various acids (e.g.
`tartaric acid, oxalic acid, fumaric acid, citric acid, malic
`acid, acetic acid, lactic acid, oleic acid, palmitic acid,
`etc.) and is preferably the tartrate (U.S. Pat. No.
`3,957,247, Japanese Patent Application Laid-open No. 65
`121273/1975).
`Analogues of TRH include peptides of the following
`formula:
`
`

`
`3
`
`4,504,470
`
`5
`
`I0
`
`15
`
`20
`
`25
`
`30
`
`35
`
`branched, and may for example be methoxy, ethoxy,
`propoxy,
`i-propoxy, butoxy, sec-butoxy,
`tert-butoxy,
`i-butoxy, pentyloxy, hexyloxy, octyloxy, nonyloxy or
`decyloxy. The hydroxyalkyl represented by A prefera-
`bly contains up to 9 carbon atoms, and may for example
`be the same alkyl represented by A which is substituted
`by hydroxy on an optional position.
`Among the compounds represented by the formula
`(II),
`‘y-butyrolactone-'y-carbonyl-L-histidyl-L-prolina-
`mide represented by the formula:
`
`97/L
`
`o
`
`0
`
`CO“NH‘-‘(III-l"'CO—N
`CH2
`/k N
`
`NH——-—"
`
`CONI-I2
`
`is preferable. In this specification,
`referred to briefly as “DN-1417”.
`The TRH analogue also includes tri-peptides of the
`general formula:
`
`the compound is
`
`‘R
`/ 3
`
`C-N
`
`CH2
`
`\ /
`
`R1
`
`/
`
`C
`/
`X
`_.
`\CH2
`(1: N CH2
`o=c/ \c
`/
`\
`/‘R2 1°“
`\
`NH —' CH-CONH-CH-—'CO'-N 1- CH-CON}-[2
`
`4
`[R is H or C1_3 alkyl; R1 is C1_3 alkyl or alkoxy; R3 is H
`or C1_3 alkyl or alkoxy; R3 is H, C1_5 alkyl or C3_6 cyclo-
`alkyl; and R4 is H or C1-5 alkyl] and salts thereof (U.S.
`Pat. No. 4,060,603, Japanese Patent Application Laid-
`open No. l15272/ 1976).
`The hydroxycarboxylic acid of 2 to 8 carbon atoms
`which is employed in accordance with this invention is
`exemplified by lactic acid, gluconic acid, malic acid.
`tartaric acid, citric acid, salicylic acid, mandelic acid,
`etc., and mandelic acid is preferable.
`The polycarboxylic acid of 2 to 8 carbon atoms is
`exemplified by oxalic acid, fumaric acid, maleic acid.
`malonic acid, succinic acid, glutaric acid, etc., and suc-
`cinic acid is preferable.
`Tropic acid is also preferably used in this invention.
`The addition level of said hydroxycarboxylic acid or
`polycarboxylic acid, based on the total weight of the
`composition, is at least about 0.1 weight percent. prefer-
`ably at least about 1 weight % and, for still better re-
`sults, at least about 3 weight %. The upper limit is about
`50 weight %, preferably about 30 weight % and. for
`still better results, about 20 weight %.
`The above-mentioned hydroxycarboxylic acid or
`polycarboxylic acid may be added in the form of a
`buffer solution. Examples of the buffer solution include
`Sdrensen
`buffer
`[Ergebniss
`der Physiologic
`l2.
`393( 1912)], Michaelis buffer [Die Wasserstoffionenl<on-
`zentration p. l86(19l4)], Kolthoff buffer [Biochemische
`Zeitschrift 179, 4l0(1926)], Mcllvaine buffer [Journal of
`Biological Chemistry 49,
`l83( 1921)] and so on.
`When said carboxylic acid is added in the form of a
`buffer solution, the amount of the acid itself should be
`within the above-mentioned range.
`The term “non-parenteral pharmaceutical prepara-
`tion” is used herein to denote any of rectal dosage forms
`(e.g. suppositories, rectal capsules, infusions, etc.), nasal
`dosage forms (e.g.
`liquids, jellies, ointments, aerosols.
`inhalants, etc.), oral cavity dosage forms (e.g.
