Note: Descriptions are shown in the official language in which they were submitted.
- 1 -
PREPARATIC)N H~VING EXCELLENT ABSORPTION PROPERTY
-
This invention relates to a novel preparation having
excellent absorption property which is intended for improvement
of absorption of a medicine poor in absorption property through the
rectum or other digestive organs ~ the body by a~is~ration of
such a medicine simultaneously with a water soluble substance
at a concentration exhibiting higher osmotic pressure than
isotonic sodium chloride solution and one or more oE an absorp~ion
promoter selected from N-acyl aminoacid derivatives or N-acyl
peptide derivatives as represented by the ~ormula: R-CO-A (13,
(wherein R is an aliphatic hydrocarbon group, an aromatic hydro-
carbon group, an aryl-substituted lower hydrocarbon group which
may optionally be substituted and A is an amino acid residue or
a peptide residue).
Absorption of a medicine through a digestive organ,
irrespective of whether it may be the stomach, small intestine,
large intestine, rectum or mouth, has heretofore been generally
believed to proceed according to pH Partition theory (Modern
Pharmaceutics, Marcel Dek~er, INC,. p. 31 - 49). Hence, a
medicine readily dissociated in the respective organs at absorption
sikes or a medicine having poor lipophilicity tends ~o be poorly
absorbed. Such difficultly absorptive medicines are administered
as injections under the present circumstances. For improvement
o~ absorption property of a medicine, there have been made various
investigations such as Prodrug, Sofdrug, utilization o~ ion pairs
or complex formation. But any of these proposals is ef~ective
speci~ically ~or individual medicines, and no universally applicable
method is known in the art ("Pharmaceutics" written by Nogami).
The present inventors have made various investigations
and consequently found that in the mechanism of membrane absorption
through digestive organs or others, which is believed to proceed
according to the pH partitions theory as mentioned above, an N-
acyl aminoacid derivative or an N-acyl p~ptide deLivative which is
represented comprehensively by the above formula ~1) causes some
r ,~- .
J.~
'~ - 2
changes in membrane permeability, whereby membrane absorption
of a medicine can b~ improved to promote successfully ~he absorption
thereof. Further, it has also been found that membrane
absorption can be markedly improved by addition of a water-soluble
subs-tance at a concentration exhibiting higher osmotic pressure
than isotonic sodium chloride solution to make the preparation
under conditions of higher tonicity than the osmotic pressure of
a body fluid. In addition to these findings, it has further been
found that a preparation obtained by use of a vehicle, additives
selected as desired and an objective medicine, for example, a
suppository to be inserted into the rectum or vagina,is a good
suppository which can excellently be absorbed through membranes
and maintain a high concentration of the medicine in blood for
a l.ong time. A very broad range of medicines may be used in the present
invention. In ~icularr the so-called water~soluble medicines
having aood solubili~y in water, for example, those with partition
coefficients of 50 or less in chloroform/water or medicines readily
dissociated into ions, are usefuI. Further, medicines applicable
only as injections in the prior art are also found to be made
excellently absorbable easily as preparations such as suppositories.
; Even a medicine with a high molecular weight such as a polypeptide
is also found as the result of this invention to be made efficient-
ly absorbable in the form of a preparation such as a suppository.
The present invention has been accomplished based on
the ahove findings, and the object of the present invention is
to provide a good prepara~ion in which a medicine can be improved
to have a markedly enhanced absorption property.
In the accompanying drawings,
Figs. 1 and 2 show disappearance curves for various
osmotic pressures of Cephalothin Na when using Cephalothin Na
as medicine, in which the percentages of Cephalothin disappeared
by absorption are plotted at various measurement times;
Fig- 3 is a curve of plasma concentration of Cephalothin
when using Cephalothin Na as medicine;
Fig. 4 is a curve of plasma concentration of Tobramycin
when using Tobramycin cas medicine;
Fig. 5 is a curve of calcium concentration in serum when
using Elcitonin as medicine; and
Fig. 6 is a curve of plasma concentration of Elcitonin
when using Elcitonin as medicine.
According to the present invention, a preparation is
provided which comprises a water-soluble substance at a concent-
ration exhibiting an osmotic pressure high than that of i~otonic sodiumchloride solution, at least one absorption promoter selected from
the group consisting of N-acyl amino acid derivatives or N-acyl
peptide derivatives represented hy the ~ormula~ ~-CO-~ (1) (~hexe
R and A are the same as those defined above)~and a therapeu~ically
effective amount of a water-soluble medicine having a partition
coefficient of 50 or less in chloroform/water.
Also provided by the present invention is a composition
for enhancing absorption of norm~lly poorly absorb3ble medicines,
comprising a water-soluble substance at a concentration exhibiting
h.igher osmotic pressure than isotonic sodium chloxide solution and one
or more of N acyl amino acid derivatives or N-acyl peptide derivatives
represented b~ the formula: R-CC-A (wherein R is an aliphatic
hydrocarbon group, an aromatic hydrocarkon group or an c~yl-substituted
lower hydrocarbon group, which may optionally be substituted; and A is
an c~mino acid residue or a peptide residue).
To speak first of a water-soluble substance to be used
in the present invention at a concentration exhibiting higher
osmotic pressure than isotonlc sodium chloride solution, it may
be preferably one which is harmless as a whole and can exhibit
;~c high osmotic pressure with as small an amount as possible.
As such a water--soluble substance t there may be included
water-soluble salts and water soluble sugars.
