0P
' 4.662.920
United States Patent {19]
[11] Patent Number:
Ten:
[45]
[54] 'ORALLY ADMINISTERED BIOLOGICALLY ~'
Inventor:
Lin-'nar L. Teng, Bothell, Wash. N.Y.
'
protein composition, comprising a sandwich complex‘ ; cally active peptide or protein with an alkyl or alkenyl
7
- sulfate having 6-24 carbon atoms and 0-3 double bonds
[21] Appl. No.: 452,493 ' [22]
Filed:
Apr. 15, 1986 v
comprising a hydrophobic core complex of a biologi- '
[73] Assignee: Research Corporation, New York, I
Date ‘of Patent: 1
[57] ABSTRACT An enterally effective, biologically active peptide or
ACTIVE PEPTIDES AND PROTEINS
‘ 1151'
4,582,820 ,
~ which form. an electrostatic complexwith a soft quater- '
nary ammonium ion of the formula NRIRZRJR‘
Dec. 23, 1982
wherein R1 represents a Ci-Cn-alkyl group; R2 and R3 ‘
[511
Int. Cl.‘ ...................... .. A61K 37/26; .C07K‘7/40
independently represent hydrogen or a C|-C|z-alkyl'
1521
U.S.>C1.
group; and R1 represents hydrogen or a radical of the. ‘
.
............................ .. 514/3; 260/112 R; ' .
260/112 T; 260/1l2.5 R;'260/1 12.5 TR;
formula
260/112.5 S; 260/112.5 E; 260/112.7; 424/95; ‘
,[58]
"
514/2; 514/12
Field of Search ‘............... "424/25, 178, 177, 329,
424/95; 514/2, 3. 12, 642; 260/112 R. 112 T, 112.5 R, 112.5 TR, 112.5 s, 112.5 E. 112.7
1561
References Cited where R5 is hydrogen, C|-C5 n-alkyl group and R6 is a
us. PATENT DOCUMENTS 2.108.165 2/1938
‘
2.694.663 11/1954; 2.844.466
and O-3 double‘ bonds; or Rl and R2 together represent
424/329
a divalent radical of the formula -CH2CI-1zCI-11CH2—,
..
-CHzCHzCI-IzCI-IzCHz—-,
424/227 '
.. 424/329
5/1979- Hirai et a1. ..... ..
'
.. 424/329
7/1958 ,
2.907.693 10/1959 3.869.550 3/1975 4.153.689
linear alkyl or alkenyl group having 6-22 carbon atoms ' I ..... .. 424/329‘
.. 424/329
OTHER PUBLICATIONS . Chemical Abstracts. 89, 1978, Abst. No. 48909t.
Chemical Abstracts.‘ 100(5), 1983, Abst. No. 30577u. , Kubinyi, Arzneim-Forsch/Drug Res.‘ 29(l1),- No. 8, 1067-1080 (1979).
Primary Examiner-Sam Rosen,
.
I
or —Cl-1zCI-1z0CI-12CH2— and R3 and R4 have the
meanings previously defined; with the proviso that R‘; R2 or R3 may optionally be substituted with a hydroxyl group or an alkoxyl group of the formula --0R4 where R‘ is an alkyl group having 1-4 carbon atoms; that ' - when RI and R2 together represent a divalent radical, f
i the radical may be substituted by methyl, hydroxyl, R1, or l-OR‘; and that when R2 is hydrogen, R1 is 'not , methyl.
’
Attorney. Agent. or Firm-Scully, ScotLMurphy & Presser
-
36 Claims, No Drawings >
r 4,582,820
2
The dif?culties of preparing other peptide and pro
ORALLY ADMINISTERED BIOLOGICALLY
tein hormones (and other biologically active peptides
ACTIVE PEPTIDES 'AND PROTEINS ~
and proteins) for oral ingestion or other types of enteral
administration parallel the problems associated with
BACKGROUND OF THE INVENTION 1. Field of the Invention
5 insulin. Accordingly, there remains a need for a compo
v
sition generally capable of effecting the oral administra
I
tionof biologically active peptides and proteins. '
The present invention relatesto pharmaceutical prep arations of biologically active peptides and proteins suitable forenteral administration.
2. Description of the Prior Art
‘
'
‘
' ’
SUMMARY OF THE INVENTION
'
It is an object of the this invention to provide an
.
effective method of orally administering biologically
As a result of recent progress in the ?eld of biochem- I
active proteins and peptides.
istry, many biologically‘active peptides and proteins are ' now available for clinical use. However, because they I '
are proteins of low lipophilicity and can be destroyed in -
the gastrointestinal tract by enzymes capable of cleav
.
'
‘
it is a further object. of this invention to provide com
positions containing biologically active proteins or pep
5 tides which are effective‘ when administered orally or
ing peptide bonds and inthe stomach by acid hydroly~ > by other enteral methods. , It is still a furtherlobjec't to provide a method of pro sis, methods of I administering these compounds orally. have not kept pace with their synthesis and identi?ca- _ ducing such compositionswhich can be‘ carried out tion. Typical of this situation is the case of insulin. It has 20 withany biologically active peptide or protein. These and other objects of the invention as will here long been established that insulin is an effective endoge inafter become morereadily apparent have been accom-' _ nous hormone useful inthe treatment of diabetes melli
pl'ished by‘ providing‘an enterally effective biologically
I tus. Furthermore, the intact insulin molec'ule’is known’
to pass through theintestinal wall of variousv animals
active peptide or protein composition, comprising:
under speci?ed conditions. However,‘ adult animals (including humans) absorb insulinlpoorlyr when it is
a sandwich complex comprising a hydrophobic core complex of a biologically active peptide or protein with
, orally administered. This isprobably due to a combina- ~
an alkyl or alkenyl sulfate having 6-24 carbon atoms:
tion of factorsrdestruction of intact insulin molecules as 2
and 0-3 double bonds which forms an electrostatic
complexmwith a soft quaternary ammonium ion of the previously discussed and slow passage of intactinsulin ‘ molecules through the intestinal wall. because of llow 30 formulav NR1R2R3R4 wherein lipophilicity. Consequently, therapeutic use of insulin is ' Rl represents a C1-C12-alkyl group;
limited by the necessity of administering it parenterally, particularly by intravenous or intramuscular injection. The desire to avoid parenteral administration‘of insu
" ' R2 and R3 independently represent hydrogen or a
C1—C|2-alkyl group; and R4 represents hydrogen or a
‘radical of the formula
lin has stimulated research efforts‘ in'oth‘er modes of 35, administration,.among which oral administration is the I most attractive. Although efforts have been made to » 7
develop oral hypoglycemic agents other-than insulin, a’ great deal of effort has also been concentratedon the modi?cation of insulin in such a way that an immuno
logically intact and metabolically competent insulin
where R5 is hydrogen,'C|—C5'n-alkyl group and R6 is a ' molecule can be absorbed through‘ the intestine so that linear alkyl or alkenyl grouphaving 6-22 carbon atoms insulin-itself or a derivative thereof may be orally ad- “ . ' and 0-3'double bonds; or ‘ ministered. The search in this area has been 'concen- , . RI and R2 together represent a divalent radical of the
trated in three directions: the development of adjuvants, the co-administration of enzymatic inhibitors, and the
formula. '
development‘of liposomes. Adjuvants used with insulin ‘ include resorcinols, non-ionic surfactants such as poly
—CHzCHzCH2CI-Iz—,
—CH=CH—N=CH-—, or —‘-CH2CH2OCI-I2CH2—- and R3 and R4 have the ’ meanings previously de?ned,
a
oxyethylene oleyl ether, andvn-hexadecyl polyethylene I ; with the provisos that R', R2 or R3 may optionally be ether. Enzyme inhibitors include pancreatic trypsin 50 substituted with a hydroxyl group or an alkoxyl group inhibitor, diisopropyl?uorophosphate' (DFP), and > of the formula —-OR4 where R4 is an alkyl group having trasylol. Liposomes include water-in-oil-in-water insu 1-4 carbon atoms, that when R1 and R2 together reprelin emulsions as well as conventional‘ liposomes.