`tablets.
`buccals, troches, etc.) and oral dosage forms (e.g. tab-
`0 lets, capsules, pills, granules, granulets, powders,
`liq-
`uids, syrups, etc.).
`Production of the non-parenteral pharmaceutical
`preparations according to this invention is conducted
`by an established procedure for producing non-parent-
`eral preparations.
`Thus, a preparation for rectal administration can be
`produced by adding said hydroxycarboxylic acid or
`polycarboxylic acid and TRH, its salt or its analogue to
`an oleaginous or aqueous basis, warming the mixture to
`a suitable temperature to dissolve or disperse them.
`pouring the resulting solution or dispersion into a mold.
`and cooling it, by way of example, all
`in the per se
`conventional manner.
`
`[X is O or S; R1, R2 and R3 each is H or lower alkyl] and
`salts thereof (U.S. Pat. No. 3,912,705, Japanese Patent
`Application Laid-open No. 108075/1974); peptides of
`the formula M1-—M2—-M3-—E [(a) M1 is selected from
`the class consisting of kio, kpc and pea, (b) M2 is se-
`lected from the class consisting of his, N3'''"-lower alkyl-
`his and N3”"-(CH2)1,—COOH—his (where b is an integer
`of 1 to 4), (c) M3 is selected from the class consisting of
`L-pip, L-pro and L-tca and (d) E is selected from the
`class consisting of —NH2 and -—OR (where R is C1_1o
`alkyl); provided, however, that (i) when E is —NH2,
`pca and L-pro do not occur concurrently in the tripep-
`tide and (ii) when E is —OR, his and L-pro do not occur
`concurrently in the tripeptide] and salts thereof [in the
`above formula, kic is 2-ketoimidazolidine; kpc is 2-
`ketopiperidine-6-carboxylic acid; pca is pyroglutamic
`acid; his is histidine; L-pip is L-2-piperidinecarboxylic
`acid; pro is proline; and tca is thiazolidine-5-carboxylic
`acid] (U.S. Pat. No. 3,959,248, Japanese Patent Applica-
`tion Laid-open No. 154247/1975); and compounds of
`the formula:
`
`Oco—NH—pHeco—N
`
`CH2
`
`0% N
`l
`H
`
`/ N
`JN
`
`/R
`
`R1
`
`R2
`3 4
`CONR R
`
`45
`
`50
`
`55
`
`60
`
`65
`
`When, for instance, an aqueous basis is used in the
`production of a rectal preparation, the desired prepara-
`tion can be obtained by dissolving or dispersing said
`hydroxycarboxylic acid or polycarboxylic acid and
`THR, its salt or analogue evenly in the aqueous basis.
`pouring the solution or dispersion into a mold and cool-
`ing the same. Examples of said aqueous suppository
`bases include polyethylene glycol, glycero-gelatin. etc.,
`and the preferred degree of polymerization of said poly-
`ethylene glycol is not less than 100. e.g. 200, 300. 400.
`1000, 4000 and 6000. Such aqueous bases may be used
`alone or in admixture, and may also contain additives
`such as methylcellulose, carboxymethylcellulose. etc.
`When an oleaginous basis is used in the production of
`a rectal preparation,
`the desired preparation can be
`
`

`
`5
`obtained by dissolving or dispersing said hydroxycar-
`boxylic acid or polycarboxylic acid in a fused mass of
`said oleaginous basis, then adding TRH, its salt or its
`analogue and dispersing the mixture evenly under ap-
`propriate heating and stirring or molding it. Alterna-
`tively, such a preparation may be produced by dispers-
`ing said hydroxycarboxylic acid or polycarboxylic acid
`in the oleaginous basis, dispersing an aqueous solution
`of TRH, its salt or its analogue, and molding the compo-
`sition. Thus, these and other per se conventional proce-
`dures can be utilized to produce the desired preparation.