- 3a -
Particularly among water-soluble salts, sodium chloride is
preferred since it is safe and its osmotic pressure is readily
controllable and further it is soluble in water rapidly at a high dissolvina
rate. Further, mannitol or glucose is pre~erred among water-soluble
sugarS~ Generally speaking, water-soluble salts may include, for
example, halides,sulfates, phosphates or carbonates of alkali
metals such as sodium, potassium or lithium, more specifically
the aforesaid sodium chloride, sodium sulfate, disodium hydrogen
phosphate, sodium dihydrogen phosphate, sodium phosphate, sodium
hydrogen carbonate, sodium carbonate, potassium chloride, potassium
-- 4
sulfate/ potassium hydrogen phosphate, potassium carbonate~
lithium chl~ride, etc. q'hese salts may be ad~usted to concent-
rations exhibitiny higher tonicity t~an the osmotic pressure of
isotonic sodium chloride solution dependin~ on the osmotic
characteristlc thereof. For example, in the case of sodium chloride
it may generally be adjusted to a concentration of 1 W/~% or
higher of the whole content. The upper limit of the concentration
is not particularly limited, but preferably the concentration
is about 2 to 30 W/W~. As preferable water-soluble sugars,
there may be employed monosaccharides or disaccharides frequently
used for adjustment of osmotic pressure in pharmaceutical
technology, including, for example, glucose, mannitol, sorbitol,
xylitol, lactose, maltose and s~lcrose. Such a sugar may be used
at a concentration with higher tonicity than isotonic sodium
chloride solution, which is generally 0.25 M or higher. These
water-soluble substances may be used in combination of two or
more kinds for adjustment of osmotic pressure, which is preferably
1~5 to 6-fold of the osmotic pressure exhibited by isotonic sodium
chloride solution.
In connection with osmotic pressure, description is
herein made by comparison with iso~onic sodium chloride solution,
but such description with the use of isotonic sodium chloride
solution as Control is merely exemplary for comparison between
osmotic pressure, and therefore may also be possible with the
use of body fluids or other solutions of salts with tolliCity
equal to such isotonic sodium chloride solution.
Referring now to the N-acyl aminoacid derivatives
or N-acyl peptide derivatives which are represented by the formula
R-CO-A (1~ ~R and A are same as those defined above) to ~e used
aS an absorption promo~er in ~his invention, they were investi-
gated by adding to~ for example, an isotonic preparation for rec-ta]
application containing a medicine for examination of increase
or decrease of membrane permeability of the medicine to accomplish
., ~
&7
~ 5 --
the present invention. The mechanism of the promotion effect
has not so far been clarified, but it seems ~Xely that the membrane
absorption mechanism may be changed through the chelating ~ction
and affinity to the membrane possessed by these absorption promoters
on the structures of cell membranes or the spaces between the
epithelial cells thereby to promote absorption.
Although the mechanism action of the absorption promoter
for increase of drug absorption through the rectum or other or~ans
may be speculated as mentioned above, such a mechanism action
is still no more than mere estimation and it is only sufficient
to employ N-acyl aminoacid derivatives or N-acyl peptide
derivatives having chelating action capable of bonding to at
least calcium ions or magnesium ions.
Further, referring to the N-acyl amino acid derivatives
or N~acyl peptide derivatives of the formula 11), these compounds
are obtained in this invention by reacting an acid compound of
the formula:
R-COOH (2)
(wherein R is the same as that defined above)
or its reactive derivative of ~he carboxyl group with a compound of
formula (3)0
H-A (3)
(wherein H~A is an amino acid or a peptide)
or its derivative havin~ a protected earboxyl group. Alternatively,
the eompound of the formula t1) may also be obtained by condensa-
tion of an acyl group of the above acid compound (2) and the amino
residue group or the imino residue group of an amino acid or a
peptide.
Carboxyl groups ean be activated by agents such as an
acid azide, an acid anhydride, an acid imidazolide, an acid
halogenide, an acti~e ester or a carbodiimide,an N,N'-carbonyl-
diimidazole or an isoxazolium salt such as Woodward's reagent.
~ . .
The preferred condensation reaction in the presentinvention is the carbodiimide, azide, active ester, halogenide
or anhydride method.
As an example of a conden5ed reaction, an amino acid or peptide
is reacted with reactive derivatives of an acid-ca~und of formula
(2) in an inert medium, for example, an organic solvent such as
chloroform, methylenechloride, ethylenechloride, ether and
hydrophilic solvent or an aqueous hydrophilic solvent such as
water, methanol, ethanol, acetone, dimethylacetamide, dimethyl-
formamide and dioxane. N-acylation is performed by the usual method,
and if necessary, a condensation agent or a base may be added
to the above solvent.
In this reaction, ~he reaction temperature is below or at
room temperature, and the acid compound of formula (2~ is used
about one mole per mole of amino acid or peptide. After the
reaction, it may be purified by preferably, a method of gel
filtration.
As an acid-compound of the formula (2), there may
preferably be used an acid in which R is an aliphatic hydrocarbon
group which may be substituted or an aromatic hydrocarbon gro~-
which may be substituted or an a~yl-substituted lower aliphatic hydrocarbon
group in which the number of car~on atoms of the lower aliphatic group
generally is in t}1e range of 1 to 10lpreferably 1 ~o 5,whlch
may be substituted.
When R is an aliphatic hydrocarbon group which may be
substituted, the hydrocarbon group may have carbon atoms generally
in the range of from 1 to 20,preferably ~ to 18. ~s alipha-tic
acids having such groups, there may be mentioned, for example,
acetic acid, propionic acid, butyric acid, valeric acid, iso~
valeric acid, hexanoic acidtenanthic acid, octanoic acid,
pelargonic acid, decanoic acid, undecylic acid, lauric acid,
myristic acid~ palmitic acid, stearic acld, acrylic acid,
c~otonic acid, vinylacetic acid, 4-pentenoic acid, oleic
acid, linoleic acid, linole~ic acid or sorbic acid as the fatty
acid and these com~ounds substituted with a lower alkyl group,
hydroxyl group, carboxylic a~id group, a]koxy group, phosphoric
acid group, amino group, imino group or a halogen.
As acids wherein ~ is an aroma~ic hydrocarbon group
which may be substitu~ed, there may be mentioned, f or example,
benzoic acid, alkylbenzoic acid, phthalic acid, isophthalic acid,
o-, m- or p-aminoben20ic acid, o-, m- or p~hydroxybenzoic
acid, o-, m- or p-alkoxyben7oic acid, dihydroxybenzoic acid and
these compounds substituted with above functional groups.
Acids wherein R is an aryl-substitu~ed lower aliphatic
hydrocarbon group which may be sub~tituted may incl~de, for example,
phenylacetic acid, phenylpropionic acid, o~,m- or p-hydroxy phenylacetic acid~
phenylmalonic acid, phenylsuccinic acicl, cinnamic acid, phenyl-
pyruvic acid, benzoylacetic acid and ~.hese compounds substituted
with the above functional groups.
