U.S. patent application number 10/237138 was filed with the patent office on 2003-10-23 for oral insulin therapy.
Invention is credited to Abbas, Richat, Arbit, Ehud, Dinh, Steven, Goldberg, Michael M., Woods, T. Cooper.
Application Number | 20030198666 10/237138 |
Document ID | / |
Family ID | 27559275 |
Filed Date | 2003-10-23 |
United States Patent
Application |
20030198666 |
Kind Code |
A1 |
Abbas, Richat ; et
al. |
October 23, 2003 |
Oral insulin therapy
Abstract
A method of attenuating the undesirable incidence of diseases
associated with chronic dosing of insulin is provided whereby the
oral administration to a patient of insulin along with a suitable
delivery agent that facilitates the absorption of insulin from the
gastrointestinal tract of the patient in a therapeutically
effective amount, for treatment of diabetes. Also disclosed are
pharmaceutical dosage forms for oral administration to a patient
for the treatment of diabetes, comprising insulin and a delivery
agent that facilitates insulin transport in a therapeutically
effective amount to the bloodstream and that result in a lower
incidence of vascular diseases associated with the repeated
administration of insulin.
Inventors: |
Abbas, Richat; (Mohegan
Lake, NY) ; Goldberg, Michael M.; (Tarrytown, NJ)
; Woods, T. Cooper; (New York, NY) ; Dinh,
Steven; (Tarrytown, NY) ; Arbit, Ehud;
(Tarrytown, NJ) |
Correspondence
Address: |
DAVIDSON, DAVIDSON & KAPPEL, LLC
485 SEVENTH AVENUE, 14TH FLOOR
NEW YORK
NY
10018
US
|
Family ID: |
27559275 |
Appl. No.: |
10/237138 |
Filed: |
September 6, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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60346746 |
Jan 7, 2002 |
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60347312 |
Jan 9, 2002 |
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60389364 |
Jun 17, 2002 |
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60368617 |
Mar 29, 2002 |
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60374979 |
Apr 23, 2002 |
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Current U.S.
Class: |
424/452 ;
424/465; 514/1.9; 514/15.4; 514/15.7; 514/16.4; 514/18.2; 514/20.8;
514/5.9; 514/6.9 |
Current CPC
Class: |
A61K 9/2013 20130101;
A61P 25/00 20180101; A61K 38/28 20130101; A61K 38/28 20130101; A61K
2300/00 20130101; A61K 9/4858 20130101; A61P 13/12 20180101; A61P
9/04 20180101; A61P 3/10 20180101; A61P 9/00 20180101; A61P 43/00
20180101; A61P 9/10 20180101; A61K 47/18 20130101 |
Class at
Publication: |
424/452 ; 514/3;
424/465 |
International
Class: |
A61K 038/28; A61K
009/48; A61K 009/20 |
Claims
What is claimed is:
1. An oral solid dosage form comprising a dose of unmodified
insulin that achieves a comparable reduction in blood glucose
concentration in human diabetic patients compared to a subcutaneous
insulin injection in those patients, while providing a lower
concentration of insulin in the peripheral blood circulation under
acute, sub-acute or chronic conditions as compared to the
peripheral blood insulin concentration obtained via the
subcutaneous injection.
2. The oral solid dosage form of claim 1, which provides a lowering
of insulin of at least about 20%.
3. An oral dosage form comprising a dose of unmodified insulin that
achieves a therapeutically effective reduction in blood glucose
after oral administration to a human diabetic patient, and which
provides a ratio of portal vein to peripheral blood insulin
concentration from about 2.5:1 to about 6:1.
4. The oral dosage form of claim 3, wherein said dosage form is
solid.
5. An oral dosage form comprising a dose of unmodified insulin that
achieves a therapeutically effective reduction in blood glucose
after oral administration to human diabetic patients, the oral
solid dosage form providing an insulin Tmax at a time point from
about 0.25 to about 1.5 hours after oral administration to said
patients, at least about 80% of the blood glucose concentration
reduction caused by said dose of insulin occurring within about 2
hours after oral administration of said dosage form.
6. An oral dosage form comprising a therapeutically effective
amount of unmodified insulin, said dosage form upon pre-prandial
oral administration to human diabetic patients causing the mean
plasma glucose concentration in said patients to be reduced for the
first hour after oral administration relative to a mean baseline
(fasted) plasma glucose concentration in said patients.
7. An oral dosage form comprising a therapeutically effective
amount of unmodified insulin, said oral dosage form upon
pre-prandial oral administration provides a mean plasma glucose
concentration which does not vary by more than about 40% for the
first hour after oral administration, relative to a mean baseline
(fasted) plasma glucose concentration in said patients, where a
meal is eaten by said patients within about one half hour of oral
administration of said dosage form.
8. The oral dosage form of claim 7, which provides a mean plasma
glucose concentration which does not vary by more than about 30%
for the first hour after oral administration.
9. The oral dosage form of claim 5, which is solid.
10. The oral dosage form of claim 7, which is solid.
11. The oral solid dosage form of claim 9, which is in the form of
a tablet or capsule.
12. The oral solid dosage form of claim 10, which is in the form of
a tablet or capsule.
13. The oral solid dosage form of claim 11, wherein the dose of
unmodified insulin contained in the dosage form is from about 50
Units to about 600 Units (from about 2 to about 23 mg).
14. The oral solid dosage form of claim 13, wherein the dose of
unmodified insulin is from about 100 Units (3.8 mg) to about 400
Units (15.3 mg) insulin.
15. The oral solid dosage form of claim 14, wherein the dose of
unmodified insulin is from about 150 Units (5.75 mg) to about 300
Units (11.5 mg).
16. The oral solid dosage form of claim 7, which provides a
T.sub.max for insulin at about 0.1 to about 1.5 hours after oral
administration.
17. The oral solid dosage form of claim 16, which provides a
T.sub.max for insulin at about 0.25 to about 0.5 hours after oral
administration.
18. The oral solid dosage form of claim 16, wherein the dosage
forms begin delivering insulin into the portal circulation (via
absorption through the mucosa of the stomach) to achieve peak
levels within about 30 minutes or less.
19. A method of providing a therapeutically effective orally
administrable unit dose of unmodified insulin, comprising combining
from about 2 to about 23 mg of unmodified insulin with from about
100 to about 600 mg of a pharmaceutically acceptable delivery agent
which facilitates absorption of said insulin from the
gastrointestinal tract of human diabetic patients, and orally
administering said unit dose to a human diabetic patient to provide
a therapeutic effect.
20. A method of treating human diabetic patients comprising orally
administering to human diabetic patients on a chronic basis an oral
insulin treatment comprising a dose of unmodified insulin together
with a delivery agent that facilitates the absorption of the dose
of insulin from the gastrointestinal tract to provide a
therapeutically effective reduction in blood glucose and a blood
plasma insulin concentration that is reduced relative to the
systemic blood insulin concentration of an equivalent
therapeutically effective reduction in blood glucose concentration
achieved by subcutaneous injection of insulin.
21. A method of reducing the incidence and/or severity of one or
more disease states associated with chronic administration of
insulin, comprising treating human diabetic patients via oral
administration on a chronic basis with a therapeutically effective
dose of a (preferably solid) pharmaceutical composition comprising
a dose of unmodified insulin and a delivery agent that facilitates
the absorption of said unmodified insulin from the gastrointestinal
tract in an effective amount such that the pharmaceutical
composition provides therapeutically effective control of mean
blood glucose concentration and a mean systemic blood insulin
concentration in diabetic patients that is reduced on a chronic
basis relative to the mean systemic blood insulin concentration
provided by chronic subcutaneous administration of insulin in an
amount effective to achieve equivalent control of mean blood
glucose concentration in a population of human diabetic
patients.
22. A method of treating diabetes and reducing the incidence and or
severity of hyperinsulinemia associated with chronic dosing of
insulin, comprising orally administering on a chronic basis to a
diabetic patient a dose of insulin and a delivery agent that
facilitates the absorption of the dose of insulin from the
gastrointestinal tract to provide therapeutically effective control
and/or reduction in blood glucose concentrations, and a mean
systemic blood insulin concentration of the diabetic patient that
is reduced relative to the mean systemic blood insulin
concentration provided by subcutaneous injection of insulin in an
amount effective to achieve equivalent control and/or reduction in
blood glucose concentration in a population of human diabetic
patients.
23. The oral solid dosage form of claim 5, further comprising an
effective amount of a delivery agent of the formula or a
pharmaceutically acceptable salt thereof, 8wherein X is hydrogen or
halogen; R is substituted or unsubstituted C.sub.1-C.sub.3
alkylene, substituted or unsubstituted C.sub.1-C.sub.3 alkenylene,
substituted or unsubstituted C.sub.1-C.sub.3 alkyl (arylene),
substituted or unsubstituted C.sub.1-C.sub.3 aryl (alkylene).
24. The oral solid dosage form of claim 23, wherein X is a
halogen.
25. The oral solid dosage form pharmaceutical composition of claim
24, wherein said halogen is chlorine.
26. The oral solid dosage form of claim 3, wherein R is C.sub.3
alkylene.
27. The oral solid dosage form of claim 23, wherein said peak
plasma delivery agent concentration occurs within two hours of oral
administration.
28. The oral solid dosage form of claim 23, wherein said delivery
agent is 4-[(4-chloro, 2-hydroxybenzoyl)amino]butanoic acid.
29. The oral solid dosage form of claim 23, which provides a peak
plasma delivery agent concentration that is from about 5,000 and
about 15,000 ng/ml within about 0.3 to about 1.5 hours after oral
administration.
30. The oral solid dosage form of claim 23 which produces a maximal
decrease in blood glucose in treated patients from about 20 and 60
minutes post oral administration.
31. The oral solid dosage form of claim 23, which produces a
maximal decrease in blood glucose in treated patients at about 40
minutes post oral administration.
32. The oral solid dosage form of claim 5, wherein said composition
produces a maximal decrease in C peptide concentration in treated
patients from about 80 and 120 minutes post oral
administration.
33. The oral solid dosage form of claim 5, which produces a lowered
serum glucose in human patients by at least 10% with in one hour
post oral administration.
34. A method of treating impaired glucose tolerance, achieving
glucose homeostasis, treating early-stage diabetes, or treating
late-stage diabetes, comprising administering the oral dosage form
of claim 5 on a chronic basis to human patients.
35. A method of treating diabetics, comprising: orally
administering to diabetic patients on a chronic basis an oral
insulin treatment comprising a dose of unmodified insulin together
with a delivery agent which facilitates the absorption of the dose
of insulin from the gastrointestinal tract to provide a
therapeutically effective reduction in blood glucose and a peak
serum insulin concentration that is reduced relative to the peak
serum insulin concentration of an equivalent therapeutically
effective reduction in blood glucose concentration achieved by
subcutaneous injection of insulin.
36. The method of claim 35, wherein the incidence of a disease
state associated with chronic insulin administration is
reduced.
37. The method of claim 36, wherein the disease state associated
with chronic insulin administration is cardiovascular disease.
38. The method of claim 36, wherein the disease state is selected
from the group consisting of a neuropathy, a nephropathy, a
retinopathy, an arteriopathy, atherosclerosis and combinations
thereof.
39. The method of claim 36, wherein the disease state is coronary
artery disease.
40. The method of claim 36, wherein the disease state is
hypertensive cardiomyopathy.
41. The method of claim 37, wherein the disease state is congestive
heart failure.
42. A method of reducing the incidence and/or severity of one or
more disease states associated with chronic administration of
insulin, comprising treating diabetic patients via oral
administration on a chronic basis of a therapeutically effective
dose of a pharmaceutical composition which comprises insulin and a
delivery agent that facilitates the absorption of insulin from the
gastrointestinal tract, such that the pharmaceutical composition
provides a therapeutically effective reduction in blood glucose and
a peak serum insulin concentration of the diabetic patient that is
reduced relative to the peak serum insulin concentration of an
equivalent therapeutically effective reduction in blood glucose
concentration achieved by subcutaneous injection of insulin.
43. The method of claim 42, wherein the method provides a reduced
expression of genes associated with vascular disease as compared to
the level of expression of genes associated with vascular disease
resulting from an equivalent reduction in blood glucose
concentration achieved in a population of patients via subcutaneous
injection of insulin.
44. The method of claim 43, wherein the genes associated with
vascular disease are selected from the group consisting of early
response genes, genes associated with cytokines, genes associated
with adhesion molecules, genes associated with lipid peroxidation,
genes associated with thrombosis and combinations thereof.
45. The method of claim 44, wherein the early response genes are
selected from the group consisting of c-myc, jun B, Egr-1, Ets-1
and combinations thereof.
46. The method of claim 35 or 42, wherein plasminogen activator
inhibitor concentrations resulting from the method are lower as
compared to the plasminogen activator inhibitor concentrations
resulting from an equivalent therapeutically effective reduction in
blood glucose concentration achieved by subcutaneous injection of
insulin.
47. The method of claim 35 or 42, wherein the pro-inflammatory
cytokine concentrations resulting from the method are lower as
compared to the pro-inflammatory cytokine concentrations resulting
from an equivalent therapeutically effective reduction in blood
glucose concentration achieved by subcutaneous injection of
insulin.
48. The method of claim 42, wherein the disease state is
cardiovascular disease.
49. The method of claim 48, wherein the disease state is selected
from the group consisting of a neuropathy, a nephropathy, a
retinopathy, an arteriopathy, atherosclerosis and combinations
thereof.
50. The method of claim 48, wherein the disease state is coronary
artery disease.
51. The method of claim 48, wherein the disease state is
hypertensive cardiomyopathy.
52. The method of claim 48, wherein the disease state is congestive
heart failure.
53. The method of claims 35 or 42, wherein the delivery agent is a
compound having the formula: 9or a pharmaceutically acceptable salt
thereof, wherein X is a halogen or hydrogen; R is substituted or
unsubstituted C.sub.1-C.sub.12 alkylene, or a substituted or
unsubstituted C.sub.1-C.sub.12 alkenylene.
54. The method of claim 53, wherein the delivery agent is
4-[(4-chloro, 2-hydroxybenzoyl)amino-butanoic acid or a derivative
or analog thereof.
55. The method of claim 35 or 42, wherein the insulin is selected
from the group consisting of recombinant human insulin, bovine
insulin, porcine insulin and functional equivalents thereof.
56. A method of treating diabetes and reducing the incidence and or
severity of hyperinsulinemia associated with chronic dosing of
insulin, comprising orally administering on a chronic basis to a
diabetic patient a dose of insulin and a delivery agent that
facilitates the absorption of the dose of insulin from the
gastrointestinal tract to provide a therapeutically effective
reduction in blood glucose and a peak serum insulin concentration
of the diabetic patient that is reduced relative to the peak serum
insulin concentration of an equivalent therapeutically effective
reduction in blood glucose concentration achieved by subcutaneous
injection of insulin.
57. A method of screening a drug for vascular injury associated
with route of administering the drug, comprising administering a
drug to a first test animal parenterally; administering the drug to
a second test animal orally, and comparing the expression of early
response genes selected from the group consisting of c-myc, c-fos,
Jun B, Erg-1 and combinations thereof for the first and second test
animal, wherein an increase in the expression of one or more early
response genes is indicative of vascular injury.
58. The method of claim 57, wherein the step of measuring the
change in expression comprises gene chip analysis.
59. The method of claim 57, wherein the step of measuring the
change in expression comprises measuring the changes in mRNA
expression.
60. A method of reducing the incidence of, the severity of, or the
incidence and severity of disease states associated with chronic
insulin administration to diabetics, comprising orally
administering an oral insulin treatment comprising a dose of
insulin together with a delivery agent which facilitates the
absorption of said insulin from the gastrointestinal tract on a
chronic basis to diabetic patients to reduce blood glucose levels
in said diabetic patients by a desired amount, such that the
concentration of insulin circulating in the blood of said diabetic
patients as a result of insulin treatment is reduced relative to
the peak serum insulin concentration of an equivalent
therapeutically effective reduction in blood glucose concentration
achieved by subcutaneous injection of insulin.
61. A method for reducing the incidence of, the severity of, or the
incidence and severity of vascular diseases associated with chronic
insulin therapy in diabetics, comprising orally administering an
oral insulin treatment comprising a dose of insulin together with a
delivery agent which facilitates the absorption of said insulin
from the gastrointestinal tract on a chronic basis to diabetic
patients to reduce blood glucose levels in said diabetic patients
by a desired amount, such that the concentration of insulin
circulating in the blood of said diabetic patients as a result of
insulin treatment is reduced relative to the peak serum insulin
concentration of an equivalent therapeutically effective reduction
in blood glucose concentration achieved by subcutaneous injection
of insulin.
62. A method of reducing the exposure of the vasculature of
diabetic patients to hyperinsulinemic conditions, comprising orally
administering an oral insulin treatment comprising a dose of
insulin together with a delivery agent which facilitates the
absorption of said insulin from the gastrointestinal tract on a
chronic basis to diabetic patients to reduce blood glucose levels
in said diabetic patients by a desired amount, such that the
concentration of insulin circulating in the blood of said diabetic
patients as a result of insulin treatment is reduced relative to
the peak serum insulin concentration of an equivalent
therapeutically effective reduction in blood glucose concentration
achieved by subcutaneous injection of insulin.
63. A method of attenuating processes resulting from the reaction
to a mild injurious stimulus in multiple areas of the response to
increases in mRNA during insulin treatment, comprising orally
administering an oral insulin treatment comprising a dose of
insulin together with a delivery agent which facilitates the
absorption of said insulin from the gastrointestinal tract on a
chronic basis to diabetic patients to reduce blood glucose levels
in said diabetic patients by a desired amount, such that the
concentration of insulin circulating in the blood of said diabetic
patients as a result of insulin treatment is reduced relative to
the peak serum insulin concentration of an equivalent
therapeutically effective reduction in blood glucose concentration
achieved by subcutaneous injection of insulin.
64. A method of treating diabetic patients, comprising orally
administering an oral insulin treatment comprising a dose of
insulin together with a delivery agent which facilitates the
absorption of said insulin from the gastrointestinal tract on a
chronic basis to diabetic patients to reduce blood glucose levels
in said diabetic patients by a desired amount, such that the
concentration of insulin circulating in the blood of said diabetic
patients as a result of said oral insulin treatment is not
substantially greater than normal physiological levels.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Patents No. 60/346,746 filed Jan. 7, 2002, No. 60/347,312 filed
Jan. 9, 2002, No. 60/389,364 filed Jun. 17, 2002, No. 60/368,617
filed Mar. 29, 2002 and No. 60/374,979 filed Apr. 23, 2002.
FIELD OF THE INVENTION
[0002] This invention relates to the oral delivery of therapeutic
proteins in a therapeutically effective amount to the bloodstream.
This invention further relates to oral administration of proteins
as active agents as part of a therapeutic regimen. This invention
further relates to the oral administration of insulin in a
therapeutically effective amount for the treatment of diabetes.
This invention further relates to compositions of a delivery agent
and insulin for oral administration that facilitates insulin
transport in a therapeutically effective amount to the bloodstream
for the treatment of diabetes. This invention further provides
methods for the preparation of a composition comprising insulin for
oral administration.
[0003] The present invention further relates to methods for
reducing adverse effects on the vascular system that are associated
with insulin therapy. More specifically, the present invention
relates to methods that reduce the incidence of diseases associated
with chronic administration of insulin. The present invention is
also directed to oral pharmaceutical dosage forms that are
administrable on a chronic basis to diabetics, in part to achieve
such results.
BACKGROUND OF THE INVENTION
[0004] Proteins, carbohydrates and other biological molecules
("biological macromolecules") are finding increasing use in many
diverse areas of science and technology. For example, proteins are
employed as active agents in the fields of pharmaceuticals,
vaccines and veterinary products. Unfortunately, the use of
biological macromolecules as active agents in pharmaceutical
compositions is often severely limited by the presence of natural
barriers of passage to the location where the active agent is
required. Such barriers include the skin, lipid bi-layers, mucosal
membranes, severe pH conditions and digestive enzymes.
[0005] Oral delivery of active agents is a particularly desirable
route of administration, because of safety and convenience
considerations. When compared to injection administration, oral
delivery can eliminate the risk of infection through the use of
needles and syringes. In addition, oral delivery provides for more
accurate dosing than multidose vials and can minimize or eliminate
the discomfort that often attends repeated hypodermic
injections.
[0006] There are many obstacles to successful oral delivery of
biological macromolecules. For example, biological macromolecules
are large and are amphipathic in nature. More importantly, the
active conformation of many biological macromolecules may be
sensitive to a variety of environmental factors, such as
temperature, oxidizing agents, pH, freezing, shaking and shear
stress. In planning oral delivery systems comprising biological
macromolecules as an active agent for drug development, these
complex structural and stability factors must be considered.
[0007] In addition, in general, for medical and therapeutic
applications, where a biological macromolecule is being
administered to a patient and is expected to perform its natural
biological function, delivery vehicles must be able to release
active molecules, at a rate that is consistent with the needs of
the particular patient or the disease process.
[0008] One specific biological macromolecule, the hormone insulin,
contributes to the normal regulation of blood glucose levels
through its release by the pancreas, more specifically by the
.beta.-cells of a major type of pancreatic tissue (the islets of
Langerhans). Insulin secretion is a regulated process which, in
normal subjects, provides stable concentrations of glucose in blood
during both fasting and feeding. Diabetes is a disease state in
which the pancreas does not release insulin at levels capable of
controlling glucose levels. Diabetes is classified into two types.
The first type is diabetes that is insulin dependent and usually
appears in young people. The islet cells of the pancreas stop
producing insulin mainly due to autoimmune destruction and the
patient must inject himself with the missing hormone. These Type 1
diabetic patients are the minority of total diabetic patients (up
to 10% of the entire diabetic population). The second type of
diabetes (type 2) is non-insulin dependent diabetes, which is
caused primarily by the resistance of patients to their own
insulin. This is the most common type of diabetes in the Western
world. Close to 8% of the adult population of various countries
around the world, including the United States, have Type 2
diabetes, and about 30% of these patients will need to use insulin
at some point during their life span due to secondary pancreas
exhaustion.
[0009] The problem of providing bioavailable unmodified human
insulin, in a useful form, to the ever increasing population of
diabetics has occupied physicians and scientists for almost 100
years. Many attempts have been made to solve some of the problems
of stability and biological delivery of this small protein. Most
diabetic patients self-administer insulin by daily subcutaneous
injections. However, the limitations of multiple daily injections,
such as inconvenience, poor patient acceptability, compliance and
the difficulty of matching postprandial insulin availability to
postprandial requirements, are well known.
[0010] In the field of insulin delivery, where multiple repeated
administrations are required on a daily basis throughout the
patient's life, it would be desirable to create compositions of
compositions of insulin that maintain protein tertiary structure so
as not to alter physiological clinical activity and stability and
do not require injections. It would also be desirable to provide
compositions of insulin that could be orally administrable, e.g.,
absorbed from the gastrointestinal tract in adequate concentrations
such that oral unit doses of insulin are bioavailable and bioactive
after oral administration. Oral absorption allows delivery directly
to the portal circulation.
[0011] Insulin exemplifies the problems confronted in the art in
designing an effective oral drug delivery system for biological
macromolecules. The medicinal properties of insulin can be readily
altered using any number of techniques, but its physicochemical
properties and susceptibility to enzymatic digestion have precluded
the design of a commercially viable delivery system.
[0012] A method of providing insulin without the need for
injections has been a goal in drug delivery. Insulin absorption in
the gastrointestinal tract is prevented by its large size and
enzymatic degradation. It would be desirable to create an oral
pharmaceutical formulation of a drug such as insulin (which is not
normally orally administrable due to, e.g., insufficient absorption
from the gastrointestintal tract), which formulation would provide
sufficient absorption and pharmacokinetic/pharmacodynamic
properties to provide the desired therapeutic effect.
[0013] Diabetes is the sixth leading cause of death in the United
States and accounted for more than 193,000 deaths in 1997. However,
this is an underestimate because diabetes contributes substantially
to many deaths that are ultimately ascribed to other causes, such
as cardiovascular disease. Complications resulting from diabetes
are a major cause of morbidity in the population. For example,
diabetic retinopathy is the leading cause of blindness in adults
aged 20 through 74 years, and diabetic kidney disease accounts for
40% of all new cases of end-stage renal disease. Diabetes is the
leading cause for amputation of limbs in the United States. Heart
disease and strokes occur two to four times more frequently in
adults with diabetes than in those who are healthy. Diabetes causes
special problems during pregnancy, and the rate of congenital
malformations can be five times higher in the children of women
with diabetes.
[0014] The main cause of mortality with Diabetes Mellitus is long
term vascular disease. Cardiovascular disease is responsible for up
to 80% of the deaths of Type II diabetic patients. See, for
example, Kirpichnikov et al., Trends Endocrinol Metab 12, 225-30
(2001); Garcia et al., Diabetes 23, 105-11 (1974); Haffner et al.,
N Engl J Med 339, 229-34 (1998); Sowers, Arch Intern Med 158,
617-21(1998); Khaw, K. T. et al., Bmj 322, 15-8 (2001). Diabetics
have a two- to four-fold increase in the risk of coronary artery
disease, equal that of patients who have survived a stroke or
myocardial infarction. See, for example, Haffner et al, N Engl J
Med 339, 229-34 (1998); Sowers, Arch Intern Med 158, 617-21 (1998).
This increased risk of coronary artery disease combined with an
increase in hypertensive cardiomyopathy manifests itself in an
increase in the risk of congestive heart failure. Stratton et al.,
Bmj 321, 405-12 (2000); Shindler, D. M. et al. Am J Cardiol 77,
1017-20 (1996). These vascular complications lead to neuropathies,
retinopathies and peripheral vascular disease. See Kirpichnikov et
al., Trends Endocrinol Metab 12, 225-30 (2001). There is a need for
diabetes treatments that will decrease the prevalence of such
vascular disease in diabetes patients.
[0015] The beneficial effects of good glycemic control on the
chronic complications of diabetes are widely accepted in clinical
practice. However, only recently it has been firmly established
that elevated blood glucose levels are a direct cause of long-term
complications of diabetes. The Diabetes Control and Complications
Trial (DCCT) and the United Kingdom Prospective Diabetes Study
(UKPDS) both showed that control of blood glucose at levels as
close to normal as possible prevents and retards development of
diabetic retinopathy, nephropathy, neuropathy, and microvascular
disease. Drug therapy of diabetes type II has consisted of oral
antidiabetic agents and insulin if and when the oral agents fail.
Insulin therapy in type I diabetes is essential and is intended to
replace the absent endogenous insulin with an exogenous insulin
supply. Because insulin is a protein drug (MW approx. 6000 Da) that
is not absorbed in the gastrointestinal tract, it ordinarily
requires parenteral administration such as by subcutaneous
injection.
[0016] Despite studies demonstrating the beneficial effects of good
glycemic control on chronic complications of diabetes, clinicians
are particularly keen on aggressive insulin therapy, and this is
widely accepted in clinical practice. The unmet challenge of
achieving good glycemic control is due, in part, to the
shortcomings of the available subcutaneous route of insulin
administration. In addition to the practical limitations of
multiple daily injections discussed above, the shortcomings of the
commonly available subcutaneous route of insulin administration
have resulted in the generally inadequate glycemic control
associated with many of the chronic complications associated with
diabetes. Elevated levels of insulin lead to increased glucose
uptake, glycogen synthesis, glycolysis, fatty acid synthesis, and
triacylglycerol synthesis leading to the expression of key genes
that result in greater utilization of glucose.
[0017] Accordingly, there is a need for a method of administering
insulin to patients in need of insulin wherein those patients are
not subject to the increased risk of vascular disease that is
normally associated with such chronic insulin treatments, as
discussed above. In other words, it is desirable to provide
compositions and methods for treating diabetes while decreasing the
incidence of such vascular diseases and other detrimental
effects.
SUMMARY OF THE INVENTION
[0018] It is one object of the present invention to provide useful
oral pharmaceutical formulations of drugs that are not considered
orally administrable due, e.g., to insufficient absorption of the
drugs from the gastrointestinal tract, which formulations are
therapeutically effective.
[0019] It is a further object of the present invention to provide
useful pharmaceutical formulations of insulin for oral
administration which are therapeutically effective.
[0020] It is a further object of the present invention to provide
delivery agents that may be orally administered together with a
drug that is not considered orally administrable due to, e.g.,
insufficient absorption of the drug from the gastrointestinal
tract, so that the drug is absorbed in adequate amounts from the
gastrointestinal tract to provide the desired therapeutic effect,
such as insulin.
[0021] It is an object of the present invention to provide
compositions comprising a delivery agent and insulin for oral
administration.
[0022] It is an object of the present invention to provide
compositions of a delivery agent and insulin for oral
administration that facilitates insulin transport in a
therapeutically effective amount to the bloodstream for the
treatment of diabetes, for the treatment of impaired glucose
tolerance, for the purpose of achieving glucose homeostasis, for
the treatment of early stage diabetes, and/or for the treatment of
late stage diabetes.
[0023] It is an object of the present invention to provide methods
for the preparation of a composition comprising insulin and
delivery agent oral administration, which result in an orally
administrable unit dose that provides a desired therapeutic
effect.
[0024] It is an object of the present invention to provide a
delivery agent(s) that can be utilized in an amount that
facilitates the preparation of an oral unit dosage form of a drug
that is not considered orally administrable by itself due to poor
absorption, etc., and results in an orally administrable unit dose
that provides a desired therapeutic effect.
[0025] It is an object of the invention to reduce the risk of
disease states associated with chronic insulin therapy.
[0026] It is another object of the invention to provide a method
for reducing the incidence in vascular diseases associated with
chronic insulin therapy in diabetics.
[0027] It is another object of the invention to delay the time to
onset of vascular diseases associated with chronic insulin therapy
in diabetics.
[0028] It is another object of the invention to reduce the severity
of vascular diseases associated with chronic insulin therapy in
diabetics.
[0029] It is another object of the invention to reduce the exposure
of the non-portal vasculature to hyperinsulinemic conditions.
[0030] It is another object of the invention to attenuate the
complex series of processes resulting from the reaction to insulin
treatment.
[0031] It is a further object of the invention to provide a method
and a pharmaceutical formulation which can reduce systemic blood
insulin concentrations while providing therapeutically effective
treatment of diabetes.
[0032] It is a further object of the invention to provide a method
and a pharmaceutical formulation which may help decrease the
instances and severity of the vascular complications and resultant
conditions (such as, e.g., retinopathy, neuropathy, nephropathy)
associated with Diabetes Mellitus.
[0033] It is a further object of the invention to lower the
exposure of the systemic vasculature to insulin during insulin
treatment.
[0034] It is a further object of the invention to reduce the
incidence and/or severity of macro- and micro-vascular
complications associated with insulin therapy in diabetics, which
leads to neuropathies, retinopathies, and peripheral vascular
disease.
[0035] In accordance with the above objects and others, the
invention is directed in part to an oral solid dosage form
comprising a dose of unmodified insulin that achieves a comparable
reduction in blood glucose concentration in human diabetic patients
compared to a subcutaneous insulin injection in those patients,
while providing a lower (e.g., 20% or greater) concentration of
insulin in the peripheral blood circulation under acute, sub-acute
and chronic conditions as compared to the peripheral blood insulin
concentration obtained via the subcutaneous injection.
[0036] The invention is also directed in part to an oral solid
dosage form comprising a dose of unmodified insulin that achieves a
therapeutically effective reduction in blood glucose after oral
administration to a human diabetic patient, and which maintains a
physiological gradient, and in certain embodiments provides a ratio
of portal vein to peripheral blood insulin concentration from about
2.5:1 to about 6:1, and preferably from about 4:1 to about 5:1.
[0037] The invention is further directed in part to an oral dosage
form comprising a dose of unmodified insulin that achieves a
therapeutically effective reduction in blood glucose after oral
administration to human diabetic patients, the oral solid dosage
form providing an insulin Tmax at a time point from about 0.25 to
about 1.5 hours after oral administration to said patients, at
least about 80% of the blood glucose concentration reduction caused
by said dose of insulin occurring within about 2 hours after oral
administration of said dosage form.
[0038] The invention is further directed in part to an oral dosage
form comprising a therapeutically effective amount of unmodified
insulin, said dosage form upon pre-prandial oral administration to
human diabetic patients causing the mean plasma glucose
concentration in said patients to be reduced for the first hour
after oral administration relative to a mean baseline (fasted)
plasma glucose concentration in said patients.
[0039] The invention is further directed in part to an oral dosage
form comprising a therapeutically effective amount of unmodified
insulin, said oral dosage form upon pre-prandial oral
administration provides a mean plasma glucose concentration which
does not vary by more than about 40% (and more preferably not more
than 30%) for the first hour after oral administration, relative to
a mean baseline (fasted) plasma glucose concentration in said
patients, where a meal is eaten by said patients within about one
half hour of oral administration of said dosage form.
[0040] In preferred embodiments of the oral dosage forms of the
invention described above, the oral dosage form is solid, and is
preferably provided incorporated within a gelatin capsule or is
contained in a tablet.
[0041] In certain preferred embodiments, the dose of unmodified
insulin contained in the dosage form is from about 50 Units to
about 600 Units (from about 2 to about 23 mg), preferably from
about 100 Units (3.8 mg) to about 400 Units (15.3 mg) insulin, and
most preferably from about 150 Units (5.75 mg) to about 300 Units
(11.5 mg), based on the accepted conversion of factor of 26.11
Units per mg.
[0042] In certain preferred embodiments, the dosage forms of the
invention provide a T.sub.max for insulin at about 0.1 to about 1.5
hours, and more preferably by about 0.25 to about 0.5 hours, after
oral administration. In certain preferred embodiments, the
T.sub.max for insulin occurs at less than about 100 minutes after
oral administration of the composition, preferably at less than
about 45 minutes, more preferably at less than about 40 minutes,
and still more preferably at about 22 minutes after oral
administration of the composition. In certain preferred
embodiments, the composition provides a T.sub.max for glucose
reduction at about 0.25 to about 1.5 hours, more preferably by
about 0.75 to about 1.0 hours, after oral administration. In
certain preferred embodiments, the T.sub.max for glucose reduction
occurs preferably at less than about 120 minutes, more preferably
at less than about 80 minutes, and most preferably at about 45
minutes, after oral administration of the composition.
[0043] In certain preferred embodiments of the invention, the
dosage forms begin delivering insulin into the portal circulation
(via absorption through the mucosa of the stomach) to achieve peak
levels within about 30 minutes or less.
[0044] In certain embodiments of the dosage forms described above,
in the absence of a delivery agent, the dose of unmodified insulin
is not adequately absorbed from the gastrointestinal tract when
administered orally to render a desired effect. In certain
preferred embodiments, in the absence of a delivery agent, the dose
of insulin is not sufficiently absorbed when orally administered to
a human patient to provide a desirable therapeutic effect but said
dose provides a desirable therapeutic effect when administered to
said patient by another route of adminstration. The invention in
such embodiments is further directed to an oral dosage form
comprising a dose of unmodified insulin together with a
pharmaceutically acceptable delivery agent in an amount effective
to facilitate the absorption of said insulin, such that a
therapeutically effective amount of said dose of insulin is
absorbed from the gastrointestinal tract of human diabetic
patients.
[0045] In certain preferred embodiments, the pharmaceutical
composition comprises from about 1 mg to about 800 mg of said
delivery agent, preferably about 50 to about 600, more preferably
from about 100 to about 300, most preferably about 200. In certain
embodiments, the composition provides a peak plasma delivery agent
concentration C.sub.max from about 1,000 and about 150,000 ng/ml,
and a T.sub.max at about 0.25 to about 1.5 hours, and more
preferably by about 0.25 to about 0.75 hours, most preferably 0.5
hours, after oral administration.
[0046] For purposes of the present invention, a preferred delivery
agent is identified via chemical nomenclature as 4-[(4-chloro,
2-hydroxybenzoyl)amino]butanoic acid. In certain preferred
embodiments, the delivery agent is a sodium salt, preferably
monosodium salt. Alternatively, the same compound is identified by
the alternative nomenclature monosodium
N-(4-chlorosalicyloyl)-4-aminobutyrate, or by the short name
"4-CNAB".
[0047] The invention is further directed in part to a method of
treatment of diabetes in humans, comprising administering one or
more unit doses of the dosage forms described above and in further
sections of the present specification.
[0048] The invention is further directed in part to a method of
treatment of impaired glucose tolerance, achieving glucose
homeostasis, early stage diabetes, and late stage diabetes in
humans, comprising administering one or more unit doses of the
dosage forms described above and in further sections of the present
specification on a chronic basis.
[0049] The invention is also related to a method of orally treating
mammals with an active agent (i.e., insulin) that is not
sufficiently absorbed when orally administered to provide a
desirable therapeutic effect but that provides a desirable
therapeutic effect when administered by another route of
adminstration, comprising orally administering said active agent
together with a delivery agent which facilitates the absorption of
insulin from the gastrointestinal tract, having one or more of the
further characteristics set forth above.
[0050] The invention is further directed to a method of providing a
therapeutically effective orally administrable unit dose of
unmodified insulin, comprising combining from about 2 to about 23
mg of unmodified insulin with from about 100 to about 600 mg of a
pharmaceutically acceptable delivery agent which facilitates
absorption of said insulin from the gastrointestinal tract of human
diabetic patients, and orally administering said unit dose to a
human diabetic patient to provide a therapeutic effect. In
preferred embodiments, the total weight of the unit dose is from
about 102 mg to about 800 mg.
[0051] The present invention is also directed in part to a method
of treating human diabetic patients comprising orally administering
to human diabetic patients on a chronic basis an oral insulin
treatment comprising a dose of unmodified insulin together with a
delivery agent that facilitates the absorption of the dose of
insulin from the gastrointestinal tract to provide a
therapeutically effective reduction in blood glucose and a blood
plasma insulin concentration that is reduced relative to the
systemic blood insulin concentration of an equivalent
therapeutically effective reduction in blood glucose concentration
achieved by subcutaneous injection of insulin.
[0052] The invention is also directed to a method of reducing the
incidence and/or severity of one or more disease states associated
with chronic administration of insulin, comprising treating human
diabetic patients via oral administration on a chronic basis with a
therapeutically effective dose of a (preferably solid)
pharmaceutical composition comprising a dose of unmodified insulin
and a delivery agent that facilitates the absorption of said
unmodified insulin from the gastrointestinal tract in an effective
amount such that the pharmaceutical composition provides
therapeutically effective control of mean blood glucose
concentration and a mean systemic blood insulin concentration in
diabetic patients that is reduced on a chronic basis relative to
the mean systemic blood insulin concentration provided by chronic
subcutaneous administration of insulin in an amount effective to
achieve equivalent control of mean blood glucose concentration in a
population of human diabetic patients.
[0053] The invention is further directed to a method of treating
diabetes and reducing the incidence and or severity of
hyperinsulinemia associated with chronic dosing of insulin,
comprising orally administering on a chronic basis to a diabetic
patient a dose of insulin and a delivery agent that facilitates the
absorption of the dose of insulin from the gastrointestinal tract
to provide a therapeutically effective reduction and/or control in
blood glucose and a mean systemic blood insulin concentration of
the diabetic patient that is reduced relative to the mean systemic
blood insulin concentration provided by subcutaneous injection of
insulin in an amount effective to achieve equivalent reduction
and/or control in a population of human diabetic patients.
[0054] The mean values of insulin concentration determination
obtained in patients who have been administered subcutaneous
insulin are well known to those skilled in the art.
[0055] The following terms will be used throughout the application
as defined below:
[0056] Diabetic patient--refers to humans suffering from a form of
diabetes.
[0057] IGT--means impaired glucose tolerance.
[0058] Diabetes--is deemed to encompasses type 1 and type 2
diabetes, unless specifically specified otherwise.
[0059] Biological macromolecule--biological polymers such as
proteins and polypeptides. For the purposes of this application,
biological macromolecules are also referred to as
macromolecules.
[0060] Delivery agent--refers to carrier compounds or carrier
molecules that are useful in the oral delivery of therapeutic
agents. "Delivery agent" may be used interchangeably with
"carrier".
[0061] Therapeutically effective amount of insulin--an amount of
insulin included in the oral dosage forms of the invention which
are sufficient to achieve a clinically significant control of blood
glucose concentrations in a human diabetic patient either in the
fasting state or in the fed state effective, during the dosing
interval.
[0062] Effective amount of delivery agent--an amount of the
delivery agent that promotes the absorption of a therapeutically
effective amount of the drug from the gastrointestinal tract.
[0063] Organic solvents--any solvent of non-aqueous origin,
including liquid polymers and mixtures thereof. Organic solvents
suitable for the present invention include: acetone, methyl
alcohol, methyl isobutyl ketone, chloroform, 1-propanol,
isopropanol, 2-propanol, acetonitrile, 1-butanol, 2-butanol, ethyl
alcohol, cyclohexane, dioxane, ethyl acetate, dimethylformamide,
dichloroethane, hexane, isooctane, methylene chloride, tert-butyl
alchohol, toluene, carbon tetrachloride, or combinations
thereof.
[0064] Peptide--a polypeptide of small to intermediate molecular
weight, usually 2 or more amino acid residues and frequently but
not necessarily representing a fragment of a larger protein.