'
,
‘
sent a divalent radical, said radical may be substituted
by hydroxyl, R1, or —-OR‘, and that when R2 is hydro
The co-administration of enzyme inhibitors has had
some degree of success, particularly whenused with
gen, R1 is not methyl.
duodenal administration. ‘Adjuvants such as hexylresor- > cinol have been administered with insulin to diabetic ‘ ‘7
'
a
DESCRIPTION OF THE PREFERRED
I EMBODIMENTS patients to give systemic,'hypoglycemic effects. How The present invention involves a method of modify ever, some adjuvants are limited to successful intra-jeju nal administration. Compared to the other types of oral 60 ing a, biologically active protein in such a way that the
insulin preparations, liposomes have been relative suc
7 protein‘ is absorbed into the systemic circulation when
cessful. Several studies have shown systemic, hypogly
administered enterally, particularly orally, while re
cemic effects after administration of a liposome contain
. maining immunologically intact and metabolically com petent. To achieve this result, the protein is coupled to .
ing insulin (e.g., Patel et al, FEBS Letters, 62, 60 (1976); Hashimoto et a1, Endocrinol., Japan, 26, 337 (1979)). 65 a protective carrier'in the form of a sandwich complex However, liposomes are still inthe development stage described in a later section. To be successful in this role, of their use as oral hypoglycemic agents and face con-' the protein complexmust meet the following criteria: tinued problems of stability, shelf-life, and so forth. (a) it must be resistant to the acidic environment in the
3
4,582,820
4
stomach; (b) it must be resistant to enzymatic degrada tion by gastric and pancreatic enzymes; (0) it must be suf?ciently lipophilic to pass the intrinsic barrier of the
thymopoietin, circulating thymic factor, and thymic
intestinal wall; and ?nally (d) the changes in physiolog
humoral factor; pancreatic hormones, such insulin, glu
ical and biological properties of insulin molecules re sulting from the modi?cation must be minimal so that
cagon, and somatostatin; gastrointestinal hormones, such as gastrin, cholecystokinin, secretin, gastric inhibi tory polypeptide, vasointestinal peptide, and motillin; chorionic (placental) hormones, such as choriogonado tropin and choriomammotropin; ovarian hormones,
their hormonal activity is maintained. While criteria (0) and (d) are basic structural requirements for all enter ally administered proteins (such as by rectal, buccal or topical routes), criteria (a) and (b) must be met in addi tion to (c) and (d) for the protein to be orally effective. More speci?cally, the present invention provides a sandwich-type complex of a biologically active peptide
mones; calcitropic (thyroid) hormones, such as parathy rin and calcitonin; thymic factors, such as thymosin,
such as relaxin; vasoactive tissue hormones, such angio tensin and brandykinin; growth factors, such as soma
tomedins, epidermal growth factor, urogastrone, and
or protein with an alkyl sulfate and a soft quaternary
nerve growth factor; hemophilia factors, such as blood clotting factors VIII and IX; enzymes, such as strepto
ammonium ion. The biologically active peptide (hereaf
kinase, ?brinolysin, deoxyribonuclease, and asparagin
ter “peptide” or “protein" will refer to both peptide and
ase; and arti?cial or pseudo peptides, such as deferox
protein molecules unless otherwise indicated) ?rst amine. Many other classes and speci?c types of peptide forms a hydrophobic core complex with the alkyl sul and protein hormones and other biologically active fate. This core complex protects the peptide molecule molecules are known. Peptide and protein hormones from acidic hydrolysis and enzymatic degradation and 20 suitable for use in the present invention are disclosed in increases the lipophilicity of the peptide molecule, Johannes Meienhofer, “Peptide and Protein Hor _ thereby allowing the intact peptide to pass through the mones”, in Burger’s Medicinal Chemistry, 4th ed., (part stomach when orally ingested and to increase the rate at II), Wolff, Ed., John Wiley and Sons (1979), which is which it is absorbed through the intestine wall. This herein incorporated by reference. Preferred hormones inner complex may be formed between the peptide 25 are those with a molecular weight of less than 7000, molecule and the alkyl sulfate. Hydrophobic complexes with insulin being especially preferred. of a peptide with an alkyl sulfate are generally rod-like The listings of peptides and proteins in this applica with a helical polypeptide chain of the peptide existing tion are not intended to be exclusive, and it may easily within a hydrophobic shell formed by the alkyl sulfate. be determined by simple experimentation if any Protein Typical of alkyl sulfate complexes with protein are 30 having biological activity can be prepared into a com those complexes formed with sodium dodecyl sulfate plex according to the invention. One simple method of (SDS). SDS has been found to bind protein molecules in testing for core complex formation involves the follow constant gram to gram ratios irrespective of nature of ing steps: (1) dissolve approximately 10 mg of the bio protein but depending on the SDS monomer concentra logically active peptide or protein in a small amount of tion. When SDS monomer concentration exceeds 35 water or buffer; (2) adds about 15 mg of an alkyl sulfate,
5>
for example sodium dedecyl sulfate, mix well and allow to stand for about 5 minutes; (3) subject the resulting
about 1.4 gram of SDS. When the SDS monomer is less
solution to agarose or acrylamide gel electrophoresis.
than 5X l0—4M, proteins form complexes with SDS in
The complex acts as an anion even at low pH (about 3
a low binding ratio where one gram of, protein binds to 40 is a good testing point) because of the sulfate groups and
about 0.4 gram of SDS. Both protein.SDS complexes assume a similar, rod-like shape with a helical polypep tide chain or protein folded back upon itself near its middle to give a double helical rod and the SDS form
migrates toward the anode. If no complex has formed, the protein will be protonated at low pH and migrate toward the cathode.
If complex formation has taken place and if the result ing a shell about the rod via hydrophobic forces. The 45 ing core complex will itself complex with a soft quater sulfate groups of SDS are on the surface of the rodlike nary ammonium ion according to the process of the
complexes as evidenced by the electrophoretic migra
present invention (infra), the biologically active peptide
tion of insulin in the presence or in the absence of SDS.
is suitable for use in the present invention.
In electrophoresis at pH 3 in the absence of SDS, insulin Complex formation between protein and a carrier is fully protonated and migrates to cathode. In the pres 50 molecule is one way to protect a protein molecule from ence of SDS (0.1%) at pH 3, insulin migrates to the acidic hydrolysis and enzymatic degradation and to anode (as if it is an anion). increase the lipophilicity of the protein molecule. De Since alkyl sulfates are themselves hydrolyzed to pending on the carrier substances selected, formation of fatty acid alcohols and a sulfuric acid salt at acidities this complex can be via columbic interaction between approximately those of the stomach, the rod-like pep 55 protein molecule and carrier substance or via hydro
tide.alkyl sulfate complex requires an additional protec tive coating for oral administration which is provided
phobic forces. In the proposed sandwich complex of proteimalkyl sulfate.soft quaternary ammonium ion,
by a soft quaternary ammonium ion, the structures of
there is a “core” complex of protein and alkyl sulfate formed via hydrophobic forces, and there is electro By utilizing the method of the invention, it is possible 60 static attraction between the sulfate groups of the alkyl to prepare peptide compositions suitable for oral admin sulfate and the soft quaternary ammonium ions. istration which contain endogenous opioid, agonists, Unlike other chemical modi?cations, complex forma such as encephalins and endorphins; hypothalmic hor tion between an agent and a carrier substance is a mo mones, such as gonadoliberin, melanostatin, melanolib lecular modi?cation of the agent without any chemical erin, somatostatin, thyroliberin, substance P, and neuro 65 alteration of the molecule itself. It is a method of modi? tensin; adenohypophyseal hormones, such as cortico cation in which the biological integrity of the molecule which are described later in detail.
tropin, lipotropin, melanotropin, lutropin, thyrotropin,
remain more or less intact. This is especially true in
prolactin, and somatotropin; neurohypophyseal hor
complexes formed via columbic forces.