As an amino acid or a peptide of the formula ~3),
natural amino acids or peptides may prefera~ly be used. They can
be in either D , L- or DL-form, and the derivatives thereof
substituted with hydroxyl, amino, carboxylic acid, lower hydro-
carbon, alkoxy, phosphoric acid or a halogen atom as well as
ethyl or methyl ester derivatives thereof are also available.
There may be mentioned, for example, various natural amino acids
(Chemistxy of Amino Acids, Vol. 1, p. 3~8 etc.) o-, m- or p~hydroxy
phenylalanine, o-, m- or p-hydroxy phenylglycine, Q~ or ~-
carboxy glutamic acidl glutamic acid Q-methylester, glutamic
acid ~-ethylester, aspartic acid ~ ~thylester, ~ or r~amino
butyric acid, iminodiacetic acid and o~hers.
As the peptide to be acylated, there may ge~erally be
3 .~
used a peptide composed of two or more amino acids, preferably
of 2-4 amino acids, which can be obtained by alkali, acid or
protease hydrolysis of natural protein such as collagen, keratin,
fibroin, albumin, globulin, gelatin and also by conventional
synthetic methods. These N-acyl amino acid deriv~tives or N-acyl
peptide derivatives preferably possess a chelating activity of
about one-thousandth or more of EDTA (ethylenediaminetetraacetic
acid).
These N-acyl amino acid derivatives or N-acyl peptide
derivatives mentioned above are preferably used in the form of
alkali metal salts such as sodium salts, potassium salts or
ammonium salts.
An N-acyl amino acid derivative or an N-acyl pepti.de
derivative which is represented by the above formula (1) is used
in the present invention as the absorption promoter.
These absorption promoters may be employed in amounts
20 of 0.01 W/W~ or more, generally in the range of from 0.1 to 30
W/W%, preferably from 1.0 to 20 W/W%. As the vehicle to be
employed for preparation of a suppository containing the above
absorption promoter, a medicine and preferably a water-soluble
salt to be added for increase of tonicity, there may suitably
be selected one from oily vehicles and water-soluble vehicles
conventionally used in the preparation of suppositories or rectal
injections, and a surfactant may also be added if desired.
As these oily vehicles or water~soluble vehicles,
there may conveniently be used those as described in "The Theory
and Practice of Industrial Pharmacy", p. 245 to 269 (1976).
The medicine to be used in the present invention is
not particularly limited, but there may be employed ordinary
pharmaceuticals, particularly preferably khe so-called water-solubl~
r ~
- 9 -
medicines which are excellently soluble in water1 such as
water-soluble medicines with a partition coefficient of 50
or less in chloroform/water or medicines readily dissociated
to ions. For example, there may be included various medicines
5 suc~ as hypnotics, tranquilizers, antiepileptics, antipyretics,
analgesics, antidepressants, muscle relaxants, antiinflammatory
agents, antiallergic agents, immunosuppressants, an~irheumatics,
vasodil~tors, antihemorrhagics~ antihypertensives, antibiotics,
antibacterial agents, urinary tract sterilizers, antitumor
10 agents, vitamins, hormones and galenicals. ~ore specifically,
typical examples are penicillin type antibiotics such as
ampicillin, hetacillin, am~xycillin, cyclacillin, cloxacillin,
dicloxacillin~ oxacillin, carindacillin, sulbenicillin,
piperacillin, apalcillin, methicillLn etc. or combined drugs
15 of ampicillin or amoxicillin with oxacillin, cloxacillin,
floxacillin or dicloxacillin; cephalosporin type antibiotics
such as cephalothin, cephaæolin, cephaloridine, cephacetorile,
cefoxitin, cefadroxil, cefatridine, cephaloglycin, cephalexin,
cephapirin, cephachlor, c~ftexol, cefuroxime, cefsulodin,
20 cefmetazole, etc. and non-toxic salts thereof such as alkali
metal salts (e.g. sodium salts or potassium sal~s), ammonium
v salts or benzylamine salts. In addition, there may also be
mentioned tetracycline type antibiotics such as doxycycline,
oxycycline, etc; aminosaccharide type antibiotics such as kanamycin,
25 sisomicin, amikacin, tobramycin, netromycin, ~entamycin, 1-N-
~s-3-amino-2-hydroxypropionyl~-gentamicin B, etc.; peptide type
antibiotics such as tuberactinomycin N, actinomycin, etc. or
non-toxic salts thexeof; further peptide hormones such as insulin,
somatostatin, calcitonin, angiotensin, kallikrein, secretin,
30 ~astrisin. parathyroid hor~one~ e~C an~ e~ ~edi~ines such
as barbital, ~heophylline, acetylsalicylic acid, mizoribin (bredinine),
5-fluorouracil, methotrexate, L-dopa, etc. The medicine may be
employed in an amount, which may suitably be selected and designed.
For example, in case of antibiotics such as ~lactam antibiotics,
2Q to 500 mg activity, generally 100 to 300 mg activity, or in
.
, , . , . ... . . , - " ~ . ,, ,, . , - . - ,
t~ 37
-- ~o --
case of peptide hormones such as insulin, 1 to 500 units may
b~ contained per gram o~ preparation. In general, the medicin~
may preferably be used in finely divided forms with 1 to 50
diameters or as an aqueous solution.
The step of forming preparations may be performed
according to csnventional methods for production of preparations
in general such as rectal suppositories, urethral suppositories or
vag ~ 1 ~suppositories,~ ointments or creams~ For example, the
absorption promoter selected, a water-soluble substance in an
. amount exhibiting higher osmotic pressure than isotonic sodium
chloride solution~and a medicine are added to a vehicle ,
optionally in combination with a surfactant, and these components
are thoroughly mixed to provide preparations.
Further, in production o these preparation, there
~ ` may also be added preservatives such as methyl- or propyl-p-
: oxybenzoate, colorants, aromas and stabili~ers~
: 20 The present invention is fur~her illustrated in detail
by referring to the following Examples, by which the presen~
invention is not limited at all but various medicines, hypertoni-
cators and absorp~ion promoters may be selected and combined in
addition to those shown in Examples.