[0065] Protein--a complex high polymer containing carbon, hydrogen,
oxygen, nitrogen and usually sulfur and composed of chains of amino
acids connected by peptide linkages. Proteins in this application
refer to glycoproteins, antibodies, non-enzyme proteins, enzymes,
hormones and peptides. The molecular weight range for proteins
includes peptides of 1000 Daltons to glycoproteins of 600 to 1000
kiloDaltons.
[0066] Reconstitution--dissolution of compositions or compositions
in an appropriate buffer or pharmaceutical composition.
[0067] Unit-Dose Forms--refers to physically discrete units
suitable for human and animal subjects and packaged individually as
is known in the art. It is contemplated for purposes of the present
invention that dosage forms of the present invention comprising
therapeutically effective amounts of insulin may include one or
more unit doses (e.g., tablets, capsules) to achieve the
therapeutic effect.
[0068] Unmodified insulin--means insulin prepared in any
pharmaceutically acceptable manner or from any pharmaceutically
acceptable source which is not conjugated with an oligomer such as
that described in U.S. Pat. No. 6,309,633 and/or which not has been
subjected to amphiphilic modification such as that described in
U.S. Pat. Nos. 5,359,030; 5,438,040; and/or 5,681,811.
[0069] As used herein, the phrase "equivalent therapeutically
effective reduction" means that a maximal reduction of blood
glucose concentration achieved by a first method of insulin
administration (e.g. via oral administration of insulin in a
patient(s)) is not more 20%, and preferably not more than 10% and
even more preferably not more than 5% different from a maximal
reduction of blood glucose concentration after administration by a
second method (e.g., subcutaneous injection) in the same patient(s)
or a different patient requiring the same reduction in blood
glucose level.
[0070] The term "AUC" as used herein, means area under the plasma
concentration-time curve, as calculated by the trapezoidal rule
over the complete dosing interval, e.g., 24-hour interval.
[0071] The term "C.sub.max" as it is used herein is the highest
plasma concentration of the drug attained within the dosing
interval.
[0072] The term "T.sub.max" as it is used herein is the time period
which elapses after administration of the dosage form at which the
plasma concentration of the drug attains the C.sub.max within the
dosing interval.
[0073] The term "multiple dose" means that the human patient has
received at least two doses of the drug composition in accordance
with the dosing interval for that composition.
[0074] The term "single dose" means that the human patient has
received a single dose of the drug composition and the drug plasma
concentration has not achieved steady state.
[0075] Unless specifically designated as "single dose" or at
"steady-state" the pharmacokinetic parameters disclosed and claimed
herein encompass both single dose and steady-state conditions.
[0076] The term "mean", when preceding a pharmacokinetic value
(e.g., mean T.sub.max) represents the arithmetic mean value of the
pharmacokinetic value unless otherwise specified.
[0077] The term "Bioavailability" as used herein means the degree
or ratio (%) to which a drug or agent is absorbed or otherwise
available to the treatment site in the body. This is calculated by
the formula 1 Rel . Bioavailability ( % ) = Dose SC Dose Oral
.times. AUC INS Oral AUC INS SC .times. 100
[0078] The term "Biopotency" as used herein means the degree or
ratio (%) to which a drug or agent is effective to the treatment
site in the body. This is calculated by the formula 2 Rel .
Biopotency ( % ) = Dose SC Dose Oral .times. AUC GIR Oral AUC GIR
SC .times. 100
[0079] The term "F.sub.rel" as used herein means the relative
bioavailability of insulin calculated by comparing dose corrected
oral insulin AUC with the dose corrected SC insulin AUC.
[0080] The term "AUC.sub.(0-x)" as used herein means the area under
the plasma concentration-time curving linear trapezoidal summation
from time 0 to time x hours post-dose.
[0081] The term "AUC.sub.(0-t)" as used herein means the area under
the plasma concentration-time curve using linear trapezoidal
summation from time zero to time t post-dose, where t is the time
of the last measurable concentration (C.sub.t).
[0082] The term "AUC.sub.(0-inf)" as used herein means the area
under the plasma concentration-time curve from time 0 to infinity,
AUC.sub.(0-inf)=AUC.sub.(0-t)+Ct/K.sub.el, where K.sub.el is the
terminal elimination rate constant calculated by linear regression
of the terminal linear portion of the log concentration vs. time
curve.
[0083] The term "AUC.sub.%Extrap" as used herein means the
percentage of the total AUC.sub.(0-inf)obtained by
extrapolation.
[0084] The term "AUEC.sub.(0-x)" as used herein means the area
under the effect-time curve calculated using the linear trapezoidal
summation from time 0 to the concentration at time x hours
post-dose.
[0085] The term "AUEC.sub.(0-t)" as used herein means the area
under the effect-time curve calculated using the linear trapezoidal
summation from time 0 to the concentration at time t hours
post-dose, where t is the time of the last measurable effect
(E).
[0086] The term "AURC.sub.(0-x)" as used herein means the area
under the response-time curve calculated using the linear
trapezoidal summation from time zero to the concentration at time x
(Baseline Subtracted AUEC).
[0087] The term "AURC(0-t)" as used herein means the area under the
response-time curve calculated using the linear trapezoidal
summation from time zero to the concentration at time t (Baseline
Subtracted AUEC), where t is the time of the last measurable
response (R).
[0088] The term "C.sup.b" as used herein means the maximum observed
plasma insulin concentration prior to intervention for
hypoglycemia.
[0089] The term "CL/F" as used herein means the apparent total body
clearance calculated as Dose/AUC.sub.(0-inf).
[0090] The term "E.sup.b" as used herein means the maximum observed
effect (baseline subtracted) prior to intervention for
hypoglycemia.
[0091] The term "E.sub.max" as used herein means the maximum
observed effect (baseline subtracted).
[0092] The term "MRT" as used herein means the mean residence time
calculated as the ratio of the Area Under the first moment of the
plasma concentration-time curve (AUMC) and the area under the
plasma concentration-time curve, (AUMC)/AUC.sub.(0-inf).
[0093] The term "R.sub.max" as used herein means the maximum
observed response (total response), i.e., minimum glucose
concentration.
[0094] The term "R.sup.b" as used herein means the maximum observed
response (total response) prior to hypoglycemic intervention.
[0095] The term "t.sup.b" as used herein means the time to reach
insulin/glucose plasma concentration prior to hypoglycemic
intervention.
[0096] The term "t.sup.c" as used herein means the time to reach
glucose concentration change from baseline prior to hypoglycemic
intervention.
[0097] The term "t.sub.Rmax" as used herein means the time to reach
maximum response.
[0098] The term "t.sub.Emax" as used herein means time of the
maximum effect (obtained without interpolation).
[0099] The term "t.sub.1/2" as used herein means the terminal
half-life calculated as ln(2)/K.sub.el.
[0100] The term "V.sub.d/F" as used herein means the apparent
volume of distribution calculated as (CL/F)/K.sub.el.
[0101] As used herein and in the appended claims, the singular
forms "a," "an," and "the," include plural referents unless the
context clearly indicates otherwise. Thus, for example, reference
to "a molecule" includes one or more of such molecules, "a reagent"
includes one or more of such different reagents, reference to "an
antibody" includes one or more of such different antibodies, and
reference to "the method" includes reference to equivalent steps
and methods known to those of ordinary skill in the art that could
be modified or substituted for the methods described herein.
[0102] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which the invention belongs. Although
any methods, compositions, reagents, cells, similar or equivalent
to those described herein can be used in the practice or testing of
the invention, the preferred methods and materials are described
herein. All publications mentioned herein are incorporated herein,
including all figures, graphs, equations, illustrations, and
drawings, to describe and disclose specific information for which
the reference was cited in connection with.
[0103] The publications discussed above are provided solely for
their disclosure before the filing date of the present application.
Nothing herein is to be construed as an admission that the
invention is not entitled to antedate such disclosure by virtue of
prior invention. Throughout this description, the preferred
embodiment and examples shown should be considered as exemplars,
rather than as limitations on the present invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0104] FIG. 1 shows C-peptide measurements for insulin delivered
orally and subcutaneously.
[0105] FIG. 2 shows the plasma insulin concentration versus time
for administration of insulin subcutaneously and orally in the
presence of 4-CNAB.
[0106] FIG. 3 shows the % decrease in plasma glucose versus time
upon delivery of placebo and administering insulin subcutaneously
and orally in the presence of 4-CNAB.
[0107] FIG. 4 shows C-peptide concentration versus time after oral
dosing of 4-CNAB alone, Placebo and 150 U human insulin alone.
[0108] FIG. 5 shows the % decrease in C-peptide versus time after
administering insulin subcutaneously and orally in the presence of
4-CNAB.
[0109] FIGS. 6A and 6B show mean (+SD) plasma insulin concentration
time profiles following the administration of 150 Units/200 mg
(Insulin/4-CNAB) (n=5), 100 Units/600 mg (n=7), 10 Units SC insulin
(n=8) and oral placebo (n=10) treatment in non-hypoglycemic
subjects.
[0110] FIGS. 7A and 7B show mean (+SD) plasma insulin concentration
time profiles following the administration of 100 Units/300 mg
(Insulin/4-CNAB) (n=7), 100 Units/450 mg (n=7), 150 Units/100 mg
(n=8), 150 Units USP oral insulin (n=8) and oral placebo (n=10)
treatment in non-hypoglycemic subjects.
[0111] FIGS. 8A and 8B show the mean (+SD) glucose
concentration-time profiles following the administration of 150
Units/200 mg (Insulin/4-CNAB) (n=5), 100 Units/600 mg (n=7), 10
Units SC insulin (n=8) and oral placebo (n=10) treatment in
non-hypoglycemic subjects.
[0112] FIGS. 9A and 9B show the Mean (+SD) glucose
concentration-time profiles following the administration of 100
Units/300 mg (Insulin /4-CNAB) (n=7), 100 Units/450 mg (n=7), 150
Units/100 mg (n=8), 150 Units USP oral insulin (n=8) and oral
placebo (n=10) treatment in non-hypoglycemic subjects.
[0113] FIG. 10 shows plasma glucose versus time for patients from
Group 1, following a minimum of 8 hour overnight fast, patients
were given one capsule containing a mixture of insulin in a
stepwise fashion (3 patients received 200 U insulin, 5 patients
received 300 U insulin and 4 patients received 400 U insulin) and a
fixed dose of 300 mg 4-CNAB as a delivery agent.
[0114] FIG. 11 shows plasma glucose versus time for patients from
Group 2, patients had a standard meal (350 kcal) after a minimum of
8 hour overnight fast. Twenty minutes prior to the ingestion of
food, the patients were administered a capsule contained 300 U or
400 U insulin (six patients received 300 U insulin and six patients
received 400 U of insulin) and 300 mg of 4-CNAB.
[0115] FIG. 12 shows glucose versus time for the study of Type I
diabetics and healthy subjects after dosing with oral insulin (300
IU insulin/400 mg 4-CNAB).
[0116] FIG. 13 shows a plot of the arithmetic means of blood
glucose excursions (i.e., differences between pre-prandial and
postprandial blood glucose concentrations) for all subjects.
[0117] FIG. 14 shows a comparison of blood glucose levels over a
time period 180 minutes following single administration of insulin
orally and subcutaneously (mean.+-.SE).
[0118] FIG. 15 shows the serum insulin levels over a time period of
180 minutes following single administration orally and
subcutaneously (mean.+-.SE).
[0119] FIG. 16 shows Glucokinase and G6Pase mRNA expression
compared to sham dosing.
[0120] FIG. 17 shows Fru-1, 6-P and
6-Phosphofructo-2-kinase/fructose-2, 6-bisphosphatase mRNA
expression compared to sham dosing.
[0121] FIG. 18 shows PEPCK mRNA expression compared to sham
dosing.
[0122] FIG. 19 shows Glycogen synthase MRNA expression compared to
sham dosing.
[0123] FIGS. 20A and 20B show early response gene mRNA expression
compared to sham dosing.
[0124] FIG. 21 shows insulin-like Growth Factor Binding Protein
mRNA expression compared to sham dosing.
[0125] FIG. 22 shows Intracellular Adhesion Molecule-1 mRNA
expression compared to sham dosing.
[0126] FIGS. 23A and 23B shows Cytokine mRNA expression compared to
sham dosing.
[0127] FIG. 24 shows Lipid Peroxidation enzyme mRNA expression
compared to sham dosing.
[0128] FIG. 25 shows Plasminogen Activator Inhibitors mRNA
expression compared to sham dosing.
[0129] FIG. 26 shows NPY, TGF-beta, ICAM-1 and 12-LO mRNA
expression compared to sham dosing.
[0130] FIG. 27 shows THY-1, VEGF-B and Integrin aE2 mRNA expression
compared to sham dosing.
[0131] FIG. 28 shows a comparison of blood glucose levels over a
time period 180 minutes following single administration of insulin
orally and subcutaneously (mean.+-.SE) in a Streptozotocin diabetic
model.
[0132] FIG. 29 shows the serum insulin levels over a time period of
180 minutes following single administration orally and
subcutaneously (mean.+-.SE) in a Streptozotocin diabetic model.
DETAILED DESCRIPTION
[0133] The present invention provides pharmaceutical compositions
that are useful as delivery agents in the oral delivery of an
active agent that is not generally considered by those skilled in
the art to be administrable via the oral route, such as insulin.
Such compositions serve to make insulin bioavailable and absorbable
through the gastrointestinal mucosa when orally administered.
[0134] Preferably, the pharmaceutical composition includes insulin
as the active agent. As used herein, "insulin" refers to insulin
from a variety of sources. Naturally occurring insulin and
structurally similar bioactive equivalents can be used. Insulin
useful in the invention can be isolated from different species of
mammal. For example, animal insulin preparations extracted from
bovine or porcine pancreas can be used. Insulin analogues,
derivatives and bioequivalents thereof can also be used with the
invention. In addition to insulin isolated from natural sources,
the present invention can use insulin chemically synthesizing using
protein chemistry techniques such as peptide synthesis. Analogues
of insulin are also suitable for the present invention.
[0135] The insulin used in the present invention may be obtained by
isolating it from natural sources or by chemically synthesizing it
using peptide synthesis, or by using the techniques of molecular
biology to produce recombinant insulin in bacteria or eucaryotic
cells. Analogs of insulin are also provided by the present
invention. Insulin from other species of mammal may also be used in
the present invention. The physical form of insulin may include
crystalline and/or amorphous solid forms. In addition, dissolved
insulin may be used. Other suitable forms of insulin, including,
but not limited to, synthetic forms of insulin, are described in
U.S. Pat. Nos. 4,421,685, 5,474,978, and 5,534,488, the disclosure
of each of which is hereby incorporated by reference in its
entirety.
[0136] The most preferred insulin useful in the pharmaceutical
compositions and methods of the present invention is human
recombinant insulin. Human recombinant insulin can be prepared
using genetic engineering techniques that are well known in the
art. Recombinant insulin can be produced in bacteria or eucaryotic
cells. Functional equivalents of human recombinant insulin are also
useful in the invention. Recombinant human insulin can be obtained
from a variety of commercial sources. For example, insulin (Zinc,
human recombinant) can be purchased from Calbiochem (San Diego,
Calif.). Alternatively, human recombinant Zinc-Insulin Crystals:
Proinsulin Derived (Recombinant DNA Origin) USP Quality can be
obtained from Eli Lilly and Company (Indianapolis, Ind.). All such
forms of insulin, including insulin analogues (including but not
limited to Insulin Lispro, Insulin Aspart, Insulin Glargine,
Insulin Detemir) are deemed for the purposes of this specification
and the appended claims are considered to be encompassed by the
term "insulin."
[0137] The present invention provides compositions of recombinant
human zinc insulin and a delivery agent as a drug for oral
administration of insulin in humans.
[0138] In yet further embodiments of the invention, the active
agent is not insulin but instead is an active agent of a biological
nature suitable for use in the present invention including, but not
limited to, proteins; polypeptides; peptides; hormones;
polysaccharides, and particularly mixtures of muco-polysaccharides;
carbohydrates; lipids; other organic compounds; and particularly
compounds which by themselves do not pass (or which pass as only a
fraction of the administered dose) through the gastro-intestinal
mucosa and/or are susceptible to chemical cleavage by acids and
enzymes in the gastro-intestinal tract; or any combination thereof.
Further examples of active agents of a biological nature include,
but are not limited to, the following, including synthetic, natural
or recombinant sources thereof: growth hormones, including human
growth hormones (hGH), recombinant human growth hormones (rhGH),
bovine growth hormones, and porcine growth hormones; growth
hormone-releasing hormones; interferons, including .alpha., .beta.
and .gamma.; interleukin-1; interleukin-2; insulin, including
porcine, bovine, human, and human recombinant, optionally having
counter ions including sodium, zinc, calcium and ammonium;
insulin-like growth factor, including IGF-1; heparin, including
unfractionated heparin, heparinoids, dermatans, chondroitins, low
molecular weight heparin, very low molecular weight heparin and
ultra low molecular weight heparin; calcitonin, including salmon,
eel, porcine and human; erythropoietin; atrial naturetic factor;
antigens; monoclonal antibodies; somatostatin; protease inhibitors;
adrenocorticotropin, gonadotropin releasing hormone; oxytocin;
leutinizing-hormone-releasing-hormone; follicle stimulating
hormone; glucocerebrosidase; thrombopoietin; filgrastim;
prostaglandins; cyclosporin; vasopressin; cromolyn sodium (sodium
or disodium chromoglycate); vancomycin; desferrioxamine (DFO);
parathyroid hormone (PTH), including its fragments; antimicrobials,
including anti-fungal agents; vitamins; analogs, fragments,
mimetics or polyethylene glycol (PEG)-modified derivatives of these
compounds; or any combination thereof.
[0139] In one embodiment of this invention, the protein active
agents have a molecular weight of less than or equal to 10,000
Daltons. In another embodiment of this invention, protein active
agents have a molecular weight of about 6,000 Daltons. In another
embodiment of this invention, protein active agents have a
molecular weight of greater than or equal to 10,000 Daltons.
According to an alternate embodiment of the present invention,
protein active agents have a molecular weight that is greater than
or equal to 20,000 Daltons. In a further embodiment, protein active
agents have a molecular weight that is greater than or equal to
30,000 Daltons. According to an alternate embodiment, protein
active agents have a molecular weight that is greater than or equal
to 40,000 Daltons. According to another alternate embodiment,
protein active agents have a molecular weight that is greater than
or equal to 50,000 Daltons.
[0140] Insulin entry into the bloodstream produces a decrease in
plasma glucose levels. Therefore, oral absorption of insulin may be
verified by observing the effect on a subject's blood sugar
following oral administration of the composition. In a preferred
embodiment of the invention, the oral dosage forms of the invention
facilitate the oral delivery of insulin, and after insulin is
absorbed into the bloodstream, the composition produces a maximal
decrease in blood glucose in treated patients from about 20 to
about 60 minutes after oral administration. In another embodiment
of the present invention, the pharmaceutical composition produces a
maximal decrease in blood glucose in treated patients from about 30
to about 50 minutes post oral administration. More particularly,
the pharmaceutical composition produces a maximal decrease in blood
glucose in treated patients at about 40 minutes after oral
administration.
[0141] The magnitude of the decrease in blood glucose produced by
insulin absorbed into the bloodstream following entry into the
gastrointestinal tract varies with the dose of insulin. In certain
embodiments of the invention, human diabetic patients show a
maximal decrease in blood glucose by at least 10% within one hour
post oral administration. In another embodiment, human diabetic
patients show a maximal decrease in blood glucose by at least 20%
within one hour post oral administration, alternatively, at least
30% within one hour post oral administration.
[0142] Normal levels of blood glucose vary somewhat throughout the
day and in relation to the time since the last meal. One goal of
the present invention is to provide oral compositions of insulin
that facilitate achieving normal levels of blood glucose throughout
the 24-hour daily cycle. In a preferred embodiment of the
invention, wherein the pharmaceutical composition includes insulin
or an insulin analog as the active agent and a delivery agent in an
amount effective to achieve a fasting blood glucose concentration
from about 90 to about 110 mg/dl. In another preferred embodiment
of the invention, wherein the pharmaceutical composition includes
insulin or an insulin analog as the active agent and a delivery
agent in an amount effective to achieve a fasting blood glucose
concentration from about 95 to about 105 mg/dl, more preferably,
the subject manifests fasting blood glucose concentrations at about
100 mg/dl.
[0143] In the time after a meal is consumed, blood glucose
concentration rises in response to digestion and absorption into
the bloodstream of glucose derived from the food eaten. The present
invention provides oral compositions of insulin that prevent or
control very high levels of blood glucose from being sustained.
More particularly, the present invention provides compositions
which facilitate achieving normal levels of blood glucose after a
meal has been consumed, i.e., post-prandial. In a preferred
embodiment of the invention, the pharmaceutical composition
includes insulin as the active agent and a delivery agent in an
amount effective to achieve a post-prandial blood glucose
concentration from about 130 to about 170 mg/dl. In another
preferred embodiment of the invention, the pharmaceutical
composition includes insulin or an insulin analog as the active
agent and a delivery agent in an amount effective to achieve a
post-prandial blood glucose concentration from about 140 to about
160 mg/dl, more preferably, the subject manifests fasting blood
glucose concentrations at less than about 160 mg/dl.
[0144] The present invention provides pharmaceutical compositions
for oral administration which includes insulin or an insulin analog
as the active agent and a delivery agent in an amount effective to
achieve pre-prandial (before a meal is consumed) blood glucose
concentration from about 95 to about 125 mg/dl. In a preferred
embodiment, the present invention provides pharmaceutical
compositions for oral administration which includes insulin or an
insulin analog as the active agent and a delivery agent in an
amount effective to achieve pre-prandial blood glucose
concentration from about 100 to about 120 mg/dl.
[0145] Blood glucose concentrations fall to their lowest levels
during the sleeping period which is usually at night. The present
invention provides pharmaceutical compositions for oral
administration which include insulin as the active agent and a
delivery agent in an amount effective to achieve blood glucose
concentrations at 3 AM from about 70 to about 120 mg/dl. In a
preferred embodiment, the present invention provides pharmaceutical
compositions for oral administration which include insulin or an
insulin analog as the active agent and a delivery agent in an
amount effective to achieve blood glucose concentrations at 3 AM
from about 80 to about 120 mg/dl.
[0146] The effect of absorption of insulin is manifested in human
patients treated with the pharmaceutical compositions of the
present invention by observing reductions in C-peptide
concentration following oral treatment. For example, in one
embodiment of the invention, the pharmaceutical composition
comprises insulin as the active agent and the compound 4-CNAB as a
delivery agent to facilitate the oral delivery of insulin, and
after insulin is absorbed into the bloodstream, the composition
produces a maximal decrease in C peptide concentration in treated
patients from about 80 and about 120 minutes post oral
administration. More particularly, the composition produces a
maximal decrease in C peptide concentration in treated patients
from about 90 and about 110 minutes post oral administration.
[0147] Absorption of insulin can be detected in subjects treated
with the pharmaceutical compositions of the present invention by
monitoring the plasma levels of insulin after treatment. The time
it takes for an active agent to reach a peak in the bloodstream
(T.sub.max) may depend on many factors such as the following: the
nature of the unit dose, i.e., solid, liquid, tablet, capsule,
suspension; the concentration of active agent and delivery agent in
the GI tract; the feeding state of the subject; the diet of the
subject; the health of the subject and the ratio of active agent to
the delivery agent. In a preferred embodiment of the invention,
wherein the pharmaceutical composition includes the compound 4-CNAB
as the delivery agent and insulin as the active agent, the
composition provides a peak plasma insulin concentration from about
0.1 to about 1 hour after oral administration. In another
embodiment, the composition provides a peak plasma insulin
concentration from about 0.2 to about 0.6 hours after oral
administration. In a preferred embodiment, the composition provides
a peak plasma insulin concentration from about 0.3 to about 0.4
hours after oral administration. In another embodiment, the
composition provides a peak plasma insulin concentration within
about 1 hour after oral administration. In certain preferred
embodiments of the invention, the pharmaceutical composition
comprises insulin as the active agent and the compound 4-CNAB as a
delivery agent to facilitate the oral delivery of insulin, and
after insulin is absorbed into the bloodstream, the plasma insulin
levels in treated patients peak at about 20 minutes post oral
administration with a second peak at about 105 minutes.
[0148] In preferred embodiments, the compositions of the present
invention include an active agent (e.g., insulin) and a delivery
agent that serves to render the active agent orally absorbable
through the mucosa of the stomach. Accordingly, the present
invention solves the problem of oral absorption of macromolecules
by providing delivery agents that facilitate transport of such
biomolecules through the gastrointestinal system and into the
bloodstream where the active agent can perform its necessary
biological role. As a result of the present invention, effective
oral drug delivery methods are provided to increase the oral
bioavailability and absorption of drugs that are currently
administered parenterally.
[0149] In other preferred embodiments, the delivery agents used in
the invention have the following structure: 1
[0150] wherein X is one or more of hydrogen, halogen, hydroxyl or
C.sub.1-C.sub.3 alkoxy, and R is substituted or unsubstituted
C.sub.1-C.sub.3 alkylene, substituted or unsubstituted
C.sub.1-C.sub.3 alkenylene.
[0151] In certain preferred embodiments, the delivery agents of the
invention preferably have the following structure: 2
[0152] wherein X is halogen, and R is substituted or unsubstituted
C.sub.1-C.sub.3 alkylene, substituted or unsubstituted
C.sub.1-C.sub.3 alkenylene.
[0153] In a preferred embodiment of the present invention, the
pharmaceutical composition includes a delivery agent wherein X is
chlorine and R is C.sub.3 alkylene. In another preferred embodiment
of the present invention, the pharmaceutical composition includes
the compound 4-[(4-chloro, 2-hydroxybenzoyl)amino]butanoic acid as
a delivery agent for the oral delivery of insulin, preferably the
monosodium salt thereof.
[0154] The delivery agents may be in the form of the carboxylic
acid or salts thereof. Suitable salts include, but are not limited
to, organic and inorganic salts, for example alkali-metal salts,
such as sodium, potassium and lithium; alkaline-earth metal salts,
such as magnesium, calcium or barium; ammonium salts; basic amino
acids, such as lysine or arginine; and organic amines, such as
dimethylamine or pyridine. Preferably, the salts are sodium salts.
The salts may be mono- or multi-valent salts, such as monosodium
salts and di-sodium salts. The salts may also be solvates,
including ethanol solvates, and hydrates.
[0155] Other suitable delivery agents that can be used in the
present invention include those delivery agents described U.S. Pat.
Nos. 5,650,386, 5,773,647, 5,776,888, 5,804,688, 5,866,536,
5,876,710, 5,879,681, 5,939,381, 5,955,503, 5,965,121, 5,989,539,
5,990,166, 6,001,347, 6,051,561, 6,060,513, 6,090,958, 6,100,298,
5,766,633, 5,643,957, 5,863,944, 6,071,510 and 6,358,504, the
disclosure of each of which is incorporated herein by reference.
Additional suitable delivery agents are also described in
International Publications Nos. WO 01/34114, WO 01/21073, WO
01/41985, WO 01/32130, WO 01/32596, WO 01/44199, WO 01/51454, WO
01/25704, WO 01/25679, WO00/50386, WO02/02509, WO00/47188,
WO00/07979, WO00/06534, WO98/25589, WO02/19969, WO00/59863,
WO95/28838, WO02/20466 and WO02/19969, and International Patent
Applications Nos. PCT/US02/06610 and PCT/US02/06295, the disclosure
of each of which is incorporated herein by reference.
[0156] Salts of the delivery agent compounds of the present
invention may be prepared by methods known in the art. For example,
sodium salts may be prepared by dissolving the delivery agent
compound in ethanol and adding aqueous sodium hydroxide.
[0157] The compounds described herein may be derived from amino
acids and can be readily prepared from amino acids by methods known
by those with skill in the art based upon the present disclosure
and the methods described in International Publications Nos. WO
96/30036, WO 97/36480, WO 98/34632 and WO 00/07979, and in U.S.
Pat. Nos. 5,643,957 and 5,650,386, the disclosure of each of which
is incorporated herein by reference. For example, the compounds may
be prepared by reacting the single amino acid with the appropriate
acylating or amine-modifying agent, which reacts with a free amino
moiety present in the amino acid to form amides. Protecting groups
may be used to avoid unwanted side reactions as would be known to
those skilled in the art.
[0158] The delivery agents may also be prepared by the methods of
International Patent Application No. PCT/US01/21073, the disclosure
of which is incorporated herein by reference.
[0159] The delivery agents may also be prepared by alkylation of
the appropriate salicylamide according to the methods of
International Publication No. WO 00/46182, the disclosure of which
is incorporated herein by reference. The salicylamide may be
prepared from salicylic acid via the ester by reaction with
sulfuric acid and ammonia.
[0160] In addition, poly amino acids and peptides comprising one or
more of these compounds may be used. An amino acid is any
carboxylic acid having at least one free amine group and includes
naturally occurring and synthetic amino acids. Poly amino acids are
either peptides (which are two or more amino acids joined by a
peptide bond) or are two or more amino acids linked by a bond
formed by other groups which can be linked by, e.g., an ester or an
anhydride linkage. Peptides can vary in length from dipeptides with
two amino acids to polypeptides with several hundred amino
acids.
[0161] The delivery agent compound may be purified by
recrystallization or by fractionation on one or more solid
chromatographic supports, alone or linked in tandem. Suitable
recrystallization solvent systems include, but are not limited to,
ethanol, water, heptane, ethyl acetate, acetonitrile, methanol and
tetrahydrofuran and mixtures thereof. Fractionation may be
performed on a suitable chromatographic support such as alumina,
using methanol/n-propanol mixtures as the mobile phase; reverse
phase chromatography using trifluoroacetic acid/acetonitrile
mixtures as the mobile phase; and ion exchange chromatography using
water or an appropriate buffer as the mobile phase. When anion
exchange chromatography is performed, preferably a 0-500 mM sodium
chloride gradient is employed.
[0162] Following oral administration of the pharmaceutical
compositions of the present invention, the delivery agent passes
though the mucosal barriers of the GI tract and is absorbed into
the blood stream where it can be detected in the plasma of
subjects. The level of delivery agent in the bloodstream as
measured in the plasma is dose-dependent. The delivery agent
facilitates the absorption of the drug (active agent) administered
therewith (either in the same dosage form, or simultaneously
therewith), or sequentially (in either order, as long as both the
delivery agent and the drug are administered within a time period
which provides both in the same location, e.g., the stomach, at the
same time).
[0163] In certain preferred embodiments of the invention, a peak
plasma concentration (C.sub.max) of the delivery agent achieved
after oral administration is preferably from about 10 to about
250,000 ng/ml, after oral administration, preferably from about 100
to about 125,000, and preferably the peak plasma concentration of
the delivery agent is from about 1,000 to about 50,000 ng/ml, after
oral administration. More preferably, the peak plasma concentration
of the delivery agents of the present invention is from about 5,000
to about 15,000 ng/ml, after oral administration.
[0164] The time it takes for the delivery agent to reach a peak in
the bloodstream (T.sub.max) may depend on many factors such as the
following: the nature of the unit dose, i.e., solid, liquid,
tablet, capsule, suspension; the concentration of delivery agent in
the GI tract; the feeding state of the subject; the diet of the
subject; the health of the subject and the ratio of delivery agent
to the active agent. The delivery agents of the present invention
are rapidly absorbed from the gastrointestinal tract when orally
administered in an immediate release dosage form, and preferably
provide a peak plasma concentration within about 0.1 to about 8
hours after oral administration, and preferably at about 0.1 to
about 3 hours after oral administration.
[0165] In preferred embodiments, the T.sub.max of the delivery
agent occurs at about 0.3 to about 1.5 hours after oral
administration. In certain embodiments, the delivery agent achieves
a (T.sub.max) within about 2 hours after oral administration, and
most preferably, within about 1 hour after oral administration.
[0166] The amount of delivery agent necessary to adequately deliver
an active agent into the blood stream of a subject needing the
therapeutic effect of that active agent may vary depending on one
or more of the following; the chemical nature of the active agent;
the chemical structure of the particular delivery agent; the nature
and extent of interaction from about the active agent and delivery
agent; the nature of the unit dose, i.e., solid, liquid, tablet,
capsule, suspension; the concentration of delivery agent in the GI
tract; the feeding state of the subject; the diet of the subject;
the health of the subject and the ratio of delivery agent to the
active agent. In a certain preferred embodiment of the invention,
the amount of the delivery agent preferred for the pharmaceutical
composition is from about 1 mg to about 2,000 mg delivery agent,
more preferably from about 1 mg to about 800 mg of said delivery
agent, more preferably from about 50 mg to about 700 mg of said
delivery agent, even more preferably from about 70 mg to about 700
mg of said delivery agent, still more preferably from about 100 to
about 600 mg.
[0167] Preferably, the delivery agent is 4-CNAB. Since the amount
of delivery agent required to deliver a particular active agent is
variable and the amount of active agent required to produce a
desired therapeutic effect is also a variable, the ratio of active
agent to delivery agent may vary for different active
agent/delivery agent combinations. In certain preferred embodiments
of the invention where the oral pharmaceutical composition includes
insulin as the active agent and the delivery agent is the compound
4-CNAB, the amount of the delivery agent included in the
pharmaceutical composition may be from about 100 mg to about 600 mg
of said delivery agent.
[0168] In certain preferred embodiments of the invention, the
pharmaceutical composition includes insulin as the active agent and
the delivery agent is the monosodium salt of 4-CNAB, the ratio of
insulin [Units] to delivery agent [mg] ranges from 10:1 [Units/mg]
to 1:10 [Units/mg], preferably, the ratio of insulin [Units] to
delivery agent [mg] ranges from 5:1 [Units/mg] to 0.5:1
[Units/mg].
[0169] Preferred insulin doses in a single administration are about
5 to about 1000 insulin units USP, preferably from about 50 to
about 400, more preferably from about 150 to about 400, and still
more preferably from about 150 to about 300 units.
[0170] The optimum ratio of insulin to delivery agent can vary
depending on the delivery agent. Optimizing the ratio of insulin to
delivery agent is within the knowledge of one skilled in the
art.
[0171] In a preferred embodiment of the invention, wherein the
pharmaceutical composition includes the compound 4-CNAB as the
delivery agent and insulin as the active agent, the composition
provides a peak plasma delivery agent concentration within about
0.1 to about 3 hours after oral administration. In certain
preferred embodiments where the pharmaceutical composition includes
the compound 4-CNAB as the delivery agent and insulin as the active
agent, the peak plasma concentration of delivery agent attained is
from about 8,000 to about 37,000 ng/ml.
[0172] The mechanism by which 4-CNAB facilitates the
gastrointestinal absorption of insulin has not yet been fully
elucidated. The current working hypothesis is that 4-CNAB interacts
with insulin non-covalently, creating more favorable
physicochemical properties for absorption. This working hypothesis
is provided for explanation purposes only and is not intended to
limit the present invention or the appended claims in any way.
[0173] A preferred embodiment of the invention provides methods for
reducing the incidence of vascular disease associated with chronic
dosing of insulin. The methods in a preferred embodiment comprise
treating human diabetic patients on a chronic basis with an oral
and a delivery agent or pharmaceutically acceptable salt thereof
that facilitates the absorption of insulin from the
gastrointestinal tract (i.e., bioavailable).
[0174] The delivery agent may be used directly by mixing one or
more such agents with the active agent (e.g., unmodified insulin)
prior to administration. The delivery agent and active agent may be
mixed in dry powder form or wet granulated together. To this
mixture, other pharmaceutically acceptable excipients may be added.
The mixture may be then tableted or placed into gelatin capsules
containing a unit dose of the active agent and the delivery agent.
Alternatively, the delivery agent/active agent mixture may be
prepared as an oral solution or suspension. The delivery agent and
active agent do not need to be mixed together prior to
administration, such that, in certain embodiments, the unit dose of
active agent (with or without other pharmaceutically acceptable
excipients) is orally administered without the delivery agents of
this invention, and the delivery agent is separately orally
administered (with or without other pharmaceutically acceptable
excipients) before, after, or simultaneously with the active
agent.
[0175] In certain preferred embodiments, the oral dosage forms of
the present invention are solid. The unmodified insulin in dry
powder form is stable, and in certain preferred embodiments is
simply mixed in a desirable ratio with the delivery agent. The dry
powder mixture may then be filled into gelatin capsules, with or
without optional pharmaceutical excipients. Alternatively, the
unmodified insulin in dry powder form may be mixed with the
delivery agent together with optional pharmaceutical excipients,
and the mixture may be tableted in accordance with standard
tableting procedures known to those having ordinary skill in the
art.
[0176] The present invention also provides methods for treating
human diabetic patients with active agents that are not inherently
bioavailable, such as for example treating diabetics with insulin.
More particularly, the present invention provides method of
treating humans with an oral dosage form of a pharmaceutical
composition, wherein the pharmaceutical composition includes the
following: first, an active agent or a pharmaceutically acceptable
salt thereof, which is not orally bioavailable when dissolved or
suspended in aqueous solution, wherein the active agent provide a
therapeutic effect when administered to a subject by another means
(e.g., via subcutaneous injection); and, second, an effective
amount of a delivery agent or a pharmaceutically acceptable salt
thereof, which renders the active agent orally absorbed (e.g.,
bioavailable). In certain embodiments, the method comprises the
following steps: first, contacting the active agent (e.g., insulin)
with said delivery agent, and thereafter orally administering the
pharmaceutical composition. Alternatively, the method comprises
administering the insulin and the delivery agent in such a manner
that the insulin and delivery agent contact each other in-vivo
(e.g., in the stomach), such that the delivery agent is available
to facilitate absorption of the insulin through the stomach
mucosa.
[0177] The dosage forms of the present invention may be produced by
first dissolving the active agent and delivery agents into one
solution or separate solutions. The solvent will preferably be an
aqueous solution, but organic solvents or aqueous organic solvent
mixtures may be used when necessary to solubilize the delivery
agent. If two solutions are used, the proportions of each necessary
to provide the correct amount of either active agent or delivery
agent are combined and the resulting solution may be dried, by
lyophilization or equivalent means. In one embodiment of the
invention, the oral dosage form may be dried and rehydrated prior
to oral administration.
[0178] The administration mixtures may be prepared, e.g., by mixing
an aqueous solution of the delivery agent with an aqueous solution
of the active ingredient, such as insulin, just prior to
administration. Alternatively, the delivery agent and the
biologically or chemically active ingredient can be admixed during
the manufacturing process. The solutions may optionally contain
additives such as phosphate buffer salts, citric acid, acetic acid,
gelatin, and gum acacia.
[0179] Stabilizing additives may be incorporated into the delivery
agent solution. With some drugs, the presence of such additives
promotes the stability and dispersibility of the agent in solution.
The stabilizing additives may be employed at a concentration
ranging from about 0.1 and 5% (W/V), preferably about 0.5% (W/V).
Suitable, but non-limiting, examples of stabilizing additives
include gum acacia, gelatin, methyl cellulose, polyethylene glycol,
carboxylic acids and salts thereof, and polylysine. The preferred
stabilizing additives are gum acacia, gelatin and methyl
cellulose.
[0180] The amount of active agent, e.g., insulin, is an amount
effective to accomplish the purpose of the particular active agent.
The amount in the composition is a therapeutically effective dose,
i.e., a pharmacologically or biologically effective amount.
However, the amount can be less than a pharmacologically or
biologically effective amount when the composition is used in a
dosage unit form, such as a capsule, a tablet or a liquid, because
the dosage unit form may contain a multiplicity of delivery
agent/biologically or chemically active agent compositions or may
contain a divided pharmacologically or biologically effective
amount. The total effective amounts can then be administered in
cumulative units containing, in total, pharmacologically or
biologically or chemically active amounts of biologically or
pharmacologically active agent.
[0181] The total amount of active agent, and particularly insulin,
to be used can be determined by those skilled in the art. However,
it has surprisingly been found that with some biologically or
chemically active agents, the use of the presently disclosed
delivery agents provides extremely efficient delivery.
[0182] The amount of delivery agent in the present composition is a
delivery effective amount and can be determined for any particular
delivery agent/active agent combination by methods known to those
skilled in the art.
[0183] The oral dosage forms of the present invention, containing a
mixture of the active agent, e.g., insulin and the delivery agent,
e.g., 4-CNAB or separately containing the active agent and the
delivery agent, may include additional materials known to those
skilled in the art as pharmaceutical excipients. Any excipient or
ingredient, including pharmaceutical ingredients or excipients.