`The above-mentioned oleaginous basis is exemplified
`by various oils and fats such as sesame oil, olive oil, corn
`oil, soybean oil, cotton-seed oil, peanut oil, castor oil,
`cacao butter, laurin fat, beef fat, lard, wool fat, squalene,
`etc.; and hydrogenolysis or fatty acid exchange reaction
`products thereof; mineral oils such as Vaseline, paraffin,
`isopar, silicone oil, etc; glycerin esters of C6_3o fatty
`acids, especially higher fatty acid esters such as glycerin
`palmitate, glycerin laurate, glycerin stearate, glycerin
`myristate, etc.; C(,_3o fatty acid esters of C2-g alcohols,
`especially waxes such as isopropyl myristate, butyl stea-
`rate, diisopropyl adipate, diethyl sebacate, etc.; and
`higher fatty acids containing 6 to 30 carbon atoms such
`as stearic acid, oleic acid, etc. These oils, fats and fatty
`acids may be used alone or in admixture. For the pro-
`duction of oleaginous suppositories, cacao butter, laurin
`fat, fatty acid exchange oil (e. g. mono-, di- and tri-glyce-
`rides of higher fatty acids such as palmitic acid, stearic
`acid, etc.), etc. are especially desirable.
`For the production of nasal dosage forms, the above-
`mentioned various components are admixed in an op-
`tional order according to the established pharmaceuti-
`cal procedure. For example, an aqueous liquid for nasal
`administration can be prepared by dissolving, suspend-
`ing or emulsifying TRH, its salt or its analogue and said
`hydroxycarboxylic acid or polycarboxylic acid in wa-
`ter, a buffer solution or an aqueous solution. An aqueous
`gel for nasal administration, for instance, can be pro-
`duced in the following manner. First, the hydroxycar-
`boxylic acid or polycarboxylic acid is dissolved in water
`and, if necessary, a pH adjusting agent, a preservative,
`etc. are added to the aqueous solution. This solution is
`divided into halves, and a gel basis is dissolved or dis-
`persed in one half, followed by heating at a suitable
`temperature or cooling to give a stable gel. TRH, its salt
`or its analogue is dissolved in the other half of said
`solution. The two solutions are evenly admixed to pro-
`vide an aqueous gel.
`Examples of the aqueous gel basis include natural
`gums (e.g. gum tragacanth, gum acasia, gum karaya,
`island moss, gum guaiac, gum xanthane,
`locust bean
`gum, etc.), cellulose derivatives (e.g. methylcellulose,
`carboxymethylcellulose, etc.), acrylic acid polymers
`(polyacrylic acid, polymethacrylic acid, etc.), vinyl
`polymers (e.g. polyvinyl pyrrolidone, polyvinyl alco-
`hol, polyvinyl methyl ether, carboxypolymethylene,
`etc.), synthetic polysaccharides (e.g. polysucrose, poly-
`glucose, polylactose, etc.), starch, dextrin, pectin, so-
`dium alginate, etc. These bases may be usedin the form
`of appropriate mixtures of two or more species.
`Oil preparations for nasal administration can also be
`produced by dissolving, suspending or emulsifying
`TRH, its salt or its analogue and said hydroxycarboxy-
`lic acid or polycarboxylic acid in an oleaginous basis.
`As examples of said oleaginous basis, there may be men-
`tioned oils and fats such as sesame oil, olive oil, corn oil,
`soybean oil, cotton-seed oil, peanut oil, etc.; mineral oils
`
`l0
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`4,504,470
`
`6
`such as Vaseline, paraffin, isopar, silicone oil, etc.; glyc-
`erin esters of C(,_3o fatty acids, especially higher fatty
`acid esters such as glycerin palmitate, glycerin laurate,
`glycerin stearate, glycerin myristate, etc.; C6_3o fatty
`acid esters of C2_g alcohols, especially such waxes as
`isopropyl myristate, butyl stearate, diisopropyl adipate,
`diethyl sebacate, etc.; and higher (C(,_3o) fatty acids,
`especially stearic acid and oleic acid. These oils, fats and
`fatty acids can be used alone or in admixture.
`Preservatives may be incorporated in nasal prepara-
`tions. Examples of such preservatives include p-hydrox-
`ybenzoic acid esters; phenolic compounds such as phe-
`nol, cresol, etc.; alcohols such as chlorobutanol, phenyl-
`ethyl alcohol, propylene glycol, etc.; invert soaps such
`as benzalkonium chloride, benzethonium chloride, etc.;
`benzoic acid, sorbic acid, dehydroacetic acid and sulfu-
`rous acid and salts thereof; acids and their salts such as
`sodium hydrogen sulfite.