The abbreviations employed Ln the Examples are as follows,
Gly: Glycine
` Pro: Proline
Phe: Phenyl alanine
Asp: Aspartic acid
Glu~ Glutamic acid
Val: Valine
Thr: Threonine
Ile: Isoleucine
Phy: Phenyl glycine
" ~
Lys: Lysine
~-Acp: -Aminocaproic acid
OEt: Ethyl ester
OMe: Methyl ester
Boc: t-Butyloxy carbonyl
HOBt~ ydroxy benzotriazole
TFA: Trifluoroacetic acid
DMF: Dimethyl formamide
WSC~: N-Ethyl, N-3-dimethylaminopropyl
carbodiimide
AcO~t: Ethyl acetate
NMM: N-Methyl morpholine
EtOH: Ethanol
HCl: Hydrochloric acid
NaOH: Sodium hydroxide
TMA: Trimethyl amine
Example 1
Absorption effects under conditions with various
tonicities were examined. Each sample solution was prepared
by adding 0.1 W/V~ cephalothin Na as a medicine together with
0.01 W/V~ N-lauroyl Gly-ONa as an absorption promoter to a
phosphate buffer of pH 7.5 conditioned with sodium chloride
to a tonicity which was varied from isotonic to twice hypertonic
than isotonic (two-fold tonicity), and to four times hypertonic than
isotonic (four-fold tonicity).
The experiment was conducted according to the following
method. Namely, Wistar-strain male rats, weighing 250 to 300 g,
were narcotized (after fast for 20 hours) with pentobarbital
(50 mg/kg~ and thereafter subjected to hypoabdominal incision
for a first cannulation at a position about 1.5 cm from the anus
and also another cannulation at a position 5 cm above said
irst cannulation. Subsequently, the rectum was internally washed
with about 20 ml isotonic sodium chloride solution kept at 38C,
~ ~i
and samples each of 10 ml were circulated through the rectum for
5 minutes (2 ml/minute) to make the concentration i.n ~he system
constant. Then, 5 ml of each sample was circula~ed at a f low
rate of 2 ml/minute, and samples each of 0.05 ml were collected
at intervals of 10 minutes from 0 minute. Each sample was
diluted to 5 ml with distill~d water and the quantity of medicine
disappeared by absorp~ion was ~etermined ~y W - ~pec~ropho~ter.
As the result, the disappearance curve of Cephalothin-
Na under the condition of 0.01 W/V~ N-lauroyl Gly-ONa was obtained
as shown in Fig. 1, in which D- O shows the result under the
l isotonic condition, ~ -~ under two-fold tonicity, ~ - ~ under
j four-fold tonicity and ~-~ under no absorp~ion promoter(Control).
'I .
Example 2
Using 0.1 W/V~ cephalothin Na as a medicine and 0.1
W/V% of N-myristoyl Pro-Pro-GlyNa as an absorption promoter
under respective osmotic pressure conditions (namely isotonic,
two fold tonicity and four fold tonicity with the use of sodium
chloride) and following otherwise the same procedure as in Example
1, quantities of Cephalo hin disappeared by absorption were
determined. The results are shown in Fig. 2, in which ~- Q
shows the result under the isotonic condition, ~- 0 unaer two-
fold tonicity, ~ . ~ under four-tonicity-and -~ - A Control.
Example 3
Quantities of 0.1 W/V% Cephalothin.~a disappeared by
absorption under isotonic and two-fold ~onic and four-fold tonic
conditions were determined, respectivelyl usincJ N~acyl amino
acids and N-acyl peptides where the N-acyl groups are aliphatic
hydrocaxbons, similarly as in Example 1. The results axe shown
in Table 1.
:
.~
.' i
. . .
- 13 -
Table 1 Ivalues after 60 minutes)
Two-~old Four-fold
Isotonic tonic tonic
condition condition condition
N-Propionoyl PheONa7.3 % - ~ 19.2 %
N-Hexanoyl PheONa 13.5 20.9 37.6
N-Octanoyl PheONa 22.5 31.7 42.1
N-Octanoyl PheOH 12.5 _ 25.5
N-Hexanoyl ValONa 4.7 - 16.2
10 N-Hexanoyl GlyONa 18.6 25.8 34.8
N-Octanoyl GlyONa 20.5 26.4 34.7
¦ N-Decanoyl GlyONa 19.5 24.6 36.2
¦ N-Myri.stoyl GlyONa10.1 - 20.4
N-Hexanoyl AspONa 21.0
15 N-Octanoyl ~spONa 19.5 - 31.0
N-Decanoyl AspONa 16.9 - 26.4
N-Lauroyl AspO~a 21.8 35.8 47.4
N-Myristoyl GlyONa10.3 1S.7 20.7
N-Decanoyl Pro-Pro~GlyONa 7.8 14.0 20.3
20 N-Lauroyl Pro-Pro-GlyONa 12.4 _ 22.4
N-Myristoyl Pro-GlyONa9.5 15.4 20.6
: No adjuvant 2.1 3.3 6.2
Example 4
25 Quantities of 0.l W/V% Cephalothin.Na disappeared
by absorption under isotonic,-two-fold tonic and four-fold
tonic cond:itions were determined by the high pressure liquid
chromatography respectively, using N-acyl amino acids and
N-acyl peptide in which the M-acyl ~roups are aromatic hydro-
carbons and aryl-substituted low hydrocarbons, similarly as
in ~xample 1. The result are shown in Table 2.