4,582,820
5
Alkyl sulfates useful in ‘the present invention ‘have ‘
'
6
q
'
I
_
decyl, undecyl, and dodecyl. Additionally, Rl and ‘R2
linear, branched, or cyclicalkyl groups having'_6'-24
together can represent a divalent, alkylene radical
carbon atoms. Of these,‘ linear alkyl‘ groups, preferably ' _which,.when taken together with the amine nitrogen, I with 8-18 carbon atoms, are preferred, especially those
‘ forms a 5- or 6-membered ring.’ Examples of suitable
having an even number of carbon atoms. vAlkenyl sul
radicals _ include -—CHzCH2CHzCHz—, —CH2Cl-l2CH2Cl-I2CHz—+ and derivatives of these ~ 1
fates having'l-3 double bonds are also suitable. Speci?c » examples of suitable alkyl and alkenyl sulftaes‘include .
radicals havingan alkyl group of formula RI in “place of
octyl, decyl, dodecyl, tetradecyl, hexadecyl, octadecyl, lO-methyloctadecyl, 4~hexylcyclohexyl, 9-octadecenyl,
one or two hydrogen atoms of said radical. Further
more, RI and R2 when taken together with the amine nitrogen may form an imidazole or morpholine having
. 9,12-octadecadienyl, 9,l2‘,l5n-octadecatrienyl, ‘and tet racosyl‘ sulfate.
.
the formula
Sulfates of the invention can be synthesized bystan- ‘
dard methods of synthesis using alcohols (e.'g., fatty alcohols) and sulfuric acid or sulfate salts. Many alkyl sulfates, e.g., sodium dodecyl sulfate, are commercially available. Suitable examples of preferred linear alkyl
sulfates include octyl sulfate, nonyl sulfate, decyl sul fate, undecyl sulfate, dodecyl sulfate, tridecyl sulfate,
tetradecyl sulfate, pentadecyl sulfate,and hexyl sulfate. Of these, decyl sulfate,~do'decyl sulfate'and tetradecyl
HC=CH
‘ Additionally, any of the substituents Rl-R3 previously 20 de?ned may be substituted with a hydroxyl or alkoxyl
sulfate are more preferred with dodecyl sulfatebeing most preferred.
‘ ‘
group, wherein the alkoxyl group has the formula —-OR4 where R4 is‘ an alkyl group having 1-4 carbon
>
The alkyl sulfates are generally present initially as _ atoms. It is’preferred that the amine contain a total of alkalimetal salts when the initial core complex is being .
3-15 carbon atoms with, 6-10 carbon atoma being more
formed. Alkali metal salts include lithium, sodium, po
preferred.‘ Speci?c examples of suitable amines include
25
tassium, rubidium and cesium salts. Of these, sodiumv ~
'trimethylamine, triethylamine, n-propylamine, methyl
and potassium are preferred, with sodium'salts- being
di'ethylamine,
most‘ preferred. Sodium and’potas’sium salts of dodecyl sulfate are especially‘preferred, with sodium, dodecyl sulfate being the most preferred alkyl sulfate salt.
diethylamine,
methylbisethoxyethyl- '
amine, methyl-4~hydroxybutylpentylamine,. N-methyl 'p'yrrolidine, N-ethylimidazole, and morpholine. Pre ferred amines include trimethylamine, triethyla'mine, tripropylarnine, morpholine, N-alkylimidazol'es, ‘and "
Tli'eweight ratio of proteinlor peptide'toalkyl sulfate is the weight ratio of the naturally forming'complex. In
. N-alkylpyrrolidines. The amines used in this invention ' are readily'available eithercommercially ‘or through
a preferred embodiment, insulin is complexed with so
"dium' dodecyl sulfate (SDS). This complexforms an '- standard' methods of synthesis. Methylamine and di insulimSDS complex‘ in a'xratio of 1:14 or 71:04 by 35 methylamine should speci?cally be avoided because of I weight (depending‘on the initial ratio present) and is a
the carcinogenic products formed when they react with ‘I
hydrophobic complex. Complexesof protein with SDS : ‘nitrites under acidic conditions (e.g., in the stomach). are preferred to othertypes of complexes because a
' When the aminesof the invention are formulated into
wide variety of proteins are reported to bind to aniden-V
the outer complex, they are presentas protonated tical amount of SDS on a gram per gram basis. See, for 40 amines. Preferred are amines protonated with mineral
example, Reynolds et al., Proc. Nat. Acad, Sci. (US), 66, 1002-1003 (1970) and ,Reynoldset al., J. Biol. Chem,
acids such‘ as, for example, sulfuric acid and hydrochlo ric'acid. However, organic acid salts, such as salts of
' 245, 5161-5165 (1970). WhenSDS forms av complex with insulin or other protein, the hydrophobic core
i voxalicor lactic acid, may also be used. Amine hydro
complex is rod-like with a helical polypeptide chain of 45 protein existing within a hydrophobic shell formed by v '
the SDS. This complex of protein with alkyl sulfate is referred to herein as a core complex. This term is not
nary ammonium ions may also be used to form the elec trostatic complex of the invention. By soft quaternary ‘ ammonium ion is meant an ion of the formula
intended to limit the present invention, but is believed to
be generally descriptive. When this core complex is itself complexed with a soft quaternary ammonium ion, anadditional layer forms on the surface of the inner
chlorides are especially preferred. In addition to protonated amines, other soft quater
50
' complex. This-latter complex is referred to as an "elec . trostatic” complex. Nevertheless, this term additionally‘,
isnot intended to be limiting‘ of the actual ‘physical 55 structure thatis present in the resulting complex. '
where‘IU-R3 have the meanings previously given, R5 is
As mentioned above, the inner complex is reacted
hydrogen, methyl or ethyl, and R6 is the alkyl or alkenyl , '
with a soft quaternary ammonium ion to form an outer _
residue of a naturally occurring fatty acid of formula R6CO2H. R6 may additionally be any‘ linear alkyl group, ~‘ having 6,-22 carbon atoms or any linear alkenyl group
complex. The phrase “soft quaternary ammonium ion" as used in this invention includes protonated, organic amines and vother amine derivatives as will later be de ?ned. A‘ suitable amine from which to form a proton, ated amine of the invention has the formula NRIRZR3 wherein R1 represents a C1-C12-alkyl group andv
having 6-22 carbon atoms and l-3 double bonds. Other quaternary ammonium ions which hydrolyze or are otherwise cleaved to release harmless organic, com-.
pounds are contemplated as equivalents. The principal wherein R2 and R3 independently- represent hydrogen 65 requirement of a soft quaternary ammonium ion is the or a Ci-Cu-alkyl group. Representative alkyl groups ability to lose its positive charge in a biological system include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, by deprotonation, hydrolysis, or enzymatic degrada
sec-butyl, pentyl, neopentyl hexyl, heptyl, octyl, nonyl,
tion.