Such pharmaceutical excipients include, for example, the following:
Acidifying agents (acetic acid, glacial acetic acid, citric acid,
fumaric acid, hydrochloric acid, diluted hydrochloric acid, malic
acid, nitric acid, phosphoric acid, diluted phosphoric acid,
sulfuric acid, tartaric acid); Aerosol propellants (butane,
dichlorodifluoro-methane, dichlorotetrafluoroethane, isobutane,
propane, trichloromonofluoromethane); Air displacements (carbon
dioxide, nitrogen); Alcohol denaturants (denatonium benzoate,
methyl isobutyl ketone, sucrose octacetate); Alkalizing agents
(strong ammonia solution, ammonium carbonate, diethanolamine,
diisopropanolamine, potassium hydroxide, sodium bicarbonate, sodium
borate, sodium carbonate, sodium hydroxide, trolamine); Anticaking
agents (see glidant); Antifoaming agents (dimethicone,
simethicone); Antimicrobial preservatives (benzalkonium chloride,
benzalkonium chloride solution, benzelthonium chloride, benzoic
acid, benzyl alcohol, butylparaben, cetylpyridinium chloride,
chlorobutanol, chlorocresol, cresol, dehydroacetic acid,
ethylparaben, methylparaben, methylparaben sodium, phenol,
phenylethyl alcohol, phenylmercuric acetate, phenylmercuric
nitrate, potassium benzoate, potassium sorbate, propylparaben,
propylparaben sodium, sodium benzoate, sodium dehydroacetate,
sodium propionate, sorbic acid, thimerosal, thymol); Antioxidants
(ascorbic acid, acorbyl palmitate, butylated hydroxyanisole,
butylated hydroxytoluene, hypophosphorous acid, monothioglycerol,
propyl gallate, sodium formaldehyde sulfoxylate, sodium
metabisulfite, sodium thiosulfate, sulfur dioxide, tocopherol,
tocopherols excipient); Buffering agents (acetic acid, ammonium
carbonate, ammonium phosphate, boric acid, citric acid, lactic
acid, phosphoric acid, potassium citrate, potassium metaphosphate,
potassium phosphate monobasic, sodium acetate, sodium citrate,
sodium lactate solution, dibasic sodium phosphate, monobasic sodium
phosphate); Capsule lubricants (see tablet and capsule lubricant);
Chelating agents (edetate disodium, ethylenediaminetetraaceti- c
acid and salts, edetic acid); Coating agents (sodium
carboxymethylcellulose, cellulose acetate, cellulose acetate
phthalate, ethylcellulose, gelatin, pharmaceutical glaze,
hydroxypropyl cellulose, hydroxypropyl methylcellulose,
hydroxypropyl methylcellulose phthalate, methacrylic acid
copolymer, methylcellulose, polyethylene glycol, polyvinyl acetate
phthalate, shellac, sucrose, titanium dioxide, carnauba wax,
microcystalline wax, zein); Colorants (caramel, red, yellow, black
or blends, ferric oxide); Complexing agents
(ethylenediaminetetraacetic acid and salts (EDTA), edetic acid,
gentisic acid ethanolmaide, oxyquinoline sulfate); Desiccants
(calcium chloride, calcium sulfate, silicon dioxide); Emulsifying
and/or solubilizing agents (acacia, cholesterol, diethanolamine
(adjunct), glyceryl monostearate, lanolin alcohols, lecithin, mono-
and di-glycerides, monoethanolamine (adjunct), oleic acid
(adjunct), oleyl alcohol (stabilizer), poloxamer, polyoxyethylene
50 stearate, polyoxyl 35 caster oil, polyoxyl 40 hydrogenated
castor oil, polyoxyl 10 oleyl ether, polyoxyl 20 cetostearyl ether,
polyoxyl 40 stearate, polysorbate 20, polysorbate 40, polysorbate
60, polysorbate 80, propylene glycol diacetate, propylene glycol
monostearate, sodium lauryl sulfate, sodium stearate, sorbitan
monolaurate, soritan monooleate, sorbitan monopalmitate, sorbitan
monostearate, stearic acid, trolamine, emulsifying wax); Filtering
aids (powdered cellulose, purified siliceous earth); Flavors and
perfumes (anethole, benzaldehyde, ethyl vanillin, menthol, methyl
salicylate, monosodium glutamate, orange flower oil, peppermint,
peppermint oil, peppermint spirit, rose oil, stronger rose water,
thymol, tolu balsam tincture, vanilla, vanilla tincture, vanillin);
Glidants and/or anticaking agents (calcium silicate, magnesium
silicate, colloidal silicon dioxide, talc); Humectants (glycerin,
hexylene glycol, propylene glycol, sorbitol); Plasticizers (castor
oil, diacetylated monoglycerides, diethyl phthalate, glycerin,
mono- and di-acetylated monoglycerides, polyethylene glycol,
propylene glycol, triacetin, triethyl citrate); Polymers (e.g.,
cellulose acetate, alkyl celloloses, hydroxyalkylcelloloses,
acrylic polymers and copolymers); Solvents (acetone, alcohol,
diluted alcohol, amylene hydrate, benzyl benzoate, butyl alcohol,
carbon tetrachloride, chloroform, corn oil, cottonseed oil, ethyl
acetate, glycerin, hexylene glycol, isopropyl alcohol, methyl
alcohol, methylene chloride, methyl isobutyl ketone, mineral oil,
peanut oil, polyethylene glycol, propylene carbonate, propylene
glycol, sesame oil, water for injection, sterile water for
injection, sterile water for irrigation, purified water); Sorbents
(powdered cellulose, charcoal, purified siliceous earth); Carbon
dioxide sorbents (barium hydroxide lime, soda lime); Stiffening
agents (hydrogenated castor oil, cetostearyl alcohol, cetyl
alcohol, cetyl esters wax, hard fat, paraffin, polyethylene
excipient, stearyl alcohol, emulsifying wax, white wax, yellow
wax); Suspending and/or viscosity-increasing agents (acacia, agar,
alginic acid, aluminum monostearate, bentonite, purified bentonite,
magma bentonite, carbomer 934p, carboxymethylcellulose calcium,
carboxymethylcellulose sodium, carboxymethycellulose sodium 12,
carrageenan, microcrystalline and carboxymethylcellulose sodium
cellulose, dextrin, gelatin, guar gum, hydroxyethyl cellulose,
hydroxypropyl cellulose, hydroxypropyl methylcellulose, magnesium
aluminum silicate, methylcellulose, pectin, polyethylene oxide,
polyvinyl alcohol, povidone, propylene glycol alginate, silicon
dioxide, colloidal silicon dioxide, sodium alginate, tragacanth,
xanthan gum); Sweetening agents (aspartame, dextrates, dextrose,
excipient dextrose, fructose, mannitol, saccharin, calcium
saccharin, sodium saccharin, sorbitol, solution sorbitol, sucrose,
compressible sugar, confectioner's sugar, syrup); Tablet binders
(acacia, alginic acid, sodium carboxymethylcellulose,
microcrystalline cellulose, dextrin, ethylcellulose, gelatin,
liquid glucose, guar gum, hydroxypropyl methylcellulose,
methycellulose, polyethylene oxide, povidone, pregelatinized
starch, syrup); Tablet and/or capsule diluents (calcium carbonate,
dibasic calcium phosphate, tribasic calcium phosphate, calcium
sulfate, microcrystalline cellulose, powdered cellulose, dextrates,
dextrin, dextrose excipient, fructose, kaolin, lactose, mannitol,
sorbitol, starch, pregelatinized starch, sucrose, compressible
sugar, confectioner's sugar); Table disintegrants (alginic acid,
microcrystalline cellulose, croscarmellose sodium, corspovidone,
polacrilin potassium, sodium starch glycolate, starch,
pregelatinized starch); Tablet and/or capsule lubricants (calcium
stearate, glyceryl behenate, magnesium stearate, light mineral oil,
polyethylene glycol, sodium stearyl fumarate, stearic acid,
purified stearic acid, talc, hydrogenated vegetable oil, zinc
stearate); Tonicity agent (dextrose, glycerin, mannitol, potassium
chloride, sodium chloride); Vehicle: flavored and/or sweetened
(aromatic elixir, compound benzaldehyde elixir, iso-alcoholic
elixir, peppermint water, sorbitol solution, syrup, tolu balsam
syrup); Vehicle: oleaginous (almond oil, corn oil, cottonseed oil,
ethyl oleate, isopropyl myristate, isopropyl palmitate, mineral
oil, light mineral oil, myristyl alcohol, octyldodecanol, olive
oil, peanut oil, persic oil, seame oil, soybean oil, squalane);
Vehicle: solid carrier (sugar spheres); Vehicle: sterile
(Bacteriostatic water for injection, bacteriostatic sodium chloride
injection); Viscosity-increasing (see suspending agent); Water
repelling agent (cyclomethicone, dimethicone, simethicone); and
Wetting and/or solubilizing agent (benzalkonium chloride,
benzethonium chloride, cetylpyridinium chloride, docusate sodium,
nonoxynol 9, nonoxynol 10, octoxynol 9, poloxamer, polyoxyl 35
castor oil, polyoxyl 40, hydrogenated castor oil, polyoxyl 50
stearate, polyoxyl 10 oleyl ether, polyoxyl 20, cetostearyl ether,
polyoxyl 40 stearate, polysorbate 20, polysorbate 40, polysorbate
60, polysorbate 80, sodium lauryl sulfate, sorbitan monolaureate,
sorbitan monooleate, sorbitan monopalmitate, sorbitan monostearate,
tyloxapol). This list is not meant to be exclusive, but instead
merely representative of the classes of excipients and the
particular excipients which may be used in oral dosage forms of the
present invention.
[0184] In the case of insulin, oral delivery may have advantages
beyond convenience, acceptance and compliance issues. Insulin
absorbed in the gastrointestinal tract mimics the physiology of
insulin secreted by the pancreas because both are released into the
portal vein and carried directly to the liver. Absorption into the
portal circulation maintains a peripheral-portal insulin gradient
that regulates insulin secretion. In its first passage through the
liver, roughly 60% of the insulin is retained and metabolized,
thereby reducing the incidence of peripheral hyper-insulinism, a
factor in diabetes related systemic complications. A feared and not
uncommon complication of insulin treatment and other oral
antidiabetic agents is hypoglycemia.
[0185] The present invention relates in part to a method of
treating human diabetics via the chronic oral administration of
insulin together with a drug delivery agent that enhances the
absorption of insulin (e.g., from the duodenum) such that a
therapeutically effective control and/or reduction in blood glucose
is achieved while effecting a reduction in the systemic blood
insulin concentration (serum insulin level) on a chronic basis
required to achieve the reduction in blood glucose concentration,
e.g., relative to the serum insulin level required to achieve
therapeutic efficacy via subcutaneous injection of insulin.
[0186] Whereas traditional subcutaneous insulin dosing shifts the
point of entry of insulin into the circulation from the natural
site (the portal vein) to the systemic circulation, the oral dosing
method of the present invention shifts the site of insulin entry
back to the portal vein. The effect of this route of dosing is two
fold. First, by targeting the liver directly, a greater control of
glucose may be achieved. Various studies have shown that
intraportal delivery of insulin can yield a comparable control of
glucose at infusion rates lower than those required by peripheral
administration. (Stevenson, R. W. et al., Insulin infusion into the
portal and peripheral circulations of unanaesthetized dogs, Clin
Endocrinol (Oxf) 8, 335-47. (1978); Stevenson, R. W. at al., Effect
of intraportal and peripheral insulin on glucose turnover and
recycling in diabetic dogs, Am J Physiol 244, E 190-5 (1983);
Shishko, P. I. et al., I. U. Comparison of peripheral and portal
(via the umbilical vein) routes of insulin infusion in IDDM
patients, Diabetes 41, 1042-9 (1992). Because the insulin will
undergo first-pass metabolism prior to entering the systemic
circulation, a lower serum concentration is achieved. This may, in
turn, alleviate any detrimental effects of insulin on non-target
tissues.
[0187] In normal healthy humans, the physiologic ratio of blood
insulin concentration in the portal vein as compared to systemic
(peripheral) blood insulin concentration is greater than about 2:1.
In contrast, administration of insulin to human diabetic patients
has been found to shift this ratio of portal vein insulin blood
concentration to systemic insulin blood concentration to about
0.75:1. By virtue of the present invention, the ratio of
concentration of unmodified insulin in the portal circulation to
systemic circulation approaches the normal physiological ratio,
e.g., from about 2:1 to about 6:1.
[0188] The present invention provides a method of attenuating
and/or reducing the incidence of diseases associated with exposure
to systemic hyperinsulinemia by the oral administration to a
patient a dosage form in accordance with the invention comprising
unmodified insulin, preferably along with a suitable drug delivery
agent that facilitates the absorption of insulin from the
gastrointestinal tract of the patient in a therapeutically
effective amount, for treatment of diabetes. Both the methods and
pharmaceutical compositions useful for oral administration of
insulin are within the scope of the invention.
[0189] The methods and oral compositions of the invention can
attenuate and/or reduce the incidence of cardiovascular disease
associated with chronic dosing of insulin. It is believed that
orally administering insulin with the compositions of the invention
will decrease the complications associated with vascular disease by
lowering the systemic vasculature's exposure to insulin that is
greater than normal physiological levels. With a first passage
through the liver, roughly 50% of the insulin is retained and
metabolized, thereby reducing the incidence of peripheral
hyper-insulinemia.
[0190] In certain embodiments, the invention provides a method of
treating diabetics comprising orally administering to diabetic
patients on a chronic basis an oral insulin treatment comprising a
dose of insulin together with a delivery agent which facilitates
the absorption of the dose of insulin from the gastrointestinal
tract to provide a therapeutically effective reduction in blood
glucose and a peak serum insulin concentration that is reduced
relative to the peak serum insulin concentration of an equivalent
therapeutically effective reduction in blood glucose concentration
achieved by subcutaneous injection of insulin. In certain
embodiments, this method can result in the reduction of the
incidence of a disease state associated with chronic insulin
administration, which disease states include, for example,
cardiovascular diseases. Cardiovascular diseases include, for
example, congestive heart failure, coronary artery disease,
neuropathy, nephropathy, retinopathy, arteriopathy,
atherosclerosis, hypertensive cardiomyopathy and combinations
thereof.
[0191] In some embodiments, the invention provides a method of
reducing the incidence and/or severity of one or more disease
states associated with chronic administration of insulin comprising
treating diabetic patients via oral administration on a chronic
basis of a therapeutically effective dose of a pharmaceutical
composition which comprises insulin and a delivery agent that
facilitates the absorption of insulin from the gastrointestinal
tract, such that the pharmaceutical composition provides a
therapeutically effective reduction in blood glucose and a peak
serum insulin concentration of the diabetic patient that is reduced
relative to the peak serum insulin concentration of an equivalent
therapeutically effective reduction in blood glucose concentration
achieved by subcutaneous injection of insulin. Disease states
associated with chronic administration of insulin for which the
incidence and/or severity can be reduced by the method described
herein include, for example, cardiovascular diseases, such as
congestive heart failure or coronary artery disease. Other such
disease states include, for example, neuropathy, nephropathy,
retinopathy, arteriopathy, atherosclerosis, hypertensive
cardiomyopathy and combinations thereof.
[0192] In some embodiments, the method of reducing the incidence
and/or severity of one or more disease states associated with
chronic administration of insulin can provide for a reduced
expression of genes associated with vascular disease as compared to
the level of expression of genes associated with vascular disease
resulting from an equivalent reduction in blood glucose
concentration achieved in a population of patients via subcutaneous
injection of insulin. The genes associated with vascular disease
can include, for example, early response genes, genes associated
with cytokines, genes associated with adhesion molecules, genes
associated with lipid peroxidation, genes associated with
thrombosis and combinations thereof. Early response genes can
include, for example, c-myc, jun B, Egr-1, Ets-1 and combinations
thereof.
[0193] The methods provided herein relating to oral administration
of insulin and oral administration of insulin on a chronic basis,
in some embodiments, provide for obtaining plasminogen activator
inhibitor concentrations that are lower as compared to the
plasminogen activator inhibitor concentrations resulting from an
equivalent therapeutically effective reduction in blood glucose
concentration achieved by subcutaneous injection of insulin. These
methods also provide for obtaining pro-inflammatory cytokine
concentrations that are lower than pro-inflammatory cytokine
concentrations resulting from an equivalent therapeutically
effective reduction in blood glucose concentration achieved by
subcutaneous injection of insulin.
[0194] In some embodiments, the invention provides a method of
treating diabetes and reducing the incidence and or severity of
hyperinsulinemia associated with chronic dosing of insulin,
comprising orally administering on a chronic basis to a diabetic
patient a dose of insulin and a delivery agent that facilitates the
absorption of the dose of insulin from the gastrointestinal tract
to provide a therapeutically effective reduction in blood glucose
and a peak serum insulin concentration of the diabetic patient that
is reduced relative to the peak serum insulin concentration of an
equivalent therapeutically effective reduction in blood glucose
concentration achieved by subcutaneous injection of insulin.
[0195] In some embodiments, the invention provides a method of
screening a drug for vascular injury associated with route of
administering the drug, comprising administering a drug to a first
test animal parenterally, administering the drug to a second test
animal orally, and comparing the expression of early response genes
selected from the group consisting of c-myc, c-fos, Jun B, Erg-1
and combinations thereof for the first and second test animal,
wherein an increase in the expression of one or more early response
genes is indicative of vascular injury. In some embodiments, the
step of measuring the change in expression is done using gene chip
analysis and can comprise measuring the changes in mRNA
expression.
[0196] In some embodiments, the invention provides a method of
reducing the incidence of and/or the severity of disease states or
of vascular diseases associated with chronic insulin administration
to diabetics, comprising orally administering an oral insulin
treatment comprising a dose of insulin together with a delivery
agent that facilitates the absorption of said insulin from the
gastrointestinal tract on a chronic basis to diabetic patients to
reduce blood glucose levels in said diabetic patients by a desired
amount, such that the concentration of insulin circulating in the
blood of said diabetic patients as a result of insulin treatment is
reduced relative to the peak serum insulin concentration of an
equivalent therapeutically effective reduction in blood glucose
concentration achieved by subcutaneous injection of insulin.
[0197] In some embodiments, the invention provides a method for
reducing the incidence of, the severity of, or the incidence and
severity of vascular diseases associated with chronic insulin
therapy in diabetics, comprising orally administering an oral
insulin treatment comprising a dose of insulin together with a
delivery agent that facilitates the absorption of said insulin from
the gastrointestinal tract on a chronic basis to diabetic patients
to reduce blood glucose levels in said diabetic patients by a
desired amount, such that the concentration of insulin circulating
in the blood of said diabetic patients as a result of insulin
treatment is reduced relative to the peak serum insulin
concentration of an equivalent therapeutically effective reduction
in blood glucose concentration achieved by subcutaneous injection
of insulin.
[0198] In some embodiments, the invention provides a method of
attenuating processes resulting from the reaction to a mild
injurious stimulus in multiple areas of the response to increases
in mRNA during insulin treatment, comprising orally administering
an oral insulin treatment comprising a dose of insulin together
with a delivery agent that facilitates the absorption of said
insulin from the gastrointestinal tract on a chronic basis to
diabetic patients to reduce blood glucose levels in said diabetic
patients by a desired amount, such that the concentration of
insulin circulating in the blood of said diabetic patients as a
result of insulin treatment is reduced relative to the peak serum
insulin concentration of an equivalent therapeutically effective
reduction in blood glucose concentration achieved by subcutaneous
injection of insulin.
[0199] In some embodiments, the invention provides a method of
treating diabetic patients, comprising orally administering an oral
insulin treatment comprising a dose of insulin together with a
delivery agent that facilitates the absorption of said insulin from
the gastrointestinal tract on a chronic basis to diabetic patients
to reduce blood glucose levels in said diabetic patients by a
desired amount, such that the concentration of insulin circulating
in the blood of said diabetic patients as a result of said oral
insulin treatment is not substantially greater than normal
physiological levels.
[0200] An aspect of the physiological response to the presence of
insulin is the stimulation of glucose transport into muscle and
adipose tissue. It has been reported that hyperglycemia (elevated
blood glucose levels) and/or hyperinsulinemia (elevated blood
concentrations of insulin) is a cause of vascular diseases
associated with diabetes. Impairment to the vascular system is
believed to be the reason behind conditions such as microvascular
complications or diseases (retinopathy (lesions in the small blood
vessels and capillaries supplying the retina of the eye);
neuropathy (impairment of the function of the autonomic nerves,
leading to abnormalities in the function of the gastrointestinal
tract and bladder, and also loss of feeling in lower extremities);
nephropathy (lesions in the small blood vessels and capillaries
supplying the kidney, which may lead to kidney disease)); or
macrovascular complications or diseases (e.g., cardiovascular
disease; etc.).
[0201] Hyperinsulinemia is caused by the administration of insulin
in a location (and manner) which is not consistent with the normal
physiological route of delivery. In normal healthy humans, insulin
is released from the pancreas into the portal vein, which transfers
the insulin to the liver. The liver utilizes a large portion of the
insulin which it receives from the portal circulation. Glucose is
the principal stimulus to insulin secretion in humans. Glucose
enters the .beta. cell by facilitated transport, and is then
phosphorylated by glucokinase. Expression of glucokinase is
primarily limited to cells and tissues involved in the regulation
of glucose metabolism, such as the liver and the pancreatic .beta.
cells. The capacity of sugars to undergo phosphorylation and
subsequent glycolysis correlates closely with their ability to
stimulate insulin release. Insulin circulates in blood as the free
monomer, and its volume distribution approximates the volume of
extracellular fluid. Under fasting conditions, the concentration of
insulin in portal blood is, e.g., about 2-4 ng/ml, whereas the
systemic (peripheral) concentration of insulin is, e.g., about 0.5
ng/ml, in normal healthy humans, translating into, e.g., a 5:1
ratio. As previously mentioned, in human diabetics who receive
insulin via subcutaneous injection, the ratio is changed to about
0.75:1. Thus, in such diabetic patients, the liver does not receive
the necessary concentrations of insulin to adequately control blood
glucose.
[0202] It has been an unmet goal in the art to imitate normal
insulin levels in the portal and systemic circulation via oral
administration of insulin. By virtue of the present invention, the
ratio of portal (unmodified) insulin concentration to systemic
(unmodified) insulin concentration approaches in human diabetic
patients approaches that which is obtained in normal healthy
humans. The chronic administration of oral dosage forms of the
present invention result in a higher portal insulin concentration
and lower systemic insulin concentration over time than that
obtained with an equi-effective dose of insulin administered
subcutaneously (i.e., which provide similar control of blood
glucose levels). By virtue of the present invention, lower levels
of hyperinsulinemia are obtained, e.g., systemic insulin
concentrations are at least about 20% lower when compared to a
comparably effective subcutaneous dose of insulin. Transient peaks
in insulin levels which may occur by virtue of the oral
administration of insulin in accordance with the present invention
is not believed to be associated with vascular diseases.
[0203] The present invention provides a method of administering
insulin and pharmaceutical compositions useful for administering
insulin such that the insulin is bioavailable and absorbable from
the gastrointestinal tract and such that the incidence of vascular
diseases normally associated with chronic dosing of insulin is
attenuated. The delivery agents of the invention enable insulin to
be orally absorbable through the mucosa of the stomach. Following
oral administration of the pharmaceutical compositions of the
present invention, the delivery agent passes though the mucosal
barriers of the gastrointestinal tract and is absorbed into the
blood stream where it can be detected in the plasma of subjects.
The level of delivery agent in the bloodstream as measured in the
plasma is dose-dependent. The delivery agent facilitates the
absorption of insulin administered therewith (either in the same
dosage form, or simultaneously therewith), or sequentially (in
either order, as long as both the delivery agent and insulin are
administered within a time period which provides both in the same
location, e.g., the stomach, at the same time). As disclosed below,
oral administration of insulin, in particular using the delivery
agents disclosed herein, effectively reduces the incidence of
vascular and other disease states that are associated with
traditional dosing of insulin, i.e., subcutaneously.
[0204] The preferred pharmaceutical compositions of the invention
comprise a combination of insulin and a delivery agent in a
suitable pharmaceutical carrier or excipient as understood by
practitioners in the art. The means of delivery of the
pharmaceutical composition can be, for example, a capsule,
compressed tablet, pill, solution, freeze-dried, powder ready for
reconstitution or suspension suitable for administration to the
subject.
[0205] The pharmaceutical compositions and method of the present
invention provide a number of advantages in addition to
convenience, acceptance and patient compliance. Insulin absorbed in
the gastrointestinal tract mimics the physiology of insulin
secreted by the pancreas because both are released into the portal
vein and carried directly to the liver. Absorption into the portal
circulation maintains a peripheral-portal insulin gradient that
regulates insulin secretion. The present invention comprises
pharmaceutical compositions and method for oral insulin delivery
that enable achieving low blood glucose without having high levels
of systemic insulin.
[0206] In certain preferred embodiments, the methods and
pharmaceutical compositions provide the pharmacokinetic parameters
set forth in U.S. Provisional Applications Nos. 60/346,746 and
60/347,312, the disclosure of each of which is incorporated herein
by reference.
[0207] The amount of delivery agent necessary to adequately deliver
insulin into the blood stream of a subject needing the therapeutic
effect of insulin can vary depending on one or more of the
following; chemical structure of the particular delivery agent; the
nature and extent of interaction of insulin and the delivery agent;
the nature of the unit dose, i.e., solid, liquid, tablet, capsule,
suspension; the concentration of delivery agent in the GI tract,
the feeding state of the subject, the diet of the subject, the
heath of the subject and the ratio of delivery agent to
insulin.
[0208] In preferred embodiments, the oral dosage forms of the
present invention comprise a mixture of insulin and a delivery
agent, e.g., 4-CNAB, or separately containing insulin and the
delivery agent.
[0209] In further embodiments of the present invention, the oral
dosage forms described herein are orally administered as described
herein in combination with an additional therapy to treat diabetes,
impaired glucose tolerance, or to achieve glucose homeostasis, said
additional therapy comprising, for example, an additional drug such
as sulfonylurea, a biguanide, an alpha-glucosidase, insulin
delivered via a different pathway (e.g., parenteral insulin),
and/or an insulin sensitizer.
[0210] In further embodiments of the invention, the oral dosage
forms described herein reduce the likelihood of hypoglycemic
events, mainly because of two reasons: (a) one cannot
hyperinsulinize the liver, because even under hyperinsulinemia the
liver uptake of glucose will be unchanged. Unlike the peripheral
tissue, the liver will only cease producing endogenous insulin and
not sequester additional glucose; and (b) the short peak of insulin
(e.g., as shown in the appended examples) shows that even if
insulin were to reach high peripheral levels, the peak drops
precipitously.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0211] The present invention comprising compositions of insulin and
the delivery agent 4-CNAB was evaluated for safety and toxicity in
a nonclinical program that included pharmacological screening,
pharmacokinetic profiling, and toxicity assessments in rats and
monkeys. In general, animal physiological responses to 4-CNAB alone
and to Insulin/4-CNAB were comparable. Pharmacokinetic studies in
mice, rats and monkeys have shown that 4-CNAB is absorbed rapidly
following oral administration, and subsequently cleared from the
body. 4-CNAB did not demonstrate potential activity in any of the
primary molecular targets evaluated in receptor binding screening
assays. Four genotoxicity studies have been conducted with 4-CNAB,
with no positive findings. Based on 14-day oral repeated dose
toxicity studies, the NOAEL (No-Adverse Effect Level) was estimated
to be 500 mg/kg in Sprague-Dawley rats, and 400 mg/kg in rhesus
monkeys.
[0212] In order that this invention may be better understood, the
following examples are set forth. These examples are for the
purpose of illustration only and are not to be construed as
limiting the scope of the invention in any manner.
[0213] The insulin for the subcutaneous injection was HUMULIN.RTM.
R injection 100 U/ml insulin from Eli Lilly and Company
(Indianapolis, Ind.).
[0214] In toxicology studies, 4-CNAB doses from 400 mg to 2000 mg
were evaluated. Following 14-day oral repeated dose toxicity
studies in rats and monkeys, the estimated No Adverse Effect Level
(NOAEL) for 4-CNAB was 500 mg/kg in Sprague-Dawley rats and 400
mg/kg in rhesus monkeys; therefore, the monkey appeared to be the
most sensitive species. The highest proposed dose of 2000 mg 4-CNAB
in man (<30 mg/kg) is 12-16 fold lower than the NOAEL in monkeys
(i.e., NOAEL=400 mg/kg 4-CNAB alone and in combination with 15 U/kg
insulin). The absolute bioavailability of insulin in monkeys was
about 1% or less. In the toxicology studies, there were no findings
in rats attributed to insulin at an oral dose level of 15U/kg in
combination with 4-CNAB doses as high as 2000 mg/kg. In monkeys, an
insulin dose of 15 U/kg was associated with a single hypoglycemic
episode in combination with a 4-CNAB dose of 1200 mg/kg in one
monkey; there were no effects at 15 U/kg insulin in combination
with lower doses. Based on the above nonclinical information, the
starting insulin dose of 150 insulin Units USP (which is about
7-fold lower than the 15 U/kg no effect dose in monkey) was
selected.
EXAMPLE 1
[0215] Preparation of the Delivery Agent 4-CNAB
[0216] The compound corresponding to the following structure may be
prepared as described below: 3
[0217] 4-Chlorosalicylic acid (10.0 g, 0.0579 mol) was added to a
one-neck 250 ml round-bottomed flask containing about 50 ml
methylene chloride. Stirring was begun and continued for the
remainder of the reaction. The coupling agent
1,1-carbonyldiimidazole (9.39 g, 0.0579 mol) was added as a solid
in portions to the flask. The reaction was stirred at room
temperature for approximately 20 minutes after all of the coupling
agent had been added and then ethyl-4-aminobutyrate hydrochloride
(9.7 g, 0.0579 mol) was added to the flask with stirring. Next,
triethylamine (10.49 ml, 0.0752 mol) was added dropwise from an
addition funnel. The addition funnel was rinsed with methylene
chloride. The reaction was allowed to stir at room temperature
overnight.
[0218] The reaction was poured into a separatory funnel and washed
with 2N HCl and an emulsion formed. The emulsion was left standing
for two days and was then filtered through celite in a flitted
glass funnel. The filtrate was put back in a separatory funnel to
separate the layers. The organic layer was dried over sodium
sulfate, which was then filtered off and the filtrate concentrated
by rotary evaporation. The resulting solid material was hydrolyzed
with 2N NaOH, stored overnight under refrigeration, and then
hydrolyzing resumed. The solution was acidified with 2N HCl and the
solids that formed were isolated, dried under vacuum, and
recrystallized twice using methanol/water. Solids precipitated out
overnight and were isolated and dried. The solids were dissolved in
2N NaOH and the pH of the sample was brought to pH 5 with 2N HCl.
The solids were collected and HPLC revealed a single peak. These
solids were then recrystallized in methanol/water, isolated, and
then dried under vacuum, yielding 4.96 g (33.0%) of 4-(4
chloro-2-hydroxybenzoyl)aminobutyric acid,
(C.sub.11H.sub.12ClNO.sub.4; Molecular weight 257.67). A melting
point of 131-133.degree. C. was determined. Combustion analysis
revealed the following content: % C: 51.27(calc.), 51.27 (found); %
H: 4.69 (calc.), 4.55 (found); % N: 5.44 (calc.), 5.30 (found).
Proton H NMR Analysis revealed: (d.sub.6-DMSO): d 13.0, s, 1H
(COOH); d 12.1, s, 1H (OH); d 8.9, t, 1H (NH); d 7.86, d, 1H (H
ortho to amide); d 6.98, d, 1H (H ortho to phenol OH); d 6.96, d,
1H, (H meta to amide); d 3.33, m, 2H (CH.sub.2 adjacent to NH); d
2.28, t, 2H (CH.sub.2 adjacent to COOH); d 1.80, m, 2H (aliphatic
CH.sub.2 beta to NH and CH.sub.2 beta to COOH).
[0219] 4-CNAB Preparation for Human Studies
[0220] 4-CNAB for the human dosings (Monosodium
N-(4-chlorosalicyloyl)-4-a- mino-butyrate) was made under good
manufacturing practices (GMP) conditions by Regis Technologies,
Inc. (Morton Grove, Ill.) according to the methods of International
Publication No. WO 00/46182 except that the starting material
4-chlorosalicylic acid (purchased from Ihara Chemical Industry Co.
Inc, Ltd., Tokyo, Japan and Aapin Chemicals Ltd., Oxfordshire, UK)
was used and converted to the amide via a methyl ester using 0.14
equivalents sulfuric acid in methanol and then about 4 equivalents
ammonia in methanol. In addition, the alkylating agent used was
ethyl-4-bromobutyrate.
[0221] The monosodium salt of 4-CNAB was made according to the
following method on a 40 kilogram scale. 4-CNAB free acid (500 g,
1.94 mol, FW=257.67) was charged to a 22 L five neck round bottom
flask. The flask was equipped with an overhead stirrer, a
thermocouple temperature read out, a reflux condenser and a heating
mantle, and was placed under nitrogen. Reagent grade acetone (13 L)
was added to the reactor and the mixture was agitated. The
4-CNAB/acetone mixture was heated to 50.degree. C. to dissolve any
solids. A hazy brown solution was achieved.
[0222] The 50.degree. C. solution was pumped through a warm
pressure filter (dressed with Whatman #1 filter paper, 5 microns,
18.5 sq. in. area) into a clean 22 L reactor to remove sodium
chloride and other insolubles. The pressure dropped across the
filter to about 20 psig at the end of filtration. The reactor
containing the clear yellow filtrate was agitated and heated. At
50.degree. C. the reactor was removed from heat.
[0223] The clear filtrate was charged with 50% sodium hydroxide
solution (155 g, 1.94 mol) as rapidly as possible, while
maintaining a vigorous agitation. (An overcharge will result in the
undesirable formation insoluble disodium salts. A slight
undercharge is preferable because the free acid is removed during
the final filtration step.) The reaction mixture exothermed to
approximately 52.degree. C. Precipitates formed and the product
gelled before becoming clear again.
[0224] After the base addition was completed and the temperature
leveled, the solution became cloudy and increased in viscosity. The
reaction was refluxed for 2 hours at 60.degree. C., while agitating
vigorously. The reaction mixture continued to thicken, forming
solid chunks. The slurry became light pink and foamed. The reactor
contents were cooled to ambient temperature over 3 to 4 hours. The
ambient temperature was held for 30 minutes. The precipitated
solids were isolated on a filter funnel. The isolated product was
not washed. The resulting 4-CNAB monosodium salt was dried in vacuo
at 40 to 50.degree. C. for 16 to 24 hours to give 490 grams (1.75
mol, 90% yield FW=279.65).
[0225] All capsules containing 200 mg 4-CNAB and 150 insulin units
USP were prepared as follows. First, the total amount of delivery
agent material necessary for filling the delivery agent alone
capsules and the delivery agent plus insulin composition capsules
was prepared by weighing 3160 g of 4-CNAB. The 3160 g 4-CNAB was
then milled in a Quadro comil, model 197S mill with screen number
2A 050 G 037 19 136 (1270 micron). Next, 1029 g of the milled
4-CNAB was passed through a #35 mesh screen. Then, the pass through
screened material was transferred into a 4 quart shell and blended
using for example, a V blender, at 25 rpm for 10.2 minutes. The
resultant blended material was used to fill capsules. In this case,
a Fast Cap Capsule Filler was used with a size 3 Fast Cap
Encapsulation tray. The empty capsules weighed approximately 48 mg
each and were filled with an average fill weight of 205.6 mg of
4-CNAB alone. Thus, the dose of the delivery agent alone capsules
was 205.6 mg.
[0226] The insulin compositions were prepared by first dispensing
31.8 g of recombinant human zinc crystalline insulin (Potency 26.18
Units per mg) (proinsulin derived (recombinant DNA origin) USP
quality) from Eli Lilly and Company (Indianapolis, Ind.) into an
appropriately sized plastic bag. Next, sequential 30 g additions of
the milled and screened 4-CNAB were added to the bag until
approximately 510 g had been added. The bag was thoroughly mixed
after each 30 g addition of 4-CNAB by shaking and inversion. In
order to add and mix the next 532.5 g of 4-CNAB, the 541.8 g
mixture of insulin and 4-CNAB was transferred to a V blender and
mixed again at 25 rpm for 10.2 minutes. Next, the remaining 4-CNAB
was added to the blender and the entire mixture was mixed in the
blender at 25 rpm for 10.2 minutes. Finally, the resulting
composition was dispensed as described above into empty capsules.
The final capsules contained an average of 5.7 mg insulin
(equivalent to 150 units insulin) and 200.5 mg of 4-CNAB or a ratio
of 1:57.3, insulin: 4-CNAB. Multiple samples of the final blend
were run on HPLC to verify uniformity and were found to be
uniform.
EXAMPLE 2
[0227] Human Subjects and Insulin Formulated with 4-CNAB
[0228] In Example 2, a single center, double-blind, randomized
placebo-controlled study of escalating single oral doses of 4-CNAB
capsules and escalating single doses of Insulin/4-CNAB capsules was
undertaken in healthy male volunteers.
[0229] Twenty-nine volunteers, divided among three groups (9 in
group 1, and 10 in each of groups 2 and 3), participated in this
study. Randomization was stratified such that any individual
subject received placebo only on a single occasion or not at all.
Two subjects in each group received placebo. Group 1 received four
escalating oral doses of 4-CNAB capsules or placebo (Table 1).
Group 2 received three escalating oral doses of Insulin/4-CNAB
capsules or placebo (Table 2). Group 3 received three oral doses of
Insulin/4-CNAB capsules or placebo and one oral dose of Insulin
capsule alone or placebo (Table 3). For all groups, there was a
washout period of at least 72 hours between treatment periods.
[0230] For Group 1, each capsule contained 200 mg 4-CNAB. For Group
2, treatment 2, each Insulin/4-CNAB capsule contained 150 Insulin
Units USP and 200 mg 4-CNAB. Group 1 and Group 2, treatment 2
capsules were made per Example 1c. Group 2, treatments 3 and 4
capsules and all Group 3 capsules were extemporaneously prepared at
the pharmacy department of Medeval Ltd. (Manchester, England). The
placebo contained Methocel E15 Premium LV
(hydroxypropylmethylcellulose). Capsules were administered with 240
mL water. After each dosing, safety, pharmacokinetic, and
pharmacodynamic parameters were measured and evaluated before
proceeding to the next dose level.
1TABLE 1 Group 1 - 4-CNAB alone (4 escalation doses) Group 1:
4-CNAB only # of Subjects & # of Subjects on Treatment: 4-CNAB
Dose placebo # of Capsules Treatment 1 7 subjects 400 mg 2 2
Treatment 2 7 subjects 800 mg 2 4 Treatment 3 7 subjects 1400 mg 2
7 Treatment 4 7 subjects 2000 mg 2 10
[0231]
2TABLE 2 Group 2 - Insulin/4-CNAB (2 doses) and SC insulin alone (1
dose) Group 2 # of Subjects & # of Insulin/4-CNAB
Insulin/4-CNAB Dose Subjects # of Treatment (Unit Insulin/mg
4-CNAB) on placebo Capsules Treatment 1 8 subjects 10 IU insulin 2
0 subcutaneous/0 mg 4-CNAB Treatment 2 8 subjects 150/200 2 1
Treatment 3 8 subjects 100/600 2 4
[0232]
3TABLE 3 Group 3 - Insulin/4-CNAB (3 doses) and Insulin alone (1
dose) # of Subjects & Group 3 Insulin/4-CNAB Dose
Insulin/4-CNAB (Unit Insulin/mg 4- # of Subjects on # of Treatment:
CNAB) placebo Capsules Treatment 1 8 subjects 100/300 2 2 Treatment
2 8 subjects 100/450 2 3 Treatment 3 8 subjects 150/100 2 1
Treatment 4 8 subjects 150/0 2 1
[0233] Healthy, adult male volunteers from about 18 and 50 years of
age, inclusive, were selected who were in good health as determined
by the investigator based on medical history, physical examination,
and clinical laboratory studies at screening. Subjects were within
the permissible deviations (+/-15%) of ideal weight according to
the 1983 tables of desirable weights issued by the adjusted
Metropolitan Life Insurance Co. All laboratory values (hematology,
serum chemistries, and urinalysis) obtained during screening were
within normal ranges. The laboratory tests were conducted in a
fasted state and glucose was measured. However, for clinical
laboratory values outside of the normal range, the laboratory test
may have been repeated once. A 12-lead electrocardiogram (ECG)
recorded within 14 days prior to the study start. Results indicated
a normal recording or a non-clinically significant abnormality.
[0234] Subjects entered the clinic the day before each study
treatment period and fasted for a minimum of 8 hours overnight.
Subjects had to fast until 6 hours postdose for a given treatment,
after which each subject was allowed to eat a full meal, including
at least two slices of bread. Water was allowed ad lib during the
study, except for 1 hour prior to through 1 hour after
administration of each treatment. Subjects were provided with
standard high carbohydrate meals (i.e. lasagne, jacket potato,
salad and a dessert such as apple pie) and snacks throughout the
study periods. Alcohol containing beverages were prohibited during
the 24 hours prior to dosing and throughout the study periods.
Subjects had to refrain from xanthine or xanthine-related agents,
grapefruit, grapefruit juice, grapefruit containing products,
Seville oranges and marmalade during the 24 hours prior to dosing
and throughout the study periods.
[0235] Blood Sampling
[0236] For treatment groups 2 and 3 subjects, blood samples (1 drop
per sample) were drawn at 15 minutes before dosing, and at 5, 10,
15, 20, 25, 30, 35, 40, and 50 minutes and 1, 1.25, 1.5, 1.75, 2,
2.5, 3, 4, 6, 8, 12, and 24 hours postdose (22 samples per
treatment for Insulin/4-CNAB dosing groups) for blood glucose
measurements (vital signs and blood sample collection times). For
the 4-CNAB alone treatment group 1, blood samples were collected
immediately before dosing (0), and at 30 minutes and at 2 hours
postdose for blood glucose measurements. The blood samples for
glucose were assayed in real time using a Glucometer.