14 -
Table 2 (values after 60 minutes3
Two~fold Four~fold
Isotonic tonic tonic
conditioncondition condition
N-Benzoyl AlaONa22.6 ~ 30.1 % 39.8
N-Benzoyl ThrONa 23.5 28 7 40.0
N-Benzoyl E-AcpONa20.7 32.3 37O7
I N-Phenacetyl LysONa 7.6 -- 17.8
¦ N-Phenacetyl IleONa 11.8 - 21.9
p-Aminobenzoyl GlyONa - 16.2 22.5
N-Phthaloyl GluONa24.1 27.3 30.4
N-Phthaloyl Glu
~-methyl ester 22.7 - 32.4
N-Phthaloyl IleONa23.1 30.4 35~1
N-Benzoyl GlyAspONa 12.7 - 25O1
Example 5
CephalothinaNa 1600 mg potency) as a medicine,
N-acyl peptides ~100 mg) as an absorption promoter and sodium
chloride (200 mg) as a hypertonicator were each pul~eri~ed
and dispersed in 1 ml of distilled water. A homogeneous
dispersion was prepared by adding the resulting mixtur~ to a
base of"Witepsol H-15"previously molten by fusion to a total
amount of 10 g. The dispersion was i.ntrarectally administered
at a dose of 30 mg/kg to Wister strain rats (male, weighing
200~250 g, our per one group) and blood sampling was performed
5 minutes, 10 minutes, 15 minutes, 20 minutes/~ 30 minutes,
45 minutes and 60 minutes after administration for measurement
of Cephalothin concentration in plasma (according to the bioassay
using Bacillus subtilis ATCC 6633). As Controls, there was also
obtained a preparation without use of the absorption promoter
(Control 1). N-Acyl peptides as the absorption promoter used
were N-MYri~toyl Pro-Pro-GlyONa, N-Lauroyl Pro~Pro-GlyONa, N-
Decanoyl Pro-Pro-GlyONa and N-Myristoyl Pro-Gl~ONa~ The result
are shown in FigD 3, wherein 3- ~ indicates plasma concentration
* Tradelrlark
..
- 15 -
curve of Cephalothin in the case o~.control, ~- ~ that in the
case of N-Myristoyl Pro-Pro-GlvONa as the absorp~ion promoter,
~ that ~ the case of N--Lauroyl Pro-Pro-GlyONa, ~ - ~ that in ~he
case of N-Decanoyl Pro-Pro-GlyONa and x - x that in the case of N-
Myristoyl Pro-GlyONa, respectively.
Example 6
Tobramycin 1200 mg potency) as a medicine, N-acyl
amino acids l10 mg) as a absorption promoter and sodium chloride
(200 mg) as a hypertonicator were each pulverized and mixed
together. The mixture was mixed with Witepsol H-15 molten by
heating to an amount of 1 g. ~he dispersion was intrarectally
administered at a dose of 20 mgP/kg and experiment was carried
out similarly as in Example 5. As Control, there was also
obtained a preparation without use of the absorption promoter.
N-acyl aminoacids as the absorption promoters used were N-
Lauroyl PheONa, N-Butyryl ~heONa, N-Lauroyl PhyONa and N-Butyryl
PhyNa. The result are shown in ~ig. 4, wherein ~ - o indicates
plasma concentration curve of Tobramycin in the case of Control,
~ - ~ that in the case o~ N-Butyryl ~heO~a, o - o that in the case of
N-Putyryl PhyONa, ~ ~ ~ that:~ the case of N-Lauroyl PheONa and
x - x N-Lauroyl PhyONa, respectively.
Example 7
Elcitonin (Asu1 7-eel calcitonin~ (10 units and 2 units),
N-acyl amino acids, N acyl peptide and sodium chloride (50 mg~
were dissolved in 1 ml of 5 ~ gelatin solution. Each solution
(0.1 ml) was administered intrarectally to SD-strain male rats
~four weeks of age) and calcium concentrations in serum were
30 measured at 30 minutes, 6Q minutes, ~0 minutes and 120 minutes
after Administration by atomic absorption method. As Control,
there was used a solution containing no absorp~ion promoter~
( djusted to 10 units of Elcitonin~. N-acyl amino acids and
N-acyl peptide as the absorption promoter used were N~Lauroyl
GlyONa, N-.~xanoyl PheOH, N-Myristoyl Pro-Pro GlyONa and
,
~ . . . .
~8i~8~7
- 16 -
N-Lauroyl AspO~a. The results are shown in Fig. 5, wherein
x - x indicates calci~ concentration in serum in ~e case of
Control, ~ - b thatin the case ofa solution containing 2 llnits
of Elcitonin and N-hexanoyl PheONa, O - O that in the case of
2 units of Elcitonin and N-~auroyl AspONa, ~ - O that in the
case of 2 units of Elcitonin and N-Lauroyl GlyONa, o - ~ that
in the case of 10 units of E~citonin and N-Myristoyl Pro Pro-
GlyONa, o - o ~hat in the case of 10 units of Elcitonin and N
Hexanoyl PheOH and ~ - ~ that in the case Of10 units of Elcitonin
with N-Lauroyl GlyONa.
Example 8
Elcitonin (Asu1 7-eel calcitonin) of 200 units, N~acyl
amino acids as the absorption promoter (50 mg) and sodium chloride
(50 mg) were dissolved in 1 ml of a 5 ~ gelatin solution. Each
solution (0.1 ml) was administered intrarectally to Wister-strain
rats (200-250 g) and Elcitonin concentration in plasma were
determined by enzyme-immunoassay method at 15, 30, 45, 60, 90
and 120 minutes after administration. N-acyl amino acids as the
absorption promoter used were N-Octanoyl GlyONa, N-Octanoyl
PheONa and N-Lauroyl GlyONa. As Control, there was used a solution
containing no absorption promoter. The results are shown in
Fiq. S, wherein O - n indicates Elcitonin concentration in plasma
in the case of ~-Lauroyl GlyONa , ~ - ~ that in the case of N-OctanoyL
GlyONa, ~ - 0 that in the c~se of N-Octanoyl PheONa and x - x that
in the case of the Control. Further, Elcitonin concentration was not
detectable in the case of the Control.
Example 9
The collagen was hydrolyzed in 0.1 N-~Cl, then frac-
tionation performed using a column of"Sephadex G-2~'and fractions
of molecular weight in the region of 900 were collected~ The
equimolar quantity of the above peptide was reacted with each
acid chloride of hexanoic acid, decanoic acid, lauric acid
and stearic acid in DMF containing TMA in an ice-bath, to give
* Trademark
.~
87
- 17 -
each hydrolyzed product of N-acyl collagen. These N-acyl peptides
were dissolved in equimolar quantities of 1N-NaOH solution,
followed by concentration and, freeze-drying ~o obtain sodium
salts of these N-acyl peptides, respectively.