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7
4,582,820
8
least several months. Since oral administration is the
In the acidic environment of the stomach, a soft qua
proteimalkyl sulfate. Depending on the rates of hydro
principal contemplated end use of the compositions of the present invention, compositions suitable for oral ingestion are preferred storage forms. Such composi tions can contain pharmaceutically acceptable carriers in addition to the previously disclosed ingredients. Suit
lysis of the sulfate to an alcohol and acid sodium sulfate and of the protein itself, an electrostatic complex may
able pharmaceutical carriers include liquid or solid car riers of pharmaceutically acceptable or otherwise inert
ternary ammonium ion of the previously given formula in a sandwich complex will hydrolyze to give a fatty acid, an aldehyde and a (protonated) substituted ammo nium ion and leave an unprotected “core" complex of
form between the “core" complex and the protonated
materials which may be used orally. Examples of liquids
substituted ammonium ion generated in situ. In other
0 are water and aqueous solutions of non-toxic salts, such
words the soft quaternary ammonium ion of the given formula is designed here not only for its added lipo philicity due to the presence of the ester moiety, but
as sterile physiological solutions of saline, or aqueous
solutions containing non-toxic organic solvents, such as ethanol, used to increase the amount of complex in solution. Dilute aqueous solutions of mineral acids hav ing a pH of less than 4 are also suitable. Phosphoric, sulfuric, and hydrochloric acids are preferred. Also
also so that once it is hydrolyzed in an acidic environ
ment, another “soft” quaternary ammonium ion, in this case, a protonated amine, takes its place to provide continued protection for the hydrophobic "core" com
suitable are emulsions, such as oil-in-water emulsions.
plex.
Solutions of non-toxic organic liquids, such as ethanol, The synthesis of a soft quaternary ammonium ion involves two steps, the preparation of an a-chloroalkyl 20 are also suitable. Solid carriers include both nutritive carriers, such as sucrose or gelatin, and non-nutritive ester, and the formation of the desired quaternary am carriers, such as cellulose or tale. A pharmaceutical monium ion: preparation of the invention may be in the form, for example, of a liquid, a capsule, a tablet, or a suppository. 25
Pharmaceutical compositions according to the pres ent invention are administered in dosages which depend upon the effect desired for the biologically active com
pound which is being administered. The determination of the effective amount of the biological compound is 30 not considered to be part of the present invention since
dosage rates are generally determined by the effect of
the composition on the particular patient taking the
a-chloroethyl ester
medication. An amount equal in dose rate (mg/kg) to the amount normally injected parenterally for known Since a soft quaternary ammonium ion is a positively charged reagent and since the alkyl sulfate is a nega 35 biologically active peptides is suitable for use in the present invention as an initial dose and may be adjusted tively charged reagent, a 1:1 molar ratio of the soft as necessary to achieve the desired effect.
quaternary ammonium ion with the alkyl sulfate is pre ferred. However, other ratios are possible and fall
A particularly preferred embodiment of this inven
tion comprises enterally administering a sandwich com within the scope of the invention. Ratios of amine to alkyl sulfate in the range from 120.3 to 1:1 are contem 40 plex of the invention containing insulin as the active
plated by the present invention. Such ratios are ob
ingredient to produce a hypoglycemic effect. The amount required will depend on the severity of the diabetes and on the condition of the patient (e.g., time since ingestion of food, type and amount of food in
tained by using an excess of the core complex or the
amine/ammonium ion component during formation of the electrostatic complex. The ?nal electrostatic complex is formed by adding
45
the soft quaternary ammonium ion to an aqueous solu
tion containing a protein.alkyl sulfate complex (i.e., the core complex). The resulting electrostatic complex comprising the entire proteinalkyl sulfate.soft quater
gested, etc.). Adjustment of the amount required to maintain the proper blood glucose level is within the capability of those of ordinary skill in the art. An orally administered sandwich complex (of insulin or any other
active peptide) is especially preferred.
nary ammonium ion complex can be isolated by extract 50 The above disclosure generally describes the present ing the aqueous solution with chloroform or another invention. A more complete understanding can be ob
non-polar solvent immiscible with water. The presence of protein in the extracted complex can be veri?ed using the Fluorescamine protein test as described in Uden
fried et al, Science, 178, 871-872 (1972), which is herein 55
incorporated by reference. when working with a previ ously untried complex, this allows easy veri?cation of the formation of the desired complex.
tained by reference to the following speci?c examples which are provided herein for purposes of illustration only and are not intended to be limiting unless other
wise speci?ed. EXAMPLE 1
An insulin sandwich complex comprising insulin:
This invention may be carried out either by preparing a pharmaceutical composition which may be stored in 60 sodium dodecyl sulfateztriethylamine hydrochloride in a weight ratio of 5:7.5:3.6 was prepared. Insulin (bovine that form or by producing the sandwich complex imme pancreas, crystalline; 25.5 u/mg; Sigma; 10 mg total) diately prior to administration. When a protonated was dissolved in a small amount of 0.005 M H3PO4, PH amine is used to form the ?nal complex, the complex 2.45, in a 5 ml volumetric ?ask. Sodium dodecyl sulfate can be stored at approximately 4° C. in 0.005 M phos phoric acid for at least 2 weeks. When a soft quaternary 65 (15 mg) was added and mixed well to give a clear solu tion. After the solution was allowed to stand for 5 min ammonium ion is used, the complex should be prepared utes, 7.2 mg of triethylamine hyrochloride was added. in deionized water at a pH of approximately 7. It can The volumetric ?ask was then filled to 5.0 ml with 0.005 then be lyophilized and stored in powder form for at
4,582,820 ~
M tarot pH 2.45 to givgthe ?nal insulinzSDSzTEA complex.
insulin-treated rats/ became comparable. After 2 hours,
W
the blood glucose level in the modi?ed-insulin-treated
EXAMPLEIZ ‘
10
hypoglycemic effects among regular-. and modi?ed rats returned to the initial level faster than in the regu
In Vivo .Tests of Sandwich Complex
5 lar-insulin-treated rats; e.g., ?ve hours after administra
tion,,inthe modi?ed insulin group 89.1% of the initial
The term "diabetic rat” in the following tests refers to rats having been treated with streptozoticin by intrave-g
. plasmaglucose concentration was observed while in the
I regular insulin group 65.7% of the initial plasma glucose
nous injection at a dosage of '75 m'g/kg at'least 4 days
prior to the experiments in question. Allrats usedin the
- concentration was found.
>
experiments had been fasting 12-18 vhours prior to the
. Among the individual rats treated with both agents,
periments, rats were awake, were able to move-freely and had access to water (but no food). Plasma glucose‘ was measured on a Beckman Glucose Analyzer 2 cm
TBA ‘complex onlyv yielded’ a slight hypoglycemic ef
fect (Table 2, 3rd row). On the other hand, rat #3 showed more pronounced hypoglycemic effect for the
ploying ‘the enzymatic reaction of B-D-glucose with oxygen and measuring oxygen consumption rather than
complex than when it was treated with regular insulin
rat #285 showed a progressive hypoglycemic effect experiments. Blood sampleswere taken at appropriate after iv'administration of regular insulin ‘(4th row. in intervals as disclosed in the tables from‘a veincannula which was implanted in a jugular vein. During the ex . Table 1), whereas iv administration of insulin:SDS:
hydrogen peroxide formation.