[0237] For insulin/4-CNAB treatment groups 2 and 3 subjects, blood
samples (3 mL in sodium heparin tube) were drawn at 15 minutes
before dosing, and at 5, 10, 15, 20, 25, 30, 35, 40, and 50 minutes
and 1, 1.25, 1.5, 1.75, 2, 2.5, 3, 4, 6, 8 and 12 hrs post-dose (21
samples per treatment) for plasma glucose, Insulin and C-peptide
measurements. For the 4-CNAB alone treatment group 1 subjects,
blood samples were collected (3 mL in sodium heparin tube) at
immediately before dosing (0), and at 30 minutes and at 2 hours
postdose for blood glucose, plasma glucose, Insulin and C-peptide
measurements. Plasma Glucose analysis was done according to
standard procedures. Human Insulin, and C-peptide analysis were
also conducted. Total blood volume (including study screening and
safety assessment) collected from each subject for the entire study
did not exceed 515 ml for subjects in groups 2 and 3, and 380 ml
for subjects in group 1.
[0238] The concentration of 4-CNAB in plasma was determined using a
combination of liquid chromatography and Mass spectrometry assay
known as LC-MS/MS. The method involves protein precipitation
followed by separation on liquid chromatography using a Hypersil
BDS column and a mobile phase consisting of methanol and acetate
buffer. The eluting peaks were quantified by MS/MS. The equipment
used for the determination of 4-CNAB in plasma comprises of an
Agilent 1100 modular HPLC system with a Micromass Quattro Micro
MS/MS detector. HPLC and AR grade chemicals and reagents were used
throughout the study.
[0239] Calibration standards were prepared in duplicate for each
batch by adding diluted standard solutions of 4-CNAB in solvent to
blank plasma, to give a range of concentrations of approximately
10.6, 20.6, 42.9, 75.2, 116.4, 174.6, 238.1, 317.5, 402.2 and 497.5
ng/ml. Calibration curves were produced for each batch by the
method of weighted (1/y.sup.2) least squares linear regression on
the 4-CNAB to internal standard peak area and the theoretical
concentrations.
[0240] Quality control standards were prepared in duplicate for
each batch by adding diluted standard solutions of 4-CNAB in
solvent to plasma, to give a range of concentrations of
approximately 30.1, 93.6, 258.7 and 443.4 ng/ml. Quality control
standards were prepared from different primary standards (i.e.
separate weighings) to those used for the preparation of the
calibration standards. The concentrations of 4-CNAB in the quality
control standards was calculated by reference to the appropriate
calibration curve.
[0241] Clinical samples were stored at -80.degree. C.
(.+-.10.degree. C.) during the course of the study until the
results were finally reported. The concentrations of 4-CNAB in the
samples were calculated by reference to the appropriate calibration
curve.
4TABLE 4 Effects of Oral Insulin Composition on Human Subjects
Basal Insulin Insulin % Max % Max Insulin Carrier # of Insulin
T.sub.max C.sub.max Glucose C-peptide (Units) (Mg) Subjects (uU/ml)
(Min) (uU/ml) Reduction Reduction 10 (SC) 0 8 4.3 .+-. 2.5 105 54.5
.+-. 25.6 32.3 .+-. 9.6 54.3 .+-. 9.8 150 (PO) 200 8 4.1 .+-. 1.9
25 26.6 .+-. 18.2 23.7 .+-. 13.2 37.5 .+-. 16.9 100 (PO) 600 8 4.4
.+-. 2.3 20 18.1 .+-. 11.3 14.6 .+-. 7.5 33.1 .+-. 13.0 100 (PO)
300 8 3.8 .+-. 3.4 25 9.5 .+-. 6.3 8.8 .+-. 10.2 21.7 .+-. 16.2 100
(PO) 450 8 5.1 .+-. 2.2 20 19.1 .+-. 9.0 14.5 .+-. 8.0 32.6 .+-.
12.7 150 (PO) 100 8 4.8 .+-. 1.8 15 14.5 .+-. 7.7 8.8 .+-. 5.1 17.9
.+-. 13.1
[0242] In the control studies, oral administration of 4-CNAB alone
had no significant effect on plasma glucose levels in human
subjects. Similarly, oral administration of 4-CNAB alone had no
significant effect on plasma C-peptide levels. The compositions
were well tolerated. There were no serious adverse events and
no-drug related adverse events.
[0243] As summarized in Table 4, insulin that has been prepared
according to the present invention can be absorbed by the
gastrointestinal (GI) tract of human subjects and produces a
decrease in plasma glucose levels in human subjects.
[0244] The present invention provides insulin in a form that can be
orally administered, absorbed through the GI tract and remains
bioavailable and bioactive. Compositions prepared according to the
present invention produce clinically relevant magnitudes of
responses in human patients at reasonable doses. The response of
the human subjects was reproducible and dose dependent response
profiles were obtained.
[0245] Table 5 shows the peak plasma concentration of the delivery
agent as C.sub.max (average of all subjects in treatment group).
The T.sub.max, the time for the delivery agent to reach a peak
concentration in the bloodstream, is also shown. The area under the
curve (AUC) is also shown.
5TABLE 5 4-CNAB C.sub.max and T.sub.max Group/Treatment 4-CNAB Dose
C.sub.max [ng/mL] T.sub.max [hr](range) AUC 1/1 400 22,314.6 .+-.
11,455.7 0.5 (0.5) 22,373.3 1/2 800 32,693.2 .+-. 16,719.5 0.5
(0.5-0.75) 42,716.6 1/3 1400 97,544.6 .+-. 15,381.2 0.5 (0.5-0.75)
144,017.1 1/4 2000 121,937.6 .+-. 63,321.7 0.75 (0.5-1.5) 212,919.3
2/1 None NA NA NA 2/2 200 9,163.3 .+-. 2,980.5 0.5 (0.25-0.5)
10,005.2 2/3 600 33,184.6 .+-. 13,303.8 0.5 (0.25-0.75) 36,659.9
3/1 300 8,656.9 .+-. 6,617.9 0.5 (0.25-0.75) 8,738.8 3/2 450
20,101.7 .+-. 9,344.9 0.5 (0.25-4.0) 25,948.0 3/3 100 5,168.4 .+-.
4,332.9 0.25 (0.25-1.0) 4,708.6 3/4 None NA NA NA
EXAMPLE 3
[0246] A single-center, open-label, randomized, active controlled,
3-period crossover study was performed in 10 patients with type 2
diabetes. Male subjects between the ages of 35 and 70 years,
inclusive with Type 2 diabetes mellitus as defined by the American
Diabetes Association (1998 Diabetes Care, 21: S5-S 19) for more
than one year were chosen. Body Mass Index was less than 36
kg/m.sup.2. Stable glycemic control was Hb.sub.AlC<11%. Patients
were off all oral hypoglycemic agents 24 hours prior to each study
dosing day and off any investigational drug for at least four
weeks. Patients refrained from strenuous physical activity
beginning 72 hours prior to admission and through the duration of
the study, and were confined to the Clinical Research Unit.
Patients provided written informed consent.
[0247] Ten patients were randomized to two possible treatment
sequences during a glucose clamp procedure:
[0248] 300 U oral insulin/400 mg 4-CNAB in two capsules,
[0249] 15 U regular subcutaneous (s.c.) insulin.
[0250] Eight of those ten patients received the following
additional treatment during a glucose clamp procedure: 150 U oral
insulin/200 mg 4-CNAB in one capsule.
[0251] In the first two treatment periods, ten patients received,
in random order and with wash-out periods, 300 U oral insulin/400
mg 4-CNAB and 15 U s.c. In the third treatment period, eight of
those original ten patients received 150 U insulin/200 mg 4-CNAB.
Each treatment was a single dose. The wash-out period between the
clamps was 1-20 days.
[0252] Patients were dosed in the morning following an overnight
fast of approximately 12 hours and following a period of 6 hours of
stabilization of the blood glucose by means of the glucose clamp.
For s.c. insulin administration, a 29 gauge needle was inserted
perpendicularly into a raised skinfold in the left lower quadrant
of the abdominal wall, approximately 10 cm from the umbilicus. For
oral insulin administration, the capsules were administered with
200 mL of water to the patients in an upright position. The total
administration time did not exceed 2.5 minutes. Subjects remained
upright for four hours after taking the study drug.
[0253] Stabilized conditions were maintained after drug
administration using a glucose clamp procedure. The glucose clamp
technique [DeFronzo, et al. 1979, Glucose Clamp Technique: A Method
of Quantifying Insulin Secretion and Resistance. Am. J. Physiol.
237: E214-E223.] was use to compare the time-action profiles of the
orally-applied insulin to s.c. insulin. This method utilizes
negative feedback from frequent blood glucose sample values to
adjust a glucose infusion to maintain euglycemia. The glucose
infusion rate becomes a measure of the pharmacodynamic effect of
any insulin administered.
[0254] Samples were collected and documented for determination of
plasma insulin concentrations, C-peptide, and plasma glucose and
glucose infusion rates from the glucose clamp procedure. The
euglycemic clamp procedure after study drug administration lasted 6
hours (+6 h baseline period for stabilization of blood glucose
concentrations at the desired clamp level).
[0255] The blood samples were centrifuged at 3000 rpm for a period
of fifteen minutes at a temperature between 2.degree. C.-8.degree.
C., within one hour of sample collection. Using a plastic pipette
and without disturbing the red cell layer, the plasma from the
collection tube was pipetted in pre-labeled polypropylene tubes for
each analysis of plasma insulin, C-peptide, and plasma glucose
(approximately 0.3-0.5 .mu.l each). The samples were stored at
-20.degree. C. until analysis.
[0256] Safety data and pharmacokinetic/pharmacodynamic data were
analyzed for all subjects. Pharmacokinetic/pharmacodynamic data
were also analyzed for the subset of 8 patients who received the
third study treatment.
[0257] Pharmacokinetic and pharmacodynamic data were statistically
analyzed for subjects that received at least the first two
treatments (visit 1 to 4) and for subjects who completed all three
treatment visits (visit 1 to 6). Pharmacodynamic parameters used
for analysis were the maximum glucose infusion rate after
application of the study drugs (GIR.sub.max), the time to maximum
glucose infusion rate (t.sub.GIRmax) time to half-maximal GIR
values before GIR.sub.max (early t.sub.50%), and the area under the
glucose infusion rate versus time curves from 0 to 60, 120, 240 and
360 minutes after dosing and from 180 to 360 min post dose
(AUC.sub.GIR 0-1 h, AUC.sub.GIR 0-2 h, AUC.sub.GIR 0-4 h,
AUC.sub.GIR 0-6 h, AUC.sub.GIR 3-6 h, respectively). In addition,
plasma concentrations of C-peptide and plasma glucose
concentrations were used for pharmacodynamic analysis. GIR.sub.max,
t.sub.max and early t.sub.50% were calculated by fitting a
polynomial function (6th order) to each individual's GIR profile
after subtraction of the mean baseline GIR. Areas under the curve
(AUCs) were calculated from the raw data using the trapezoidal
rule. Pharmacokinetic parameters were calculated accordingly but
without curve fitting. Pharmacokinetic parameters determined
included the maximum plasma insulin concentration (C.sub.max), time
to maximum insulin concentration (T.sub.max) and the area under the
plasma insulin concentration versus time curve from 0 to 1,2 and 6
hours after application of the study drugs (AUC.sub.INS 0-1 h,
AUC.sub.INS 0-2 h, AUC.sub.INS 0-4 h and AUC.sub.INS 0-6 h
respectively). The calculation of AUCs from time of dosing until
return to the baseline concentration (AUC.sub.INS 0-t') was omitted
as in some patients insulin concentrations did not return to
baseline measurement within 360 minutes post dosing. The
pharmacokinetic and pharmacodynamic data obtained were used for
comparative analysis of the treatments with 15 IU s.c. insulin and
300 IU oral insulin. All tests were performed against a 2-sided
alternative hypothesis, with a significance level of 5% (a=0.05).
The tests were declared statistically significant if the calculated
p-value was <0.050. In view of the small sample size and some
outliers a first analysis was done using non-parametric tests only
(signed Wilcoxon rank tests and Kruskal-Wallis tests).
[0258] However, the Kolmogorov-Smirnov test indicated normal
distribution for all PK and PD parameters. Therefore, parametric
tests (paired t-tests and ANOVAs) were performed in addition.
Although there were no substantial differences between the results
of the non-parametric and the parametric tests it was decided that
the non-parametric results are used for the presentation of the
data.
[0259] No statistical comparisons (apart from ANOVA post-hoc tests
and Kruskal-Wallis test) were performed with the treatment with 150
IU oral insulin as only 8 out of 10 subjects participated in this
study arm, and as it was not performed in random order.
[0260] Results
[0261] Table 6 shows the results of this study. The pharmacokinetic
and pharmacodynamic parameters are given as the average (plus or
minus standard deviations) of the measured value for all the
subjects receiving the particular treatment.
6TABLE 6 Comparisons of Pharmacokinetic and Pharmacodynamic
Parameters Mean .+-. SD oral 300 oral 150 SC Parameter (n = 10) (n
= 8) (n = 10) Insulin AUC.sub.0-1 (mU .multidot. mL.sup.-1
.multidot. min) 2559.25 .+-. 1831.45 1099.58 .+-. 1221.15 542.31
.+-. 296.26 AUC.sub.0-2 (mU .multidot. mL.sup.-1 .multidot. min)
2926.58 .+-. 2104.23 1337.14 .+-. 1407.11 1801.97 .+-. 789.43
AUC.sub.0-3 (mU .multidot. mL.sup.-1 .multidot. min) 3046.75 .+-.
2169.16 1463.59 .+-. 1443.01 3122.81 .+-. 1242.82 AUC.sub.0-4 (mU
.multidot. mL.sup.-1 .multidot. min) 3106.67 .+-. 2212.98 1649.43
.+-. 1541.90 4576.31 .+-. 1817.96 AUC.sub.0-5 (mU .multidot.
mL.sup.-1 .multidot. min) 3172.83 .+-. 2262.81 1819.01 .+-. 1593.02
5916.31 .+-. 2208.85 AUC.sub.0-6 (mU .multidot. mL.sup.-1
.multidot. min) 3225.33 .+-. 2319.66 1949.64 .+-. 1623.949 7003.81
.+-. 2440.251 C.sub.max (mU/mL) 93.44 .+-. 71.18 37.90 .+-. 39.23
32.7 .+-. 10.59 T.sub.max (min) 27.00 .+-. 9.49 22.50 .+-. 7.07
160.5 .+-. 82.78 Glucose Infusion Rate AUC.sub.0-1 (mg/kg) 172.63
.+-. 85.54 58.08 .+-. 39.99 27.38 .+-. 32.22 AUC.sub.0-2 (mg/kg)
297.11 .+-. 142.73 102.62 .+-. 88.94 136.54 .+-. 106.54 AUC.sub.0-3
(mg/kg) 321.19 .+-. 146.29 116.96 .+-. 78.26 271.43 .+-. 191.04
AUC.sub.0-4 (mg/kg) 343.31 .+-. 140.47 142.00 .+-. 85.76 421.33
.+-. 264.76 AUC.sub.0-5 (mg/kg) 364.40 .+-. 135.36 160.28 .+-.
99.64 548.83 .+-. 342.71 AUC.sub.0-6 (mg/kg) 374.10 .+-. 134.70
190.77 .+-. 133.38 650.70 .+-. 380.16 GIR.sub.max (mg/kg/min) 4.35
.+-. 2.23 2.12 .+-. 0.89 3.57 .+-. 1.79 T.sub.GIRmax (min) 39.80
.+-. 16.00 131.63 .+-. 146.04 255.30 .+-. 108.15 Early t50% (min)
13.40 .+-. 6.48 103.50 .+-. 140.90 150.40 .+-. 87.44 Late t.sub.50%
(min) 114.70 .+-. 78.74 #NV .+-. #NV #NV .+-. #NV (n = 7) (n =
6)
[0262] The oral treatment patients showed a faster and higher rise
in insulin concentrations and showed a faster onset of action than
the s.c treatment (AUC.sub.0-1 oral 300 vs. s.c. 15: 2559.+-.1831
vs. 542.+-.296 mU.mL-1 .min, p<0,01; C.sub.max oral 300 vs. s.c.
15: 93.+-.71 vs. 33.+-.11 .mu./U/ml, p<0,01; T.sub.max oral 300
vs. s.c. 15: 27.+-.9 vs. 161.+-.83 min, p<0,01). Accordingly,
pharmacodynamic results showed a significantly faster onset of
action of the oral treatment (AUC.sub.0-1 oral 300 vs. s.c. 15
173.+-.86 vs. 27.+-.32, p<0,01; T.sub.GIRmax oral 300 vs. s.c.
15 40.+-.16 vs. 255.+-.108, p<0,01; early t.sub.50% oral 300 vs.
s.c. 15 13.+-.6 vs. 150.+-.87, p<0,01). The maximum glucose
infusion showed no statistically significant difference.
[0263] Relative bio-potency (based on PD results) is listed in the
Table 7. Respective values for bio-availability (based on PK
results) are listed in Table 8.
7TABLE 7 Biopotency Time interval n mean SD SEM Max Min Median
Biopotency (%): 300 IU oral vs. 15 IU s.c. 0-60 7 54.90 91.93 34.74
261.44 5.54 19.90 0-120 9 11.70 9.21 3.07 32.76 3.65 8.12 0-180 9
5.76 3.41 1.14 13.56 2.03 4.85 0-240 10 21.14 54.94 17.37 177.45
2.19 3.56 0-300 10 47.53 140.38 44.39 447.03 1.56 3.12 0-360 10
31.01 89.44 28.28 285.54 1.10 2.69 Biopotency (%): 150 IU oral vs.
15 IU s.c. 0-60 7 79.19 166.92 63.09 455.95 0.00 15.88 0-120 9
10.00 13.06 4.35 40.76 0.00 4.82 0-180 9 5.18 6.09 2.03 19.53 0.00
3.41 0-240 10 3.23 3.57 1.13 10.67 0.00 1.95 0-300 10 2.83 3.20
1.01 9.74 0.00 1.56 0-360 10 2.52 2.78 0.88 7.93 0.00 1.76
[0264]
8TABLE 8 Bioavailability Time interval n mean SD SEM Max Min Median
Bioavailability (%): 300 IU oral vs. 15 IU s.c. 0-60 10 43.7 60.5
19.1 198.2 7.2 18.3 0-120 10 13.0 20.1 6.4 69.1 2.9 7.0 0-180 10
7.8 12.7 4.0 43.5 1.8 3.9 0-240 10 4.9 7.3 2.3 25.4 1.3 2.6 0-300
10 3.4 4.1 1.3 14.6 1.0 2.1 0-360 10 2.7 3.0 1.0 11.0 0.8 1.8
180-360 10 0.3 0.3 0.1 1.1 0.0 0.1 Bioavailability (%): 150 IU oral
vs. 15 IU s.c. 0-60 8 21.4 19.8 7.0 53.3 0.4 14.7 0-120 8 8.4 6.8
2.4 16.8 0.1 7.8 0-180 8 5.4 4.3 1.5 10.3 0.1 5.7 0-240 8 4.0 3.1
1.1 7.6 0.0 4.6 0-300 8 3.4 2.7 0.9 6.8 0.0 3.8 0-360 8 3.1 2.4 0.9
6.2 0.0 3.4 180-360 8 1.5 1.6 0.6 3.7 0.0 1
[0265] FIG. 1 shows a plot of C-peptide [nmol/l] vs. time for 15 IU
s.c., 300 IU oral and 150 IU oral. As shown in FIG. 1, C-peptide
measurements showed no significant changes during the treatment
periods.
[0266] The SC injection and the oral insulin treatments were all
well tolerated. There were no adverse events and no clinically
relevant findings for clinical laboratory evaluations, vital signs,
physical examination, or ECG.
EXAMPLE 4
[0267] Plasma Delivery Agent Design and Efficiency
[0268] Delivery agents 1-3 were investigated for their ability to
penetrate the GI mucosa. The plasma concentrations of each delivery
agent was measured in human subjects after oral administration of
delivery agent loaded capsules as a measure of each delivery agents
penetration efficiency. See Tables 9 and 10.
9TABLE 9 Structures of Delivery Agents 1-3 Delivery Agent 1 (SNAC)
4 Delivery Agent 2 (SNAD) 5 Delivery Agent 3 (4-CNAB) 6
[0269]
10TABLE 10 Delivery Agent Plasma Concentrations in Humans Variables
Delivery Agent Delivery Agent X n Dose (Mg) AUC (ng .multidot.
hr/ml) 1 (SNAC) H 7 750 3499 2 (SNAD) H 9 750 2037 3 (4-CNAB) Cl 3
800 47478
[0270] Blood sampling for plasma delivery agent concentration
determination (2 mL in sodium heparin tube) were drawn 15 minutes
before dosing, and at 5, 10, 15, 30, and 45 minutes and 1, 1.5, 2,
3, 4, 6, 8, and 12 hours post-dose (14 samples per treatment) for
delivery agent measurements in all treatment groups.
[0271] Two 18-gauge IV lines were situated prior to dosing; one for
blood sampling, and the other for potential infusion of 20% glucose
for subjects in groups 2 and 3. The subjects in group 1 only had
one cannula inserted. The blood samples were centrifuged at 3000
rpm for a period of fifteen minutes at a temperature from about
2.degree. C. to 8.degree. C., within one hour of sample collection.
Using a plastic pipette and without disturbing the red cell layer,
the plasma from the collection tube was pipetted in duplicate for
each analysis, blood glucose, Human Insulin, C-peptide, delivery
agent into pre-labeled polypropylene tubes. The samples were stored
at -70.degree. C. until analysis.
[0272] The indicated doses were ingested by healthy human
volunteers and the plasma concentrations of the delivery agents
were monitored over time and the area under the curve (AUC)
calculated. Surprisingly, as provided in Table 10, oral
administration of 800 mg delivery agent number 3 with X as chlorine
and n equal to 3 alkyl produced an approximately 13.5 fold greater
penetration of the GI mucosa in humans than did oral administration
of 750 mg of delivery agent 1 having n equal 7 alkyl. Similarly,
oral administration of 800 mg of delivery agent number 3 produced
more than a 23 fold greater penetration of the GI mucosa in humans
than did oral administration of 750 mg of delivery agent 2 having n
equal 5 alkyl.
[0273] Similar results were obtained when delivery agents 1-3 were
administered orally to monkeys and the plasma concentrations of the
delivery agents monitored over time and the AUC calculated. As
provided in Table 11, oral administration of 300 mg of delivery
agent number 3 with X as chlorine and n equal to 3 alkyl produced a
more than 11 fold greater penetration of the GI mucosa in monkeys
than did oral administration of 300 mg of delivery agent 1 having n
equal to 7 alkyl. See Table 11. Further, 300 mg of delivery agent 3
displayed a more than 6 fold greater penetration of the GI mucosa
in monkeys than did oral administration of 300 mg of delivery agent
2 having n equal to 5 alkyl. See Table 11.
11TABLE 11 Delivery Agent Plasma Concentrations in Monkeys Delivery
Agent Delivery Agent X n Dose (Mg) AUC (ng .multidot. hr/ml) 1
(SNAC) H 7 300 45 2 (SNAD) H 9 300 82 3 (4-CNAB) Cl 3 300 499
EXAMPLE 5
[0274] Comparison of the Delivery Efficiency of Delivery Agents
1-3
[0275] Next, delivery agents 1-3 were compared for the ability to
efficiently transport insulin across the GI mucosa in a
biologically active form by determining the relationship between
delivery agent dose, insulin dose and the glucose response. See
Table 12. The effective dose of delivery agent necessary to deliver
a therapeutic dose of insulin and produce a therapeutic effect was
measured. See Table 12. The therapeutic effect was determined by
the ability of the delivery agent/insulin combination to lower
serum glucose by at least 10% within one hour post
administration.
12TABLE 12 Effective Clinical Dose of Delivery Agent in Humans
Delivery Agent Delivery Agent X N Dose (Mg) 1 (SNAC) H 7 2400 2
(SNAD) H 9 1500 3 (4-CNAB) Cl 3 200
[0276] Again, as shown in Table 12, delivery agent 3 with X as
chlorine and n equal to 3 alkyl was approximately 12 fold more
efficient in facilitating insulin transit across the GI mucosa in a
biologically active form than was delivery agent 1 having n equal
to 7 alkyl. Similarly, delivery agent no. 3 was 7.5 fold more
efficient in facilitating transport of insulin across the GI mucosa
in a biologically active form than was delivery agent 2 having n
equal to 5 alkyl. See Table 12.
[0277] Most importantly, only delivery agent 3 is efficient enough
at facilitating transport of biologically active insulin to allow
packaging of a therapeutically effective dose of insulin plus
delivery agent into a single capsule.
EXAMPLE 6
[0278] A double-blind, randomized placebo-controlled study was done
in order to assess the safety and tolerability of escalating single
oral doses of 4-CNAB capsules and insulin/4-CNAB capsules. The
objectives of this study were to (1) evaluate the safety and
tolerability of single oral doses of 4-CNAB and of Insulin/4-CNAB
capsules in healthy subjects, and (2) assess the pharmacokinetic
(PK) of 4-CNAB and insulin. The effect of study drug on blood
glucose was also evaluated.
[0279] Twenty-nine volunteers, divided among three groups (9 in
group 1, and 10 in each of groups 2 and 3), participated in this
study. Randomization was stratified such that any individual
subject received placebo only on a single occasion or not at all.
Two volunteers in each group received placebo. Group 1 received 4
escalating oral doses of 4-CNAB capsules or placebo. In Group 2,
volunteers received a single subcutaneous (SC) dose of insulin or
placebo, a single oral dose of Insulin/4-CNAB or placebo followed
by another single oral dose of Insulin/4-CNAB or placebo. Group 3
received another 3 single oral doses of Insulin/4-CNAB capsules or
placebo followed by a single oral dose of insulin. For all groups
there was a washout period of at least 72 hours between treatment
periods.
[0280] The volunteers were all healthy, adult male volunteers
between 18 and 50 years of age, inclusive, and were determined to
be in good health, within the permissible deviations (+/-15%) of
ideal weight. Laboratory values (hematology, serum chemistries, and
urinalysis) obtained during screening were within normal
ranges.
[0281] Test Product:
[0282] 4-CNAB 200 mg Capsules, Oral administration,
[0283] 4-CNAB Sodium Salt, Oral administration,
[0284] 4-CNAB/Insulin-200 mg/1 50 Units Insulin Units Capsules,
Oral administration,
[0285] Human Insulin, Oral Administration, Human Insulin, Purchased
Commercially.
[0286] Reference Product:
[0287] Placebo 200 mg Methocel E15 Premium LV--Capsules, Oral
administration,
[0288] Sodium Chloride 0.9% Injection.
[0289] The subjects first completed a screening period between 2
and 21 days prior to study start. The evening before each treatment
period, subjects entered the clinical research center for check-in
assessments and to begin a pre-dose fast of at least 8 hours. Study
data was collected through to 12 hours post-dose, and the subjects
remained in the unit under observation for a further 12 hours for
Groups 2 and 3.
[0290] On the basis of plasma concentrations of 4-CNAB and insulin,
pharmacokinetic ("PK") analysis was performed using
non-compartmental PK methods as implemented in WinNonlin
Professional version 3.2. The profiles for insulin and C-peptide
corrected insulin were evaluated up to 6 hours as food was given 6
hours following insulin treatment. The following parameters were
derived for 4-CNAB: C.sub.max, t.sub.max, AUC.sub.(0-t),
AUC.sub.(0-inf), AUC.sub.%Extrap, K.sub.el, t.sub.1/2, CL/F, Vd/F,
MRT.sub.(0-t), and MRT.sub.(0-inf). The following parameters were
derived for insulin and C-peptide corrected insulin: C.sub.max,
t.sub.max, AUC.sub.(0-2), AUC.sub.(0-6), AUC.sub.(0-t) and
concentrations (C.sup.b) and corresponding time (t.sup.b)
immediately prior to intervention for hypoglycemia.
[0291] On the basis of plasma concentrations of glucose,
pharmacodynamic ("PD") analysis was performed using WinNonlin.TM.
Professional version 3.2. The profiles for glucose, glucose change
from baseline and glucose percentage change from baseline were
evaluated up to 6 hours as food was given 6 hours following insulin
treatment. The following PD parameters were computed for plasma
glucose concentration: AURC.sub.(0-2), AURC.sub.(0-t), R.sub.max
(minimum value), t.sub.Rmax and concentrations (R.sup.b) and
corresponding time (t.sup.b) immediately prior to intervention for
hypoglycemia. The following PD parameters were computed for plasma
glucose concentration change from baseline: AUEC.sub.(0-2),
AUEC.sub.(0-t), E.sub.max (baseline subtracted), t.sub.Emax,
percent change from baseline and concentrations (E.sup.c) and
corresponding time (t.sup.c) immediately prior to intervention for
hypoglycemia.
[0292] Concentrations and both PK and PD parameters were summarized
with descriptive statistics. No formal statistical analysis was
done.
[0293] There were no serious adverse effects (SAEs) or deaths. No
subject withdrew from the study for study drug related reasons.
There were no clinically significant abnormal results as assessed
by vital signs, ECG, clinical laboratory parameters (except blood
glucose) and physical examination. A total of 65 AEs were reported
by 23 subjects. All but one (a headache of mild intensity
experienced by Subject 19) were considered to be
treatment-emergent. The most common treatment-emergent AEs reported
during the study were hypoglycemia (26 in 13 subjects), headache
(12 in 9 subjects) and dizziness (5 in 5 subjects). Of the 26
events of hypoglycemia, subjects received intervention i.e.
food/drink such as a chocolate/glucose syrup bar, banana or apple
juice on 20 occasions, in order to raise their blood glucose. The
greatest number of AEs was reported after 10 Units SC insulin (17
in 8 subjects), 16 of which were thought to have a causal
relationship to the study drug.
[0294] Drug Substances:
[0295] The recombinant human insulin used in the nonclinical
studies discussed in the above examples commercially obtained from
Calbiochem (San Diego, Calif.). The recombinant human insulin used
in the clinical studies is Zinc-Insulin Crystals Human: Proinsulin
Derived (Recombinant DNA Origin) USP Quality obtained from Eli
Lilly and Company (Indianapolis, Ind.). No modifications or
processing were done to the bulk drug substance in the manufacture
of the clinical Recombinant Human Insulin/4-CNAB oral dosage
form.
[0296] Monosodium N-(4-chlorosalicyloyl)-4-aminobutyrate (4-CNAB),
whose chemical structure is shown below, was produced at a
cGMP-compliant manufacturing facility. The mechanism by which
4-CNAB facilitates the gastrointestinal absorption of insulin has
not yet been fully elucidated. While not wishing to be bound by
theory, one hypothesis is that 4-CNAB interacts with insulin
non-covalently, creating more favorable physicochemical properties
for absorption. 7
[0297] Insulin is administered parenterally, usually by
subcutaneous injection. It is not absorbed to any extent through
the gastrointestinal tract, presumably due to its size and
potential for enzymatic degradation. Monosodium
N-(4-chlorosalicyloyl)-4-aminobutyrate (4-CNAB) is a novel compound
discovered by Emisphere Technologies, Inc. When combined with
insulin, 4-CNAB has been shown to enhance the gastrointestinal
absorption of Human Insulin following oral administration to rats
and primates.
EXAMPLE 7
[0298] Previous Non-clinical Studies with 4-CNAB and
Insulin/4-CNAB
[0299] 4-CNAB was previously evaluated in a non-clinical program
that included pharmacological screening, pharmacokinetic profiling,
and toxicity assessments in rats and monkeys. In general, animal
physiological responses to 4-CNAB alone and to Insulin/4-CNAB were
comparable. Pharmacokinetic studies in mice, rats and monkeys have
shown that 4-CNAB is absorbed rapidly following oral
administration, and subsequently cleared from the body. 4-CNAB did
not demonstrate potential activity in any of the primary molecular
targets evaluated in receptor binding screening assays. Four
genotoxicity studies have been conducted with 4-CNAB, with no
positive findings. Based on 14-day oral repeated dose toxicity
studies, the NOAEL (No Adverse Effect Level) was estimated to be
500 mg/kg in Sprague-Dawley rats, and 400 mg/kg in rhesus monkeys.
Developmental and reproductive toxicology studies have not yet been
conducted.
[0300] Non-clinical studies in rats and monkeys demonstrated that,
over the range tested, insulin absorption increases with increasing
doses of 4-CNAB. Similarly, for a fixed oral dose of 4-CNAB,
insulin absorption increases with increasing doses of insulin. Oral
insulin absorption was evaluated in rats at varying doses of both
insulin and 4-CNAB. Significant increases in serum insulin
concentrations were observed following the administration of
insulin at doses of 4.55, 6.5, 9.75, and 13 Units/kg in the
presence of a fixed 4-CNAB dose (200 mg/kg). The mean peak serum
insulin levels were 31, 44, 85, and 132 .mu.U/mL respectively.
Insulin absorption was dose dependent and increased as the dose of
insulin increased. Oral administration of aqueous solutions of
insulin alone (13 Units/kg) or 4-CNAB alone (200 mg/kg) did not
result in any significant increases in serum insulin levels.
Significant increases in serum insulin concentrations were also
observed following the administration of 4-CNAB at doses of 50,
100, 200, and 300 mg/kg in the presence of a fixed insulin dose (13
Units/kg). The mean peak serum insulin levels were 9, 39, 103, and
157 .mu.U/mL, respectively. Insulin absorption was dose dependent
and increased as the dose of 4-CNAB increased.
[0301] Based on previous nonclinical studies, the starting insulin
dose of 150 insulin Units USP (which is about 7-fold lower than the
15 U/kg no effect dose in monkeys) was selected.
[0302] This present study was designed to provide safety,
tolerability and pharmacokinetics data following various oral
4-CNAB or Insulin/4-CNAB doses administered as capsules. These data
will provide safety information for the continued clinical
development of an oral Insulin/4-CNAB solid dosage form.
[0303] A subcutaneous (s.c.) insulin treatment group was added to
allow comparison of the combined treatment against an existing
standard treatment and an oral insulin alone treatment group was
also included to further evaluate the effect of 4-CNAB on oral
insulin absorption. This study evaluated the safety and
tolerability of single oral doses of 4-CNAB and of Insulin/4-CNAB
capsules in healthy subjects and assessed the PK of 4-CNAB and
Insulin and the effect of study drug on blood glucose.
[0304] This study was a single center, double-blind, partially
randomized placebo-controlled study of escalating single oral doses
of 4-CNAB capsules, escalating single doses of Insulin/4-CNAB
capsules, a single oral dose of insulin and a s.c. administration
of insulin in healthy male volunteers.
[0305] The 29 volunteers were divided into three groups. In Group
1, there were four separate treatments as shown in the following
Table 13. For each treatment, seven subjects received active
treatment and 2 received placebo, according to the pre-prepared
randomization code. Randomization was stratified such that any
individual subject received placebo only on a single occasion or
not at all.
13TABLE 13 Group 1 Treatments (4-CNAB alone - 4 escalating doses)
4-CNAB only # of Subjects & # Placebo Treatment: 4-CNAB Dose
Subjects # of Capsules Treatment 1 7 subjects 400 mg 2 2 Treatment
2 7 Subjects 800 mg 2 4 Treatment 3 7 Subjects 1400 mg 2 7
Treatment 4 7 Subjects 2000 mg 2 10
[0306] In Group 2, there were three separate treatments as shown in
the following Table 14. For each treatment 8 subjects received
active treatment and two received placebo, according to the
pre-prepared randomization code.
14TABLE 14 Group 2 Treatments (Insulin/4-CNAB - 2 escalation doses
and SC insulin dose) # of Subjects & Insulin/4-CNAB
Insulin/4-CNAB Dose # Placebo # Treatment (Unit Insulin/mg 4-CNAB)
Subjects of Capsules Treatment 1 8 Subjects (10/0) 2 0 (SC only)
Treatment 2 8 Subjects (150/200) 2 1 Treatment 3 8 Subjects
(100/600) 2 4
[0307] In Group 3, there were four separate treatments as shown in
the following Table 15. For each treatment 8 subjects received
active treatment and two received placebo, according to the
pre-prepared randomization code.
15TABLE 15 Group 3 Treatments (Insulin/4-CNAB - 3 escalation doses)
# of Subjects & Insulin/4-CNAB Insulin/4-CNAB Dose # Placebo #
Treatment: (Unit Insulin/mg 4-CNAB) Subjects of Capsules Treatment
1 8 Subjects (100/300) 2 2 Treatment 2 8 Subjects (100/450) 2 3
Treatment 3 8 Subjects (150/100) 2 1 Treatment 4 8 subjects (150/0)
2 1
[0308] Capsules were administered with 240 mL water. A washout
period of at least 72 hr was observed between doses. After each
dosing, safety data (i.e. vital signs, blood glucose, and available
4-CNAB plasma concentrations) were collected and evaluated before
proceeding to the next dose level.
EXAMPLE 8
[0309] There was also a parallel group, partially randomized double
blind clinical trial. The study allowed the investigation of the
effect of 4-CNAB on oral insulin PK and PD to be studied across a
range of doses and to be compared with SC and oral insulin alone
treatments. Simultaneous measurement of C-peptide protein allowed
the correction for endogenous insulin. Food given at 6 h post-dose
also allowed its effect on insulin and glucose to be observed.
Parallel groups were used due to the number of treatments
administered and also reduced the length of the study. The
double-blind nature of subject and physician ensured minimal
bias.
[0310] Male subjects aged between 18 and 50 years were recruited
and were chosen to be representative of the general healthy
population, which was deemed suitable for such a study. Selection
criteria (inclusion and exclusion) were chosen to ensure that the
subjects were healthy and therefore at minimal risk from the study
procedures and to side effects of Insulin/4-CNAB. The subjects were
healthy, adult male volunteers between 18 and 50 years of age,
inclusive and were in good health as determined by the Investigator
based on medical history, physical examination, and clinical
laboratory studies at screening. The subjects were within the
permissible deviations (+/-15%) of ideal weight according to the
1983 tables of desirable weights issued by the adjusted
Metropolitan Life Insurance Co.
[0311] All laboratory values (hematology, serum chemistries, and
urinalysis) obtained during screening were generally within normal
ranges. The laboratory tests were conducted in a fasted state and
glucose measured. However, for clinical laboratory values outside
of the normal range, the laboratory test was repeated once. A
volunteer with one or more laboratory values outside the normal
range was still eligible for the study if the abnormality was not
clinically significant, and the Investigator and Sponsor's
representative approved the eligibility of the participant.
[0312] The subjects had 12-lead ECG recorded within 14 days prior
to the study start, and results must have indicated a normal
recording or a non-clinically significant abnormality.
[0313] Thirty subjects were randomly assigned to three groups.
Within each treatment period in each group, 8 subjects were planned
to receive active treatment and 2 subjects to receive placebo. In
Group 1, there were 4 escalating single doses of 4-CNAB (400, 800,
1400 and 2000 mg and each subject received either all four of these
escalating doses or three escalating single doses and one dose of
placebo. In Group 2 there were 3 treatments (10 Units of SC insulin
and 2 escalating oral doses of Insulin/4-CNAB; 150 Units/200 mg,
100 Units/600 mg) and each subject received either all three of
these treatments or two of these treatments and one placebo
treatment. In Group 3 there were 3 escalating oral doses of
Insulin/4-CNAB (100 Units/300 mg, 100 Units/450 mg and 150
Units/100 mg) and one SC dose of 150 Units of insluin. Each subject
received either all four of these treatments or three of these
treatments and one placebo treatment.
[0314] All capsules for each treatment were administered with 240
mL water. Escalating doses were not administered until the
Investigator and the Sponsor assessed the safety and tolerability
of the previous dose. Dose escalation continued until two subjects
per treatment exhibited a blood glucose level of less than 54 mg/dl
(3.0 mmol/L) after which no further dosing took place pending
discussion with the Sponsor. Once this dose had been identified an
adaptable approach to exploring changing doses of insulin and
4-CNAB using capsules prepared at Medeval was implemented. The next
chosen insulin dose was no higher than the insulin dose that caused
the blood glucose level of 54 mg/dl (3.0 mmol/L).
[0315] The 4-CNAB alone and Insulin/4-CNAB capsules were prepared
by AAIPharma Inc., Wilmington N.C., and shipped prior to the study
start. The 4-CNAB used for the capsules was manufactured under cGMP
compliance. The Insulin used to prepare the capsules was
Zinc-Insulin Crystals Human: Proinsulin Derived (Recombinant DNA
Origin) USP Quality obtained from Eli Lilly and Company
(Indianapolis, Ind.). Insulin used for the SC dosing was provided
by Medeval Ltd. This insulin was zinc-insulin crystals human:
proinsulin derived (recombinant DNA origin) equivalent to Humulin R
(The trade name is Humulin S injection 100 Units/mL).Both 4-CNAB
capsules and Insulin/4-CNAB capsules were provided in high-density
polyethylene (HDPE) bottles. Each bottle contained 40 capsules and
a polyester coil, had a heat-induction seal and a child-resistant
cap, and was stored frozen at or below minus 10.degree. C. On the
day of dosing, the appropriate number of bottles were removed from
the freezer and brought to room temperature (between 15 and
30.degree. C.) for about one hour. Once the bottles were at room
temperature, the caps were opened and the seals broken. The
polyester coils were removed and the capsules dispensed. Capsules
were used within 4 hours of dispensing. Unopened bottles were not
left at room temperature for more than 4 hours. Each bottle was
intended for single use, and the unused capsules were not to be
used for subsequent dosing.