1- Using the N~lauroyl derivative of ~he hydrolyzed product of
collagen as an a~sorption promoter, each rectal suppository
containing 0.1 W/W~, 2.5 W/W%, 5 W/W% and 7.5 WfW~ of the
absorption promoter together with ampicillinNa as a medicine
and'~itepsol H-15l'as a suppository base. These preparations
were intrarectally administered at a dose of 15 mg/kg to male
rabbits and ampic,il,lin,concentr,a~ions in plasma were assayed
according, to the bioassay method using ATCC 6633.
'l5 As the result, peak plasma concentrations of ampicillin
were 1.4 y/ml (no absorption promoter), 2.5 y/ml (addition of
0.1 W/W~, 4.4 y/ml (addition of 2.5 W/W~, 7.0 y/ml (addition
of 5.0 W/W%) and 5.8 y/ml (addition of 7.5 W/W~,respectively.
From the result, it can be seen ~hat ~he absorption promoters
may preferably be employed in ~he range of from 0.1 W~W~ to
5 W/W~.
2. The rectal suppository containing each 5 W/W~ of
N~acyl derivative of hydrolyzed produc~ of collagen together
with ampicillin-Na as a medicine and'Wi~epsol~as a base. Then
the samples were administered intrc~rectally each at a dose of
15 mg/kg to male rabbits and the concentrations of ampicillin
in plasma were assayed. The results are'shown in Table 3.
From the ~esult, it can be seen that the N-stearoyl d rivative of the
hydrolyzed product of collagen is preferably effective. Fur~her,
using 0.5 W~W% of N-stearoyl derivative of hydrolyzed product
of collagen, other ~-lactam an~ibiotics such as cephalothin-Na,
cephazolin.Na and cephapirin.Na were examin~d under the same
conditions as mentioned above~ ~5 the result, the peak concen-
trations of cephalothin was 12.8 r/ml, tha~ of cephazolin 35~8
* Trad~k
. .
- 18 -
r/ml and that of cephapLrin l9o0 ~ml, respectively. Qn the
other hand, in the case of no absorption promoter, the concen
tration was not de~ectable for cephalothin and cephaæolin, or
4.5 r/ml for cephapirin.
Table 3
i
Chain length Plasma Level (y/ml~
fatty acid Time (min.)
1 o _ _¦ 1 ol 20 ¦ 40 60 ~ 90
C18¦3.0 8.0 10.2 ¦ 5-0 2.9 11.9
__ ~
C125.2 6.8 5.3 3O1 1O8 0.79
C10_ 6~7 ,7.2 4-4 ¦ 1-6 0.63
C84.8 5.1 3.4 7.5 0.61
__ ~ _. _
No addition 1.1 1.2 0.95 d . 68 0.52
3. And the next, absorption effects under ccnditions
with various tonicities were examined as follows. Each sample
was prepared by adding 5 mg/ml of ampicillin-Na as a medicine
~; together with 5 W/W% or 0.1 W/W~ of N stearoyl derivative of
hydrolyzed product of collagen peptides and with 1.7 W/W% oE
NaCl (twice hypertonic condition), 10 W/W~ of glucose (twice
hypertonic condition) or 0~85 W/W% of NaCl (isotonic condition).
,~
The experiment was carried out using Recirculating Perfusion
Method IWis-tar strain rats~. The plasma concentrations were
determined during recirculation of 10 ml each of above solution
and the results are shown in Table 4.
. . , , . - . . . . , , . ~ . . .. . . . .. . .
- 19 -
Table 4
. N~acyl dexlvativé Plasma level (y/ml)
of hydrolyzed
: product of collagen Ti.me (min~
10 ¦ 20 ¦ 40 ¦ 60 90
NaCl 5.0 W/W% 4.5 32.7 59.2¦ 320520.2
.~ 1.7W/W% __ ~__
: 0.1 W/W% 3~728.5 45.0 25.6 14.3
:: _ . .... _ _
Glucose 5.0 W/W% 5.2 33.954.6 34.4 25.1
10 W/W~ 0.1 W/W~ 3.0 25.743.3 30.2 21.5
_~ .. _ _ ~ ~ _
NaCl 5.0 W/W~ 3.9 7.314.9 11~6 10.0
0.85W/W~ 0.1 W/W~ 4.47.6 6.3 4.2
4. The experiment was performed similarly as in
Example 9-3 except that N-decanoyl collagen peptide was used
as an absorption promoter instead of N-stearoyl collagen
peptide~ The results are shown in Table 5.
From the results shown in Tables4 and 5, it may be
concluded that the absorption of ampicillin Na can be improved
to promote successfully under the hypertonic conditions by
addition of NaCl or saccharide. Fu.rther, sim.ilar results were
obtained for other ~-lactam an~ibiotics such as cephalothin
Na, cephazolin Na arld cephapirin Na.
Table S
Plasma level ~y/ml)
Time ~min.)
20 40 _ _ r
1.7 W/W% 3.9 22.7 32.9 16.0 ~ 1
Glucose 3.6 2002 34.2 7705 ¦ l O.Z
. ~ ....
0.85 W/W%_ 2.6 6.6 8.5 7.4 6.9
~,
. . .
- 20 -
':
Example 10
Cephalothin~Na (200 g potency), N-lauroyl ~lyONa
(50 g3 and sodium chloride (50 g), each being pulverized,
were mixed and the resulting mixture was dissolved in 2 ~
gelatin solution to a volume of one liter, which was then
~ filled into injection cylinders in aliquots each of 1 ml to
i provide intrarectal injection preparations.
Example 11
1 10 Gentamycin (100 g po~encyj, N-Decanoyl AspONa l50 g)
I and mannitol 1250 g), each being pulverized, were mixed and
the mixture was homogeneously dispersed in 5 % yelatin solution
to a volume of one liter, which was then filled into injection
cylinders in aliquots each of 1 ml to provide intrarectal
injection preparations.