'
g
‘
1
.
whereas the reverse was true in the later hours (see 5th . ‘The data do indicate that insulin in a sandwich com
20 row in Table 2 vs 6th row in Table 1).
A. Intravenous (iv) Administration of Regular and Modi?ed Insulin in Diabetic (Rats
?rst 90min when the rat was treated with the insulin
-
plexof insulin SDS TEA with a weight ratio of 5:7.5:3.6
The results of iv administration of regular and modi ?ed insulin in diabetic rats are shown inTablesl and '2
1 .remains physiologically effective. Compared with the
hypoglycemic effect of regular-insulin in diabetic rats, respectively. Both regular and ‘modified insulin yielded progressive, systemic, hypoglycemic‘effects for live 25 the hypoglycemic effect of the modi?ed insulin is not as
sustaining.- In ‘all likelihood, the presence of a protein
hours in all animals. From 15 min to 45 min after iv
v denaturing'agent, SDS, in the sandwich complex proba ‘ bly reduces the physiological effectiveness of insulin to
administration, the inducedhypoglycemic effect was more pronounced in regular-insulin-treated aminals.
Then from 60 min to 2 hrs after iv administration, the l a certain extent. TABLE 1 Intravenous Administration of Insulin (0.5 tt/kg) Into Diabetic Rats % Initial plasma glucose conc: (Actual 277.9 Glucose mg %) At Time Intervals After Administration
Bd. Wt. ' Rat No.
(g)
4
7 312.5
8
300.5
' 0
15 min
100 (362)
46.1 (197) ‘
' 100
274.5
. 285
45 min 7
43.9 (159)
36.5 (132) .' >
,
60 min
38.4 i (139)
90 min
120 min
180 min
300 min
45.4 (168)
42.3 (153)
58.8 (213)
60.8 (220)
63.8
53.6
59.1
57.6
53.6
58.0
59.8
58.1
(176)
. (148)
(163)
(159)
(148)
(160)
(165)
(167)
100
57.7
g 16.2,
31.0
27.2
50.4
56.9
76.3
89.2
(371)
(214)
(60)
(84)
( 101)
(187)
(211)
(283)
(331)
(276) 10 ,
30 min
230
100
84.5
69.7
56.2
51.8
46.1
35.1
37.9
46.6
A 1
290
(425) 100
(3 59) 47.9
_ (296) 36.9
. (239) 41.5
(220) 48.3
(196) 51.7
(149) 60.6
(161) 67.8
(198) 90.7
(236)
(I13)
(87)
(98)
(114)
(122)
(143)
( 160)
(214)
3
260
100 (451) 100
82.5 (372) ‘ 63.8 i
79.4 (358) 50.0 i
65.3 (296) 48.3 i-
71.6 (323) 49.2 i- 15.4
70.7 (319) 53.0 i 9
47.8 (215) 50.1 :1: 10.1
45.9 (207) 57.8 :t
(339 i 85)
16.6 (239 :1: 104)
22.9 (185 i 118) '
13.8 (169 i 83)
(176 i 83)
(190 i: 69)
(177 :l: 32)
14.0 (196 :1: 48)
48.6 (219) 65.7 :1: 19.6 (225 :l: 56)
1
I Mean i
277:9 i 29.9
Stand. Deviat.
,
.
‘TABLE 2 .
Intravenous Administration of Insulin Complex Into Diabetic RatsԤ0.5 E/kg) '
Bd. Wt. Rat # “
(g)
283 .
272
284 '
>
289
. 285
249
286
337.5 i
3
9
_
' % Initial plasma glucose Cone. (Actual plasma glucose cone. in mg %) at designated time interval
263
283
r 282.3 1- 30.6
0 100
15 min i
. 30 min
76.7
‘
69.7
(31 1)
45 min i
120 min
180 min
60.1
' 60 min 53.6,
90 min
51.4
64.1
76.5
(269)
(239)
(229)
(286)
(341)
(446)
(342)
100
.68.4'
69.8
71.5
53.0
43.9
43.0
68.7
(421)
(288)
(294)
(301)
(223)
(195)
(181)
(289)
'
300 min
100
110
77.0
79.8
72.9
‘ 103.7
70.7
97.8
(321)
(356)
(247)
(256)
(234)
(333)
(277)
(314)
‘
(304)
94.7
100 (129)
68.2 (88) 63.8
60.5 (78) 51.5
‘ 58.9 (76) 57.1 .
24.0 (31) 60.1
—' _ 63.8
.-—' . 74.2
—‘
100
52.7 (68) 39.3
87.1
(163)
(64)
(104)
(84)
(93)
(98)
(104)
(121)
(142)
100
72.9
47.4
53.4 '
44.7
57.9
53.7
79.7
97.9
(383)
(277)
(180)
(203)
(170)
(220)
(204)
(303)
(372)
100
70.0 :1:
66.0 i
62.8 i
56.7 i 9.3
57.9 i 29.4
56.5 :t
76.9 :1:
89.1 1: ~
(311 i 135)
24.1 (233 i
10.1 ‘(204 :t
' 10.9 (199 i
(173 i- 73)
(175 i 116)
10.9 (199 i
13.1 (263 :1:
9.5 (290 i
4,582,820
11
12
TABLE 2-continued Bd. Wt. (g)
Rat #
Intravenous Administration of Insulin Complex Into Diabetic Rats (0.5 u/kg) % Initial plasma glucose Conc. (Actual plasma glucose cone. in mg %) at designated time interval 0
15 min
30 min
45 min
132)
95)
96)
60 min
90 min
120 min
180 min
300 min
64.0)
80)
102)
‘Died of convulsion
least ?ve hours. The streptozotocin treated rats respond
B. Oral Administration of Regular and Modi?ed In sulin (at BOu/kg) in Normal and Diabetic Rats. In order to prove that the systemic hypoglycemic effect observed after oral administration of in sulinzSDSzTEA in diabetic (or normal) rats, if any, is
to modi?ed insulin to a much greater extent than do
normal rats; i.e., the glucose level is about 20% lower
(compare Tables 3 and 5).
In contrast, the oral administration of regular insulin does not result in a systemic, hypoglycemic effect in due to the complex per se and not due to the presence of SDSzTEA nor to nonspeci?c protein:SDS:TEA com 15 diabetic rats (see Table 6). The same conclusion can be drawn from other control studies. Oral administration plexes, oral administration of insulin:SDS:TEA in dia of the electrostatic complex of SDSzTEA does not yield betic rats were run against three controls. The three
a systemic, hypoglycemic effect in normal (see Table 4)
controls were regular insulin, a carrier complex of SDSzTEA, and a complex of albuminzSDSzTEA in the same weight ratio as than in the insulin complex; i.e.,
and diabetic (see Table 7) rats. Nor does the oral admin istration in diabetic rats of a sandwich complex of a
protein which does not have hypoglycemic effect per
a1bumin:SDS:TEA=5:7.5:3.6.
se. Likewise, the oral administration of a sandwich
The results of oral administrations of modi?ed insulin
complex of albuminzSDSzTEA in the weight ratio of 5:7.5:3.6 in diabetic rats fails to produce a systemic amount equivalent to that in the sandwich complex of 25 hypoglycemic effect. insulin calculated based on 80p.of insulin per kg of rat in The experimental results reflected in the above data normal rats are shown in Tables 3 and 4. The results of show that by forming a complex with a lipophilic sub oral administrations of modi?ed insulin, regular insulin, stance like TEA in the form of a sandwich complex of carrier complex and the sandwich complex of albumin insulinzSDSzTEA, the lipophilicity of insulin is im (albuminzSDSzTEA in a weight ratio of 5:7.5:3.6) in
and carrier complex (SDS:TEA=I:1 by mole) in the
proved. By doing do, metabolically competent insulin
diabetic rats are shown in Tables 5, 6, 7 and 8 respec 30
molecules are able to pass through the lipid barrier of the gastrointestinal tract and thence into the circulation
tively.