[0316] Placebo capsules consisted of Size 3 hard gelatin capsules
filled with 200 mg of Methocel E15 Premium LV. Stability data were
not required for this extemporaneously prepared placebo as the
placebo capsules only contained Methocel, which is a stable
excipient. The placebo capsules were stored at room temperature
between 15 and 30.degree. C. in a dry place.
[0317] Each 4-CNAB capsule contained 200 mg of 4-CNAB and was
stored frozen at or below minus 10.degree. C. The bottle was
brought to room temperature (between 15 and 30.degree. C.) before
opening and was not left bottle at room temperature for more than 4
hours. Each Insulin/4-CNAB capsule contained 150 Insulin Units USP
of Human insulin and 200 mg of 4-CNAB. Each capsule was stored
frozen at or below minus 10.degree.C., was brought to room
temperature (between 15 and 30.degree. C.) before opening, and was
not left at room temperature for more than 4 hours
[0318] The subjects were assigned a randomization number
consecutively in the order of their inclusion into the study from 0
to 030 after they had given their informed consent and had
successfully completed screening tests. Thirty subjects were to be
enrolled into three separate groups. Within each treatment period
of Group 1, seven subjects were randomized to receive active
treatment and two subjects to receive placebo. In Groups 2 and 3,
eight subjects were randomized to receive active treatment and two
subjects to receive placebo. In each group any individual received
placebo only on one occasion or not at all. Subjects who withdrew
from the study were not replaced. From the screening visit until
the allocation of a randomization number, the subjects were
identified by their initials and date of birth.
[0319] The doses selected for this study were based on results of
pre-clinical toxicology studies of 4-CNAB and Insulin conducted in
Sprague-Dawley rats and in rhesus monkeys to determine the no
effect adverse event level (NOAEL). The estimated NOAEL level of
4-CNAB in monkeys was 400 mg/kg. The highest proposed dose of
4-CNAB in man (2000 mg [28.5 mg/kg in 70 kg man]) was 12-16 fold
lower than the NOAEL in monkeys. In one monkey, an insulin dose of
15 U/kg in combination with a 4-CNAB dose of 1200 mg/kg was
associated with a single hypoglycemic episode but no effects were
observed at 15 U/kg in combination with lower doses. Taking this
information into account, 15 insulin Units USP was chosen as the
starting dose of insulin in man for this study. Escalating doses
would only be administered once all safety and tolerability data
had been reviewed.
[0320] 4-CNAB alone and Insulin/4-CNAB were administered according
to the randomization code and the following dosing regimen (Tables
1-3): Group 1 treatments were 4 escalating oral doses of 4-CNAB
capsules, with each subject receiving four active treatments or
three active treatments and one placebo treatment. Group 2
treatments were SC doses of insulin and 2 escalating oral doses of
Insulin/4-CNAB capsules, with each subject receiving three active
treatments or two active treatments and one placebo treatment.
Group 3 treatments were another 3 escalating oral doses of
Insulin/4-CNAB and a single oral dose of insulin, with each subject
receiving four active treatments or three active treatments and one
placebo treatment Between each treatment period subjects had a
washout period of at least 72 hr.
[0321] On day one of each study treatment period, study medication
(capsules or SC dose) were administered at approximately 8:00 AM
following an 8-hour minimum overnight fast. The capsules were
administered with 240 mL of water with subjects in an upright
position. The total administration time did not exceed 2.5 minutes.
The SC dose of insulin solution or placebo (saline) was injected in
the abdominal wall as a single bolus administration. Each treatment
period lasted between 12 and 24 h.
[0322] The appearance of the prepared active and placebo study
treatments was identical and therefore maintenance of blinding from
treatment appearance was not an issue in this study.
[0323] No concomitant medications, including over-the-counter
medications, vitamins or nutritional supplements were permitted
during the study period or during the 14 days preceding the study
period. Acetominophen (paracetamol) was allowed during the study to
treat minor AEs such as headaches. In the event that concomitant
medications were taken during the course of the study, the
continued participation of that particular subject was determined
by the Investigator and the medical monitor.
[0324] Administration of the medication was supervised by the
Investigator or his deputy. After oral drug administration, a mouth
and hand inspection took place. The time of drug administration was
recorded and checked by a second member of staff. Therefore,
non-compliance was not an issue in this study.
[0325] Subjects fasted for a minimum of 8 hours prior to morning
dosing until 6 hours after dosing, after which each subject ate a
full meal, including at least two slices of bread. Subjects were
provided with standard high carbohydrate meals (i.e. lasagna,
jacket potato, salad and a dessert such as apple pie) and snacks.
Water was allowed ad libitum, except for 1 hour prior through to 1
hour after administration of each treatment (apart from that
required for dosing). Subjects were asked to refrain from xanthine
or xanthine related agents from 24 hours prior to dosing, and
throughout the study periods. Subjects also refrained from
grapefruit, grapefruit juice, grapefruit containing products,
Seville oranges and marmalade during the 24 hours prior to dosing
and throughout the study periods. No concomitant medication, apart
from acetominophen (paracetamol), was allowed during the study. No
alcohol was allowed for 24 hours prior to admission and while
resident in the Clinical Unit. Non-smokers or smokers who smoked up
to five cigarettes a day were recruited. Smoking was not allowed
while resident in the Clinical Unit. Subjects were asked to avoid
strenuous physical activity and contact sports from 48 hours prior
to Day -1 until the end of the residential period.
[0326] For Insulin/4-CNAB treatment Groups 2 and 3, subjects blood
samples (1 drop per sample) were drawn at 15 minutes before dosing,
and at 5, 10, 15, 20, 25, 30, 35, 40, and 50 minutes and 1, 1.25,
1.5, 1.75, 2, 2.5, 3, 4, 6, 8, 12, and 24 hr post-dose (22 samples
per treatment for Insulin/4-CNAB dosing Groups) for blood glucose
safety measurements. For the 4-CNAB alone treatment group, blood
samples were collected before dosing (-15 minutes), and at 30
minutes and at 2 hr post-dose for blood glucose measurements. The
blood samples for glucose were assayed in real time-using a
Glucometer. These measurements were used for detection of onset of
hypoglycaemia, so rescue action could be taken where necessary.
[0327] Pharmacokinetic and Pharmacodynamic Assessments
[0328] Plasma glucose was measured based on a timed-endpoint method
using a BECKMAN Synchron CX system that mixes exact proportions of
reagents that catalyse the phosphorylation of glucose. The Synchron
CX measures changes in the absorbance spectrum at 340 nm at a fixed
time interval. The change in absorbance is directly proportional to
the concentration of glucose in the sample.
[0329] Plasma C-peptide was measured using a DELFIA C-peptide kit,
based on a solid phase, two-site fluoroimmunometric assay, which
used the direct sandwich technique in which two monoclonal
antibodies are directed against antigenic determinants on the
C-peptide molecule. Reagent dissociates europium ions from the
labeled antibody, which form fluorescent chelates with the reagent.
The fluorescence is directly proportional to the concentration of
C-peptide in the sample.
[0330] For Insulin/4-CNAB treatment Groups 2 and 3, subjects blood
samples (3 mL in sodium heparin tube) were drawn at 15 minutes
before dosing, and at 5, 10, 15, 20, 25, 30, 35, 40, and 50 minutes
and 1, 1.25, 1.5, 1.75, 2, 2.5, 3, 4, 6, 8 and 12 hr post-dose (21
samples per treatment) for plasma glucose, Insulin and C-peptide
measurements. For the 4-CNAB alone treatment group, subjects, blood
samples were collected (3 mL in sodium heparin tube) at immediately
before dosing (0), and at 30 minutes and at 2 hr post-dose for
plasma glucose, insulin and C-peptide measurements. Plasma Glucose
analysis was done at Medeval according to standard procedures.
Human Insulin, and C-peptide analyses were also conducted. For the
insulin alone control group (minimum 3 subjects), blood samples (3
mL in sodium heparin tube) were drawn at 15 minutes before dosing,
and at 10, 20, 30, 40, 50 minutes and 1, 1.25, 2, 2.5, 3 and 6 hr
post-dose (12 samples per treatment). Plasma glucose, insulin and
C-peptide measurements were done. Plasma glucose analysis was done
at Medeval Ltd. according to standard procedures.
[0331] Plasma 4-CNAB Concentration:
[0332] Blood sampling for plasma 4-CNAB concentration determination
(2 mL in sodium heparin tube) was drawn at 15 minutes before
dosing, and at 5, 10, 15, 30, and 45 minutes and 1, 1.5, 2, 3, 4,
6, 8, and 12 hr post-dose (14 samples per treatment) for 4-CNAB
measurements in all treatment groups, except for the SC insulin
group and insulin alone control group as there was no need to
analyze for 4-CNAB.
[0333] Two 18-gauge IV lines were situated prior to dosing; one for
blood sampling, and the other for potential infusion of 20% glucose
for subjects in Groups 2 and 3, if required for the treatment of
hypoglycaemia. The subjects in Group 1 only had one cannula
inserted. Blood samples for 4-CNAB analysis were centrifuged at
3000 rpm for a period of 15 minutes at a temperature between
2.degree. C.-8.degree. C., within one hour of sample collection.
Using a plastic pipette and without disturbing the red cell layer,
the plasma from the collection tube was pipetted into pre-labelled
polypropylene tubes and stored at -70.degree. C.
[0334] Blood samples for plasma glucose, insulin and C-peptide
analyses were stored between 2.degree. C. and 8.degree. C.
immediate after sampling and prior to centrifugation. The samples
were centrifuged at 3000 rpm for a period of 15 minutes at a
temperature between 2.degree. C. and 8.degree. C., within one hour
of sample collection. Using a plastic pipette and without
disturbing the red cell layer, the plasma from the collection tube
was pipetted into pre-labelled polypropylene tubes without delay.
The samples were stored at -70.degree. C. until analysis.
[0335] Total blood volume (including study screening and safety
assessment) collected from each subject for the entire study did
not exceed 625 mL for subjects in Group 3, and 487 mL for subjects
in Group 2, and 380 mL for subjects in Group 1.
[0336] Plasma glucose measurements were used to compute various
pharmacodynamic parameters. The following parameters were planned
to be derived for 4-CNAB and insulin: AUC.sub.(0-t),
AUC.sub.(0-inf), K.sub.el, t.sub.1/2, C.sub.max, t.sub.max, CL/F,
Vd/F, MRT and F.sub.rel. The following PD parameters were computed
for plasma glucose and plasma glucose change from baseline:
AUEC.sub.(0-t) (baseline subtracted), AURC.sub.(0-t) (total
response), E.sub.max (baseline subtracted), R.sub.max (total
response) and t.sub.Emax, (obtained without interpolation). The
above parameters were to be summarized using descriptive statistics
(mean, standard deviation (SD), coefficient of variation (CV%),
standard error of the mean (SE), minimum (Min), median, maximum
(Max), and sample size (N) by treatment group. Individual data was
reported by dose, by subject.
[0337] Individual vital signs were listed per treatment, study day
and measurement time and summarized descriptively. Where vital sign
measurements were repeated due to measurement error, the repeated
value was used for calculation of summary statistics, otherwise the
original value was used.
[0338] 12-lead ECG assessments were listed per dose level,
treatment, study day and measurement time. Clinical laboratory
parameters were listed. Laboratory values outside the laboratory's
normal ranges were listed separately together with associated
repeats and comments as to their clinical significance.
[0339] Plasma glucose concentrations were also to be presented as
percent change (decrease or increase) from the baseline, where
baseline was taken as the pre-dose concentration. Thus, 3 Glucose %
Change from Baseline = ( Glucose Conc . - Baseline Glucose Conc .
Baseline Glucose Conc . ) .times. 100
[0340] where Glucose Change from Baseline=(Glucose Conc.-Baseline
Glucose Conc.).
[0341] Dose escalation within each group continued until two
subjects per treatment exhibited a blood glucose level of less than
54 mg/dl (3.0 mmol/L), after which no further dosing took place
pending discussion with the Sponsor. Once this dose had been
identified there was an adaptable approach to exploring changing
the ratios of insulin and 4-CNAB using capsules prepared at
Medeval. The chosen insulin dose was no higher than the insulin
dose that caused the blood glucose level of 54 mg/dL (3.0 mmol/L).
The dose of 4-CNAB was not higher than that already given.
[0342] Subjects first completed a screening period between 2 and 21
days prior to study start. The evening before each treatment
period, subjects entered the clinical research center for check-in
assessments and to begin a pre-dose fast of at least 8 hours. Study
data was collected through 12 hr post-dose. Subjects from Groups 2
and 3 remained under observation in the unit for a further 12
hours.
[0343] Safety assessments included physical examinations, medical
history, vital signs, 12-lead electrocardiogram (ECG) monitoring,
laboratory evaluations and checking for adverse events (AEs).
Activity parameters included blood glucose, insulin, C-peptide, and
4-CNAB plasma concentration measurements.
[0344] Blood sampling for PK/pharmacodynamic (PD) parameters was
done throughout the study. An 18-gauge IV line was situated in each
arm prior to dosing. One IV line was used for blood sampling, and
the second IV line was to be used for potential infusion of 20%
glucose (Group 2 and 3 only) in case of hypoglycaemia. Blood
samples (2 mL in sodium heparin tubes) were drawn at specified
timepoints described in Section 9.7.2 for blood glucose, plasma
glucose, insulin and C-peptide, and 4-CNAB measurements in all
treatment groups.
[0345] For the PK analysis of insulin, analysis was conducted for
both measured insulin and C-peptide corrected insulin
concentrations. This was done using concentrations of the precursor
C-peptide and the following equation:
Corrected Insulin Concentration=Insulin Concentration-(Baseline
Ratio.times.C-peptide)
[0346] where 4 Baseline Ratio = Insulin Concentration C - peptide
Concentration
[0347] at each time point
[0348] Similarly, in order to take into account baseline levels of
glucose, PD analysis was conducted based on the change in glucose
concentration from pre-dose levels, rather than absolute values
only. The glucose concentration percent change from baseline values
were also calculated, and profiles were tabulated and plotted.
[0349] The additional parameters AUC.sub.(0-2) for insulin and
C-peptide corrected insulin and AURC.sub.(0-2) and AUEC.sub.(0-2)
for glucose and glucose change from baseline, respectively, were
calculated because the maximum change in insulin and glucose
appeared to occur during the first 2 hours following dosing. For
insulin no extrapolation was possible for the vast majority of
subjects therefore elimination half-life rate constant K.sub.el and
hence AUC.sub.(0-inf) could not be calculated. Since food was given
at 6 hours following dosing AUC, AURC and AUEC were calculated up
to 6 hours for insulin and glucose respectively which gave a more
accurate measure of the effect of Insulin/4-CNAB on insulin and
glucose concentrations. For glucose change from baseline E.sub.max
was taken as the maximum reduction up to 6 hours post-dose.
[0350] A number of subjects experienced hypoglycemia during
treatments and were given food (chocolate, fruit or orange juice)
in order to raise blood glucose levels. These subjects were
excluded from the concentration-time summary statistics and the
summary PK and PD parameters were presented separately from
subjects who did not require hypoglycemic rescue. For those
subjects who experienced hypoglycemia, additional parameters of
C.sup.b and t.sup.b values for insulin, R.sup.b and t.sup.b values
for glucose and E.sup.c and t.sup.c values for glucose change from
baseline were recorded that reflected the concentration and time
immediately prior to hypoglycemic rescue.
[0351] Following single oral placebo administration Subject 15
displayed a baseline glucose concentration of 1.2 mmol/L. This was
considered unreasonably low and the baseline value for this
treatment in this subject taken to be the same as the concentration
measured at the subsequent time point (5.2 mmol/L).
EXAMPLE 9
[0352] A total of 29 healthy male subjects were successfully
screened and enrolled onto the study. The 29 subjects were divided
into three treatment groups. The disposition of the subjects who
were enrolled in the study is given in FIG. 1 below.
16TABLE 16 Subject Disposition Group 1: N = 9 Subjects Group 2: N =
10 Subjects Group 3: N = 10 Subjects N = 7 received 400 mg 4- N = 8
received 10 Units SC N = 8 received 100 Units CNAB Insulin
Insulin/300 mg 4-CNAB N = 7 received 800 mg 4- N = 8 received 150
Units N = 8 received 150 Units CNAB Insulin/200 mg 4-CNAB
Insulin/450 mg 4-CNAB N = 7 received 1400 mg 4- N = 8 received 100
Units N = 8 received 150 Units CNAB Insulin/600 mg 4-CNAB
Insulin/100 mg 4-CNAB N = 7 received 2000 mg 4- N = 4 received Oral
Placebo N = 8 received 150 USP Insulin CNAB N = 2 received SC
Placebo N = 7 received Placebo* N = 8 received Placebo
[0353] Subject 27 (in Group 3) Did Not Receive Placebo
Treatment
[0354] The plasma concentration-time profiles for 4-CNAB were
evaluated in those subjects who received 4-CNAB or Insulin/4-CNAB
treatments. Insulin, C-peptide and glucose concentration-time
profiles following administration of all treatments were evaluated
for the 20 subjects in Groups 2 and 3. PK parameters for 4-CNAB,
insulin and C-peptide corrected insulin and PD parameters for
glucose concentration change from baseline, respectively were
calculated for subjects whether they required hypoglycemic rescue
or not. Concentrations from subjects who required food/drink due to
hypoglycemia were excluded from descriptive statistics. PK and PD
parameters were summarized separately for these subjects, except
following SC insulin where descriptive statistics were provided for
all 8 subjects who required intervention with food/drink due to
hypoglycemia.
[0355] Individual plasma concentrations of 4-CNAB after 4-CNAB
alone and Insulin/4-CNAB treatments are tabulated in Appendix
16.2.8.3 and individual 4-CNAB plasma concentration-time profiles
on linear and log-linear scales are included in Appendix 16.2.8.4.
Mean (+SD) plasma concentration-time profiles of 4-CNAB following
the administration of 4-CNAB or Insulin/4-CNAB capsules to healthy
male volunteers are shown below in FIGS. 2 and 3 respectively.
[0356] 4-CNAB Pharmacokinetics
[0357] The mean 4-CNAB concentration-time profiles following
escalating oral doses of 4-CNAB alone showed rapid absorption with
peak concentrations achieved at median times of approximately 0.62
h. After reaching the maximum concentration, 4-CNAB concentrations
rapidly declined in a biphasic manner. The maximum concentrations
C.sub.max and exposure (i.e. AUCs) clearly increased with
increasing dose. When the combination capsules of Insulin/4-CNAB
were administered, 4-CNAB maximum levels were reached at
approximately the same time as with 4-CNAB treatments alone and
4-CNAB peak concentrations clearly increased with increasing
amounts of 4-CNAB in combination with insulin. The combined
treatment of 100 Units Insulin/600 mg 4-CNAB which contained the
highest dose of 4-CNAB, resulted in the highest mean peak
concentration while the lowest mean peak concentration was observed
following the administration of lowest dose of 150 Units
Insulin/100 mg 4-CNAB.
[0358] Mean values.+-.SD, and ranges in parenthesis for t.sub.max,
for each treatment for 4-CNAB in plasma following 4-CNAB alone and
Insulin/4-CNAB treatments are given below together with descriptive
statistics.
[0359] Pharmacokinetic parameters of 4-CNAB following oral
administration of capsules of 4-CNAB alone are summarized in Table
17 below for (Group 1)
17 TABLE 17 4-CNAB Dose Parameter 400 mg 800 mg 1400 mg 2000 mg
C.sub.max (ng/mL) 22315 .+-. 11456 38011 .+-. 15804 103321 .+-.
14590 135199 .+-. 86565 t.sub.max (h) 0.50 (0.50-0.50) 0.50
(0.50-0.75) 0.75 (0.50-0.77) 0.75 (0.50-1.50) AUC.sub.(0-t) (ng
.multidot. h/mL) 22232 .+-. 6760 44343 .+-. 14478.sup.c 143620 .+-.
33809 204136 .+-. 102850 AUC.sub.(0-inf) 26708 .+-. 12209 49409
.+-. 15057.sup.b 153815 .+-. 38110 192013 .+-. 37147.sup.a (ng
.multidot. h/mL) K.sub.el (l/h) 0.11 .+-. 0.78 0.12 .+-. 0.04.sup.b
0.18 .+-. 0.14 0.06 .+-. 0.03.sup.a t.sub.1/2 (h) 12.0 .+-. 13.4
5.90 .+-. 1.55.sup.b 6.3 .+-. 4.1 15.3 .+-. 6.9.sup.a Cl/F (mL/min)
294 .+-. 122 290 .+-. 88.sup.b 160 .+-. 38 178 .+-. 32.sup.a Vd/F
(L) 220 .+-. 130 156 .+-. 82.sup.b 81 .+-. 50 245 .+-. 131.sup.a
MRT.sub.(0-t) (h) 1.60 .+-. 0.23 1.75 .+-. 0.22.sup.c 1.60 .+-.
0.20 1.73 .+-. 0.31 MRT.sub.(0-inf) (h) 7.30 .+-. 11.55 2.60 .+-.
0.53.sup.b 3.06 .+-. 1.71 7.45 .+-. 2.36.sup.a
[0360] Values are given as Mean.+-.standard deviation (except
t.sub.max, where median (range) is given) as N=7 except: .sup.a=3,
.sup.b=4 and .sup.c=5
[0361] The Pharmacokinetic parameters of 4-CNAB following the
administration of 4-CNAB in combination with insulin (Groups 2 and
3) are summarized in Table 18 below:
18 TABLE 18 Insulin (Units)/4-CNAB(mg) Parameter 100/300 100/450
100/600 150/100 150/200 C.sub.max (ng/mL) 8904 .+-. 6353 23183 .+-.
7933 35790 .+-. 12291 6195 .+-. 3605 10143 .+-. 5094 t.sub.max (h)
0.50 0.50 0.50 0.50 0.50 (0.27-0.75) (0.27-4.00) (0.25-0.77)
(0.25-1.00) (0.25-0.55) AUC.sub.(0-t) 8745 .+-. 5517 25988 .+-.
4408 36636 .+-. 5764 4675 .+-. 1076 10018 .+-. 1894 (ng .multidot.
h/Ml) AUC.sub.(0-inf) 9238 .+-. 5938 26831 .+-. 4564 37571 .+-.
5432 4918 .+-. 1100 10281 .+-. 2078 (ng .multidot. h/mL) K.sub.el
(l/h) 0.19 .+-. 0.12 0.1852 .+-. 0.06 0.19 .+-. 0.06 0.22 .+-. 0.14
0.25 .+-. 0.09 t.sub.1/2 (h) 5.3 .+-. 3.3 4.1 .+-. 1.2 4.1 .+-. 1.2
4.8 .+-. 3.4 3.2 .+-. 1.4 Cl/F (mL/min) 834 .+-. 527 287 .+-. 49
271 .+-. 38 352 .+-. 68 338 .+-. 75 Vz/F (L) 324 .+-. 239 101 .+-.
35 96 .+-. 35 148 .+-. 102 89 .+-. 35 MRT.sub.(0-t) (h) 1.51 .+-.
0.16 1.79 .+-. 0.50 1.45 .+-. 0.17 1.42 .+-. 0.56 1.59 .+-. 0.23
MRT.sub.(0-inf) (h) 2.43 .+-. 0.82 2.30 .+-. 0.46 1.90 .+-. 0.58
2.39 .+-. 1.39 1.97 .+-. 0.39
[0362] Values are given as Mean.+-.standard deviation (except
t.sub.max, where median (range) is given). Mean is of 8
Subjects.
[0363] As can be seen from the mean concentration-time profiles and
the resulting values of C.sub.max and AUC.sub.(0-inf) following
4-CNAB alone doses, levels (C.sub.max) of 4-CNAB and exposure
(AUCs) generally increased with 4-CNAB doses, as both C.sub.max and
AUC values increased in a dose-dependent manner for the 400 mg and
800 mg doses. In the 4-CNAB alone treatment groups, the C.sub.max
ranged between 22315.+-.11456 ng/mL and 135199.+-.86565 for doses
of 400 mg and 2000 mg, respectively. The time of maximum 4-CNAB
concentration was consistent across all doses with median values
ranging between 0.50-0.75 h. Mean elimination half-life values for
4-CNAB were variable and ranged between 5.90 and 15.3 h due to the
variability in the terminal elimnation phase and difficulty in
estimating the elimination rate constants. However, the MRT values
were more consistent and ranged from 1.4 to 1.8 hours.
[0364] The mean 4-CNAB concentration-time profiles and parameters
C.sub.max and AUC shown in Table 14 above for 4-CNAB following 100
Units Insulin/4-CNAB and 150 Units Insulin/4-CNAB combinations
indicate increasing 4-CNAB absorption with increasing 4-CNAB dose.
Generally, 4-CNAB absorption increased with increasing dose of
4-CNAB with the exception of the 100 Units Insulin/300 mg 4-CNAB
treatment. In the Insulin/4-CNAB combination group, the highest
values for C.sub.max occurred following 100 Units Insulin/600 mg
4-CNAB (35790.+-.12291 ng/mL) and the lowest following 150 Units
Insulin/100 mg 4-CNAB (6195.+-.3605 ng/mL). Mean values of
elimination half-life for 4-CNAB were less variable for the
combination capsules and ranged between 3.2 hours and 5.3 hours
between the dose group.
[0365] Insulin Pharmacokinetics
[0366] Individual plasma-concentrations of insulin following all
treatments were tabulated, and individual plasma insulin
concentration-time profiles were prepared. Mean (.+-.SD) plasma
insulin concentration-time profiles for non-hypoglycemic subjects
following the administration of treatments are shown in FIGS. 6A,
6B, 7A and 7B.
[0367] FIG. 6A and 6B show Mean (+SD) plasma insulin
concentration-time profile following the administration of 150
Units/200 mg (Insulin/4-CNAB) (n=5), 100 Units/600 mg (n=7), 10
Units SC insulin (n=8) and oral placebo (n=10) treatment in
non-hypoglycemic subjects (Group 2).
[0368] FIGS. 7A and 7B show Mean (+SD) plasma insulin
concentration-time profiles following the administration of 100
Units/300 mg (Insulin/4-CNAB) (n=7), 100 Units/450 mg (n=7), 150
Units/100 mg (n=8), 150 Units USP oral insulin (n=8) and oral
placebo (n=10) treatment in non-hypoglycemic subjects (Group
3).
[0369] Mean insulin concentrations in non-hypoglycemic subjects
reached peak levels between 0.4 and 0.6 hours following dosing
before declining steeply to return to baseline levels after
approximately 1 h. Increases in insulin absorption and exposure
were observed with increasing doses (when changing from the 150
Units Insulin/100 mg 4-CNAB treatment to the 150 Units Insulin/200
mg 4-CNAB treatment) as expected. Based on C.sub.max and AUC values
it was clear that the combined Insulin/4-CNAB treatments enabled
insulin absorption significantly compared to 150 Units oral insulin
alone and oral placebo. There was no insulin absorption when 150
Units insulin was dosed alone. Following 10 Units SC insulin, the
mean profile for insulin concentrations was erratic, with two peak
concentrations of approximately 300 pmol/L occuring at around 2 and
4 hours post-dose.
[0370] Individual PK parameters of insulin in plasma following all
treatments for hypoglycemic and non-hypoglycemic subjects are
listed in Tables 14.6.2-3 to 14.6.2-11 together with descriptive
statistics. Mean values.+-.SD of insulin PK parameters for all
subjects, non-hypoglycemic subjects and hypoglycemic subjects for
each treatment are given in the Tables 19 and 20 below.
19TABLE 19 Pharmacokinetic parameters of insulin following dosing
of subjects in Group 2 Insulin (Units)/4-CNAB (mg) Parameter
150/200 100/600 10 Units SC Oral Placebo.sup.c All Subjects N 8 8 8
11 C.sub.max.sup.a (pmol/L) 226.9 .+-. 174.8 159.3 .+-. 125.6 374.8
.+-. 141.7 90.7 .+-. 91.3 t.sub.max.sup.a (h) 0.42 (0.17-1.75) 0.33
(0.25-1.50) 1.88 (1.58-4.00) 0.50 (0.00-6.00) AUC.sub.(0-2) (pmol
.multidot. h/L) 180.68 .+-. 173.6 106.9 .+-. 101.6 414.3 .+-. 181.5
66.0 .+-. 22.5 AUC.sub.(0-6) (pmol .multidot. h/L) 366.8 .+-. 367.1
212.4 .+-. 187.0 1228.5 .+-. 378.1 222.9 .+-. 127.4
Non-hypoglycemic N 5 7 -- 10 Subjects C.sub.max.sup.a (pmol/L)
114.6 .+-. 75.7 120.1 .+-. 64.2 NA 77.0 .+-. 83.5 t.sub.max.sup.a
(h) 0.33 (0.17-0.45) 0.33 (0.25-0.42) NA 0.50 (0.00-6.00)
AUC.sub.(0-2) (pmol .multidot. h/L) 57.2 .+-. 16.5 72.5 .+-. 30.5
NA 66.4 .+-. 23.6 AUC.sub.(0-6) (pmol .multidot. h/L) 103.3 .+-.
30.9 149.7 .+-. 63.5 NA 202.9 .+-. 114.7 Hypoglycemic N 3 1 8 1
Subjects C.sub.max .sup.a(pmol/L) 414.0 .+-. 106.7 NA 374.8 .+-.
141.7 NA t.sub.max.sup.a (h) 0.58 (0.42-1.75) NA 1.88 (1.58-4.00)
NA AUC.sub.(0-2) (pmol .multidot. h/L) 386.6 .+-. 56.6 NA 414.3
.+-. 181.5 NA AUC.sub.(0-6) (pmol .multidot. h/L) 805.8 .+-. 84.6
NA 1228.5 .+-. 378.1 NA C.sup.b (pmol/L) 62.0 .+-. 25.5 NA 168.3
.+-. 77.5 NA t.sup.b (h) 1.00 (0.62-1.00) NA 1.00 (0.75-1.25)
NA
[0371] In Table 19: values are given as Mean.+-.SD (except
t.sub.max, where median (range) is given).
[0372] .sup.a Maximum concentration and corresponding time insulin
concentration up to 6 hr.
[0373] .sup.b Insulin concentration and corresponding time
immediately prior to recovery from hypoglycemia.
[0374] .sup.c Oral placebo values combined for Groups 2 and 3.
[0375] NA=not applicable; either 2 subjects or less.
20TABLE 20 Pharmacokinetic parameters of insulin following dosing
of subjects in Group 3 Insulin (Units)/4-CNAB (mg) 150 Oral Insulin
Units Oral Parameter 100/300 100/450 150/100 Alone Placebo.sup.b
All Subjects N 8 8 8 8 11 C.sub.max.sup.a 91.0 .+-. 63.4 152.5 .+-.
123.5 95.6 .+-. 43.1 38.4 .+-. 12.8 90.7 .+-. 91.3 (pmol/L)
t.sub.max.sup.a (h) 0.29 (0.08-1.53) 0.38 (0.25-3.02) 0.25
(0.18-0.28) 0.43 (0.17-3.00) 0.50 (0.00-6.00) AUC.sub.(0-2) 80.5
.+-. 55.6 108.5 .+-. 91.9 69.1 .+-. 20.5 51.8 .+-. 15.5 66.0 .+-.
22.5 (pmol .multidot. h/L) AUC.sub.(0-6) 182.0 .+-. 73.3 279.2 .+-.
309.0 163.8 .+-. 56.9 135.2 .+-. 47.1 222.9 .+-. 127.4 (pmol
.multidot. h/L) Non- N 7 7 8 8 10 Hypoglycemic C.sub.max.sup.a 69.7
.+-. 21.4 112.4 .+-. 53.1 95.6 .+-. 43.1 38.4 .+-. 12.8 77.0 .+-.
83.5 Subjects (pmol/L) t.sub.max.sup.a (h) 0.25 (0.08-0.42) 0.33
(0.25-0.42) 0.25 (0.18-0.28) 0.43 (0.17-3.00) 0.50 (0.00-6.00)
AUC.sub.(0-2) 61.3 .+-. 12.6 78.3 .+-. 36.2 69.1 .+-. 20.5 51.8
.+-. 15.5 66.4 .+-. 23.6 (pmol .multidot. h/L) AUC.sub.(0-6) 167.3
.+-. 62.3 173.4 .+-. 83.1 163.8 .+-. 56.9 135.2 .+-. 47.1.sup.c
202.9 .+-. 114.7 (pmol .multidot. h/L)
[0376] In Table 20: values are given as Mean.+-.SD (except
t.sub.max, where median (range) is given).
[0377] .sup.a Maximum concentration and corresponding time insulin
concentration up to 6 hr.
[0378] .sup.b Oral placebo values combined for Groups 2 and 3.
[0379] .sup.c Value corresponds to AUC.sub.(0-t) for 6 h sampling
schedule.
[0380] NA=not applicable; either 2 subjects or less.
[0381] Mean C.sub.max insulin values in non-hypoglycemic subjects
following 100 Units Insulin/300 mg 4-CNAB, 100 Units Insulin/450 mg
4-CNAB and 100 Units Insulin/600 mg 4-CNAB were 69.9.+-.21.4
pmol/L, 112.4.+-.53.1 pmol/L and 120.1.+-.64.2 pmol/L,
respectively. The times of peak insulin concentrations ranged
between approximately 0.1 and 0.4 h for all Insulin/4-CNAB combined
treatments. The insulin concentration was lowest following 150 USP
Units oral insulin alone indicating no absorption of insulin when
oral administration of insulin alone was administered.
[0382] Mean C.sub.max insulin values for all subjects
(non-hypoglycemic and hypoglycemic subjects) were highly variable.
Following 100 Units Insulin/300 mg 4-CNAB, 100 Units Insulin/450 mg
4-CNAB and 100 Units Insulin/600 mg 4-CNAB mean C.sub.max values
were 91.0.+-.63.4 pmol/L, 152.5.+-.123.5 pmol/L, and 159.3.+-.125.6
pmol/L, respectively. The median times of peak insulin
concentrations ranged between approximately 0.25 and 0.4 h for all
Insulin/4-CNAB combined treatments.
[0383] Pharmacokinetic parameters of C-peptide corrected insulin
following dosing of subjects in Group 2 are summarized below in
Table 21.
21 TABLE 21 Insulin (Units)/4-CNAB (mg) Parameter 150/200 100/600
10 Units SC Oral Placebo.sup.c All Subjects N 8 8 8 11
C.sub.max.sup.a (pmol/L) 186.9 .+-. 137.2 118.7 .+-. 87.7 304.5
.+-. 130.9 56.3 .+-. 90.4 t.sub.max.sup.a (h) 0.42 0.33 1.88 0.30
(0.17-1.75) (0.33-1.50) (1.50-4.00) (0.00-6.00) AUC.sub.(0-2) (pmol
.multidot. h/L) 116.1 .+-. 126.5 50.0 .+-. 60.15 355.2 .+-. 170.2
7.4 .+-. 25.0 AUC.sub.(0-6) (pmol .multidot. h/L) 146.9 .+-. 193.0
47.4 .+-. 75.3 942.8 .+-. 333.0 53.5 .+-. 132.5 Non- N 5 7 -- 10
hypoglycemic C.sub.max.sup.a (pmol/L) 97.5 .+-. 75.8 95.0 .+-. 61.0
NA 46.3 .+-. 88.7 Subjects t.sub.max.sup.a (h) 0.33 0.33 NA 0.30
(0.17-0.45) (0.25-0.42) (0.00-6.00) AUC.sub.(0-2) (pmol .multidot.
h/L) 25.9 .+-. 20.0 29.4 .+-. 15.5 NA 5.44 .+-. 25.4 AUC.sub.(0-6)
(pmol .multidot. h/L) 11.1 .+-. 39.2 21.8 .+-. 22.1 NA 37.1 .+-.
127.3 Hypoglycemic N 3 1 8 1 Subjects C.sub.max.sup.a (pmol/L)
335.8 .+-. 34.16 NA 304.5 .+-. 130.9 NA t.sub.max.sup.a (h) 0.58 NA
1.88 NA (0.42-1.75) (1.50-4.00) AUC.sub.(0-2) (pmol .multidot. h/L)
266.5 .+-. 31.0 NA 355.2 .+-. 170.2 NA AUC.sub.(0-6) (pmol
.multidot. h/L) 373.4 .+-. 65.2 NA 942.8 .+-. 333.0 NA C.sup.b
(pmol/L) 44.2 .+-. 31.25 NA 159.5 .+-. 75.6 NA t.sup.b (h) 1.00 NA
1.00 NA (0.62-1.00) (0.75-1.25)
[0384] In Table 21, values are given as Mean.+-.SD (except
t.sub.max, where median (range) is given) and
[0385] .sup.a Maximum concentration and corresponding time
C-peptide corrected insulin concentration up to 6 hr.
[0386] .sup.b C-peptide corrected Insulin concentration and
corresponding time immediately prior to recovery from
hypoglycemia.
[0387] .sup.c Oral placebo values combined for Groups 2 and 3.
[0388] NA-not applicable; either 2 subjects or less.
[0389] Pharmacokinetic parameters of C-peptide corrected insulin
following dosing of subjects in Group 3 are summarized below in
Table 22:
22 TABLE 22 Insulin (Units)/4-CNAB (mg) 150 Oral Insulin Parameter
100/300 100/450 150/100 Units Alone Oral Placebo.sup.b All Subjects
N 8 8 8 7 11 C.sub.max.sup.a 63.8 .+-. 61.1 109.4 .+-. 83.2 66.6
.+-. 41.7 16.0 .+-. 7.5 56.3 .+-. 90.4 (pmol/L) t.sub.max.sup.a (h)
0.42 0.38 0.25 2.00 0.30 (0.17-1.53) (0.25-3.02) (0.18-6.02)
(0.17-2.50) (0.00-6.00) AUC.sub.(0-2) 32.9 .+-. 57.4 47.7 .+-. 64.8
15.2 .+-. 16.2 10.8 .+-. 16.6 7.4 .+-. 25.0 (pmol .multidot. h/L)
AUC.sub.(0-6) 49.7 .+-. 92.2 81.5 .+-. 185.6 10.9 .+-. 35.2 21.5
.+-. 42.0 53.5 .+-. 132.5 (pmol .multidot. h/L) Non- N 7 7 8 7 10
Hypoglycemic C.sub.max.sup.a 45.1 .+-. 33.5 84.1 .+-. 46.1 66.6
.+-. 41.7 16.0 .+-. 7.5 46.3 .+-. 88.7 Subjects (pmol/L)
t.sub.max.sup.a (h) 0.42 0.33 0.25 2.00 0.30 (0.17-0.50)
(0.25-0.50) (0.18-6.02) (0.17-2.50) (0.00-6.00) AUC.sub.(0-2) 15.4
.+-. 31.4 25.7 .+-. 19.8 15.2 .+-. 16.2 10.8 .+-. 16.6 5.44 .+-.
25.4 (pmol .multidot. h/L) AUC.sub.(0-6) 37.4 .+-. 92.2 16.8 .+-.
33.0 10.9 .+-. 35.2 21.5 .+-. 42.0 37.1 .+-. 127.3 (pmol .multidot.
h/L)
[0390] In Table 22, values are given as Mean.+-.SD (except
t.sub.max, where median (range) is given) and:
[0391] .sup.a Maximum concentration and corresponding time insulin
concentration up to 6 hr.
[0392] .sup.b Oral placebo values combined for Groups 2 and 3.
[0393] Mean C.sub.max values of C-peptide corrected insulin for all
subjects achieved following 150 Units/200 mg and 100 Units/600 mg
treatments were 186.9.+-.137.2 pmol/L and 118.7.+-.87.7 pmol/L,
respectively. For non-hypoglycemic subjects following treatments
containing 100 Units/300 mg, 100 Units/450 mg and 100/600 mg there
appeared to be a dose-dependent relationship in terms of C-peptide
corrected insulin exposure based on C.sub.max and AUC.sub.0-2
values with increasing doses of 4-CNAB. An increase in insulin
exposure was also observed when changing from the 150 Units
Insulin/100 mg 4-CNAB treatment to the 150 Units Insulin/200 mg
4-CNAB treatment.
[0394] Following administration of 10 Units of SC insulin, all 8
subjects required hypoglycemic recovery with food/drink and hence
it was impossible to calculate relative bioavailability and potency
for the combined oral Insulin/4-CNAB treatments. Typically, the
median times of peak concentrations were around 0.3-0.4 h for all
treatments except for 150 USP oral insulin alone and for SC
insulin, which had median t.sub.max times of approximately 2.00 h.
From these data, it was clear that little of the 150 USP oral
insulin was absorbed. The lack of insulin absorbed following 150
USP oral insulin alone compared to the Insulin/4-CNAB treatments
indicates the effectiveness of the oral delivery agent 4-CNAB on
oral absorption of human insulin.
[0395] For the Insulin/4-CNAB combined treatments containing 100
Units insulin, there appeared to be a dose-dependent relationship
in terms of C-peptide corrected insulin absorption and exposure
based on C.sub.max and AUC.sub.(0-2) values with increasing doses
of 4-CNAB. Increases in insulin absorption and exposure were also
observed for the 150 Units insulin doses (when changing from the
150 Units Insulin/100 mg 4-CNAB treatment to the 150 Units
Insulin/200 mg 4-CNAB treatment) as expected. Based on C.sub.max
and AUC values it was clear that the combined Insulin/4-CNAB
treatments enabled significant insulin absorption as compared to
150 Units oral insulin alone and oral placebo.
[0396] Based upon the above data, the following pharmacokinetic
conclusions can be drawn:
[0397] The exposure to 4-CNAB increased with increasing doses of
4-CNAB alone.