Example 12
One thousand units of Elcitonin, 50 g of N-Hexanoyl
PheONa and 250 g of mànnitol were each pulverized and mixed
together. The resulting mixture was dispersed homogeneously
in 5 % gelatin solution to a volume of one liter, which was
then filled into injection cylinders in aliquots each of 1 ml
I to provide injection preparation5 for va~inal suppositories.
Example 13
One thousand units of Elcitonin, 50 ~ of N Myristoyl
Pro-GlyONa and 5 g of sodium chloride were dissolved in 100 ml
of distilled water and the solu~.ion was added to"Witepsol H-5"*
containing 1 ~Span 60~produced by Kao-Atlas Co.) to an amount
of 500 g, followed further by homogeneous emulsifying. The
emulsion was filled in suppository containers in aliquots each
of 1 g to provide rectal suppositories.
Example 14
Cefoxitin-Na (200 g potency), ~-phthaloyl ~lyONa ~50 g)
* Trademark.
** Trademark for soxbitan monost~a~ate; it is a nonionic s ~ actant.
... . .. ... , . , , . . , ~ . . .. .. . . . .
- 21 -
and soaium chloride (50 g) each being pulverized were mix~d
and dispQrsed in'~itepsol H-5"molten by heating to an amount
of 1 kg, which was hen illed in suppository containers in
aliquots each of 1 g to provide suppositories.
Example 15
Example 14 was repeated except that Cefazolin~Na
(200 g potency) was employed in place of Cefoxitin-Na to obtain
suppositories.
Example 16
1-N-(s-3-amino-2-hydroxypropionyl) gentamycin B (100 g
potency), N-Hexanoyl GlyONa (10 gl and sodium chloride (50 g)
were each pulverized and mixed. The mixture was mixed and
homogeneously dispersed wi~h"Witepsol H-5"molten by heating to
an amount of 1 kg. The dispersion was molded in suppository
containers to provide suppositories each of 1 g.
Example 17
Ampicillin Na ~25 g potency), NaCl (3.4 g~ and N-
stearoyl derivative of hydrolyzed pxoduct of collagen (5 g),
each being pulverized, were mixed and the mixture was homogeneous-
ly dispersed in Witepsol H~15 molten by heating at 45C to an
amount of 100 g. The emulsion was filled in suppository containers
to provide rectal suppositories under cool conditions.
Example 18
Ampicillin Na (20 g potency)l NaCl (205 g) and N-
stearoyl derivative of hydrolyzed product of collagen, each
being pulverized, were dispersed in peanut oil containing
Emulgen 408 ~3.5 g, Kao Atlas Co., Ltd.; polyoxyethylene
oleylalcohol ether~ and was made up to an amount of 100 y.
Each 1.5 g o~ dispersion was ~illed in gelatin rectal capsules
to provide rectal capsules.
* Trademar~
- 22 -
Example 19
Ampicillin Na (25 g potency), glucose (10 g ) and
N-stearoyl d~rivative of hydrolyzed product of collagen
; ~5 g), each being pulverized, were mixed and the mixture
was homogeneously dispersed in ~it~psol H-15 molt~n by
heating to an~amount of 100 g. Th~ emulsion was filled in
suppository containers to provide rectal suppositories~
Example 20
Example 19 was repeated using glucose (10.9 g) and
; N-stearoyl derivative of hydrolyzed product of collagen ~0.1 g3
in place of glucose (10 g) and N-stearoyl derivative of hydrolyzed
product of collag2n (5 g) to provide suppositories.
~'
Example 21
Example 20 was repeated using NaCl 13.4 g~ in place
of glucose to provide suppositories.
Example 22
Exa~ple 19 was repeated using cephalothin~Na as a
medicine in place of ampicilline.Na and using NaCl (3.4 y~
instead of glucose to provide suppositories.
Example ~3
~5 Cephazolin-Na (20 g po~ency), glucose (10 g) and sodium
salt o N-stearoyl derivative of hydrolyzed product of collagen
(2.5 g) being pulverized were dispersed in peanut oil containing
NIKKOL BC-20 TX (4.5 g, Nikko Chemical Co., Ltd.: polyoxyethylene
cetylalcohol ether) to an amoun~ of 100 g, which was filled into
gelatin rectal capsules in aliquots each of 1.5 ml to provide
rectal capsules.
Example 24
Example 23 was r~peated except that sulbenicillin-Na
(20 g) was employed in place of cephazolin-Na to provide rectal
capsules.
* Trademark
-
- 23 -
Example 25
Example 23 was repeated ~xcep~ that Enviomycin sulfate
(20 g potency) was employed in place of cephazolin-Na to provide
rectal capsules.
Example 25
Mannitol (10 g) and N-ste~royl derivative of hydrolyzed
product of collagen (2.5 g~, each being pulverized together with
12500 Units of Elcitonin were mixed and the mixture was homo-
geneously dispersed in"Witepsol ~-15"molten by heating to an
amount of 100 g. Then, the emulsion was filled in suppository
containers to provide rectal suppositories.
Example 27
15Example 26 was repeated except that" Witepsol S-55"was
used instead of'~itepsol H-15"and homogeneously dispersed. The
emulsion was filled in vaginal suppository containers to provide
each 2 g of vaginal suppositories.
Exampl~ 28
The pulverized dicloxacillin Na (5 g po~ency) and
"Mygriol 812ll(13.8 g with moisture contents of 0.13 %) were mixed
and the above mixed solution was added to ampicillin txihydrate
(10 g potency), NaCl (3.4 g1 and N-stearoyl derivative of
hydrolyzed product of collagen sodium salt (2.5 g), each being
pulveriæed, then homogeneously dispersed in Witepsol H-5 molten
by heating to an amount of 100 g. The emulsion was filled in-to
gelatin rectal capsules to provide 1 g each of rectal suppositories.
Example 29
Pulverized glucose ~10 g) and N-stearoyl derivative
of hydrolyzed product of collagen in the form of sodium salt
(5 g) and 12500 Units of Elcitonin were added to O.S W/W~ of
"Wakogel 103"a~djusted to pH 6.0 (Wako ~ure Chemical Industories
Co., Ltd.; carboxy vinyl polymerj to an amount of 100 gO T~en,
,,
i- * Trademark
~`J~** Trademark
J~7
- 2~ -
the mixture was filled into injection cylinders in aliquots
each of 1 ml to provide intrarectal injec~ion preparations.