As shown in Tables 3 and 5, oral administration of to produce a progressive, systemic hypoglycemic effect modi?ed insulin in both diabetic and normal rats give a in normal and diabetic rats. systemic, hypoglycemic effect in rats which lasts at TABLE 3 Oral Administration of Modi?ed Insulin in Normal Rats (80 tL/kg) Glucose Level (% initial level) at Time Interval After Administration (min.)
0 (Initial Bd. Wt.
# Rat
Glucose
15
30
45
60
I20
180
300
211
486.9
100(134)
67.9
64.2
76.9
73.2
88.8
85.1
57.5
(91)
(86)
(103)
(98)
(119)
(I14)
(77)
212
507
100(116)
99.1
97.4
69.8
106.9
--
87.1
89.7
(115)
(113)
(81)
(I24)
(101)
(104)
214 215 230
(g)
455
mg/100 ml)
100(171)
537.6 411
100(95) 100(123)
72.8
88.9
108.8
26.9
43.9
68.4
77.2
(133)
(I52)
(I86)
(46)
(75)
(117)
(132)
56.8
50.5
49.5
49.5
84.2
115.8
131.6
(54)
(48)
(47)
(47)
(80)
(I10)
(125)
—
72.4
—
92.7
74.0
91.1
85.4
(114)
(91)
(112)
(105)
--
108.9
92.2
95.6
107.8
78.1 i 25.0
(9B) 76.3 i 33.4 (87 i 26)
(83) 76.6 i 19.5 (90 :t: 17)
(36) 90.6 i 15.4 (107 t 11)
(98) 91.5 i 25.6
(89) 231
410
100(90)
Mean 1 Std. Dev.
' 100 (122 i 29)
—
107.8
74.1 i 18.2
(97) 80.0 i 21.7 (98 z 34)
(122 t 51)
(104 i 59)
(107 i 20)
TABLE 4 Oral Administration of Carrier Complex In Normal Rats (T he amount of carrier comnlex is equal to that in complex insulin - SDS - TEA at 80 u/kg but without ‘
" L
% Initial plasma Glucose conc. (actual Plasma glucose mg/ 100 ml) at Time Interval After Administration (min) Rat
Bd. Wt. (g)
Glucose mg/ 100 ml
42 43
474 415
15
30
45
60
120
I80
300
100(80) 100(129)
157.5(126) 124(160)
142.5(114) 132.6(171)
175(140) 128.7(166)
152.5(122) 131.0(169)
156.3(125) 127.1(164)
152.5(122) 101.6(131)
141.3(113) 77.5(100)
45
46
‘ 445
100(106)
129.3(137)
117.9(125)
105.1(122)
84.9(90)
102.8(109)
90.6(96)
476
100(110)
125.5(138)
115.5(127)
105.5(116)
101.8(112)
90.0(99)
91.8(101)
—'
47 48
553 461
100(128) 100(124)
121.6(186) 124.2(154)
119.5(153) 105.7(131)
115.6(148) 100(124)
110.9(142) 102.4(127)
98.4(126) 100(124)
106.3(136) 92.7(115)
—' -'
Mean 1‘
470.1 i
Std.
46.2
—'
100(113 i
130.4 : 13.4
122.3 : 13.2
121.7 i 28.1
113.9 1 24.1
112.4 1 24.9
105.9 i
109.4 :
18.7)
(150 i 21)
(137 :t 21)
(136 i 9)
(127 i 27)
(125 t 22)
23.7
45.1
13
4,582,820 I
14
TABLE 4-continued
Oral Administration of Carrier Complex 1n Normal Rats ' (The amount of carrier complex is equal to that in complex insulin - SDS - TEA at 80 1.1/kg but without insulin
% Initial plasma Glucose conc. (actual Plasma glucose mg/100 ml) 1 at Time Interval After Administration (min) Bd. Wt.
R31
Glucose
(g)
mg/IOOml
Dev.
I
'
1s‘
'
'
30
'
45-
.
'60
>
120
'
‘
'
‘
'
'
-
~
130
300
(117 i 16)
(107 :9)
‘Plasma samples were not stored in the Freezer ( — 20' C.). but left at room temperature for three days
TABLE 5 Oral Administration of Modi?ed Insulin to Diabetic (Strept‘ozotocin-treated) Rats (80 u/kg)
Glucose Level (% initial level)at-Time Interval After Administration Bd. Wt.
0 (Initial Glucose
# Rat
(g)
mg/100 ml)
15
220
496
100(133)
60.2
30 .
‘ (82)
222
3, 425
100(108)
'
45
40.6 ‘
'
-
(54)‘
_
I
60
120
180
37.1
31.6
27.6
(49)
(42)
(37)
300 —
I
123.6
106.9
119.4
72.2
79.6
87.0
(140)
(137)
(129)
(78)
(86)
(94)
(87) -
88.6 I
38.3
36.9
29.2
21.1
23.2
29.5
i .
80.6
226
379
100(298)
(264)
(1 14)
(l 10)
(87)
(63)
(69)
(88)
227
424.5
100( 124)
62.1
44.4
50.8
35.5
46.8
55.5
83.1 (103)
441
100(292)
69.9
100(166)
(204) 98.2 (163)
100 (187 i- 86)
75.8 i 16.8 (155 i- 72)
(77) 218
-
_ 219 ~
519
Mean i Std. Dev.
v
(55)
‘
(63)
(44)
(53)
(70)
32.5 I
~ 21.6
12.0
16.1
44.9
(95) 72.3 (120)
' (63) 75.9 (126)
(35) -73.7 (I22)
(47) 68.7 (I14)
(131) ‘ 71.1 (118)
38.2 i 22.7 (72 i 29)
61.8 i 23.6 (105 i 19)
37.3
.
(109) 77.1 (128)
‘
,
57.4 i 28.1 (100 i 36)
.
62.4 i 35.5 45.3 i 23.0 44.1 d: 27.8 (103 :t 26) ' (75 i 30)’ (68 i 32)
TABLE 6 Oral Administration of Unmodi?ed Insulin to Diabetic (Streptozotocimtreated) Rats (80 p/kg). Glucose Level (% initial level) at Time Interval After Administration 0 (11111131) #
Bd. Wt.
R31 ‘(3) 222
Glucose
I
mg/100 ml)
415.5 '
100(122)
361
100(231)
'
15
30
119
_
I45 3 -
(14s)
226 213
364
I
‘
100(351)
‘
155.2
122.6
(311)
(293)
-
-
496
100(146)
.
-
‘
223
314.5
100(313)
' 229
386.3
.
100(401)
Mean 2':
100(264 i ,
115)
'
(309)
'
132.4
'
(421)
113.5‘2
. _
(340)
134 i- 20
(122)
‘
~
123.9
121.3
(296)
(290)
11.5'I
(321 :1: 32)
(1119)
268.5
243.3
234.2
(392)
(502)
(342)
129.2
132.1
123.0
(411)
(422)
(391)
'
5131*2
104.1 :1: 19.3 Y‘ 105.63: 31.2
5313'l
(226)
225.3
' (410)
11.3'2
64.5"
(251)
(329)
128.9
(290)
(213 i 112) (325 :1: 23).
120.5
(233)
11.5‘!I
,
(343)
.
- Std. Dev.