[0398] The exposure to 4-CNAB appeared to increase with increasing
doses of 4-CNAB in the combined Insulin/4-CNAB treatments with the
exception of 100 Units Insulin/300mg 4-CNAB treatment.
[0399] The exposure to C-peptide corrected insulin increased with
increasing proportions of 4-CNAB as part of the treatment.
[0400] There was no absorption of insulin following oral
administration of 150 Units oral insulin alone.
[0401] Oral absorption of insulin was greatest following
administration of the combined treatments containing 150 Units
Insulin/200 mg 4-CNAB and 100 Units Insulin/600 mg 4-CNAB.
[0402] Pharmacodynamic Results
[0403] Individual C-peptide plasma concentrations following
treatments were tabulated and presented graphically. Mean (+SD)
plasma C-peptide concentration-time profiles for non-hypoglycemic
subjects following the administration of the various treatments to
healthy male volunteers are shown in FIGS. 8A, 8B, 9A and 9B.
[0404] FIGS. 8A and 8B show the mean (+SD) glucose
concentration-time profiles following the administration of 150
Units/200 mg (Insulin/4-CNAB) (n=5), 100 Units/600 mg (n=7), 10
Units SC insulin (n=8) and oral placebo (n=10) treatment in
non-hypoglycemic subjects (Group 2). FIGS. 9A and 9B show the Mean
(+SD) glucose concentration-time profiles following the
administration of 100 Units/300 mg (Insulin /4-CNAB) (n=7), 100
Units/450 mg (n=7), 150 Units/100 mg (n=8), 150 Units USP oral
insulin (n=8) and oral placebo (n=10) treatment in non-hypoglycemic
subjects (Group 3).
[0405] Mean C-peptide concentrations declined almost immediately
following dosing of the combined Insulin/4-CNAB treatments
achieving the greatest decline between 0.5 and 1.0 hours.
Concentrations then appeared to return to baseline levels after
approximately 2 hours following dosing. Following oral placebo and
150 Units oral insulin alone there was little change in C-peptide
concentrations from baseline. The mean profile following 10 Units
SC insulin is for subjects who experienced hypoglycemia (8 out of 8
subjects) and were given food/drink in order to increase their
blood glucose levels. C-peptide is a byproduct of endogenous
insulin excretion. After giving oral insulin, the oral insulin
surpresses the production of endogenous insulin, and it leads to
deline of c-peptide production. The decrease of the c-peptide
levels after oral administration of insulin/4-CNAB, and litter
change of c-peptide levels after insulin alone or placebo dosing
clearly demonstrated effective absorption of human insulin in this
study.
[0406] Mean glucose concentrations began to decline after
approximately 0.2 h following dosing of the combined Insulin/4-CNAB
treatments, Rmax of the combined Insulin/4-CNAB treatments was
observed to be between 0.5 and 1.0 h following dosing.
Concentrations then appeared to return to baseline levels after
approximately 2 hours following dosing. Following oral placebo and
150 Units oral insulin alone there was only a slight change in
glucose concentrations indicating little absorption of insulin. The
mean profile following 10 SC Units of insulin is for subjects who
experienced hypoglycemia (8 out of 8 subjects) and were given
food/drink in order to increase their blood glucose levels.
[0407] Individual PD parameters of glucose following all treatments
for hypoglycemic and non-hypoglycemic subjects were listed together
with descriptive statistics. Mean values .+-.SD of glucose PD
parameters for all subjects, non-hypoglycemic subjects and
hypoglycemic subjects for each treatment are given in Tables 23 and
24 below.
23TABLE 23 Pharmacodynamic parameters of plasma glucose for
subjects in Group 2 Insulin (Units)/4-CNAB (mg) Parameter 150/200
100/600 10 Units SC Oral Placebo.sup.c All Subjects N 8 8 8 11
R.sub.max.sup.a (mmol/L) 3.95 .+-. 0.80 4.65 .+-. 0.30 3.31 .+-.
0.42 5.03 .+-. 0.23 t.sub.Rmax.sup.a (h) 0.63 0.58 1.00 6.0
(0.58-3.00) (0.50-6.00) (0.67-3.00) (0.33-6.00) AURC.sub.(0-2)
(mmol .multidot. h/L) 11.2 .+-. 1.8 10.85 .+-. 0.68 10.40 .+-. 1.36
11.10 .+-. 0.72 AURC.sub.(0-6) (mmol .multidot. h/L) 33.3 .+-. 2.6
31.88 .+-. 1.23 32.02 .+-. 4.66 32.55 .+-. 2.41 Non- N 5 7 None 10
hypoglycemic R.sub.max.sup.a (mmol/L) 4.40 .+-. 0.45 4.66 .+-. 0.32
NA 5.02 .+-. 0.24 Subjects t.sub.Rmax.sup.a (h) 0.58 0.58 NA 6.0
(0.58-3.00) (0.50-6.00) (0.33-6.00) AURC.sub.(0-2) (mmol .multidot.
h/L) 10.42 .+-. 2.19 10.66 .+-. 0.43 NA 10.95 .+-. 0.55
AURC.sub.(0-6) (mmol .multidot. h/L) 31.60 .+-. 0.87 31.78 .+-.
1.28 NA 31.94 .+-. 1.40 Hypoglycemic N 3 1 8 1 Subjects
R.sub.max.sup.a (mmol/L) 3.20 .+-. 0.69 NA 3.31 .+-. 0.42 NA
t.sub.Rmax.sup.a (h) 1.00 NA 1.00 NA (0.58-1.00) (0.67-3.00)
AURC.sub.(0-2) (mmol .multidot. h/L) 12.47 .+-. 2.81 NA 10.40 .+-.
1.36 NA AURC.sub.(0-6) (mmol .multidot. h/L) 36.03 .+-. 1.88 NA
32.02 .+-. 4.66 NA R.sup.b (mmol/L) 3.23 .+-. 0.75 NA 3.45 .+-.
0.41 NA t.sup.b (h) 1.00 NA 1.00 NA (0.62-1.00) (0.75-1.25)
[0408] In Table 23, values are given as Mean.+-.SD (except
t.sub.max, where median (range) is given) and
[0409] .sup.a Minimum concentration and corresponding time in blood
glucose concentration up to 6 hr post-dose.
[0410] .sup.b Glucose concentration and corresponding time
immediately prior to recovery from hypoglycemia.
[0411] .sup.c Oral placebo values combined for Groups 2 and 3.
[0412] NA--not applicable; either 2 subjects or less.
24TABLE 24 Pharmacodynamic parameters for subjects from Group 3
Insulin (Units)/4-CNAB (mg) 150 Oral Insulin Parameter 100/300
100/450 150/100 Units Alone Oral Placebo.sup.b All Subjects N 8 8 8
8 11 R.sub.max.sup.a 4.93 .+-. 0.55 4.58 .+-. 0.49 5.01 .+-. 0.20
5.08 .+-. 0.20 5.03 .+-. 0.23 (mmol/L) t.sub.Rmax.sup.a (h) 0.63
0.67 0.71 6.00 6.0 (0.08-6.00) (0.50-6.00) (0.50-6.00) (0.62-6.00)
(0.33-6.00) AURC.sub.(0-2) 11.10 .+-. 0.66 10.77 .+-. 0.81 11.02
.+-. 0.47 11.06 .+-. 0.69 11.10 .+-. 0.72 (mmol .multidot. h/L)
AURC.sub.(0-6) 32.6 .+-. 1.75 32.34 .+-. 2.90 32.39 .+-. 0.92 32.15
.+-. 1.41 32.55 .+-. 2.41 (mmol .multidot. h/L) Non- N 7 7 8 8 10
Hypoglycemic R.sub.max.sup.a 5.07 .+-. 0.39 4.71 .+-. 0.32 5.01
.+-. 0.20 5.08 .+-. 0.20 5.02 .+-. 0.24 Subjects (mmol/L)
t.sub.Rmax.sup.a (h) 0.67 0.67 0.71 6.00 6.0 (0.08-6.00)
(0.50-6.00) (0.50-6.00) (0.62-6.00) (0.33-6.00) AURC.sub.(0-2)
11.19 .+-. 0.69 10.57 .+-. 0.64 11.02 .+-. 0.47 11.06 .+-. 0.69
10.95 .+-. 0.55 (mmol .multidot. h/L) AURC.sub.(0-6) 32.91 .+-.
1.50 31.47 .+-. 1.61 32.39 .+-. 0.92 32.15 .+-. 1.41.sup.c 31.94
.+-. 1.40 (mmol .multidot. h/L)
[0413] In Table 23, values are given as Mean.+-.SD (except
t.sub.max, where median (range) is given) and
[0414] .sup.a Minimum concentration and corresponding time in
plasma insulin concentration up to 6 hr post-dose.
[0415] .sup.b Oral placebo values combined for Groups 2 and 3.
[0416] .sup.c Value corresponds to AURC.sub.(0-t) for 6 h sampling
schedule.
[0417] Individual plasma glucose concentration changes from
baseline were tabulated, and individual glucose concentration
change from baseline-time profiles were prepared. Mean (+SD)
glucose concentration change from baseline-time profiles for
non-hypoglycemic subjects were also prepared.
[0418] Following the combined Insulin/4-CNAB treatments, plasma
glucose concentrations declined rapidly until approximately 0.75 h,
then gradually increased to return to baseline levels after about
2.00 hour post-dose. From the profiles above, the maximum glucose
concentration change from baseline for the Insulin/4-CNAB
combination occurred following 150 Units Insulin/200 mg 4-CNAB. The
treatment that had the next greatest effect on glucose appeared to
be the 100 Units/600 mg 4-CNAB treatment. These findings correlated
well with the peak concentrations of C-peptide corrected insulin
levels achieved following these two treatments (See Table 14).
Subjects who received 10 Units of SC insulin experienced the
greatest decline in glucose concentrations, which hence lead to
hypoglycemic response and recovery, by intervention with food/drink
intake.
[0419] Individual PD parameters for glucose change from baseline
following all treatments for hypoglycemic and non-hypoglycemic
subjects were prepared together with descriptive statistics. Mean
values .+-.SD of glucose change from baseline PK parameters for all
subjects, non-hypoglycemic subjects and hypoglycemic subjects for
each treatment are given in the Tables 25, 26 and 27 below. In
addition, the mean maximum percentage change from baseline up to 6
hours post-dose is also given in these tables.
25TABLE 25 Pharmacodynamic parameters for the change in plasma
glucose from the baseline for subjects in Group 2 Insulin
(Units)/4-CNAB (mg) Parameter 150/200 100/600 10 Units SC Oral
Placebo.sup.c All Subjects N 8 8 8 11 E.sub.max.sup.a (mmol/L)
-1.65 .+-. 1.08 -0.88 .+-. 0.48 -2.28 .+-. 0.40 -0.54 .+-. 0.21 %
Change.sup.b -28.64 .+-. 17.80 -15.92 .+-. 7.30 -39.82 .+-. 8.26
-8.05 .+-. 6.23 t.sub.Emax.sup.a (h) 0.68 0.58 1.00 6.00
(0.58-3.00) (0.50-0.83) (0.75-3.00) (0.33-6.00) AUEC.sub.(0-2)
(mmol .multidot. h/L) -0.01 .+-. 1.59 -0.25 .+-. 0.86 -0.71 .+-.
1.32 0.05 .+-. 0.52 AUEC.sub.(0-6) (mmol .multidot. h/L) -0.35 .+-.
1.72 -2.11 .+-. 5.24 -1.40 .+-. 4.41 -0.65 .+-. 2.07 Non- N 5 7 --
10 hypoglycemic E.sub.max.sup.a (mmol/L) -1.02 .+-. 0.70 -0.89 .+-.
0.51 NA -0.54 .+-. 0.22 Subjects % Change.sup.b -18.34 .+-. 11.85
-16.08 .+-. 7.87 NA -9.49 .+-. 3.67 t.sub.Emax.sup.a (h) 0.67 0.58
NA 6.00 (0.58-3.00) (0.50-0.83) (0.33-6.00) AUEC.sub.(0-2) (mmol
.multidot. h/L) -0.42 .+-. 0.63 -0.49 .+-. 0.59 NA -0.09 .+-. 0.27
AUEC.sub.(0-6) (mmol .multidot. h/L) -0.94 .+-. 1.37 -1.66 .+-.
1.74 NA -1.22 .+-. 0.93 Hypoglycemic N 3 1 8 -- Subjects
E.sub.max.sup.a (mmol/L) -2.70 .+-. 0.66 NA -2.28 .+-. 0.40 NA %
Change.sup.b -45.80 .+-. 11.02 NA -39.82 .+-. 8.26 NA
t.sub.Emax.sup.a (h) 1.00 NA 1.00 NA (0.62-1.00) (0.75-3.00)
AUEC.sub.(0-2) (mmol .multidot. h/L) -2.70 .+-. 0.66 NA NA NA
AUEC.sub.(0-6) (mmol .multidot. h/L) 1.00 NA 1.00 NA 0.62-1.00
(0.75-1.25) E.sup.c (mmol/L) 0.67 .+-. 2.64 NA -0.71 .+-. 1.32 NA
t.sup.c (h) 0.63 .+-. 2.06 NA -1.40 .+-. 4.41 NA
[0420] In Table 25, values are given as Mean.+-.SD (except
t.sub.max, where median (range) is given) and:
[0421] .sup.a Maximum change from baseline and corresponding time
up to 6 h=(Glucose Concentration-Baseline Concentration).
[0422] .sup.b Maximum % change from baseline up to 6 h=(Glucose
Concentration-Baseline Concentration/Baseline
Concentration*100).
[0423] .sup.c Glucose concentration change from baseline and
corresponding time immediately prior to recovery from
hypoglycemia.
[0424] .sup.d Oral placebo values combined for Groups 2 and 3.
[0425] NA--not applicable; either 2 subjects or less.
26TABLE 26 Pharmacodynamic parameters for the change in glucose
concentration change from baseline for subjects in Group 3 Insulin
(Units)/4-CNAB (mg) 150 Oral Insulin Parameter 100/300 100/450
150/100 Units Alone Oral Placebo.sup.b All Subjects N 8 8 8 7 11
E.sub.max.sup.a -0.75 .+-. 0.50 -0.94 .+-. 0.46 -0.66 .+-. 0.21
-0.79 .+-. 0.43 -0.54 .+-. 0.21 (mmol/L) % Change.sup.b -14.19 .+-.
9.98 -16.98 .+-. 8.28 -11.62 .+-. 3.28 -12.41 .+-. 6.48 -8.05 .+-.
6.23 t.sub.Emax.sup.a (h) 0.69 0.67 3.42 6.00 6.00 (0.08-6.02)
(0.50-6.00) (0.50-6.02) (0.67-6.00) (0.33-6.00) AUEC.sub.(0-2)
-0.26 .+-. 0.41 -0.26 .+-. 0.63 -0.29 .+-. 0.34 -0.63 .+-. 0.77
0.05 .+-. 0.52 (mmol .multidot. h/L) AUEC.sub.(0-6) -1.50 .+-. 1.41
-0.73 .+-. 2.67 -1.59 .+-. 1.16 -3.01 .+-. 2.44.sup.d -0.65 .+-.
2.07 (mmol .multidot. h/L)
[0426]
27TABLE 27 Pharmacodynamic parameters for the change in glucose
concentration from baseline for subjects in Group 3 Insulin
(Units)/4-CNAB (mg) 150 Oral Insulin Oral Parameter 100/300 100/450
150/100 Units Alone Placebo.sup.b Non- N 7 7 8 7 10 Hypoglycemic
E.sub.max.sup.a -0.26 .+-. 0.96 -0.80 .+-. 0.27 -0.66 .+-. 0.21
-0.79 .+-. 0.43 -0.54 .+-. 0.22 Subjects (mmol/L) % Change.sup.b
-12.64 .+-. 9.70 -14.47 .+-. 4.60 -11.62 .+-. 3.28 -12.41 .+-. 6.48
-9.49 .+-. 3.67 t.sub.Emax.sup.a (h) 4.00 0.67 3.42 6.00 6.00
(0.10-6.00) (0.50-6.00) (0.50-6.02) (0.67-6.00) (0.33-6.00)
AUEC.sub.(0-2) -0.31 .+-. 0.41 -0.46 .+-. 0.32 -0.29 .+-. 0.34
-0.63 .+-. 0.77 -0.09 .+-. 0.27 (mmol .multidot. h/L)
AUEC.sub.(0-6) -1.54 .+-. 1.51 -1.61 .+-. 0.98 -1.59 .+-. 1.16
-3.01 .+-. 2.44.sup.d -1.22 .+-. 0.93 (mmol .multidot. h/L)
[0427] In Tables 26 and 27:
[0428] .sup.a Maximum change from baseline and corresponding time
up to 6 h=(Glucose Concentration-Baseline Concentration).
[0429] .sup.b Maximum % change from baseline up to 6 h=(Glucose
Concentration-Baseline Concentration/Baseline Conc).
[0430] .sup.c Oral placebo values combined for Groups 2 and 3.
[0431] .sup.d Value corresponds to AUEC.sub.(0-t) for 6 h sampling
schedule.
[0432] The greatest change from baseline of glucose based on
E.sub.max was produced by the 150 Units/200 mg followed by the 100
Units/600 mg and 100 Units/450 mg treatments giving values of
-1.0.+-.0.7 mmol/L, -0.9.+-.0.5 mmol/L and -0.8.+-.0.3 mmol/L,
respectively in non-hypoglycemic subjects. The effect of insulin on
maximum glucose change from baseline appeared to increase with
increasing doses of 4-CNAB and plasma concentrations of C-peptide
corrected insulin, ranging between -0.3.+-.1.0 mmol/L and
-0.9.+-.0.5 mmol/L for 100 Units Insulin/300 mg 4-CNAB and 100
Units Insulin/600 mg 4-CNAB treatments, respectively. In general
there was a good correlation between increasing Insulin/4-CNAB
doses and C-peptide corrected plasma insulin concentrations and
maximum plasma glucose percent reduction from baseline.
[0433] The greatest percent reduction in plasma glucose
concentration was achieved after oral dosing of the 150 Units/200
mg treatment followed by the 100 Units/450 mg and 100 Units/600 mg
treatments giving maximum percent reduction values of
-28.64.+-.17.80%, -16.98.+-.8.28% and -15.92.+-.7.30%,
respectively. For these treatments, similar AUEC.sub.(0-2) values
were observed, all approximately -0.4 mmol.h/L as well as similar
t.sub.Emax median times of approximately 0.6 h. Maximum % reduction
in glucose concentrations from baseline increased with increasing
doses of 4-CNAB, with values ranging between -12.6 and -16.1% for
the 100 Units Insulin/300 mg 4-CNAB and 100 Units Insulin/600 mg
4-CNAB treatments, respectively.
[0434] A similar good correlation was observed between maximum
glucose percent change (reduction from baseline) and Insulin/4-CNAB
doses and C-peptide corrected insulin concentrations. The greatest
percent change (decline) in glucose levels was produced in subjects
receiving 10 Units SC insulin (-39.82.+-.8.26%),
(E.sub.max:-2.28.+-.0.5 mmol/L) which led to the need for
hypoglycemic recovery, and in subjects receiving oral dose of 150
Units/200 mg (-28.64.+-.17.80%).
[0435] Individual plasma glucose concentration percent changes from
baseline were tabulated, and individual glucose concentration
percent change from baseline-time profiles were created. Mean (+SD)
glucose concentration percent change from baseline-time profiles
for non-hypoglycemic subjects following the administration of
treatments to healthy male volunteers were created, except for SC
insulin where mean profile is for hypoglycemic subjects.
[0436] Based upon the above data, the following pharmacodynamic
conclusions can be drawn:
[0437] The effect of insulin on the mean maximum glucose
concentration change from baseline (Emax) appeared to increase with
increasing doses of 4-CNAB as part of the combined treatments.
[0438] In general an increasing effect on glucose concentration
change from baseline and glucose concentration percent change from
baseline were observed with increasing Insulin/4-CNAB doses and
C-peptide corrected insulin concentrations.
[0439] Based on the mean maximum glucose concentration change from
baseline (Emax), 100 Units Insulin/600 mg 4-CNAB and 150 Units
Insulin/200 mg 4-CNAB appeared to elicit a greater PD response
compared to oral placebo and 150 Units oral insulin alone,
indicating the effectiveness of the deliver agent in delivering
insulin.
[0440] The effect of any of the oral Insulin/4-CNAB combinations in
lowering glucose levels was less than that observed for SC
insulin.
[0441] Discussion and Overall Conclusions
[0442] The primary study objective of this single center,
double-blind, randomized, placebo-controlled study of escalating
single oral doses of Insulin/4-CNAB capsules in healthy male
volunteers (discussed above in Examples 7-9) was to evaluate the
safety and tolerability of single oral doses of the oral delivery
agent 4-CNAB alone (4-CNAB capsules) and in combination with
insulin (Insulin/4-CNAB capsules) in healthy subjects. The
secondary objectives were to assess the PK of 4-CNAB when given
alone and when given as part of the Insulin/4-CNAB combination, to
assess the PK of insulin and the effect of increasing proportions
of 4-CNAB on insulin PK and to assess the effects on blood glucose
following single oral doses of 4-CNAB alone or Insulin/4-CNAB.
Control treatments of 10 Units SC insulin, 150 USP Units oral
insulin alone, oral placebo and SC placebo allowed the
tolerability, PK and effects of 4-CNAB and Insulin/4-CNAB to be
evaluated effectively.
[0443] There were no deaths or SAEs in this study. Twenty-nine
subjects passed screening and completed the study. There were a
total of 42 AEs following treatments that were thought to be
related to study drug. The safety profiles following 4-CNAB alone
were very good with very few AEs. Most treatment-related AEs were
reported following 150 Units Insulin/200mg 4-CNAB (n=7) and after
100 Units Insulin/450 mg 4-CNAB (n=6) in addition to 16 events
following 10 Units SC insulin. Forty-one of the total treatment
related 42 AEs were classified as mild in severity. The most common
treatment-emergent AEs reported during the study were hypoglycemia
(26), headache (12) and dizziness (5). During the 26 episodes of
hypoglycemia, subjects required rescue treatment on twenty
occasions, i.e. food/drink such as a chocolate bar, banana or apple
juice, in order to raise their blood glucose. Of these, 12 were
following 10 Units SC insulin and 3 were following 150 Units
Insulin/200 mg 4-CNAB.
[0444] Mean 4-CNAB peak plasma concentrations and AUC appeared to
increase with increasing doses of 4-CNAB either when given alone or
as part of the Insulin/4-CNAB treatments, with the exception of the
100 Units Insulin/300 mg 4-CNAB treatment. The median time of
maximum 4-CNAB concentration was similar (around 0.6 h) for 4-CNAB
alone and when given as the combined Insulin/4-CNAB treatment. The
half-life of 4-CNAB was highly variable when given alone but more
consistent with a half-life value of around 4 h when given with
insulin. The variability in the half-life was due to the variable
in the terminal elimnation phase and difficulty in estimating the
elimination rate constants. However, the MRT values were more
consistent and ranged from 1.4 to 1.8 hours for all treatments
[0445] The oral absorption and exposure of insulin based on
C.sub.max and AUC.sub.(0-2) increased with increasing doses of
4-CNAB, indicating effective absorption of insulin with increasing
levels of 4-CNAB. The absorption and exposure to insulin following
Insulin/4-CNAB treatments was clearly greater than when given 150
USP oral insulin alone. Mean C-peptide corrected insulin Cmax
ranged between 45.1.+-.33.5 pmol/L, 95.0.+-.61.0 pmol/L, and
97.5.+-.75.8 pmol/L for doses of 100 Units Insulin/300 mg 4-CNAB,
100 Units Insulin/600 mg 4-CNAB, and 150 Units Insulin/200 mg
4-CNAB respectively. Unfortunately, because all 8 subjects required
rescue with food after 10 Units SC insulin due to hypoglycemia, it
was difficult to obtain an accurate measure of insulin relative
bioavailability of the Insulin/4-CNAB treatments compared to SC
dosing.
[0446] Increases in mean E.sub.max and AUEC.sub.(0-2) were seen
with increasing levels of 4-CNAB as part of Insulin/4-CNAB
treatments and with increasing plasma concentrations of C-peptide
corrected insulin.Mean maximum glucose % reduction from baseline
ranged between -12.64.+-.9.7% and -14.47.+-.4.6% for the 100 Units
Insulin/300 mg 4-CNAB and 100 Units Insulin/600 mg 4-CNAB
treatments, respectively. The values for AUEC(0-2) ranged between
-0.31.+-.0.41 mmol.h/L and -0.49.+-.0.59 for the 100 Units
Insulin/300 mg 4-CNAB and 100 Units Insulin/600 mg 4-CNAB
treatments, respectively.
[0447] Conclusions
[0448] Oral administration of 4-CNAB alone was well tolerated.
[0449] Overall the safety profiles for combined treatments were
good, but 3 subjects following 150 Units Insulin/200 mg 4-CNAB
required food/drink due to hypoglycemia. All 8 subjects required
rescue treatments due to hypoglycemia following 10 Units SC
insulin.
[0450] The exposure to 4-CNAB appeared to increase with increasing
doses of 4-CNAB when given alone.
[0451] C-peptide corrected plasma insulin increased with increasing
doses of 4-CNAB demonstrating effective oral delivery and
absorption of human insulin.
[0452] The effect of insulin on the mean maximum plasma glucose
concentration change from baseline (E.sub.max) increased with
increasing doses of 4-CNAB and was significantly greater than oral
insulin alone and placebo for all combined treatments indicating
the effectiveness of the delivery agent in delivering insulin.
[0453] In general an increasing effect of oral insulin on plasma
glucose concentration change from baseline and percent change from
baseline was observed with increasing Insulin/4-CNAB doses.
[0454] Based on the mean maximum percent plasma glucose reduction,
the treatments 100 Units Insulin/600 mg 4-CNAB and 150 Units
Insulin/200 mg 4-CNAB appeared to elicit a greater PD response
compared to oral placebo or 150 Units oral insulin alone indicating
the effectiveness of the delivery agent in delivering insulin and
producing a subsequent effect.
EXAMPLE 10
[0455] Comparison of Pharmacodynamic and Pharmacokinetic Properties
of Oral Insulin vs. Subcutaneous (Sc) Regular Insulin in Type 2
Diabetic Patients
[0456] A single-center, open-label, randomized, active controlled,
3-period crossover study was conducted in ten 10 patients with type
2 diabetes in order to compare the pharmacodynamic (PD) and
pharmacokinetic (PK) characteristics of an oral insulin formulation
with SC regular insulin using the glucose clamp technique and in
order to get a first impression about the metabolic effect of oral
insulin in the main target population.
[0457] The glucose clamp technique was applied to compare the
time-action profiles of the orally applied insulin in comparison to
SC regular insulin. This method utilizes negative feedback from
frequent blood glucose sample values to adjust a glucose infusion
to maintain a defined and constant blood glucose level. The glucose
infusion rate therefore becomes a measure of the pharmacodynamic
effect of any insulin administered.
[0458] A primary objective of this study was to compare the PK and
PD effect of two doses of an oral insulin capsule formulation (300
U Insulin/400 mg 4-CNAB in 2 capsules, and 150 U Insulin/200 mg
4-CNAB in one capsule) with that of 15 U SC injected regular
insulin. Relative bioavailability and biopotency of the two oral
formulations vs. SC injection was determined, inter-subject
variability was investigated for selected PD and PK parameters.
[0459] Male subjects between 35 and 70 years old, inclusive, with
type 2 diabetes mellitus as defined by the American Diabetes
Association (1998 Diabetes care, 21: S5-S 19) for more than one
year were chosen. Subjects included in the study had BMI<36
kg/m.sup.2, had stable glycemic control (HbA.sub.1c<11%), were
off all oral hypoglycemic agents 24 hours prior to each study
dosing day and off any investigational drug for at least four (4)
weeks prior to Visit 1, refrained from strenuous physical activity
beginning 72 hrs prior to admission and through the duration of the
study, and were confined to the clinical research unit as required
by the protocol. Subjects maintained a constant body weight (+/-2
kg).
[0460] At Visit 1, the subjects arrived at the clinic in a fasted
state (no calorie intake for at least 12 hours). The subjects'
height, weight, body mass index (BMI), vital signs and medical
history were recorded, and a physical examination was done. An
electrocardiogram (ECG) was performed for all subjects as well as
local screening laboratory tests.
[0461] The oral treatment provided was Insulin/4-CNAB (Monosodium
N-(4-chlorosalicyloyl)-4-aminobutyrate (4-CNAB). The insulin used
to prepare the capsules was Zinc-Insulin Crystals Human: Proinsulin
Derived (Recombinant DNA Origin) USP Quality obtained from Eli
Lilly and Company (Indianapolis, Ind.). Each Insulin/4-CNAB capsule
contained 150 Insulin Units USP and 200 mg 4-CNAB, and was prepared
by AAI Pharma, Inc., Wilmington, N.C. Two tablets were given to
those who received the 300 U oral Insulin/400 mg 4-CNAB
treatments.
[0462] Insulin/4-CNAB capsules were provided in HDPE bottles, each
of which contained 40 capsules and a polyester coil. Each bottle
had a heat-induction seal and a child-resistant cap, and were
stored frozen at or less than minus 10.degree. C. On the day of
dosing, the appropriate number of capsules was removed from the
freezer and brought to room temperature (between 15 and 30.degree.
C.) for about one hour. Capsules were used within four hours of
dispensing, and unopened bottles were not left at room temperature
for more than four hours.
[0463] The SC injection was U-100 human regular insulin
(Humulin.RTM. R from Eli Lilly and Company, at a dose of 15 U,
supplied in 1.5-mL cartridges-100 units/mL, provided by Profil. The
Insulin was stored in the refrigerator within a temperature range
of 5-8.degree. C.
[0464] At Visit 2, each subject was randomized to one of the two
possible treatment sequences. Each subject received one of the two
treatments during a glucose clamp procedure: an oral treatment
(treatment A) of 300 U oral Insulin/400 mg 4-CNAB (in 2 capsules)
or one SC treatment (treatment B) of 15 U regular SC insulin.
[0465] At Visit 3, the subjects received the alternative treatment
A or B, i.e., the one they did not receive in Visit 2, in
conjunction with a glucose clamp procedure according to their
randomization sequence. Only subjects having received both
treatments by the end of Visit 3 were regarded as completers. A
final examination (Visit 4) was performed after Visit 3, preferably
immediately after the glucose clamp procedures were completed, but
no longer than 14 days after Visit 3.
[0466] All the subjects were invited to attend a third treatment
day (Visit 5), on which they received another oral treatment
(treatment C ) of a single dose of 150 U of the oral Insulin/200 mg
4-CNAB (one capsule). Eight of ten patients received the treatment
C. A "second" final medical examination (Visit 6) was performed
after Visit 5, preferably immediately after the glucose clamp
procedures were completed, but no longer than 14 days after Visit
5.
[0467] The assignment of the treatments within each sequence is
described in Table 28 below:
28 TABLE 28 Treatment Treatment Period Sequence 1 (Visit 2) 2
(Visit 3) 3 (Visit 5) 1 A B C (optional) 2 B A C (optional)
[0468] All treatment periods started in the morning after an
overnight fast of at least 12 hours. Dosing was performed following
a period of 6 hrs of stabilization of the blood glucose by means of
the glucose clamp. The subjects received the oral insulin capsules
with 200 mL of water in an upright position.
[0469] During each study visit, stabilized individual blood glucose
concentrations were maintained after drug administration using a
glucose clamp procedure. Blood samples were collected from each
patient during each treatment period for determination of plasma
insulin concentrations, plasma C-peptide, and plasma glucose.
Glucose infusion rates and blood glucose measurements were measured
continuously during the glucose clamp procedure. All treatments
were identical in their sample collection and monitoring periods
for all visits. After a 6 hours pre-dose baseline period for
stabilization of blood glucose concentrations at the desired clamp
level, the clamp procedure after study drug administration lasted 6
hours.
[0470] The patients' fasting blood glucose concentration at Visit 2
(measured before the baseline insulin infusion was established) was
the target level for the glucose clamp experiments. At the
following clamps, the fasting blood glucose concentrations were not
allowed to differ more than 4 mmol/L (72 mg/dL) from this
individualized clamp level, otherwise the visits were postponed for
at least 24 hrs.
[0471] The clamp level was adjusted by a variable intravenous
insulin infusion and glucose infusion rate during a 6 hrs baseline
period before dosing. During the last 2 hours before administration
of the study medication, the insulin infusion was set to a rate of
0.2 mU/kg/min, which rate was not changed until the end of the
experiment. At t=0 minutes, exogenous insulin was administered by
oral administration or by SC injection. The PD response elicited by
the study medication was registered for another 6 hrs. Serial blood
samples were collected after study drug administration for
measurement of plasma insulin concentrations, plasma C-peptide, and
plasma glucose, providing information for PK/PD determinations.
[0472] Safety was monitored by vital sign measurements and
documentation of adverse events (AE) during all visits throughout
the study. No food intake was permitted until the clamp procedure
was completed but the patients were allowed to drink water ad
libitum. After the last blood sample had been obtained, subjects
were provided with a meal.
[0473] Since the three treatments were single dose administrations,
a cross-over design was the most appropriate in order to keep
patient numbers low and to reduce data variability. SC injection of
15 U regular insulin is a common standard treatment and was
therefore used as control. Two oral insulin doses were chosen to
demonstrate a dose dependency of PK and PD parameters and to
investigate whether or not the suppressive effect on hepatic
glucose production could be seen also at the reduced oral dose of
150 U.
[0474] During the study, insulin therapy and the chronic use of all
agents that, in the evaluation of the investigator might have
interfered with the interpretation of trial results or were known
to cause clinically relevant interference with insulin action,
glucose utilization or recovery from hypoglycemia, were prohibited.
Intake of all oral hypoglycemic agents was stopped 24 hrs prior to
each study dosing day and was not resumed until the end of the
clamp procedures.
[0475] The SC insulin dose of 15 U was selected to fall within a
range typical for type 2 diabetic patients. The oral dose of 150 U
insulin (combined with 200 mg 4-CNAB) estimated to be equivalent to
the SC dose was a 10-fold scale-up compared with the SC dose, based
on previous investigational studies. The oral dose of 300 U insulin
(combined with 400 mg 4-CNAB) was a 20-fold scale-up compared with
the SC dose.
[0476] In each treatment arm, all patients received the same SC or
oral insulin dose. The patients' fasting blood glucose
concentration, measured before the baseline insulin infusion was
established, was the target level for the glucose clamp
experiments. During the consecutive glucose clamp experiments, the
fasting blood glucose concentration was not allowed to differ more
than 4 mmol/L (72 mg/dL) from this individualized clamp level,
otherwise the visits were postponed for at least 24 hrs.
[0477] The clamp level was adjusted by a variable intravenous (IV)
insulin infusion and glucose infusion rate during a 6 hrs baseline
period. During the last 2 hrs before administration of the study
medication, the insulin infusion was set to a rate of 0.2
mU/kg/min, which rate was not changed until the end of the
experiment. At t=0 minutes, exogenous insulin was administered by
oral administration or by SC injection. The PD response elicited by
the study medication was registered for another 6 hrs. No food
intake was allowed during this period, but water could be consumed
as desired.
[0478] The primary PD endpoint of the study was the area under the
glucose infusion rate curve (AUC.sub.GIR) in the first hr after
drug administration (AUC.sub.GIR 0-1 h). The secondary endpoints
for pharmacodynamic assessment were the following parameters:
Maximum glucose infusion rate (GIR.sub.max), time to GIR.sub.max
(t.sub.GIRmax), area under the glucose infusion rate curve in
defined time-intervals (AUC.sub.GIR 0-2 h, AUC.sub.GIR 0-3 h,
AUC.sub.GIR 0-4 h, AUC.sub.GIR 0-5 h, AUC.sub.GIR 0-6 h), time to
early and late half-maximum glucose infusion rate (early and late
T.sub.GIR 50%), and maximum reduction of C-peptide
concentrations.
[0479] The secondary endpoints for pharmacokinetic assessment were
the following parameters: Maximum plasma insulin concentrations
(C.sub.INSmax), time to C.sub.INSmax (t.sub.INSmax), area under the
insulin concentration curves in defined time-intervals (AUC.sub.INS
0-1 h, AUC.sub.INS 0-2 h, AUC.sub.INS 0-3 h AUC.sub.INS 0-4 h,
AUC.sub.INS 0-5 h AUC.sub.INS 0-6 h). Inter-subject variability was
investigated for selected PD and PK parameters.
[0480] During each study period, blood samples were collected for
the determination of plasma insulin concentrations, plasma
C-peptide and plasma glucose concentration. Sampling occurred from
6 hrs before dosing and continued for 6 hrs after the dose was
administered. Blood samples were collected via a venous cannula.
Blood samples were collected relative to the administration of the
study drug (1) prior to study of drug administration at -1 and -0.5
hrs, (2) immediately after study drug administration (time 0), and
(3) post administration of the study drug at 10, 20, 30, 40, 50
min, and 1, 1.25, 1.5, 1.75, 2, 2.5, 3, 3.5, 4, 5, and 6 hrs
[0481] The glucose clamp procedure was performed using a Biostator
(glucose controlled insulin infusion system--GCIIS, Life Science
Instruments, Elkhart, Ind., USA). A 17-gauge PTFE catheter was
inserted into an antecubital vein for blood sampling and kept
patent with 0.15 mol/L saline. A dorsal hand vein or lateral wrist
vein of the same arm was cannulated in retrograde fashion for
insertion of an 18-gauge PTFE double lumen catheter connected to
the glucose sensor of the Biostator. The catheterized hand was
placed in a hot box and warmed to an air temperature of 55.degree.
C. On the contralateral arm, a third vein was cannulated with an
18-gauge PTFE catheter to infuse a glucose solution (20% in water).
Into the same vein, an insulin solution (regular human insulin in
0.15 mol/L saline diluted with 2 mL of the patient's blood per 100
mL) was continuously infused by means of a syringe pump (Perfusor
Secura FT, Braun, Melsungen, Germany).
[0482] The clamp level was adjusted by a variable intravenous
insulin infusion and glucose infusion rate during a 6 hour baseline
period. During the last 2 hours before administration of the study
medication, the insulin infusion was set to a rate of 0.2
mU/kg/min, which rate was not changed until the end of the
experiment. At t=0 minutes, exogenous insulin was administered by
oral administration or by SC injection. The PD response elicited by
the study medication was registered for another 6 hrs. No food
intake was allowed during this period, but water could be consumed
as desired.
[0483] Blood glucose concentration and GIR determinations were made
continuously from -6 hrs prior to administration of the study drug
up to 6 hrs post-administration of the study drug by the Biostator.
These data were recorded at 1-minute intervals throughout the
treatment period. Safety assessments included AEs, laboratory data,
vital signs, physical examinations and ECGs.
[0484] Plasma concentrations of insulin, as shown in Table 29
below, were determined by a good laboratory practice (GLP)
validated microparticle enzyme immunoassay (MEIA).
29TABLE 29 Summary of Plasma Insulin Concentrations (pmol/L) 300 U
150 U 15 U SC oral oral Time Point N Mean SD N Mean SD N Mean SD
Time 0 10 4.6 7.3 10 9.2 9.9 8 0.3 0.9 10 minutes 10 5.7 10.8 10
50.3 62.4 8 59.6 44.7 20 minutes 10 13.7 18.3 10 429.6 474.6 8
188.3 162.6 30 minutes 10 41.2 32.5 10 409.5 268.1 8 192.5 250.6 40
minutes 10 94.9 63.1 10 366.2 258.2 8 114.3 158.9 50 minutes 10
109.4 75.6 10 214.2 185.8 8 79.8 121.7 60 minutes 10 116.4 63.2 10
122.1 108.2 8 50.2 69.5 75 minutes 10 137.4 86.5 10 48.2 37.3 8
30.3 38.6 90 minutes 10 116.5 50.9 10 20.6 28.2 8 19.5 25.9 105
minutes 10 132.2 72.4 10 7.5 12.9 8 11.5 14.9 120 minutes 10 119.1
70.2 10 19.1 22.1 8 17.1 15.7 150 minutes 10 129.8 51.1 10 10.0
14.1 8 8.3 11.6 180 minutes 10 149.6 61.3 10 9.1 13.6 8 17.0 23.4
210 minutes 10 146.8 71.8 10 4.9 7.0 8 17.7 24.9 240 minutes 10
138.2 64.6 10 5.1 5.9 8 22.0 31.1 300 minutes 10 129.8 42.5 10 8.1
17.2 8 12.0 15.4 360 minutes 10 87.7 56.7 10 2.4 4.5 8 14.2
16.4
[0485] In Table 29, baseline corrected values were used, i.e.,
pre-dose baseline values were subtracted, and in case of a negative
result, the value was set to zero.