`Oral cavity and oral dosage forms can be produced
`by the per se conventional procedures. Taking tablets as
`an example, the desired preparation can be produced by
`mixing TRH, its salt or its analogue, said hydroxycar-
`boxylic acid or polycarboxylic acid and a lubricating
`agent
`into an excipient mixture and, after thorough
`mixing, compressing the composition into tablets. The
`excipients which can be used for this purpose include,
`among others, spray-dried lactose, starch, microcrystal-
`line cellulose, hydroxypropylcellulose, polyvinylpyr-
`rolidone, etc., and the lubricating agent may be selected
`from among the agents commonly used in the produc-
`tion of tablets, such as stearic acid compounds (e.g.
`magnesium stearate, calcium stearate, stearic acid, etc.),
`talc and so on. The quantitiesand kinds of such excipi-
`ents and lubricating agents are selected from within
`those ranges of strength and disintegration characteris-
`tics which warrant practically useful tablets.
`For example, tablets may be produced by mixing 2
`mg of TRH, its salt or its analogue and 1 mg of magne-
`sium stearate into a mixture of 80 mg of spray-dried
`lactose, 9 mg of starch and 18 mg of microcrystalline
`cellulose and compression-molding the composition to
`give 110 mg tablets.
`Production of granules may be effected, for example,
`by charging a mixer with TRH, its salt or its analogue,
`adding a starch solution prepared by heating a 10%
`dispersion of corn starch at a suitable temperature,
`kneading the mixture, drying the same at a suitable
`temperature in vacuum and milling the dry mixture to
`give granules.
`To produce granules, granular sugar, corn starch,
`hydroxypropylcellulose (HPC), etc. are admixed with
`TRH, its salt or its analogue in a mixer and with a 50:50
`(v/v) mixture of water and ethyl alcohol being sprayed
`from a nozzle, the composition is kneaded and granu-
`lated, followed by drying in a fluidized bed dryer.
`PH of preparations according to this invention is
`adjusted to 2 to 6. The pH of such preparations is mea-
`sured as follows. In the case of aqueous preparations
`such as aqueous solutions, aqueous gels, etc., a single
`dose unit is added to 10 ml of distilled water and the pH
`of the solution is measured. In the case of tablets, cap-
`sules, granules, granulets, powders and aqueous suppos-
`itories, for instance, a single dosage unit is dissolved in
`10 ml of distilled water and the pH of the solution is
`measured. In the case of oil preparations inclusive of oil
`suppositories, a single dosage unit is added to 10 ml of
`distilled water, dispersed and dissolved under stirring
`
`

`
`4,504,470
`
`8
`Germany) suppository, 50 mg of either suppository was
`deposited 1.5 cm from the anus.
`After administration, 0.2 ml of blood was taken from
`the cordal vein at timed intervals and the plasma radio-
`activity was measured with a scintillation counter. Sep-
`aration of the unmetabolized compound from metabo-
`lites was performed by thin-layer electrophoresis. For
`the evaluation of absorption rates,
`the area under a
`curve of the plasma concentration of DN—l4l7 over a
`period of 6 hours after administration [AUC (0-6 hr. )]
`was compared with the corresponding value obtained
`by subcutaneous administration to calculate the bi-
`oavailability of the drug. The results are presented in
`Table 1.
`
`TABLE 1
`
`
`7
`and centrifuged, and the pH of the aqueous layer is
`measured at room temperature.
`Adjustment of the pH of preparations can be effected
`by adding an acid, a base, a buffer solution or the like in
`the course of production of the preparations. As exam-
`ples of the acid, there may be mentioned inorganic acids
`(e.g. hydrochloric acid, boric acid, phosphoric acid,
`carbonic acid, bicarbonic acid, etc.), amino acids and
`organic acids (e.g. monocarboxylic acids), and the hy-
`droxycarboxylic acids and polycarboxylic acids re-
`ferred to hereinbefore. The base is exemplified by so-
`dium hydroxide, potassium hydroxide, sodium hydro-
`gen carbonate, sodium carbonate, etc. The buffer solu-
`tion is exemplified by Sorensen buffer [Ergebniss der
`Physiology 12, 393(l912)], Clark-Lubs buffer [Journal
`of Bacteriology 2, (1), 109, l91(l971)], Mcllvaine buffer
`[Journal Biological Chemistry 49, l83(1921)l» Michaelis
`buffer
`[Die Wasserstoffionenkonzentration,
`p.