(210)
.-
101.9
- '
~
129.3
a
(363)
—
(212) v
_
_ 115
300
-
’
-
- '
(214)
130
100
113.0
‘
146.6‘ 5
120
124.6
(152)
'
219
»
60
,
3216*2
51.11“.
‘
4112'2
(239)
(151)
(211)
(196)
124.1 : 51.9
121.4 i 19.4
123.3 :1: 16.1
116.1 d: 15.0
(235 i 31)
(210 i 113)
(331 i 121)
(232 5; 89)
"Blood sample was taken under stress condition-jugular cannula did not work. Every sample was withdrawn from the tail artery when the rat was under ether anesthiza tion.
'
I
I
I
,
.
'
‘zlnsulin solution (1.10 ml of 1.1 mg insulin/l ml 0005M H3P04. pH Z45) was probably administered into the respiratory system of the rat instead of the stomach. Rat looked rather sick for 30 min. after administration.
-
,
.
‘
.
TABLE 7.
.
Oral Administration of Carrier Complex in Diabetic (Streptozotocin Treated) Rats. (The amount of carrier complex is equal to that in -
‘
complex insulin --SDS - TEA at 30 u/kg without insulin.) 3
~
'
'
Glucose Level 1% initiallevel) at Time Interval After Administration~(min) Bd.
# Rat
0 (Initial
Wt.
Glucose
I
(g)
mg/100 ml) 15
'
251
440
100(91)
253
456
100(97)
256
497.5
257
553.5
272
273 Mean 1 Std.
Dev.
._
340.0
' 324 435 -_t89
‘
‘128.6(117)
. _
30
45
‘102.2(93)
'
_
'
60
j.
v90
>
120
‘
180 I
300
106.6(97)
103.3(94)
100(91) '
103.3(94) ‘
100(91)
117.5(114) ' 106.2(103)
101(98)
78.4(76) '
82.5(80)
99.0(96)
129.9(126)
106.2(103)
100(84)
127.4(107)
148.8(125)
154.7(130) ' 158.3(133) ' 156(131)
163(137)
126.2(106)
91.7(77)
100(94)
116(109)
117.7(105) I 98.9(93)
87.2(82)
69.2(65)
103.2(97)
123.6(498)
99.8(402)
100(403)
100.3(404) ‘ 115.6(466) I 100(403)
100(341)
101.2(343)
103(354)
100 (185 :1:
115.2 : 12.3
114.6 i 17.5
146)
(199 i
(208 i
116(105)
101.8(347) I
110.5 1. 21.8
(195 i
'
109(441)
87.4(298)
‘93.6(88) 118.9(449)
108.9(439)
79.5(271)
82.7(282)
108.7 1- ( ~ 28 ~
105.1 i 28.7
107.4 :t 29
- (191 i
(193 i
(188 :1:
I
'
103.3(94)
73.6(251)
76.3(260)
103.8 i 27.2
96.8 :1: 11.2
(190 :1:
(I72 :1:
I
4,582,820
15
16
TABLE 7-continued Oral Administration of Carrier Complex in Diabetic (Streptozotocin Treated) Rats. (The amount of carrier complex is equal to that in corrlLlex ’
"
- SDS ' TEA at 30 tt/kg without '
"
)
Glucose Level (% initial level) at Time Interval After Administration (min)
# Rat
Bd.
0 (Initial
Wt. (g)
Glucose mg/IOO ml) 15
137)
30
45
6O
90
I20
I80
300
161)
141)
147)
166)
144)
164)
164)
TABLE 8 Oral Administration of Albumin Complex of Albumin - SDS ~ TEA in a weight ratio of
5:75:3.6 in Diabetic (Streptozotocin Treated) Ratsv Glucose Level (% initial level) at Time Interval After Administration (min) 0 (Initial Bd. Wt.
Glucose
# Rat
(g)
mg/IUO ml)
15
30
45
60
90
120
I80
300
253
441
100(85)
114.1
107.1
91.8
96.5
112.9
85.9
80
90.6
(97)
(9|)
(73)
(32)
(96)
(73)
(53)
(77)
254
489
100(103)
107.8
98.1
67
73.8
65.1
87.4
109.7
89.3
(111)
(101)
(69)
(76)
(67)
(90)
(113)
(92)
255
579
100(91)
118.7
85.7
85.7
92.3
84.6
78.0
86.8
92.3
261
464
100(281)
278
294
100(391)
281
282
274
100(664)
309
100(331)
Mean i 407.1 i
Std. Dev.
116
(108)
(78)
(78)
(84)
(77)
(7|)
(79)
(84)
89.3
93.2
99.6
95.0
103.2
103.2
104.3
100
(282)
(251)
(262)
(280)
(267)
(290)
(290)
(293)
111.8
111.0
103.1
97.2
96.4
92.6
99.5
97.2
(437)
(434)
(403)
(380)
(377)
(362)
(389)
(377)
96.4
99.3
96.4
98.2
100
97.9
100
(642)
(659)
(640)
(652)
(664)
(650)
(664)
110.6
96.1
102.4
88.2
105.4
108.5
101.8
97.6
(366)
(318)
(339)
(292)
(349)
(359)
(337)
(323)
100
106.9 1-
98.6 i
92.3 i 12.7
91.6 + 8.6
95.4 -_t-
93.4 i 10.6
97.4 i
94.5 i 43
(278 i 211)
104 (287 i
8.5 (278 ~_+-
(270 i 214)
(262 i 210)
15.9 (274 i
(271 i 213)
10.4 (278 :1:
(205 1 137)
206)
215)
218)
EXAMPLE 3
35
Four a-chloroethyl esters have been prepared and
isolated puri?ed in good yield; i.e.,
214)
(singlet, 3H), 2.48 (triplet, 2H), 1.98 (doublet, 3H), 1.48 (broad singlet, 18H), 0.98 (triplet, 31-1). The invention now being fully described, it will be
0
CH3
apparent to one of ordinary skill in the art that many 40 changes and modi?cations can be made thereto without departing from the spirit or scope of the invention as set forth herein. What is claimed as new and desired to be secured by
0
CH3
45
a-chloroethyl n-octanoate (Cg);
Letters Patent of the United States is:
a-chloroethyl ri-nonanoate (C9);
1. An enterally effective, biologically active peptide or protein composition comprising: a complex comprising a hydrophobic core complex of a
biologically active peptide or protein complexed with
u-chloroethyl n-decanoate (C10); 0
CH3
50
a-chloroethyl n-dodecanoate (C12); 0
CH3
55
Two
soft
quaternary
ammonium
salts,
[l-(n
and [l—(n-dodecanoyloxy)ethyl]-3-methylimidazolium chloride (B), have been prepared by the method previ ously described in the speci?cation. The toxicity of 60 l-methylimidazole is very slight (e.g., oral Mice LD50,
(singlet, 1H), 7.88 (doublet, 2H), 7.28 (quartet, 1H), 4.28
group or an alkoxyl group of the formula —OR4 where R4 is an alkyl group having l-4 carbon atoms;
R2 and R3 independently represent hydrogen, a C|—C|.
dodecanoyloxy)ethyl]triethyl ammonium chloride (A)
1500 mg/kg; ipr. Mice LD50, 380 mg/kg.) 1H NMR (CDC13) of ammonium ion (A): 6.68 (quar tet, 1H), 3.28 (quartet, 6H), 2.38 (triplet, 2H), 1.98 (dou blet 2H), 1.58 (triplet, 9H), 1.38 (broad singlet, 18H), 0.98 (triplet, 3H). 1H NMR (CDC13) of imidazonium ion (B): 10.38
an alkyl or alkenyl sulfate having 6-24 carbon atoms and 0-3 double bonds, said core complex forming an electrostatic complex with a soft quaternary ammo nium ion of the formula NRIRZR3R4 wherein R1 represents a C1-C12-alkyl group of substituted C1-C1; alkyl group said substituents being a hydroxyl
65
2-alkyl group or a substituted C1-C1; alkyl group said substituents being a hydroxyl group or an alkyl group of the formula —OR4 where R4 is an alkyl group
having 14 carbon atoms; and R4 represents hydrogen or a radical of the formula
4,582,820 .