Example 3 0
GlvOEt,HCl (29.31 g), Boc-Pro (43.05 g~, ~OBt (28.37 g~
in DMF ~150 ml) was treated wi~h WSCD 138.43 ml~ in an ice-ba~h,
and stirred for ~ hrs. at 0GCs furthex overnight at roo~ t~mpera-
ture. The xeaction mixture was ~vaporated in vacuo~ The residue
was dissolved in 1 Q of ~cOE~ and washed with 5 ~ aqueous sodium
bicarbonate solution, aqueous NaCl solution, 1-N HCl, 1-N NaO~I
and distilled water in this order. The solvent was evaporated
in vacuo after dehydration to give a slightly yellowish oily
product. ~Boc-Pro-GlyOEt; 70.2 g).
The oily product in methylene chloride (20 ml) was
treated with 70 ml of TF~ at 0C and stirred for 30 minutes
at room temperature. The solvent was evaporated and resulting
oily residue ~as added to NMM for neutralization at 0C.
Myristic acid ~7.54 g) in THF (50 ml) was treated with WSCD
(6.59 ml) at 0C, then stirred for 1 hr. and added to ~bove
neutralized ~olution and stirred overnight at room temperature.
This reaction mixture was concentrated and dissolved in chloroform
~300 ml) and washed with 5 ~ sodium bicarbonate, 1N-HCl, IN-NaOH
and distilled water in ~his order. The solution was concentrated
after dehydration with sodium s~l~ate and applied to a "Sephadex
I,-20"column (4 x 120 cm), and eluted with Benzane~AcOEt (1:1).
The fraction was applied to q'LC on silica gel by the following
solvent system (CHCl3:MeOH~AcOH 95:5:3), the part o R=0.30
was collected and extracted with CHCl3.This solution was evaporated
in vacuo and an oily product was obtained (N-myristoyl Pro-GlyOEt).
~ield 76. 8~.
The product was dissolved in EtOH ~30 ml) and 1N~NaOH
(33 ml~ was added to the solution at 0C. The resultant solu~ion
was stirred for 1 hrs. at room temperature and evaporated in
` 35 vacuo. The concentrated solution was-applied to a column o~
:. .
.
~ * Tra~mark
:` . '
`
- 25 -
:~ *
Sephadex L-20"(4 x 120 cm). The UV absorption at 230 nm was
determined in each fraction (12.5 ml each). The fraction
corresponding to the main peak (tube No. 95-111) were combined
and freez,e dried after concentration. Whi-te powders are
obtained (N Myristoyl Pro-GlyONa). Yield 56.6 ~, m,p. 145-148~o
Rf=0.80 (n-Butanol: Acetic acid: H2O 3:1:1). ~mino acid ratio
in a 6-N-HCl hydrolysate: Proline 0.90, Glycine 1.00
Calcd. for: C, 59.70; H, 9.31; N, 6.92
Found: C, 59.61; H, 9.21; N, 7.22
1 0
Example 31
Using GlyOMe HC1 (30.2 ~), Boc-Proline (4.3 g),
HOBt (28.37 g) and WSCD (38.43 ml~, the process was carried
out similarly as in Example 30, whereby, a slightly yellowish
oily product was obtained (BOc-Pro-GlyOMe). The product
(10.31 g) in dioxane ~5 ml) was added to ~.32 N-HCl in dioxane
(30 ml) in an ice bath, then stirred for 30 min. at room
temperature. The solution ~7as evaporated and dried in vacuol
thereafter dissolved in D~F (30 ml) and neutralized by addition
of NMM (0.5 ml). Boc-Proline (6.46 g), HOBt (4.05 g) in DMF
(30 ml) was treated with r~SCD (5.~9 ml) and stirred for 30 min.
in an ice bath. The solution was added to above neutralized
solution and stirred for 2 hrs~ at 0C and further overnight
at room temperature. The solution was evaporated and dissolved
in CHC13(300 ml), then washed with 5 % aqueous sodium bicarbonate
solution, 1~-HCl, lN-NaOH in this order. The solution was
dehydrated and concentrated to give an oily product (16.84 g~
~Boc-Pro-Pro-GlyOMe). ~f=0.65 lCHCl3: ~tOH: AcOEt 5:2:5)
The product was acylated to N-Myristoyl Pro-Pro-
GlyOMe (8.50 g) in the same manner as in Example 30O The oily
substance dissolved in EtOH ~30 ml) was added to ~-NaOH solution
(20.08 ml) and stirred for 30 min., then applied ~ a S~phadex
L-~0 column. The main fraction was collected, concentrated
and freeze dried to obtain white powders (4.61 g) (N-Myristoyl
Pro-Pro-GlyONa).
*Trademark
(~ 7
- 26 -
Yield: 54.9~, R=0.66 (n-BuOH:AcOH:H2O 3:1:1)
Calcd. for: C, 60.10; H, 8.92; N, 8.09
Found: C, 59.89; H, 9~20; N, 7.~2
Amino acid ratio in a 6N-HCl hydrolysate: Proline 1.90,
Glycine 1.00
Example 32
The process was carried out similarly as in Example
31~ using capric acid instead of myristic acid, then N Decanoyl
~ro-Pro-GlyONa (7.22 g) was obtai~ed. Yield: 81.0%, Rf=0.66
(n-BuOH:AcOH:H2O 3:1:1)
Amino acid ratio in a 6N-HCl hydrolysate: Proline 1.8, Glycine 1.O
Calcd. for: C, 54.87; H, 8.38; N, 8.73
Found: C, 55~03, H. 8.62; N, 8.39
Example 33
The process was carried out similarly as in Example
31, using lauric acid instead of myristic acid, whereby N-lauroyl
Pro-Pro-GlyONa (5.29 g) was obtained. Yield: 73.4 %, Rf-0u66
(n-BuOH:AcOH:H2O 3:1:1)
Amino acid ratio in a 6N-~Cl hydrolysate: Proline 1.9, Glycine 1.O
Calcd. for: C, 58.64; H, 8.61; N, 8.55
Found: C, 59.01; H, 8.90; N, 8.20