`l86(l914)], Kolthoff buffer [Biochemische Zeitschrift
`179, 4l0(1926)] and so on.
`The dosage of any of the pharmaceutical preparations
`according to this invention varies with the kind of the
`active medicament, dosage form, subjects to be treated
`(e.g. mouse, rat, horse, cattle, man and other warm-
`blooded mammals) and the object of administration.
`Taking man as an example, a unit dose as the active
`medicament is 2 to 20 mg in the case of nasal or rectal
`administration, and 2 to 40 mg in the case of oral cavity
`or oral administration.
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`This invention has the following advantageous fea-
`tures.
`
`(l) A sufficient medical effect can be realized at a low
`dose level with high efficiency. When it is investi-
`gated whether an increased absorption of the drug
`leads to its increased bioavailability which, in turn,
`results in an improved antagonistic action to pen-
`tobarbital-induced sleep in rats,
`it is clear that an
`improved bioavailability leads to an improved effi-
`cacy.
`(2) It causes little pain at the site of administration and
`can be used expediently.
`(3) For repeated-dose administration, self-administra-
`tion and, hence, home therapy are feasible.
`(4) As TRH, its salt or its analogue is released very
`gradually from preparations, the blood level and,
`hence,
`the clinical efficacy of the drug are sus-
`tained for a long time.
`The following experimental and working examples
`are intended to illustrate this invention in further detail.
`It should be understood that all percents (%) are by
`weight (w/w %) unless otherwise specified.
`EXPERIMENTAL EXAMPLE 1
`
`Male rats of SD strain weighing ca. 300 g, which had
`been fasted for 24 hours, were closed with an amount of
`14C-DN-1417 corresponding to 2 mg/kg of DN-1417 by
`the rectal, nasal, oral cavity and oral routes. The dosage
`form for nasal, oral cavity or oral administration was
`either an aqueous solution or an oil solution, and for
`nasal administration, 0.02 ml of the solution was given
`with a micropipette; for oral cavity application, a wad
`of cotton soaked with 0.02 ml ofthe solution was sublin-
`gually administered; and for oral administration, 1 ml of
`the solution was given by gavage. For rectal administra-
`tion, 0.1 ml of an aqueous solution was given using a
`pipette. In the case of a polyethylene glycol suppository
`and Witepsol W-35 (Dynamit Nobel Chemicals, West
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`Absorption ratio of DN-I417 (2 mg/kg)
`administered by various routes to rats
`AUC (0-6 hr.) Eg - ml gn = 3)
`Subcu-
`Test material
`Rectal
`Nasal
`Oral
`Laneous
`
`Physiological saline
`(pH 3.8)
`5.6
`Water
`(pH 3.8)
`5% Acetic acid
`(pH 2.4)
`5% Citric acid
`(pH 1.9)
`5% Lactic acid
`(pH 2.0)
`5% Gluconic acid
`(pH 2.5)
`5% l-Malic acid
`(pH 1.8)
`5% dl-Mandelic
`(pH 2.1)
`acid
`2% dl-Tropic acid
`Witepsol W-35
`5% dl-Mandelic
`acid +
`Witepsol W-35
`
`3.2
`3.0
`
`3.4
`
`0.5
`1.5
`L6
`
`2.0
`3.0
`
`1.4
`-1.3
`3.9
`4.7
`3.3
`4,3
`0.1
`
`5.1
`2.1
`4.2
`
`(pH 2.3)
`
`It will be apparent from Table 1 that, as compared
`with oral administration, rectal and nasal applications
`resulted in greater bioavailability and efficient absorp-
`tion. However, these bioavailabilities are as low as 25%
`and 21%, respectively, as compared with subcutaneous
`application and it is clear that an enhancement of ab-
`sorption is necessary. Keeping the pH of the solutions
`on the acidic side is, by itself, not sufficient to enhance
`absorption in any appreciable measure, and the addition
`of amino acids, lecithin, etc. does not result in any sig-
`nificant
`improvement of absorption.