I
17
’
'
v
'
.
o
I
‘amount of the sandwich complex of claim 13 sufficient
is a linear alkyl or alkenyl group having a 6-22 carbon
to produce a hypoglycemic effect.
atoms and 0-3 double bonds; or formula
ally administering to a diabetic human or animal an '
amount'of the sandwich complex of claim 19 sufficient
-,—-CH2CH1CH2CH2—, 5
‘
7
26. A method of treating diabetes comprising enter
I R1 and R2 together represent a divalent radical or the
—'CHzCl-lzCl-l¢CH1_CH2-, —CH=CHv-'-N=CH-, 0r —CHzCHzOCHzCH2-;
toproduce a hypoglycemic effect.
g
g
ing is orally administering. '
28. The method of claim 26, wherein said administer
ing is orally administering.
by hydroxyl, R1, or —OR‘; and that when R2 is hy drogen; R‘ is not methyl.
_
27. The method of claim 25, wherein said administer
'
with the proviso that when R1 and R2 together repre sent a divalent radical, said radical may be substituted
29. A method of producing an enterally effective,
,
biologically active peptide or protein ‘composition,
2. The composition of claim 1, wherein said sulfate is .
18
wherein R5 is hydrogen, C|-C5 n-alkyl group and R6
an alkyl sulfate.
comprising the steps of:
'
‘
‘
'
3. The composition of claim 2, wherein said alkyl' . dissolvinga biologically active peptide or protein in an is aqueous solvent to form a solution; sulfate contains from ‘10m 14 carbon atoms. adding an alkyl or alkenyl sulfate having 6-24 carbon 3 4. The composition or claim 2, wherein said alkyl atoms and 0-3 double bonds‘ to said solution to form
. sulfate is dodecyl sulfate.
a hydrophobic core complex; adding a soft quaternary ammonium ion to the solution 20 of said core complex, wherein said ammonium ion has
5. The composition of claim 1, wherein said soft qua ternary ammonium ion is a‘ protonated‘ trialkylamine I "containing from 6 to '10 total carbon atoms. '
. the formula NR'RlR-‘R‘i wherein ..
6. The composition of claim 5, therein said trialkyla mine contains 6 total carbonatoms. I
_ C1-C1; alkyl group said substituents being a hydroxyl V _
7. The composition of claim 6, wherein said trialkyla mine is triethylamine.
,
I
'
>
8. The composition of claim 1, wherein the ratio of said alkyl sulfate to said quaternary ammonium ion is'a 1:1 molar ratio.
7
'
‘group or an alkoxyl ' group of the formula --OR4' 25
where R4 is an alkyl‘ group having 14 carbon atoms;
R2 and R3 independently represent hydrogen, a C1-C1; alkyl group, or a substituted C1-C1; alkyl group said
i
9. The composition of claim 1, wherein said peptide or protein is present in a weight ratio of about 1:04 with ’ respect to said alkyl or'alkenyl sulfate.
,
RI represents a C|-C|2-alkyl group or a' substituted
_
substituents being a hydroxyl group or an alkoxyl
group to the formula —-OR4 where R4 is and alkyl 30
’ group having 14 carbon atoms; and
R4 represents hydrogen or a radical of the formula
10. The composition of claim 1, wherein saidpeptide or protein is present ina weight ratio of about 1:].4 with respect to said alkyl or alkenyl sulfate.
‘
‘
11. The composition of claim 2, wherein said biologi .35 cally active peptide or protein is a peptide hormone. 12. The composition of claim 11, wherein said hor mone has a molecular weight of less than 7000.
' where R5 is hydrogen or C|-C5 n-alkyl group and R6
13. The composition of claim 2, wherein said peptide or protein is insulin.
‘
isa linear alkyl or alkenyl group having 6-22 carbon
f
atoms and 0-3 double bonds; or
14. The composition of [claim 13, wherein said alkyl sulfate contains l0, 12 or 14 carbon atoms.
formula
15. The composition of claim 14, wherein said alkyl sulfate is dodecyl sulfate.
16. The composition or claim 15, wherein ‘the weight ratio of insulin to dodecyl sulfate is about 1:1.4.
—CH2CH1CH2CH2-,
with the proviso that when R1 and R2 together repre
45
sent a divalent radical, said radical may be substituted
17. The composition of claim 13, wherein the weight ratio of insulin to dodecyl sulfate is about 1:0.4.
.
R‘ and R2 together represent a divalent radical ‘of the
by hydroxyl, R1, of —OR4 and that when R2 is hydro
‘
gen, R1 is not methyl;
‘
18. The composition of claim 16, wherein said quar thereby forming a complex comprising said hydropho ternary ammonium ion is present in a 1:1 molar ratio to 50 ‘ bic core ‘complex in an electrostatic complex with said alkyl sulfate.
.
~
0
"
i
said ammonium ion.
g
19. The composition of claim 18, wherein said soft
30. The method of claim 29, wherein said sulfate is an 2
quaternary ammonium‘ ion is protonated triethylamine. 20. A pharmaceutical composition, comprising: the
alkyl sulfate. ‘
31. The method of claim 30, wherein said sulfate is j sandwich complex of claim '1 and a pharmaceutically 55 dodecyl sulfate. acceptable carrier.
-
'
- 32. The method of claim 29, wherein said soft quater
'
21. The composition of claim 20, wherein
carrier
nary ammonium ion is a protonated trialkyl amine. 33. The method of claim 32, wherein said'amine is
is water or an aqueous solution of a non-toxic salt. ‘
22. The composition of claim 20, wherein/said carrier is a dilute aqueous solution of a mineral acid having a pH of less than 4.
g g _
* triethylamine.
34. The method of claim .29,‘ wherein said peptide or
60 .
e
23. The composition of claim 22, wherein .said carrier is 0.005v M phosphoric acid.i
7, protein is a peptide hormone. ' 35. The method of claim 34, wherein said hormone is ' , insulin.
24. The composition of claim 20, wherein said com- I
‘ 36. The method of claim 29, wherein said sulfate is H
plex is lyophilized and said ‘carrier is asolid.
dodecyl sulfate, said soft quaternary ammonium ion is a
25. A method of treating diabetes comprising enter ally administering to ‘a diabetic human or ‘animal an
trialkylamine, and said peptide or protein is. insulin. ‘
.
t
i
ll
3
i
UNITED STATES PATENT AND TRADEMARK OFFICE
CERTIFICATE OF CORRECTION PATENTNO. ; 4.582.820 DATED
: April 15., 1986
INVENTUMS) :
Lin-nar L. Teng
It is certified that error appears in the above-identified patent and that said Letters Patent is hereby corrected as shovim below:
Column 1, line 4, before BACKGROUND OF THE INVENTION, insert the
following:
—-"This invention was made with government
support under grant number ROl HL 22035 awarded by the National Institutes of Health. rights in the invention. "-
The Government has certain
Signed and Sealed this Fifth Day of October, 1993
Attest:
6%“ W BRUCE LEHMAN
Arresting O?‘icer
Commissioner of Parents and Trademarks