[0486] Table 30 below shows a Summary of C-Peptide Levels
(nmol/L):
30TABLE 30 Summary of C-Peptide Levels 15 U SC 300 U oral 150 U
oral Time Point N Mean SD N Mean SD N Mean SD -60 minutes 10 1.02
0.42 10 0.95 0.37 8 0.86 0.33 -30 minutes 10 1.05 0.40 10 0.98 0.36
8 0.86 0.30 Time 0 10 1.04 0.39 10 0.99 0.30 8 0.95 0.35 10 minutes
10 1.05 0.42 10 1.00 0.31 8 1.00 0.38 20 minutes 10 1.07 0.47 10
1.01 0.34 8 1.02 0.37 30 minutes 10 1.09 0.46 10 1.02 0.38 8 1.01
0.34 40 minutes 10 1.11 0.47 10 1.02 0.36 8 0.97 0.28 50 minutes 10
1.05 0.43 10 1.01 0.36 8 0.99 0.36 60 minutes 10 1.04 0.39 10 0.97
0.35 8 1.00 0.36 75 minutes 10 1.07 0.45 10 1.00 0.41 8 0.99 0.34
90 minutes 10 1.03 0.44 10 1.04 0.38 8 0.92 0.30 105 minutes 10
1.01 0.43 10 1.06 0.36 8 0.94 0.33 120 minutes 10 0.99 0.45 10 1.04
0.36 8 0.95 0.36 150 minutes 10 1.01 0.50 10 1.04 0.34 8 1.01 0.38
180 minutes 10 0.99 0.44 10 1.07 0.38 8 1.08 0.46 210 minutes 10
0.97 0.40 10 1.04 0.40 8 1.03 0.38 240 minutes 10 0.94 0.31 10 1.11
0.45 8 1.00 0.37 300 minutes 10 0.96 0.36 10 1.07 0.44 8 0.99 0.43
360 minutes 10 0.92 0.34 10 1.07 0.39 8 0.97 0.42
[0487] The parameters assessed in this study were standard
measurements appropriate for comparing the PK and PD properties of
insulin absorption after oral administration and SC injection. The
use of a glucose clamp with a Biostator minimized the likelihood of
the onset of symptomatic hypoglycemia. Calculations based upon the
plasma insulin concentrations or glucose infusion rates from the
glucose clamp procedure reflect standard PK or PD calculations
commonly used for the glucose clamp technique.
[0488] The primary PD endpoint of the study was the area under the
glucose infusion rate curve (AUC.sub.GIR) in the first hr after
drug administration (AUC.sub.GIR 0-1 h).
[0489] PK and PD data were statistically analyzed for subjects that
received at least the first 2 treatments (Visit 1 to 4) and for
subjects who completed all three treatment visits (Visit 1 to
6).
[0490] PD parameters used for analysis were the maximum glucose
infusion rate after application of the study drugs (GIR.sub.max),
the time to maximum glucose infusion rate (t.sub.GIRmax), time to
half-maximum GIR values before GIR.sub.max (early t.sub.GIR 50%),
time to half-maximum GIR values after GIR.sub.max (late t.sub.GIR
50%), and the area under the glucose infusion rate versus time
curves from 0 to 60, 120, 180, 240, 300, and 360 min after dosing,
and from 180 to 360 min post dose (AUC.sub.GIR 0-1 h, AUC.sub.GIR
0-2 h, AUC.sub.GIR 0-3 h AUC.sub.GIR 0-4 h, AUC.sub.GIR 0-5 h,
AUC.sub.GIR 0-6 h, AUC.sub.GIR 3-6 h, respectively). In addition,
plasma concentrations of C-peptide and plasma glucose
concentrations were used for PD analysis. GIR.sub.max, t.sub.GIRmax
and early and late t.sub.GIR 50% were calculated by fitting a
polynomial function (6th order) to each individual's GIR profile
after subtraction of the mean baseline GIR. Areas under the curve
(AUCs) were calculated from the raw data using the trapezoidal
rule.
[0491] PK parameters were calculated using non-compartmental
methods. PK parameters determined included the maximum plasma
insulin concentration (C.sub.INSmax), time to maximum insulin
concentration(t.sub.INSmax), and the area under the plasma insulin
concentration versus time curves from 0 to 1, 2, 3, 4, 5 and 6 hrs
after application of the study drugs (AUC.sub.INS 0-1 h,
AUC.sub.INS 0-2 h, AUC.sub.INS 0-3 h, AUC.sub.INS 0-4 h,
AUC.sub.INS 0-5 h and AUC.sub.INS 0-6 h, respectively). The
calculation of AUCs from time of dosing until return to baseline
concentration (AUC.sub.INS 0-t') was omitted as in some patients
insulin concentrations did not return to baseline measurement
within 360 min post dosing.
[0492] The PK and PD data obtained were used for comparative
analysis of the treatments with 15 U SC insulin and 300 U oral
insulin. All tests were performed against a 2-sided alternative
hypothesis, with a significance level of 5% (.alpha.=0.05). The
tests were declared statistically significant if the calculated
p-value was <0.05. In view of the small sample size and some
outliers, a first analysis was done using non-parametric tests only
(signed Wilcoxon rank tests and Kruskal-Wallis tests). However, the
Kolmogorov-Smirnov test indicated normal distribution for all PK
and PD parameters. Therefore, parametric tests (paired t-tests and
ANOVAs) were performed in addition. Although there were no
substantial differences between the results of the non-parametric
and the parametric tests, it was decided that the non-parametric
results were used for the presentation of the data.
[0493] Safety data included AEs, laboratory data, vital signs,
physical examinations, and ECGs. Vital signs (systolic and
diastolic blood pressure, respiration rate, heart rate, and body
temperature) for each treatment group were summarized at baseline
(defined as the -30 min time point) and at the end of each study
day.
[0494] Ten patients with type 2 diabetes were planned (5 patients
assigned to each of 2 sequences) with complete data for analysis.
One patient dropped out prior to any treatment and was replaced as
per protocol. Therefore, the number of enrolled patients was 11.
Ten patients completed the study with the originally planned 2
treatments. Eight of these 10 patients accepted the offer to attend
an additional oral treatment of 150 U Insulin/200 mg 4-CNAB due to
protocol amendment, and this additional treatment was not performed
in random order. PK/PD data were also analyzed for the subset of 8
patients who received the second oral study treatment.
[0495] Analysis of Pharmacokinetics and Pharmacodynamics
[0496] PK and PD data were analyzed for the 10 patients who
received the study treatments A and B (oral 300 U Insulin/400 mg
4-CNAB, SC 15 U regular insulin) and had evaluable data. PK and PD
data were also analyzed for the amended group of 8 patients who
received the second oral study treatment (treatment C: oral 150 U
Insulin/200 mg 4-CNAB). All included patients with treatment
received at least 2 treatments (one oral and one SC treatment) as
planned in the protocol.
[0497] The following tables summarize the pharmacokinetic and
pharmacodynamic parameters calculated for the different
treatments.
31TABLE 31 Comparisons of the PK and PD Parameters Mean .+-. SD
Oral 300 U Insulin/400 mg 4- Oral 150 U SC 15 U CNAB Insulin/200 mg
4-CNAB Regular Insulin Parameter (n = 10) (n = 8) (n = 10) Insulin
AUC.sub.0-1h (.mu.U .times. mL.sup.-1 .times. min) 2559.25 .+-.
1831.45 1099.58 .+-. 1221.15 542.31 .+-. 296.26 AUC.sub.0-2h (.mu.U
.times. mL.sup.-1 .times. min) 2926.58 .+-. 2104.23 1337.14 .+-.
1407.11 1801.97 .+-. 789.43 AUC.sub.0-3h (.mu.U .times. mL.sup.-1
.times. min) 3046.75 .+-. 2169.16 1463.59 .+-. 1443.01 3122.81 .+-.
1242.82 AUC.sub.0-4h (.mu.U .times. mL.sup.-1 .times. min) 3106.67
.+-. 2212.98 1649.43 .+-. 1541.90 4576.31 .+-. 1817.96 AUC.sub.0-5h
(.mu.U. .times. mL.sup.-1 .times. min) 3172.83 .+-. 2262.81 1819.01
.+-. 1593.02 5916.31 .+-. 2208.85 AUC.sub.0-6h (.mu.U .times.
mL.sup.-1 .times. min) 3225.33 .+-. 2319.66 1949.64 .+-. 1623.949
7003.81 .+-. 2440.251 C.sub.max (.mu.U/mL) 93.44 .+-. 71.18 37.90
.+-. 39.23 32.7 .+-. 10.59 t.sub.max (min) 27.00 .+-. 9.49 22.50
.+-. 7.07 160.5 .+-. 82.78 Glucose Infusion Rate AUC.sub.0-1h
(mg/kg) 172.63 .+-. 85.54 58.08 .+-. 39.99 27.38 .+-. 32.22
AUC.sub.0-2h (mg/kg) 297.11 .+-. 142.73 102.62 .+-. 88.94 136.54
.+-. 106.54 AUC.sub.0-3h (mg/kg) 321.19 .+-. 146.29 116.96 .+-.
78.26 271.43 .+-. 191.04 AUC.sub.0-4h (mg/kg) 343.31 .+-. 140.47
142.00 .+-. 85.76 421.33 .+-. 264.76 AUC.sub.0-5h (mg/kg) 364.40
.+-. 135.36 160.28 .+-. 99.64 548.83 .+-. 342.71 AUC.sub.0-6h
(mg/kg) 374.10 .+-. 134.70 190.77 .+-. 133.38 650.70 .+-. 380.16
GIR.sub.max (mg/kg/min) 4.35 .+-. 2.23 2.12 .+-. 0.89 3.57 .+-.
1.79 t.sub.GIRmax (min) 39.80 .+-. 16.00 131.63 .+-. 146.04 255.30
.+-. 108.15 Early t.sub.50% (min) 13.40 .+-. 6.48 103.50 .+-.
140.90 150.40 .+-. 87.44 Late t.sub.50% (min) 114.70 .+-. 78.74 NA
NA
[0498]
32TABLE 32 Comparisons of Relative Bioavailability and Biopotency
(Mean .+-. SD) Oral 300 U Insulin/400 mg 4-CNAB Oral 150 U
Insulin/200 mg 4-CNAB TIME (n = 10) (n = 8) INTERVAL
Bioavailability (%) Biopotency (%) Bioavailability (%) Biopotency
(%) 0-1 hr 43.7 .+-. 60.5 54.9 .+-. 91.9 (n = 7) 21.4 .+-. 19.8
110.9 .+-. 193.4 (n = 5) 0-2 hrs 13.0 .+-. 20.1 11.7 .+-. 9.2 (n =
9) 8.4 .+-. 6.8 12.9 .+-. 13.6 (n = 7) 0-3 hrs 7.8 .+-. 12.7 5.8
.+-. 3.4 (n = 9) 5.4 .+-. 4.3 6.7 .+-. 6.2 (n = 7) 0-4 hrs 4.9 .+-.
7.3 21.1 .+-. 54.9 4.0 .+-. 3.1 4.0 .+-. 3.6 0-5 hrs 3.4 .+-. 4.1
47.5 .+-. 140.4 3.4 .+-. 2.7 3.5 .+-. 3.2 0-6 hrs 2.7 .+-. 3.0 31.0
.+-. 89.4 3.1 .+-. 2.4 3.2 .+-. 2.8
[0499] The pharmacokinetic and pharmacodynamic parameters discussed
here are the averages and standard deviations of the individual
values. The oral dose of 300 U Insulin/400 mg 4-CNAB showed a
faster and higher rise in plasma insulin concentrations indicating
a faster onset of action than the SC treatment (AUC.sub.INS 0-1 h
oral 300 U vs. SC 15 U: 2559.+-.1831 vs. 542.+-.296
.mu.U.times.mL.sup.-1.times.min, p<0.01; C.sub.INS max oral 300
U vs. SC 15 U: 93.+-.71 vs. 33.+-.11 .mu.U/mL, p<0.01;
t.sub.INSmax oral 300 U vs. SC 15 U: 27.+-.9 vs. 161.+-.83 min,
p<0.01).
[0500] Accordingly, this trend is mirrored in the pharmacodynamic
(GIR) results, as expected, with a significantly faster onset of
the PD effect after the oral treatment compared to the SC treatment
(AUC.sub.GIR 0-1 h oral 300 U vs. SC 15 U: 173.+-.86 vs. 27.+-.32
mg/kg, p<0.01; t.sub.GIRmax oral 300 U vs. SC 15 U: 40.+-.16 vs.
255.+-.108 min, p<0.01; early t.sub.50% oral 300 U vs. SC 15 U:
13.+-.6 vs. 150.+-.87 min, p<0.01). The maximum glucose infusion
rates showed no statistically significant difference.
[0501] Relative bioavailability and biopotency of the two oral
insulin doses in comparison to the SC administration were
calculated as defined hereinabove. Relative bio-availability of
oral insulin is listed in the Table 33. Respective values for
bio-potency for oral insulin are listed in Table 34.
33TABLE 33 Summary of Relative Bioavailability of Insulin Time
interval n Mean SD SEM Max Min Median Bioavailability (%): 300 U
oral vs. 15 U SC 0-60 10 43.7 60.5 19.1 198.2 7.2 18.3 0-120 10
13.0 20.1 6.4 69.1 2.9 7.0 0-180 10 7.8 12.7 4.0 43.5 1.8 3.9 0-240
10 4.9 7.3 2.3 25.4 1.3 2.6 0-300 10 3.4 4.1 1.3 14.6 1.0 2.1 0-360
10 2.7 3.0 1.0 11.0 0.8 1.8 180-360 10 0.3 0.3 0.1 1.1 0.0 0.1
Bioavailability (%): 150 U oral vs. 15 U SC 0-60 8 21.4 19.8 7.0
53.3 0.4 14.7 0-120 8 8.4 6.8 2.4 16.8 0.1 7.8 0-180 8 5.4 4.3 1.5
10.3 0.1 5.7 0-240 8 4.0 3.1 1.1 7.6 0.0 4.6 0-300 8 3.4 2.7 0.9
6.8 0.0 3.8 0-360 8 3.1 2.4 0.9 6.2 0.0 3.4 180-360 8 1.5 1.6 0.6
3.7 0.0 1.0
[0502]
34TABLE 34 Summary of Relative Biopotency of Oral Insulin Time
interval n Mean SD SEM Max Min Median Biopotency (%): 300 U oral
vs. 15 U SC 0-60 7 54.90 91.93 34.74 261.44 5.54 19.90 0-120 9
11.70 9.21 3.07 32.76 3.65 8.12 0-180 9 5.76 3.41 1.14 13.56 2.03
4.85 0-240 10 21.14 54.94 17.37 177.45 2.19 3.56 0-300 10 47.53
140.38 44.39 447.03 1.56 3.12 0-360 10 31.01 89.44 28.28 285.54
1.10 2.69 Biopotency (%): 150 U oral vs. 15 U SC 0-60 5 110.86
193.40 86.49 455.95 11.74 23.52 0-120 7 12.86 13.59 5.14 40.76 3.23
6.94 0-180 7 6.66 6.17 2.33 19.53 1.58 6.28 0-240 8 4.03 3.56 1.26
10.67 0.00 2.86 0-300 8 3.55 3.22 1.14 9.74 0.00 2.52 0-360 8 3.15
2.77 0.98 7.93 0.00 2.40
[0503] Relative biopotency (based on PD results) of 300 U oral
Insulin/400 mg 4-CNAB was as high as 54.9.+-.91.9% in the first hr
after application, and 31.0.+-.89.4% over 6 hrs. Respective values
for bioavailability (based on PK results) were 43.7.+-.60.5%, and
2.7.+-.3.0%. The unexpected increase in mean relative biopotency
for the time intervals 0-4, 0-5 and 0-6 hrs accounts for Patient
101 whose values were calculated only for these three time periods
and which were up to 100-fold higher than those found for the other
patients (177.45%, 447.03%, and 285.54%, respectively).
[0504] The oral dose of 150 U Insulin/200 mg 4-CNAB also showed a
faster rise in plasma insulin concentrations compared to the SC
treatment (AUC.sub.INS 0-1 h oral 150 U vs. SC 15 U: 1100.+-.1221
vs. 542.+-.296 .mu.U.times.mL.sup.-1.times.min; t.sub.INSmax oral
300 U vs. SC 15 U: 23.+-.7 vs. 161.+-.83 min), whereas the observed
maximum plasma concentrations were similar for both treatments
(C.sub.INSmax oral 150 U vs. SC 15 U: 38.+-.39 vs. 33.+-.11
.mu.U/mL).
[0505] Accordingly, GIR results for the oral 150 U insulin dose
showed a faster onset of the PD effect (AUC.sub.GIR 0-1 h oral 150
U vs. SC 15 U: 58.+-.40 vs. 27.+-.32 mg/kg; t.sub.GIRmax oral 150 U
vs. SC 15 U: 132.+-.146 vs. 255.+-.108 min; early t.sub.50% oral
150 U vs. SC 15 U: 104.+-.141 vs. 150.+-.87 min). The maximum
glucose infusion rate was lower after the oral than after the SC
treatment (GIR.sub.max. oral 150 U vs. SC 15 U: 2.1.+-.0.9 vs.
3.6.+-.1.8 mg/kg/min).
[0506] These findings indicate that suppression of hepatic glucose
production can be achieved also by the lower dose of 150 U oral
insulin.
[0507] Relative biopotency of 150 U oral Insulin/200 mg 4-CNAB was
110.9.+-.193.4% in the first hour after application, and
3.2.+-.2.8% over 6 hours. Respective values for bioavailability
were 21.4.+-.19.8%, and 3.1.+-.2.4%. The abnormal high mean
relative biopotency of 110.9% in the first hr results from an
extremely high value of 455.95% found for Patient 102 and the fact
that values of only 5 patients were available. Because of the
mentioned distortion of the biopotency means, the medians are
considered to be a more suitable representation of the data.
[0508] Comparison of the PK and PD data of the two oral insulin
doses suggests a nearly linear dose relationship of the PK
parameters AUC.sub.INS and C.sub.INS max. The PD response, as
represented by AUC.sub.GIR and GIR.sub.max, also increases with
dose but in a less clear fashion.
[0509] Pharmacokinetic/Pharmacodynamic Conclusions
[0510] This first glucose clamp study demonstrated that orally
applied insulin exhibits a pronounced metabolic effect. In view of
the presented PD and PK properties, and the advantages of an oral
administration (high portal insulin concentrations, convenience of
administration), Insulin/4-CNAB seems to be a very attractive
candidate for pre-prandial (before meal) insulin therapy in type 1
and type 2 diabetic patients.
[0511] All treatments evaluated during the study were safe and well
tolerated. No adverse events were observed following oral
administration of Insulin/4-CNAB capsules or subcutaneous injection
of regular insulin.
EXAMPLE 11
[0512] Safety of Orally Administered 4-CNAB/Insulin
[0513] An open-label, single-dose, crossover study was conducted in
order to compare the safety, pharmacokinetics, and pharmacodynamics
of orally administered 4-CNAB/Insulin in fasting type 2 diabetic
patients serving as their own controls, and to compare blood
glucose, insulin and C-peptide levels following a standard meal in
type 2 patients given their regular medication, with that of blood
glucose, insulin and C-peptide levels following a standard meal
with 4-CNAB/Insulin.
[0514] Twenty-four (24) volunteers, between 46 and 70 years old,
each with type 2 diabetes mellitus were studied. Patients had a
body mass index of between 21 and 35 and had stable glycemic
control (HgA1c ranged from 5.9 to 11.6%). Fifteen patients were on
antidiabetic medication (either Metformin or Acarbose), and 9
patients controlled their diabetes by diet alone. All participants
who were on medication did not take their antidiabetic drug 24
hours prior to study
[0515] The diabetic volunteers were divided into two groups--in one
group, 12 patients were studied on fasting state, and in a second
group, 12 patients were studied before and during standard meal.
Every patient served as his own control and was tested without
getting the insulin-4-CNAB mixture.
[0516] With respect to Group 1, following a minimum of 8 hour
overnight fast, subjects were given one capsule containing a
mixture of insulin in a stepwise fashion (3 patients received 200 U
insulin, 5 patients received 300 U insulin and 4 patients received
400 U insulin) and a fixed dose of 300 mg 4-CNAB as a delivery
agent. In the control session, a placebo was administered to these
same patients. See FIG. 10.
[0517] With respect to Group 2, subjects had a standard meal (350
kcal) after a minimum of 8 hour overnight fast. Twenty minutes
prior to the ingestion of food, the patients were administered a
capsule contained 300 U or 400 U insulin (six patients received 300
U insulin and six patients received 400 U of insulin) and 300 mg of
4-CNAB. In the control session, these same patients took their own
regular medication, either 850 mg Metformin or 100 mg Acarbose.
Subjects who control their diabetes on diet alone had their meal
(47 g of carbohydrates (54%) and 350 kcal total calories) without
any drug. See FIG. 11.
[0518] Due to the fact that blood glucose level was not reduced by
30% (in average of the first three fasting subjects), the dose was
increased to 300 U insulin. Then, since the blood glucose level was
not reduced by 30% with 300 U insulin (in average of the 3
subjects), in both groups (fasting and standard meal), the dose of
insulin was increased to 400 U insulin.
[0519] The delivery agent 4-CNAB was supplied by Emisphere
Technologies Inc., of Tarrytown, N.Y., and was stored at room
temperature desiccated until use. Recombinant Human Zinc Insulin
was shipped directly by Eli Lilly and Company, and was stored at
-20 C. Standard capsules were made of gelatin, size 00C, and were
prepared by a pharmacist at Hadassah Medical Organization.
[0520] A catheter was inserted into the antecubital arm vein of
each patient. Blood was withdrawn at baseline twice, 5 to 10 min.
apart, and at timed intervals after the administration of the
capsule. In group 1, the fasting group, blood was withdrawn during
the first hour every 5 minutes and thereafter every 10-20 minutes.
In group 2, the standard meal group, blood was withdrawn during the
first two hours every 10 minutes and thereafter every 20 minutes.
In both groups, blood samples were withdrawn until blood glucose
reached basal levels. All plasma samples were analyzed for glucose,
insulin, C-peptide and the delivery agent 4-CNAB. Blood glucose
levels were measured in real time using two Elite Glucometers
(Bayer corporation, Elkhart, Indianapolis, Ind., USA). At the end
of the trial, plasma glucose concentrations were measured, using an
Enzymatic UV test of Roche Diagnostics (Roche Diagnostics
Indianapolis, Ind., USA). Plasma insulin and C-peptide were
determined using radio-immunoassay kits produced by Linco Research,
Inc. St. Charles, Mo., USA.
[0521] Results and Conclusions
[0522] While the results were highly variable, there was a clear
trend indicating in most subjects the absorption of insulin and its
biological effect causing either hypoglycemia or a suppressed
elevation of blood glucose, following meals. In the meal session,
when the effect of the insulin administered was compared to the
effect of the anti-diabetic drug given, there was only a small
difference in the blood glucose values, demonstrating the fact that
oral insulin was biologically effective even during meals. As in
previous examples provided herein employing non-diabetic
volunteers, the rise in insulin levels appeared 10-30 minutes after
the capsule was swallowed and preceded the drop in blood glucose
levels (when there was a drop.)
[0523] The C-peptide levels were measured in order to evaluate the
extent of enteral absorption of insulin. The absorption of insulin
caused a drop in the C-peptide levels, particularly in the standard
meal group, indicating a decrease in endogenous insulin secretion
due to the absorption of the insulin and the resulting
hypoglycemia.
[0524] Fasting diabetic type 2 volunteers given increasing dose of
insulin (200 U to 400 U insulin) orally with 300 mg delivery agent
4-CNAB demonstrated a decrease in glucose levels and a moderate
increase in insulin levels. In the standard meal group, the insulin
capsule caused higher insulin levels and a decrease in C-peptide
level. In most cases, following the ingestion of the capsule, there
was a decrease in plasma glucose levels, and the nadir appeared
after 10-30 minutes in the fast group. In patients with the
standard meal who received their regular antidiabetic agent, the
4-CNAB/Insulin capsule "covered" their meal no less than, and
sometimes even better than, the Metformin or the Acarbose. In most
of the patients in the standard meal group, the C-peptide levels
were suppressed, pointing to the fact that the secretion of
endogenous hormone was partially abolished.
[0525] Plasma insulin levels were elevated in most of the subjects
in the fasting group. These levels were not always followed by a
reduction in the glucose levels.
[0526] No adverse events were detected during the trial or a few
weeks later, except for one subject who complained of mild
headaches five minutes after ingestion of the capsule, probably not
associated with the trial mixture.
[0527] It is concluded that this oral insulin preparation is safe
and efficient. There is, however, a need for further improvement in
absorption of the biologically active insulin.
EXAMPLE 12
[0528] A single-center, open label, randomized, single dose, 3-way
cross-over study was conducted in type 1 diabetes mellitus patients
to investigate the effect of food on the absorption and
pharmacokinetics of insulin after a single dose of oral
4-CNAB/insulin, and to determine the effect of food on the
pharmacodynamics of glucose and C-peptide after a single oral dose
of 4-CNAB/insulin.
[0529] For the diabetic volunteers, male or postmenopausal female
subjects between 18 and 65 years old, inclusive, each with type 1
diabetes mellitus as defined by the American Diabetes Association
(1998 Diabetes care, 21: S5-S19) were studied. Subjects had a body
mass index of between 18 and 30 kg/m2 and had glycemic control
HgA1c at screening <10%. Patients also had negative test for
antibodies against insulin at screening, fasting blood glucose at
screening <12.0 mmol/l, and fasting C-peptide at screening
<0.2 nmol/ml. For the healthy control volunteers, male subjects
between 18 and 65 years old, inclusive, for more than one year were
chosen. Subjects included in the study had between 18 and 30
kg/m2.
[0530] For diabetic patients, the study consisted of an eligibility
screening period, three study periods and a follow-up exam at the
conclusion of the last period. The three study periods included the
following: administration of single doses of 4-CNAB/insulin
followed by fasting (treatment A), followed by an ADA breakfast 20
min after dosing (treatment B), and followed by an ADA breakfast 20
min after dosing (treatment C). The study was conducted using an
open label, randomized, crossover design with an interval of at
least 7 days between treatments. The patients fasted overnight. The
type 1 diabetics were randomized to treatment A or treatment B in
periods 1 and 2. In period 3 all diabetics received treatment C. A
total of 8 type 1 diabetic patients were enrolled. As a control
group 2 healthy volunteers were enrolled.
[0531] For healthy control subjects, the study consisted of an
eligibility screening period, one study period and a follow-up exam
at the conclusion of the period. The healthy control subjects were
not receiving any medication but served as a control for the effect
of breakfast on insulin production. Blood sampling and safety
assessments followed the same schedule as for the diabetics. The
healthy control subjects received the standard ADA breakfast at the
same time as the type 1 diabetics in one study period (treatment
D).
[0532] The study design is presented in Tables 35 below
35TABLE 35 Study design for Type I diabetics End of week -3 third
to -1 Treatment A Treatment B Treatment C peroid Eligibility 4- 4-
4-CNAB/insulin follow- screening CNAB/insulin CNAB/insulin ADA
breakfast up fasting ADA breakfast 20 min after 30 min after dosing
dosing
[0533]
36TABLE 36 Study Design for Healthy volunteers week -3 to -1
Treatment D End of period Eligibility screening no dosing follow-up
ADA breakfast
[0534] This study tested the effect of a standard ADA breakfast
administered 30 or 20 minutes after dosing on the absorption and
pharmacokinetics of 4-CNAB/insulin administered as oral capsules. A
control group of healthy subjects received a standard ADA breakfast
in one period to measure the amount of insulin produced for this
breakfast in healthy control subjects. The type I diabetic
patientss were taken off their regular long-acting insulin 24 hrs
prior to dosing and their glucose levels were controlled prior to
dosing by overnight insulin infusion.
[0535] The following treatments were administered to the type 1
diabetics according to the randomization schedule (see below)
[0536] a) 400 mg 4-CNAB/300 IU insulin followed by fasting;
[0537] b) 400 mg 4-CNAB/300 IU insulin followed by an ADA breakfast
30 minutes after dosing;
[0538] c) 400 mg 4-CNAB/300 IU insulin followed by an ADA breakfast
20 minutes after dosing.
[0539] Prior to dosing, the patients fasted overnight. The patients
received an insulin infusion overnight. The study drug was
administered 30 minutes after infusion was stopped (dosing at
approximately 09:00 am). In one period, the oral dose was followed
by fasting until 3 hours after dosing. In the two other periods,
the oral dose is followed by intake of a standard ADA-breakfast 30
or 20 minutes after dosing. On day one of each study period, study
medication was administered to subjects. Only an ADA breakfast was
administered to the healthy volunteers.
[0540] The type I diabetics stopped their regular long acting
insulin 24 hrs prior to dosing. They were allowed to use their
immediate acting insulin up to their entry into the clinic around
15:00 hrs on day 1. They received 4-6 units (depending on their
weight) of regular insulin subcutaneously (s.c.) at approximately
17:30 hrs on day 1. Thirty minutes after the administration of s.c.
insulin, the diabetics received a standard dinner. Between 20:30
and 21:00 hrs, the diabetics received a snack. At approximately
21:00 hrs on day-1 an i.v. infusion of insulin was started at the
infusion rate indicated in Table 38. The composition of the insulin
infusion and the infusion rate were dependent on the patient's
weight and blood glucose concentration as described in Tables 37
and 38.
37TABLE 37 Composition of insulin infusate Insulin (U/L) Patient
weight (kg) 80 60-65 88 65-70 96 70-75 104 75-80 112 80-85 124
85-90 140 90-95 180 >95
[0541]
38TABLE 38 Infusion algorithm Plasma glucose conc. [mmol/liter
Infusion rate (mg/dL)] (ml/h) <5.5 (<99).sup.a 0 5.5-6.6
(100-119) 5 6.7-7.7 (120-139) 10 7.8-8.8 (140-159) 15 8.9-9.9
(160-179) 20 10-13.3 (180-239) 40 >13.3 (>240) 60
[0542] Infusion rate was adjusted, if necessary, based on the
results of blood glucose measurements done every 60 minutes. A
blood sample (one drop) for assessment of real-time blood glucose
using a Glucocard.RTM. was taken from an indwelling cannula. The
blood glucose concentration was adjusted to remain between 6 and 8
mmol/l. The insulin infusion was stopped 30 minutes before drug
administration at approximately 09:00 hrs on day 1. At times when
no insulin was needed, only normal saline is administered.
[0543] The 4-CNAB (Sodium
N-[4-(4-chloro-2-hydroxybenzoyl)amino]butyrate) was manufactured by
Emisphere Technologies, Inc. of Tarrytown, N.Y. in 400 mg strength
oral capsules. Glucose stabilization prior to dosing was done with
Actrapid, manufactured by Novo Nordisk and having an active
compound of insulin, at 100 U/ml strength, via i.v. infusion. The
insulin for subcutaneous injection was Actrapid, manufactured by
Novo Nordisk, at 100 U/ml strength.
[0544] The type I diabetics stopped their regular long acting
insulin 24 hrs prior to dosing. They were allowed to use their
immediate acting insulin up to their entry into the clinic around
15:00 hrs on day-1. They received 4-6 units of regular insulin s.c.
at approximately 17:30 hrs on day-1. Thirty minutes after the
administration of s.c. insulin, the diabetics received a standard
dinner (see section 13.4). Between 20:30 and 21:00 hrs, the
diabetics received a snack then they were fasted until the next
morning. At approximately 21:00 hrs an i.v. infusion of insulin was
started. The insulin infusion was stopped 30 minutes before drug
administration at approximately 09:00 hrs on day 1.
[0545] On day 1 of each study period, study medication was
administered to subjects in the upright position. The study drug
was swallowed with 200 mL of water. The study drug was not chewed.
Subjects will not lie down for three hours after dosing. Depending
on the treatment given, the patients received a standard ADA
breakfast 30 or 20 minutes after dosing or they continued fasting.
After the 3 hr blood sample was drawn, patients were allowed to
resume their normal pattern of meals and resume using their regular
long or immediate acting insulin. Water was allowed ad libitum
during the study, except for 1 hour prior to up to 1 hour after
drug administration in each treatment.
[0546] The healthy control subjects did not receive any medication
but received a standard ADA breakfast at the same time as the
diabetics (approximately 09:30 hrs on day 1) after an overnight
fast. The controls may resume normal meals after the 3-hr blood
sample has been taken.
[0547] 9.4.6 Prior/Concomitant Medication and Other Restrictions
During Study
[0548] Study participants did not take any prescription or non
prescription medication (with the exception of paracetamol,
(acetaminophen) and topical medication) for 14 days prior to
entrance into the clinical research facility and for the duration
of the study period. The exception to this rule follows: type 1
diabetics continued their insulin therapy and fixed comedication
which was used unaltered during the last 6 months.
[0549] Methylxanthine-containing beverages or food (coffee, tea,
coke, chocolate), grapefruit juice, and alcohol were not allowed
from 48 hours (2 days) prior to entrance into the clinical research
center and during the study.
[0550] During each period of the study a series of blood samples
were taken for 4-CNAB and insulin Pharmacokinetic analyses.
[0551] Blood samples for Pharmacokinetic analysis of 4-CNAB and
insulin were drawn 30, 15 and 5 minutes prior to and at 10, 20, 30,
40, 50, 60, 75, 90, 105, 120, 150 and 180 minutes after drug
administration (15 samples per subject per period). The blood
samples (6 mL each) were taken via an indwelling Venflon.RTM.
catheter or by direct venepuncture into sodium heparin-containing
tubes. The blood samples were centrifuged at 1500.times.g for
fifteen minutes at a temperature between 2.degree. C. and 8.degree.
C., within one hour of sample collection. The total volume of about
450 mL (type 1 diabetics) or about 180 mL (healthy control
subjects) blood was taken during the study.
[0552] Blood samples for plasma glucose will be drawn at: 30, 15
and 5 min prior to and at 10, 20, 30, 40, 50, 60, 75, 90, 105, 120,
150 and 180 minutes after drug administration (15 samples per
subject per period). Samples will be drawn into 2 ml lithium
heparin tubes. The blood samples were centrifuged at 1500.times.g
for fifteen minutes at a temperature between 2.degree. C.-8.degree.
C., within one hour of sample collection.
[0553] Blood samples drawn for insulin analysis were analyzed for
C-peptide at the following time points: 30 and 5 min prior to and
at 30, 60, 90, 120 and 180 minutes after drug administration (7
samples per subject per period).
[0554] Blood samples for real-time glucose were drawn every 60 min
during i.v. insulin infusion on day -1 and day 1, at pre-dose and
at 30, 60, 90, 120, 150 and 180 after each drug administration on
day 1 (10 samples per subject per period). The blood samples were
taken from the indwelling canula (with obturator), one drop per
assessment. The samples were analyzed for glucose in real time
using a Glucocard.RTM..
[0555] The pharmacokinetic parameters to be determined or
calculated from the plasma concentration time data for 4-CNAB and
insulin are C.sub.max (maximum plasma concentration), t.sub.max
(time to attain maximum plasma concentration), k.sub.el
(elimination rate constant), t.sub.1/2 (elimination half-life),
AUC.sub.last (area under the plasma concentration-time curve up to
time t, where t is the last time point with concentrations above
the lower limit of quantitation (linear trapezoidal rule)),
AUC.sub.0-.infin. (total AUC after extrapolation from time t to
time infinity, where t is the last time point with a concentration
above the lower limit of quantitation),
AUC.sub.last+c.sub.last/k.sub.el, and %AUC.sub.extra (percentage of
estimated part for the calculation of AUC.sub.0-.infin.:
(AUC.sub.0-.infin.-AUC.sub.last)/AUC.sub.0-.infin.)*100%)).
[0556] The following pharmacodynamic parameters were computed from
the plasma concentration-time data of glucose and C-peptide (both
original and baseline subtracted data) using non-compartmental
analysis: E.sub.max, t.sub.emax, and AUEC.sub.last (area under the
effect-time curve calculated using linear trapezoidal summation
from time zero to time t, where t is the time of the last
measurable effect (E).
[0557] The process of administration for the diabetic patients was
as follows:
[0558] 1) Patients received 4-6 units (depending on their weight)
of regular insulin subcutaneously (s.c.) at approximately 17:30 hrs
on day 1.
[0559] 2) Insulin infusion started at 21:00 on day 1, and the pump
stopped 30 min before dosing on day 1.
[0560] 3) Dosing at approximately 09:00 am.
[0561] 4) A blood sample (one drop) was analyzed for real-time
glucose every 60 min. during the time of insulin infusion and at
pre-dose, 30, 60, 90, 120, 150, 180 and 240 min after each drug
administration on day 1 (8 samples per subject per period).
[0562] 5) Blood samples for Pharmacokinetic analysis of 4-CNAB and
insulin were drawn 30, 15 and 5 min prior to and at 10, 20, 30, 40,
50, 60, 75, 90, 105, 120, 150, 180, 210, and 240 min after drug
administration (17 samples per subject per period).
[0563] 6) Blood samples for plasma glucose were drawn at: 30, 15
and 5 min prior to and at 10, 20, 30, 40, 50, 60, 75, 90, 105, 120,
150, 180, 210, and 240 min after drug administration (17 samples
per subject per period).
[0564] 7) Blood samples for C-peptide were drawn at: 30 and 5 min
prior to and at 30, 60, 90, 120,180 and 240 min after drug
administration (8 samples per subject per period).
[0565] 8) Pre-dose and 1, 2, 4 and 6 hrs after drug
administration.
[0566] 9) Around 09:00 a.m.
[0567] 10) The last day of the third period
[0568] The process of administration for the healthy control
subjects was as follows:
[0569] 4) A blood sample (one drop) will be analyzed for real-time
at pre-dose, 30, 60, 90, 120, 150, 180 and 240 min after each drug
administration on day 1 (8 samples per subject per period).
[0570] 5) Blood samples for Pharmacokinetic analysis of 4-CNAB and
insulin will be drawn 30, 15 and 5 min prior to and at 10, 20, 30,
40, 50, 60, 75, 90, 120, 150, 180, 210, and 240 min after drug
administration (16 samples per subject per period).
[0571] 6) Blood samples for plasma glucose will be drawn at: 30, 15
and 5 min prior to and at 10, 20, 30, 40, 50, 60, 75, 90, 120, 150,
180, 210, and 240 min after drug administration (16 samples per
subject per period).
[0572] 7) Blood samples for C-peptide will be drawn at: 30 and 5
min prior to and at 30, 60, 90, 120,180 and 240 min after drug
administration (8 samples per subject per period).
[0573] 8) Pre-dose and 1, 2, 4 and 6 hrs after drug
administration.
[0574] 9) Around 09:00 am.
[0575] 10) The last day in clinic
[0576] The term pre-dose refers to the time that the group of
diabetics receives 4-CNAB/insulin. The healthy control subjects do
not receive medication.
EXAMPLE 13
[0577] Comparison between Oral Insulin and s.c. Short Acting
Postprandial Blood Glucose Excursions
[0578] A randomized, 3-period crossover, double-blind, double-dummy
study was conducted in order to compare the effect (i.e., the
postprandial pharmacokinetic and pharmacodynamic profiles) of an
oral insulin formulation with that of s.c. administered short
acting insulin on postprandial blood glucose excursions in Type 2
Diabetic subjects without any antidiabetic medication.
[0579] A primary objective of this study was to compare the effect
of an oral insulin formulation (300 U insulin combined with 400 mg
4-CNAB in 2 capsules, each capsule containing 150 U insulin/200 mg
4-CNAB) with that of 12 U subcutaneous (s.c.) injected short acting
insulin [Humalog.RTM. injection 100 U/ml from Eli Lilly and
Company] on postprandial blood glucose excursions. The postprandial
blood glucose excursions were assessed after a standardized
breakfast intake.
[0580] Fifteen male subjects between 35 and 70 years old,
inclusive, with type 2 diabetes mellitus as defined by the American
Diabetes Association (1998 Diabetes care, 21: S5-S19) for more than
one year were chosen. Subjects included in the study had BMI<36
kg/M.sup.2, had stable glycemic control (HbA.sub.lC<11%), were
off all oral hypoglycemic agents 24 hours prior to each study
dosing day and off any investigational drug for at least four (4)
weeks prior to Visit 1, refrained from strenuous physical activity
beginning 72 hrs prior to admission and through the duration of the
study, and were confined to the clinical research unit as required
by the protocol. Subjects maintained a constant body weight (+/-2
kg).
[0581] At visit 1, each subject was randomized to one of six
possible treatment sequences, such that each received one of six
possible treatments at each treatment visit day, as determined by
the randomization sequence. Subjects were randomly assigned to one
of six sequence groups:
[0582] Oral Insulin [OI]; Short acting Insulin [SI]; No
supplemental Insulin [NI]
[0583] Oral Insulin [OI]; No supplemental Insulin [NI]; Short
acting Insulin [SI]
[0584] Short acting Insulin [SI]; Oral Insulin [OI]; No
supplemental Insulin [NI]
[0585] Short acting Insulin [SI]; No supplemental Insulin [NI];
Oral Insulin [OI]
[0586] No supplemental Insulin [NI]; Short acting Insulin [SI];
Oral Insulin [OI]
[0587] No supplemental Insulin [NI]; Oral Insulin [OI]; Short
acting Insulin [SI]
[0588] On two separate occasions, subjects received one of the two
possible treatments prior to a standardized breakfast intake: 300 U
oral insulin/400 mg 4-CNAB in 2 capsules (each capsule contained
150 U insulin/200 mg 4-CNAB), 12 U short acting insulin
(Humalog.RTM. injection 100 U/ml from Eli Lilly and Company). At a
third experiment the meal intake was performed without supplemental
insulin. The wash-out period between the experiments was 1-20 days.