`In contrast.
`the
`addition of a hydroxycarboxylic acid results in a re-
`markable improvement in absorption of DN-I417 and
`the effect of dl-mandelic acid is especially notable. Ab-
`sorption from the Witepsol W-35 suppository is also
`high. While the addition of lower fatty acids such as
`acetic acid leads to a marked increase of absorption.
`there is the problem of acetic acid odor. Moreover.
`local
`irritations are expected, so that these acids are
`undesirable in practical use.
`
`EXPERIMENTAL EXAMPLE 2
`
`Using male SD-strain rats weighing ca. 300 g which
`had been fasted for 24 hours in groups of 3 animals. an
`amount of 3H-TRH equivalent to 2 mg/kg of TRH was
`administered by the rectal, nasal and oral routes.
`the
`The method and volume of administration.
`method of collecting blood samples and the method of
`determination were all the same as described in Experi-
`mental Example 1. Evaluation of absorption ratio was
`made using as a parameter the plasma level of TRH.
`The results are shown in Table 2.
`
`

`
`4,504,470
`
`9
`TABLE 2
`Absorption ratio of TRH (2 mg/kg)
`administered by various routes to rats
`AUC (0-2 hr.) Hg - hr/ml in = 3)
`
`OralNasalRectalTest material 5
`
`
`
`0.7
`1.0
`0.7
`Water
`(pH 3.2)
`5% dl-Mandelic
`(pH 2.1)
`2.4
`2.2
`1.1
`acid
`
`
`10
`were added, followed by thorough stirring. The result-
`ing composition was poured into a 1-gram suppository
`mold and cooled gradually to give a 1-gram supposi-
`tory. This suppository (1 unit) was put in 10 ml of dis-
`tilled water, melted by warming, and after stirring, was
`allowed to cool to room temperature. After centrifuga-
`tion, the pH of the water layer was measured with a
`Horiba F-7LC pH meter (Horiba Seisakusho Japan).
`The pH found was 4.1.
`
`It will be apparent from Table 2 that the absorption of 10
`TRH from the rectal and nasal mucosa is enhanced by
`the addition of dl-mandelic acid.
`
`EXPERIMENTAL EXAMPLE 3
`
`Witepsol W-35 suppositories containing 2 mg/kg
`equivalent of DN-1417 and 5 w/v % of one of the ab-
`sorption promoting agents mentioned below were rec-
`tally administered to groups of 10 male rats of SD strain
`(4 weeks old) weighing about 80 grams, and the expel
`ratio of the suppositories and the changes in the epithe-
`lium of the local mucous membrane of the rectum after
`10 days of administration (once daily) were investigated
`by scanning electron microscopy.
`The results of the expel ratio test using as a control a
`similar suppository which does not contain any absorp-
`tion promoting agent are shown in Table 3.
`TABLE 3
`
`Expel ratio of rectal suppositories
`in rats
`After
`After
`After
`30 min.
`20 min.
`10 min.
`:._____.__Z_.__._____.___.__—_._._.____
`2/10
`2/ 10
`2/ 10
`Witepsol W-35 suppository
`Suppository with
`1/10
`3/10
`3/10
`5% dl-mandelic acid
`3/10
`3/10
`1/10
`Suppository with
`5% 1-malic acid
`N/10: The number of animals which excreted the suppositories per 10 animals.
`
`15
`
`20
`
`25
`
`30
`
`35
`
`The scanning electron microscopic findings after 10
`consecutive days of administration are not different 40
`from those for the control Witepsol W-35 suppository.
`It is, therefore, apparent that the above acids are not
`specially irritant to the local mucous membrane of the
`rectum and are highly safe absorption enhancing agents.
`EXAMPLE 1
`
`45
`
`9.316 Grams of Witepsol W-35 was weighed into a
`mortar in which it was melted at 40° to 45° C. Then, 500
`mg of dl-mandelic acid was added and the mixture was
`stirred well under warming. Thereafter, 183.6 mg (120
`mg as DN-1417) of crystals of DN-1417 citrate were
`added, followed by thorough stirring. The composition
`was then poured into a 1-gram suppository mold and
`cooled gradually to give a 1-gram suppository.
`This suppository (one unit) was put in 10 ml of dis-
`tilled water, melted by warming, and after stirring, was
`cooled to room temperature. It was then centrifuged
`and the pH of the water layer was measured with a
`Horiba H-7Lc pH meter (Horiba Seisakusho Japan).
`The pH found was 2.6.
`EXAMPLE 2
`
`50
`
`55
`
`60
`
`8.906 Grams of Witepsol W-35 was weighed into a
`mortar in which it was melted by warming at 40°—45° C.
`Then, 500 mg of dl-mandelic acid and 410 mg of 65
`disodium phosphate were added. The mixture was
`stirred thoroughly under warming. Thereafter, 183.6
`mg (120 mg as DN-1417) of crystals of DN-l417.citrate
`
`EXAMPLE 3
`
`The procedure of Example 1 was repeated under the
`conditions indicated in Table 4 to produce 1-gram sup-
`positories containing 18.36 mg of DN-1417 citrate.
`TABLE 4
`Suppos-
`The level of
`The kind of
`itory
`addition of
`pH
`acid added
`No.
`the acid (mg)
`2.41
`Lactic acid
`3-1
`50
`. 2.62
`Gluconic acid
`3-2
`50
`2.41
`Malic acid
`3-3
`50
`2.46
`Tartaric acid
`3-4
`50
`2.50
`Citric acid
`3-5
`50
`2.40
`Salicylic acid
`3-6
`50
`2.15
`Oxalic acid
`3-7
`50
`2.4
`Fumaric acid
`3-8
`50
`2.17
`Maleic acid
`3-9
`50
`2.39
`Malonic acid
`3-10
`50
`2.74
`Succinic acid
`3-11
`50
`
`
`Glutaric acid 503-12 2.84
`
`
`EXAMPLE 4
`
`The procedure of Example 2 was repeated under the
`conditions indicated in Table 5 to produce 1-gram sup-
`positories containing 18.36 mg of DN-1417 citrate.
`TABLE 5
`
`The level of
`Suppos-
`itory
`The kind of
`addition of
`
`No.
`acid added
`acid (mg)
`pH
`4-1
`Lactic acid
`50
`3.8
`4-2
`Gluconic acid
`50
`4.4
`4-3
`Malic acid
`50
`3.8
`4-4
`Tartaric acid
`50
`3.9
`4-5
`Citric acid
`50
`4.0
`4-6
`Salicylic acid
`50
`3.8
`4-7
`Oxalic acid
`50
`3.6
`4-8
`Fumaric acid
`50
`3.8
`4-9
`Maleic acid
`50
`3.6
`4-10
`Malonic acid
`50
`3.9
`4-11
`Succinic acid
`50
`4.3
`4-12
`Glutaric acid
`50
`4.4
`
`
`EXAMPLE 5
`
`9.316 Grams of a mixture of 75 w/w % of polyethyl-
`ene glycol (PEG) 1000 and 25 w/w % of PEG 4000
`was put in a mortar and melted by warming at 50° to 60°
`C. Then, dl-mandelic acid and DN-1417 citrate were
`added and the resultant composition was treated as in
`Example 1 to produce 1-gram suppositories. This prepa-
`ration (1 unit) was put in 10 ml of distilled water and the
`pH was measured. The pH found was 2.8.
`EXAMPLE 6
`
`8.906 Grams of a mixture of 75 w/w % of polyethyl-
`ene glycol (PEG) IOOO and 25 w/w % of PEG 4000
`was put in a mortar and melted by warming at 50°-60°
`C. After addition of dl-mandelic acid, disodium phos-
`phate and DN-1417 citrate, the composition was treated
`
`

`
`11
`in the manner as Example 2 to produce l-gram supposi-
`tories. This preparation (1 unit) was put in 10 ml of
`distilled water and the pH was measured. The pH found
`was 4.4.