The study drugs were given after an overnight fast of approximately
12 hours. The oral administration or the injection was given in the
morning.
[0589] The 4-CNAB used for the capsules was manufactured under GMP
compliance. The Insulin used to prepare the capsules was
Zinc-Insulin Crystals Human: Proinsulin Derived (Recombinant DNA
Origin) USP Quality obtained from Eli Lilly and Company
(Indianapolis, Ind.). The Insulin/4-CNAB capsules contained 150
Insulin Units USP and 200 mg 4-CNAB. The insulin/4-CNAB capsules
were prepared by aaiPharma Inc., Wilmington N.C.
[0590] Insulin/4-CNAB capsules were provided in HDPE bottles, each
of which contained 40 capsules and a polyester coil. Each bottle
had a heat-induction seal and a child-resistant cap, and were
stored frozen at or less than minus 10.degree. C. On the day of
dosing, the appropriate number of capsules was removed from the
freezer and brought to room temperature (between 15 and 30.degree.
C.) for about one hour. Capsules were used within four hours of
dispensing, and unopened bottles were not left at room temperature
for more than four hours.
[0591] Samples were collected for determination of plasma insulin
concentrations, 4-CNAB concentration and c-peptide. Blood glucose
concentrations were determined immediately after sample collection
and documented. All experiments were identical in their sample
collections and monitoring period for all visits. The experimental
procedure after the meal intake will last 6 hours (+2 h baseline
period for stabilization of blood glucose concentrations at the
desired preprandial blood glucose level).
[0592] The study subjects will be released at the end of each
treatment period. At visits 3 and 4, the study subjects will return
to the clinic to receive the alternative treatments in conjunction
with the standardized breakfast according to their randomization
sequence. Only subjects having received all three treatments by the
end of visit 4 will be regarded as completers. A final examination
(visit 5) will be performed after visit 4, preferably immediately
after the experimental procedures were completed, but no longer
than 14 days after visit 4.
[0593] The subjects ingested the meal fifteen minutes after drug
administration. Blood glucose concentrations were monitored for 6
hours after glucose ingestion, and serial blood samples were
collected in regular intervals for measurement of insulin
concentration, 4-CNAB concentration, c-peptide, and blood glucose
as outlined in Table 39, providing information for pharmacokinetic
and pharmacodynamic determinations.
39TABLE 39 Sampling Schedule Plasma Insulin/c- 4CNAB peptide Blood
glucose Study Concentrations concentrations concentrations drug
Time (2 ml per (5 ml per (0.25 ml per adminis- (minutes) sample)
sample) sample) tration -120 x Samples will -60 x be taken -30 X x
every 5-15 0 x min. x 10 X x Altogether, 20 X x the number of 30 X
x samples will 40 X x not exceed 50 x 144 per 60 X x experiment 75
x 90 X x 105 x 120 X x 150 x 180 x 210 x 240 X x 300 x 360 X X
TOTAL 20 100 36 Blood loss
[0594] NOTE: These times are approximate. The sample times may be
changed or deleted. Sampling will not exceed the numbers listed.
Furthermore, x=sample collected.
[0595] The calculations for total blood loss are as follows: Blood
volume requirement for blood samples is about 10 ml for visit 1,
468 ml for visit 2, 3, and 4, and 10 ml for visit 5. Thus, total
blood loss for this study will be approximately 488 ml over a
minimum of 4 days and a maximum days.
[0596] The studies started in the morning. A 17-gauge PTFE catheter
was inserted into a vein of one arm for blood sampling for
measurement of blood glucose, plasma insulin and 4CNAB
concentrations. The line was kept patent with 0.15-mol/L (0.9%)
sterile saline. The catheterized arm was warmed to an air
temperature of approximately 55.degree. C. The first measured
fasting blood glucose of every patient was in the range of 110-180
mg/dl on all treatment days. The first measured blood glucose value
for a single patient was not allowed to differ for more than 40
mg/dl at visit 3 and visit 4 compared to visit 2. If the first
measured blood glucose values were out of this range, the visit was
postponed. A repetition was only be done once for each patient. In
case a second repetition became necessary, the patient was regarded
as having dropped out and was replaced. Blood glucose was checked
in 15 minute intervals before dosing.
[0597] At time-point -15, exogenous insulin was administered by
oral insulin administration or by subcutaneous injection at two of
the three experimental days. At time point 0, subjects ingested a
standardized breakfast at every study day (visits 2 through 4). The
pharmacodynamic response elicited was studied by measurements of
blood glucose concentrations in 5 minute intervals for another 6
hours, and no food intake was allowed during this period, although
water was consumed as desired.
[0598] In case of a hypoglycemia (defined as blood glucose
concentrations below 60 mg/dl), a blood glucose concentration of 60
mg/dl was maintained by means of a variable-rate intravenous
infusion of 20% glucose. The glucose infusion rate was adopted, if
necessary, in relation to the blood glucose concentrations measured
to maintain this blood glucose level. In case of blood glucose
values exceeding 350 mg/dl for more than 60 minutes, the
experiments were aborted and the subject was treated with
additional s.c. insulin to normalize his blood glucose
concentrations.
[0599] Blood samples for the determination of plasma insulin
concentrations, 4CNAB and c-peptide were collected at defined
intervals (see Table 39). Plasma samples were stored at
approximately -20.degree. C. (4CNAB at -70.degree. C.) until
determination by immunoassay is performed. After the end of the
sampling period, the study subjects were released from the
clinic.
[0600] Inter-subject variability for selected pharmacodynamic and
pharmacokinetic parameters was assessed. Incidence of postprandial
hypoglycemia was assessed for each subject and across the study
population.
[0601] Blood glucose excursions (i.e., differences between
pre-prandial and postprandial blood glucose concentrations), the
arithmetic means of which are shown in FIG. 13, registered after
the ingestion of the meal were used to evaluate pharmacodynamic
parameters of the two insulin administration routes and compared
with the same data obtained for the study day without any
supplemental insulin. From these measurements, the area under the
glucose infusion rate versus time curve from 0-6 hours (and other
time intervals), the maximal blood glucose excursion, and time to
the maximal blood glucose excursion were used.
[0602] The pharmacodynamic endpoint was the area under the blood
glucose excursion curve (AUC.sub.BG) in the first two hours after
meal intake (AUC.sub.BG 0-2 h). For pharmacodynamic assessment the
following parameters were calculated: Maximal blood glucose
excursion (BG.sub.max), time to BG.sub.max (TBG.sub.max), Area
under the blood glucose excursion curve in defined time-intervals
(AUC.sub.BG 0-1 h, AUC.sub.BG 0-2 h, AUC.sub.BG 0-3 h, AUC.sub.BG
0-4 h, AUC.sub.BG 0-6 h), maximal absolute blood glucose
concentrations (BGabs.sub.max), time to BGabs.sub.max
(TBGabs.sub.max).
[0603] For pharmacokinetic assessment the following parameters were
calculated: Maximal plasma insulin concentrations (INS.sub.max),
time to INS.sub.max (TINS.sub.max), Area under the glucose infusion
rates in defined time-intervals (AUC.sub.Ins 0-1 h, AUC.sub.Ins 0-2
h, AUC.sub.Ins 0-3 h, AUC.sub.Ins 0-4 h, AUC.sub.Ins 0-6 h) and
maximum reduction of C-peptide concentrations
[0604] Plasma insulin concentrations were be subjected to
appropriate pharmacokinetic analyses. Parameters to be determined
include maximum plasma concentrations (C.sub.max), time to maximum
plasma concentrations (t.sub.max), and the area under the plasma
concentration versus time curve from the time of dosing until a
return to the baseline measurement (AUC.sub.0-t'), where t' is the
time that the level of plasma insulin concentration returns to the
baseline. In addition, other pharmacokinetic parameters, such as
half-life (t.sub.1/2), elimination rate constant (.lambda..sub.z)
and partial AUC values, may be calculated if considered
appropriate. Parameters will be calculated for each individual
subject enrolled within the study.
ADDITIONAL EXAMPLES
[0605] In order that the method of reducing the incidence and/or
severity of one or more disease states associated with chronic
administration of insulin may be better understood, the following
examples are set forth. These examples are for the purpose of
illustration only and are not to be construed as limiting the scope
of the invention in any manner.
[0606] The delivery agent 4-CNAB was prepared by Emisphere
Technologies, Tarrytown, N.Y. Insulin (Zinc, Human Recombinant) was
purchased from Calbiochem (San Diego, Calif.). The Insulin potency
was approximately 26 USP units/mg. Insulin was stored as a
lyophilized solid at -20.degree. C. In solution, it was stored as
frozen aliquots (15 mg/mL) that were subjected to only one
freeze-thaw cycle.
[0607] An aqueous insulin stock solution was prepared (at pH 7.5)
with a final insulin concentration of approximately 15 mg/mL.
Delivery agents were dissolved in water with subsequent additions
of sodium hydroxide or hydrochloric acid to both dissolve the
delivery agent and to titrate the dosing solution to pH 7.5-8.5.
The required amount of insulin was added to the delivery agent
solution before dosing.
[0608] Insulin levels in the rats were assayed using the Insulin
ELISA Test Kit [DSL, Webster, Tex. Cat. #DSL-10-1600]. The assay
covered the range 3.125 to 250 mU/mL. Blood glucose levels in the
rats were measured using a glucometer, One-Touch Basic Blood
Glucose Monitoring System, manufactured by Lifescan Inc. (Milpitas,
Calif.).
[0609] Animal Model
[0610] A total of 60 male Sprague-Dawley rats were fasted for 24
hours and then anesthetized with Thorazine (1.5 mg/kg, im) and
Ketamine (44 mg/kg, im). They were then divided into the following
5 treatment groups:
[0611] 1. H.sub.2O (p.o. 1 mL/kg)
[0612] 2. Carrier (p.o., 1 mL/kg; 200 mg/kg)
[0613] 3. Insulin (p.o., 1 mL/kg; 0.5 mg/kg)
[0614] 4. Insulin and Carrier (p.o., 1 mL/kg; 0.5 mg/kg Insulin and
200 mg/kg 1182)
[0615] 5. Insulin (s.c., 0.05 mg/kg)
[0616] There were twelve animals per group, with three being
sacrificed at 30, 60, 120 and 180 minutes. For serum insulin and
blood glucose monitoring, 0.4 mL of blood was drawn from the tail
artery. Following euthanasia, an aorta sample was removed and
snap-frozen in liquid nitrogen. All animal studies where conducted
in accordance with the IACUC approved protocols.
[0617] Animals received streptozotocin (65 mg/kg, iv) after
acclimation to the facility. Blood glucose was measured at 24, 48,
and 72 hours after injection. Those animals with blood glucose
greater than 150 mg/dl were fasted 12 hours and received treatment
as described.
[0618] Hybridization Sample Preparation
[0619] Total RNA was prepared from the frozen tissue sample
following the protocol for use of Trizol Reagent (Invitrogen, Inc.,
Carlsbad, Calif.). The samples were then further cleaned up by use
of the Qiagen Midi Kit (Valencia, Calif.). Quality of each RNA
sample was assessed using agarose gel electrophoresis and UV
absorbance at 260 and 280. Acceptable RNA samples were pooled in
equal quantities on a mass basis. This pooled sample was used to
prepare cDNA following the protocol provided by Affymetrix. This
sample was then used as template in an in vitro transcription
labeling and amplification reaction using the Enzo BioArray High
Yield RNA Transcript Labeling Kit (Affymetrix Inc., Santa Clara,
Calif.). 15 .mu.g of labeled transcript was then fragmented and
used to prepare a hybridization solution as described in the
Affymetrix GeneChip Protocol, Affymetrix, Inc., 2000.
[0620] GeneChip Analysis.
[0621] The samples were hybridized to a Test Array and 5' to 3'
ratios, detection limit, and image quality were assessed to ensure
the quality of the labeled sample. Acceptable samples were then
hybridized to an Affymetrix Rat U34A array. Washing and staining of
these arrays was performed using the standard Affymetrix protocols.
Array quality was assessed in the same manner as the Test array.
Acceptable samples were analyzed both in the expression patterns
seen across the group as well as pair wise in the following
manner:
[0622] 1. Group 2 vs. Group 1
[0623] 2. Group 3 vs. Group 1
[0624] 3. Group 4 vs. Group 1
[0625] 4. Group 5 vs. Group 1
[0626] 5. Group 4 vs. Group 5
[0627] 6. Group 4 vs. Group 2
EXAMPLE 14
[0628] Fold change were determined by the Affymetrix Microarray
Suite Software package and values below 2 fold were considered
insignificant. This software package analysis compares the
individual members of each probe set to determine a Difference
Call. In this report all calculated fold changes are used in the
figures, however in the results and discussion only those fold
changes that received an Increasing or Decreasing call by the
Affymetrix software were used to draw conclusions. These data are
included in Table 1 below.
40TABLE 1 Fold Change Data From Genechip Analysis of Oral and S.c.
Dosing of Insulin Subcutaneously Direct P.O. to S.C. Orally Dosed
Dosed Comparison Time (minutes) 60 120 180 60 120 180 30 60 120 180
12-LO 2.7 -3.4 -2.1 5.8 4.3 -6.4 -1.6 -2.2 -14.3 3
6-Phosphofructo-2- 1.2 3.3 1.8 1.2 3.2 1.4 -1.3 -1 -1.1 1.3
kinase/fructose-2,6- bisphosphatase a-actin -1.2 1.6 3.8 -1.2 1.6
3.8 1.1 -1.1 1.1 2 c-myc -2.4 -1.6 1.9 1 -1 3.8 -1.1 -2.6 -1.6 1.1
desmin -15.7 -2.8 8.4 -15.7 -2.8 8.4 -1.4 -28.2 6.6 -3 Egr-1 -2.1
1.1 5.6 2.3 1.6 6.9 1.6 -4.7 -1.4 -1.2 Fru-1,6-P -1.7 -1.4 -3.3
-2.2 1 -2.9 -1.3 -1.8 -1.4 -2.2 G6Pase -25.1 -12.2 2.8 1.2 -8.1
17.1 7.1 -35 -6.8 -8.7 Glycogen -14.2 -4.1 1.6 4.6 -33.4 1.8 -1.4
-57.6 10.5 -1.1 Phosphorylase Glycogen synthase -1.1 9.4 -1.4 -2.3
5.3 -1.2 -2.3 2.2 1.8 -1.2 gp 130 3.7 1.3 1.7 3.3 1.8 1.3 1.4 1.2
-1.6 1.4 GSK3 beta -2.5 -1.4 1.5 -4.2 -1 1.4 1.8 1.9 -1.4 1.2
Hexokinase II 1.1 9.8 2.2 -1.6 5.3 1.6 2.8 2.1 2 1.2 HO-1 -4.9 -1.5
2.5 -1.6 5.8 1.8 1.8 -3.3 -6 1.4 ICAM-1 -2.5 -1.1 1.6 -1.9 1.8 2.9
1.9 -1.3 -1.9 -1.8 IGFBP-1 3 -39.2 -2 2.4 -3.1 -1.6 -1.1 1.4 -6.3
-1.2 IGFBP-2 -1.1 -15.8 1.5 -1.3 -6.9 1.1 1.7 1.2 -2.3 1.4 IGFBP-3
-2.5 1.4 2 -1.4 1.6 1.1 -1.2 -1.6 -1.1 1.8 IL-6 -1.7 1.9 -1.7 -2
3.8 1.4 -1.4 1.8 -1.6 -2.4 Jun B -6.1 -6.6 -2.6 -1.3 1.3 1.9 1.3
-3.3 -4 -6.5 PAI-1 -2.6 1.5 -1 1.1 2.6 1.8 -1.1 -2.8 -1.7 -1.6
PAI-2 4.8 2.1 -2 2.3 2.1 -1.4 2.6 2.3 1.3 -1.4 PEPCK -1.6 2.1 -1.4
-1.4 2.1 -1.8 2.8 -1.1 -1 1.3 SM22 1.1 1.1 2.1 1.1 1.1 2.1 -1.5 1.1
1 1.2 vimentin -1.6 3.1 1.3 -1.5 2.8 -1 -1.1 -1 1.1 1.3
[0629] The numbers in bold in Table 1 indicate values that the
Affymetrix Microarray Suite software gave an Increasing or
Decreasing call.
EXAMPLE 15
[0630] Pharmacokinetics and Pharmacodynamics
[0631] FIG. 14 shows a graph of blood glucose (mg/mL) over time
following a single administration with subcutaneous and oral
delivery. This figure shows that the administration of insulin
orally, using 4-CNAB as a carrier, yielded approximately 95% of the
glucose depression seen with the traditional subcutaneous dosing.
However, as shown in FIG. 15, which shows the serum insulin (mU/mL)
over time using the administrations of FIG. 13, the serum insulin
C.sub.max required to achieve this depression however, in the
orally administered animals was approximately 30% of those
receiving subcutaneous injections. The T.sub.max was also about 15
minutes later for the subcutaneous sample. The depression in blood
glucose was likely eliminated by the continued administration of
anesthesia in order to continue blood sampling.
EXAMPLE 16
[0632] Glucose Regulation
[0633] Glycolysis/Gluconeogenesis occurs through three main cycles
that can be driven in both a glycolytic and gluconeogenic
direction. From a glycolytic standpoint, the first cycle is the
Glu/Glu-6-Pase Cycle, which converts glucose to Glc-6-P. This is
followed by the Fru-6-P/Fru-1, 6-P.sub.2 Cycle, and the
Pyruvate/PEPCK Cycle.
Glu/Glu-6-Pase Cycle
[0634] In muscle tissue, glucose is converted to Glc-6-P by
hexokinase. See Granner et al., J Biol Chem 265, 10173-6 (1990). In
the studies, both subcutaneous and orally administered insulin
yielded elevations in the mRNA levels of the enzyme Hexokinase II.
FIG. 16 shows Glucokinase and G6Pase mRNA expression compared to
sham dosing. As shown in FIG. 16, despite the lower serum insulin
levels, the orally dosed animals showed a 2-fold higher level of
hexokinase II at 120 minutes. Direct comparison of the arrays from
the orally dosed and subcutaneously dosed animals indicates a
2.8-fold higher mRNA level at 30 minutes.
Fru-6-P/Fru-1, 6-P.sub.2 Cycle
[0635] The bi-functional enzyme 6-phosphofructo-2-kinase/fructose
2,6-bisphosphatase serves as a switch between gluconeogenesis and
glycolysis. Insulin administration has been shown to drive
increases in this enzyme. Granner et al., J Biol Chem 265, 10173-6.
(1990); Lemaigre et al.,. Biochem J 303, 1-14. (1994); Denton. et
al., Adv Enzyme Regul 36, 183-98 (1996). FIG. 4 shows Fru-1, 6-P
and 6-Phosphofructo-2-kinase/f- ructose-2, 6-bisphosphatase mRNA
expression compared to sham dosing. In our studies and as shown in
FIG. 17, this enzyme showed a nearly identical pattern of
expression between the two routes of administration with no
significant differences in gene expression being observed.
[0636] The enzyme Fructose 1,6-bisphosphatase catalyzes the
conversion of Fru-1, 6-P.sub.2 to Fru-6-P, the gluconeogenesis side
of this cycle. This mRNA is induced by diabetes and starvation and
reduced by insulin administration. As shown in FIG. 17 and similar
to 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase, the
pattern of expression for this enzyme is nearly identical in both
sets of test animals.
Pyruvate/PEP Cycle
[0637] Phosphoenolpyruvate carboxykinase (PEPCK) is a key enzyme in
the gluconeogenesis pathway converting oxaloacetate to
phosphoenolpyruvate. It is known to be down regulated by insulin.
See, for example, Granner et al., J Biol. Chem. 265, 10173-6.
(1990); Lemaigre et al,. Biochem J 303, 1-14. (1994); Denton, R. M.
et al., Adv Enzyme Regul 36, 183-98 (1996); Gabbay et al., J Biol
Chem 271, 1890-7 (1996). FIG. 18 shows PEPCK mRNA expression
compared to sham dosing. As shown in FIG. 18, little difference is
seen in the expression levels of this mRNA.
Glycogen Synthesis
[0638] Insulin is also known to increase the rate of glucose
conversion to glycogen. This is performed by linking glucose-1-P
molecules into a branched chain. This chain then serves as a store
of glucose to be utilized in hypoglycemic states.
[0639] Glycogen Synthase enzyme is responsible for extending the
chain of glucose molecules. Administration of insulin is known to
up-regulate this enzyme. Vestergaard et al., Dan Med Bull 46, 13-34
(1999). FIG. 19 shows glycogen synthase mRNA expression compared to
sham dosing. As shown in FIG. 19, oral dosing and subcutaneous
dosing produced nearly identical patterns of expression in the
levels of this enzyme, with oral dosing yielding nearly twice the
increase in mRNA at 120 minutes.
[0640] The enzyme Glycogen Synthase Kinase 3 is involved in the
inhibition of glycogen synthesis through the phosphorylation of
glycogen synthase. As shown in FIG. 6, the expression pattern of
this enzyme in the two dosing samples was very similar, exhibiting
an initial decrease that returns to sham levels at 120 and 180
minutes. Subcutaneous dosing achieved a slightly stronger down
regulation at 60 minutes; however, this difference was not seen in
the direct comparison between the two GeneChips.
[0641] The enzyme Glycogen Phosphorylase is responsible for the
breakdown of the glycogen chain. Insulin is known to normalize
phosphorylase levels in diabetic animals. In our studies, as shown
in FIG. 19, a dramatic difference in the levels of this enzyme was
observed. The oral dosing achieved an immediate decrease in mRNA
levels that slowly increased to sham levels at 180 minutes. The
subcutaneous dosing yielded an early up regulation that was
reversed dramatically at 120 minutes and returned to near sham
levels at 180 minutes. The differences seen between the oral and
subcutaneous dosings were observed in comparison to sham as well as
to each other.
EXAMPLE 17
[0642] Vascular Response to Injury
[0643] Vascular diseases are commonly described as a response to
injury. The vessel is exposed to a stimulus (injury) that leads to
a progression of responses designed to repair damage to the vessel
wall. This injury may be in several forms, including oxidative
stress, mechanical stress, viral infection and changes in shear
stress. Though the injury itself is variable, the response to
injury has many common aspects. Early response genes are up
regulated leading to the transcription of genes for cellular
migration and proliferation as well as the recruitment of
inflammatory cells to the site of injury. As the response
continues, enzymes that lead to matrix remodeling will be
expressed. The result is generally the thickening of the arterial
wall through smooth muscle proliferation and atherosclerotic plaque
formation. The clinical result is the arteriopathies associated
with diabetes. In this application, a method for examining the mRNA
levels of genes associated with various forms of vascular injury is
described.
[0644] Vascular diseases are a complex set of processes that
involve numerous changes in mRNA levels. While the mRNA markers of
vascular injury presented here were seen in this specific study,
several others are likely to exist. These include early response
genes (i.e. c-myb and c-fos), cytokines (i.e. interleukins, and
chemokines), growth factors and their receptors (i.e. fibroblast
growth factor, vascular endothelial growth factor, and transforming
growth factor beta), adhesion molecules (i.e. selectins, and
integrins), extracellular matrix proteins (i.e. collagen and
actin), matrix metalloproteinases and their inhibitors, cell cycle
proteins (i.e. cyclins and cyclin dependent kinases), and protein
kinases (i.e. mitogen activated protein kinases, and protein kinase
C), some of which are presented here. This list continues to grow
as vascular disease becomes better understood, and which markers
are in a particular sample may vary.
[0645] Early Response Genes
[0646] One of the initial markers of arterial injury is the
expression of transcription factors in control of the subsequent
expression of proteins responsible for potentiating the vascular
response to injury. These early response genes include c-myc,
c-fos, jun, and Egr-1. FIGS. 20A and 20B show early response gene
mRNA expression compared to sham dosing. In our studies, and as
shown in FIGS. 20A and 20B, a differential expression in the levels
of Egr-1, c-myc, Jun B and Ets-1 was observed.
[0647] Egr-1 is associated with several elements of the vascular
response to injury. Its expression is very low in uninjured vessels
but increases with mechanical or oxidative injury. Egr-1 has been
demonstrated to drive increases in mRNA levels of cytokines,
adhesion molecules, growth factors and members of the coagulation
cascade. In our study and as shown in FIGS. 20A and 20B, Egr-1 is
immediately up regulated by subcutaneous insulin administration to
a level 4.7-fold higher than with oral. Oral administration does
not induce an early increase in Egr-1 mRNA levels. Instead, levels
are maintained at near sham levels until at 180 minutes when they
are elevated to slightly below that of the subcutaneously
animals.
[0648] Balloon injury to rat aortae leads to a rapid increase in
mRNA for Jun B. Jun and Fos bind to form the heterodimeric
transcription factor AP-1. This factor leads to the expression of
adhesion molecules, cytokines, and other factors involved in the
response to injury. As shown in FIGS. 20A and 20B, Jun B expression
remained near sham levels at all time points; however, direct
comparison between arrays indicates significantly higher levels in
the subcutaneously dosed samples at 120 and 180 minutes. Though
both levels are near control at 120 and 180 minutes, a comparison
between the two arrays shows a significant decrease in expression
in the orally dosed sample.
[0649] The switch in the cell cycle state from the dormant G.sub.0
to the proliferative G.sub.1 is accompanied by increased levels of
c-myc. Vascular damage induces expression of c-myc, and inhibition
of c-myc through antisense oligonucleotides prevents intimal
hyperplasia following balloon injury in the rat and porcine. It is
therefore a critical marker of vascular injury. As shown in FIGS.
20A and 20B, subcutaneous dosing and not oral dosing lead to a
significant increase in the mRNA levels of c-myc at 180 minutes.
The orally dosed samples remained at near sham levels; however no
significant difference between the two dosing routes was seen when
compared directly.
[0650] Ets-1 mRNA levels for subcutaneous and oral dosing are shown
in FIG. 20A.
EXAMPLE 18
[0651] Insulin-Like Growth Factor Family
[0652] Insulin-like growth factor (IGF) I and II are a single chain
polypeptides sharing homology with proinsulin. They play an
important role in systemic glucose metabolism but have also been
shown to effect cell cycle progression, mitogenesis, cell migration
and apoptosis. Much of IGF's function is regulated by IGF-binding
proteins (IGFBPs). In general, IGFBPs bind to IGF, preventing its
binding to the IGF receptor. IGFBP-3 is the most prevalent in this
respect, binding >90% of the IGF in adult serum. Though their
primary function is the regulation of IGF, IGFBPs have been shown
to have a biological effect at sites of vascular injury. IGFBP-1
stimulates the migration of vascular smooth muscle cells (VSMC)
independent of IGF-1. IGFBP-1 to -5 have been shown to be expressed
in restenotic tissue suggesting a role in the arterial response to
vascular injury.
[0653] FIG. 21 shows IGFBP mRNA expression compared to sham dosing.
As shown in FIG. 21, no significant difference in the expression of
IGF-1, IGF-2 or the IGF receptor was seen between the two dosing
routes. However, there were drastic differences seen with the
IGFBPs. IGFBP-1 was reduced 39-fold as opposed to 3-fold, and
IGFBP-2 was reduced 15-fold compared to 6-fold at two hours
compared to the sham dosing. In both cases the IGFBP expression is
decreased, opposite to the effect seen in vascular injury. The
mechanism driving this change may be beyond that of a direct effect
of insulin on the VSMC's. Nonetheless, it is clear that the level
of IGFBP-1 and -2 expression is higher in the subcutaneously dosed
animals and this correlates with an increased response to injury.
Little change was observed in IGFBP-3.
EXAMPLE 19
[0654] Adhesion Molecules
[0655] One of the initial steps in arteriopathies is the adhesion
of inflammatory cells to the vessel wall. This is mediated by
adhesion molecules, such as intercellular adhesion molecule-1
(ICAM-1), vascular cellular adhesion molecule-1 (VCAM-1), the
selectins and the integrins. Changes in the levels of mRNA for
these genes were examined, and FIG. 22 shows intercellular adhesion
molecule-1 mRNA expression compared to sham dosing. As shown in
FIG. 22, no significant effect was seen in either dosing group
except in the case of ICAM-1. ICAM-1 was increased at 180 minutes
in the subcutaneously dosed animals. Increased expression of ICAM-1
is seen in several different forms of vascular injury, and is
associated with the recruitment of inflammatory cells to the site
of injury. This difference is seen both in the comparison to sham
and in the direct comparison of the arrays from the two dosing
groups.
EXAMPLE 20
[0656] Cytokines
[0657] Sites of vascular injury communicate their inflamed state
via the expression of pro-inflammatory cytokines that through both
autocrine and paracrine effects regulate the expression of growth
factors, cytokines, adhesion factors, and matrix
metalloproteinases. Of these one found commonly at sites of
vascular disease is interleukin-6 (IL-6). VSMCs are not initially
susceptible to IL-6 stimulation as they do not express either the
IL-6 receptor or glycoprotein 130 (gp130), both of which allow IL-6
signaling. VSMCs are the first cells in which gp130 has been shown
to be up regulated. FIGS. 23A and 23B show cytokine mRNA expression
compared to sham dosing. In our studies and as shown in FIGS. 10A
and 10B, gp130 was seen to be equally increased in both
subcutaneously and orally dosed animals. However, a significant
increase in IL-6 mRNA was seen only in the subcutaneously dosed
group, as shown in FIGS. 23A and 23B. This is a critical difference
as it shows the aorta of the both groups getting "primed" for a
response to injury, but only the subcutaneous dosing actually
drives significant production of the expected signal. Cytokine data
are represented graphically also for the cytokines Eotaxin, MCP-1,
IL-12 and EL-13 in FIG. 23A.
EXAMPLE 21
[0658] Lipid Peroxidation
[0659] Several proteins associated with the metabolism of lipids
and the oxidation of LDL have been implicated in the progression of
atherosclerosis. It has been suggested that the oxidation of LDL
produces agents that recruit monocytes, promote their adhesion to
the endothelium, and inhibit macrophages from migrating. These
steps lead to the formation of foam cells and the fatty streak
found in atherosclerotic lesions. 12-lipoxygenase (12-LO) has been
demonstrated to drive atherosclerotic lesion formation, and it has
also been documented to be significantly up regulated in the
vascular response to injury. It therefore is of critical importance
in this setting.
[0660] FIG. 24 shows lipid peroxidation mRNA expression compared
with sham dosing. As shown in FIG. 24, at 60 minutes, there is a
5.8-fold increase in the subcutaneously dosed samples as compared
to a 2.7-fold in the orally dosed. In the orally dosed samples,
this up-regulation is reversed at 120 and 180 minutes with 3.4- and
2.1-fold reductions in mRNA levels compared to sham. In the
subcutaneously dosed animals, the mRNA levels remain high until 180
minutes, at which time a 6.4-fold decrease is observed. It is
important to note that the values at 120 minutes represent greater
than 14-fold higher levels of this mRNA in the subcutaneously dosed
samples.
[0661] Heme Oxygenase-1 (HO-1) is induced by mildly oxidized LDL.
It serves a protective antioxidant function through elimination of
heme and the further antioxidant capabilities of its reaction
products. As shown in FIG. 24, the mRNA levels of this gene are
seen to be 6-fold higher in the subcutaneously dosed animals when
compared to the orally dosed animals at 60 minutes. Though the
function of this enzyme is protective, its up regulation represents
a response to injury and may well be in response to the increased
levels of 12-LO or its stimulation of LDL oxidation.
EXAMPLE 22
[0662] Thrombosis
[0663] Fibrin deposition within the arterial wall is believed to
play a major role in atherosclerosis. Through their fibrinolytic
activity, the plasminogen activators block this from occurring.
These protective actions are blocked by the plasminogen activator
inhibitors (PAI-1 and -2). FIG. 25 shows plasminogen activator
inhibitors mRNA expression compared to sham dosing. In our study,
as shown in FIG. 25, PAI-1 levels were elevated in the subcutaneous
samples only. A 2.6-fold increase over sham and a 2.8-fold increase
over oral were seen at 120 minutes. A 2.6-fold decrease in these
levels was seen in the oral samples at 60 minutes, which returned
to sham levels at 120 and 180 minutes. PAI-2 expression was similar
in both sets of dosing. At 60 minutes, the orally dosed samples
exhibited a significantly greater (4.8-fold) level of PAI-2. This
elevation is not present at 120 and 180 minutes. There was no
significant difference between the two dosing routes for this
mRNA.
EXAMPLE 23
[0664] Additional markers are illustrated in FIG. 26, which
compares the effects of subcutaneous delivery of insulin and oral
delivery of insulin on the mRNA expression of NPY, TGF-beta, ICAM-1
and 12-LO.
EXAMPLE 24
[0665] Comparison of mRNA expression between subcutaneous delivery
of insulin and oral delivery of insulin are shown for the markers
THY-1, VEGF-B and Integrin aE2 in FIG. 27. For the oral delivery
data, the effects on mRNA expression of two different dosages are
shown.
EXAMPLE 25
[0666] Pharmacokinetics and Pharmacodynamics in a Streptozotocin
Diabetic Model
[0667] FIG. 28 shows a graph of blood glucose (mg/mL) over time
following a single administration with subcutaneous and oral
delivery for a streptozotocin diabetic model. Two different oral
dosages of insulin are demonstrated. FIG. 29 shows the serum
insulin levels (mU/mL) over time using the administrations of FIG.
28.
[0668] Controls for Gene Chip Studies
[0669] As control groups, the carrier and insulin were orally
administered separately. The results of these GeneChips were
analyzed to identify any possible activities of the individual
components of the composition. The carrier alone samples generated
no consistent and significant changes in mRNA levels in the aorta.
The same is true for the insulin alone samples.
[0670] Discussion
[0671] The examples discussed above demonstrate the ability of an
oral composition of insulin to alleviate the undesirable effects on
the vasculature of the traditional subcutaneous dosing at the level
of messenger RNA regulation, and document changes in glucose
metabolism caused by altering the dosing route. The pharmacodynamic
data demonstrates the ability of the orally dosed composition to
achieve a glucose depression similar to that of the traditional
dosing method. Though the pharmacodynamic data is similar, the
pharmacokinetic data shows a greatly lower serum insulin level in
the orally dosed composition compared with that of the traditional
dosing method. . The difference in serum insulin level must be a
result of direct administration of the insulin to the liver. The
liver reacts to the bolus of insulin in two ways. First, it
accelerates glycolysis, glycogen synthesis and other mechanisms
associated with hyperinsulinemia. Second, first-pass metabolism
decreases the level of insulin reaching the systemic circulation.
The result is a rapid decrease in blood glucose and a decrease in
the level of insulin to which the systemic circulation is exposed.
In achieving a similar glucose control as subcutaneous dosing while
lowering the exposure of the peripheral circulation to insulin, the
undesirable effects of insulin on non-target tissues can be
prevented.
[0672] Although not considered a major site of glucose metabolism,
VSMCs do possess glucose regulatory capacity and therefore yield
insight into differences in the peripheral response to changing the
dosing route. What is surprising is that despite drastically lower
levels of circulating insulin, little difference in the mRNA levels
of key enzymes involved in glucose regulation is observed. In fact,
the levels seen for hexokinase II and glycogen synthase suggest a
stronger response to the oral composition. We conclude that natural
regulation of glucose involves the liver controlling peripheral
glucose metabolism and utilization through a messenger other than
insulin. The fact that higher circulating levels of insulin can
compensate for the loss of this natural process may simply be due
to the fact that the two proteins bind the same or similar
receptors.
[0673] The IGF system is a prime candidate for such secondary
signaling and is known to exhibit glucose regulatory activity.
IGFBP-1 and -2 were down regulated in both sets of data. This is
contrary to published data on vascular injury and may not be
associated with a vascular injury response so much as playing a
role in glucose control. The data previously reported on the
liver's response to changes in dosing route of insulin does not
demonstrate a differential response in either the IGF's or their
binding proteins. This does not rule out this pathway, as no data
is available on the proteases responsible for degradation of the
IGFBP's. It is quite possible that the liver responds to elevated
insulin levels by releasing IGFBP proteases that then degrade
IGFBP's freeing IGF to drive a reduction in glucose. Further study
is required to determine if this is the case, but the liver and
aorta gene array data supports this hypothesis. If correct, this
hypothesis supports the use of an oral composition of insulin
simply based on its ability to mimic the natural glucose control
pathway.
[0674] There are numerous disease states related to diabetes,
including associated neuropathies, nephropathies and retinopathies.
These may be due, at least in part, to the degradation of the
microvasculature after chronic dosing of insulin. Because orally
administered insulin can achieve a greater glucose depression with
a lower systemic level of insulin, a lower incidence of diabetes
related disorders results.
[0675] Using gene micro-arrays, we were able to monitor numerous
areas of the vascular response to injury in animals receiving our
oral composition and the traditional subcutaneous insulin. The
first step is to assess any effects from administration of the
carrier alone. It is believed that, upon entering the systemic
circulation, the carrier and insulin no longer interact due to the
dilution effect. An exhaustive analysis of the array data from
animals receiving the carrier without insulin was performed
including adding a second three-hour experiment to try to further
identify any response. The analysis yielded no mRNA's whose levels
appear to be affected by administration of the carrier. To clarify,
any response seen in the carrier alone samples was also seen in the
animals receiving insulin orally without carrier suggesting that
the effect was due to an experimental parameter not accounted for
in the sham dosing. This study is not designed to identify specific
genes regulated by the carrier, as such a study would require
multiple animals at each time point. Nonetheless, this data
supports the view that the carrier has a minimal effect on the
vasculature.
[0676] The vascular response to injury is a complex set of
processes that occur over an extended period of time. Some of
these, such as atherosclerotic plaque formation, occur over years
or even decades, while the more rapid examples, such as Restenosis,
occur on the order of months. Thus, the time scale for studying
vascular damage in animal models is on the order of days or weeks
and not hours. In this study, we aimed to identify any signs of
vascular injury induced by a single dose of insulin and to document
any effect changing the route of administration had on these
markers. While this may have been a rather optimistic approach,
since the type of injury is mild and the time course very short
compared to standard models of vascular injury, the results quite
remarkably demonstrate qualitatively that oral dosing of insulin
beyond simply mimicking the natural route of entry, also attenuates
the injury to the vasculature.
[0677] It was determined that subcutaneous insulin dosing lead to
higher levels of three key early response genes, while a
significant increase in only one of these genes was seen with oral
dosing. Likewise, elevated levels of IL-6 and ICAM-1 were seen only
in the subcutaneously dosed animals. These genes represent the
early signs of the cell proliferation as well as the start of an
inflammatory response, and their expression can trigger a cascade
of events leading to deterioration of the vessel wall. Subcutaneous
dosing also generated higher levels of 12-lipoxygenase, PAI-1,
PAI-2 and heme oxygenase-1. This second set of genes can be
responsible for creating further injury to the vasculature in the
form of thrombus and oxidized LDL. Repeated expression of these
genes could lead to atherosclerosis and thrombosis. It was found
that oral dosing of insulin prevented elevations in all of these
genes, except an elevation of PAI-2 that was not significantly
different from that seen with subcutaneous dosing. Together, this
data suggest the clear advantage of oral dosing of insulin over
subcutaneous dosing of insulin of lessened incidence of vascular
diseases.
[0678] The data from the subcutaneously dosed animals presents a
picture of a healthy aorta at the earliest stages of an extended
vascular response to injury. The data from the orally dosed animals
clearly indicates a dramatic attenuation of this response. By
administering insulin orally, elevations in the levels of genes
associated with cellular proliferation and migration, inflammatory
cell recruitment, and atherosclerotic plaque formation were almost
entirely avoided.
[0679] It is remarkable that this difference is so clear even
following only a single administration of insulin. It was initially
believed that multiple dosings would be required before a clear
difference in aorta mRNA levels was achieved. In light of this
data, it is easy to see how chronic subcutaneous dosing can lead to
the increased incidence of vascular diseases and their associated
clinical complications. Ongoing studies are currently being
conducted which support the increased incidence of vascular disease
in chronic subcutaneous dosing. Furthermore, the data suggests that
the peripheral glucose metabolism may be similar despite a decrease
in circulating insulin levels.
[0680] Our results show that returning insulin delivery to its
natural site of entry into the circulation and consequently
lowering the peripheral insulin levels can achieve a lower
incidence of the diseases associated with diabetes. While we have
described a number of embodiments of this invention, various
modifications of the invention in addition to those shown and
described herein will become apparent to those skilled in the art
from the foregoing description. Such modifications are intended to
fall within the scope of the appended claims.
[0681] In the preceding specification, the invention has been
described with reference to specific exemplary embodiments and
examples thereof. It will, however, be evident that various
modifications and changes may be made thereto without departing
from the broader spirit and scope of the invention as set forth in
the claims that follow. The specification and drawings are
accordingly to be regarded in an illustrative manner rather than a
restrictive sense.
[0682] While we have hereinbefore described a number of embodiments
of this invention, it is apparent that our basic constructions can
be altered to provide other embodiments which utilize the processes
and compositions of this invention. Therefore, it will be
appreciated that the scope of this invention is to be defined by
the claims appended hereto rather than by the specific embodiments
which have been presented hereinbefore by way of example.
* * * * *