`
`4,504,470
`
`EXAMPLE 7
`
`In 10 ml of physiological saline were dissolved 500
`mg of dl-mandelic acid and 410 mg of disodium phos-
`phate. Then, 367.2 mg of DN-1417 citrate was dissolved
`to produce a nasal preparation (12 mg/0.5 ml as DN- 10
`1417). One dosage unit (0.5 ml) of this preparation was
`dissolved in 10 ml of distilled water and the pH of the
`solution was measured. The pH found was 4.0.
`EXAMPLE 8
`
`15
`
`In the manner as Example 7, nasal preparations con-
`taining 18.36 mg of DN-1417 citrate were produced
`under the conditions indicated in Table 6. The single
`dosage unit of each preparation was 0.5 ml.
`
`TABLE 6
`Prepara-
`The level of
`tion
`The kind of
`addition of
`
`No.
`acid added
`the acid (mg)
`pH
`
`8-1
`8-2
`8-3
`8-4
`8-5
`8-6
`
`Lactic acid
`Gluconic acid
`Malic acid
`Tartaric acid
`Citric acid
`Salicylic acid
`
`25
`25
`25
`25
`25
`25
`
`3.8
`4.1
`4.0
`3.8
`3.8
`3.8
`
`3.9
`25
`Fumaric acid
`8-8
`3.5
`25
`Oxalic acid
`8-7
`3.6
`25
`Maleic acid
`8-9
`3.8
`25
`Malonic acid
`3-10
`3-11
`Succinic acid
`25
`4.3
`3-12
`Glutaric acid
`25
`4.3
`
`
`20
`
`25
`
`30
`
`35
`
`EXAMPLE 9
`
`40
`
`500 Milligrams of dl-mandelic acid and 410 mg of
`disodium phosphate were dispersed in 10 ml of Miglyol
`812 (Dynamit Nobel, West Germany) under warming at
`about 40° C. Then, 368.2 mg of DN-1417 citrate was
`dispersed to produce a nasal preparation (12 mg/0.5 ml
`as DN-1417). A single dosage unit (0.5 ml) of this prepa-
`ration was added to 10 ml of distilled water, dispersed
`under shaking, and centrifuged. The pH of the water 45
`layer was 4.1.
`'
`The above procedure was repeated except that ses-
`ame oil was used in lieu of Miglyol to produce a nasal
`preparation. The pH of this preparation as measured in
`the manner as above was 4.1.
`
`50
`
`EXAMPLE 10
`
`12.5 Milligrams of dl-mandelic acid was milled in a
`mortar, followed by addition of 176.3 mg of a 3:7 mix-
`ture of starch and lactose. After thorough mixing, 61.2
`mg of DN-1417 citrate was added. To the mixture was
`further added 0.75 mg of magnesium stearate and after
`thorough mixing, the composition was tableted to give
`tablets for oral/oral cavity administration. One of the
`tablets was dissolved in 10 ml of distilled water and the 60
`pH of the solution was measured. The pH found was
`3.2.
`
`55
`
`EXAMPLE 11
`
`The procedure of Example 10 was repeated under the 65
`conditions set forth in Table 7 to produce oral and oral
`cavity preparations containing 61.2 mg of DN-1417
`citrate.
`
`12
`TABLE 7
`
`The level of
`Tablet
`The kind of
`addition of
`
`No.
`acid added
`the acid (mg)
`3H
`11-1
`Lactic acid
`22.5
`3.02
`11-2
`Gluconic acid
`12.5
`I. 16
`11-3
`Malic acid
`12.5
`.'-.10
`11-4
`Tartaric acid
`12.5
`2.97
`11-5
`Citric acid
`12.5
`2.99
`11-6
`Salicylic acid
`12.5
`5.97
`11-7
`Oxalic acid
`12.5
`2.7
`11-8
`Fumaric acid
`12.5
`2.98
`11-9
`Maleic acid
`12.5
`3.75
`11-10
`Malonic acid
`12.5
`2.94
`11-11
`Succinic acid
`12.5
`1.33
`11-12
`Glutaric acid
`12.5
`5.37
`
`
`EXAMPLE 12