Weight loss abstract
The present invention provides methods for using human placental
alkaline phosphatase or an active derivative to reduce blood glucose
level in a mammal. Treatment regimens provided by the invention
maybe used to control Type 1 and Type 2 forms of diabetes in humans.
The methods and treatment regimens maybe effective to maintain the
human's blood glucose level below about 10 mM, and preferably within
the normal range of 4 mM to 7 mM. The methods and treatment regimens
may be used in combination with administration of known anti-diabetic
medicaments. Also provided by the invention is a method for inducing
weight loss or reducing an expected weight gain caused by or associated
with obesity or Type 2 diabetes. The invention further provides
a preparation for administration to a human, the preparation comprising
homogeneous purified human placental alkaline phosphatase in a physiologically
acceptable carrier.
Weight loss claims
What is claimed is:
1. A method for treating a human to induce weight loss or to reduce
weight gain comprising regularly administering purified human placental
alkaline phosphatase, or an active derivative, to the human in an
effective amount to induce weight loss, to reduce an expected weight
gain, or to maintain a constant body weight for the human over time.
2. The method of claim 1, wherein the human is afflicted with Type
2 diabetes.
3. The method of claim 1, wherein the human is obese.
4. The method of claim 1, wherein the human is obese and non-diabetic,
but has an elevated blood glucose level prior to regular administration
of placental alkaline phosphatase or active derivative.
5. The method of claim 1 wherein the step of regular administration
includes administration of human placental alkaline phosphatase
or active derivative about once per two weeks.
6. The method of claim 1 wherein the step of regular administration
includes administration of human placental alkaline phosphatase
or active derivative about once per week.
7. The method of claim 1 wherein the step of regular administration
includes administration of human placental alkaline phosphatase
or active derivative about twice per week.
8. The method of claim 1 wherein the step of regular administration
includes administration of human placental alkaline phosphatase
or active derivative about once per day.
9. The method of claim 1 wherein the effective amount is in the
range of about 0.2 grams to about 3 grams per square meter of calculated
surface area for the human.
10. The method of claim 1 wherein the effective amount is in the
range of about 0.2 grams to about 1 gram per square meter of calculated
surface area for the human.
11. The method of claim 1 wherein the effective amount is in the
range of about 1 gram to about 3 grams per square meter of calculated
surface area for the human.
12. The method of claim 1 wherein the step of administering is
performed by injection of a preparation comprising: a) a physiologically
acceptable carrier; and b) human placental alkaline phosphatase
or active derivative, dissolved or dispersed in the carrier.
13. The method of claim 12 wherein the mode of injection is selected
from intravenous, subcutaneous, intraperitoneal, intramuscular,
and intradermal.
14. The method of claim 12 wherein the preparation comprises homogeneous
purified human placental alkaline phosphatase.
Weight loss description
TECHNICAL FIELD
The present invention relates to methods for using human placental
alkaline phosphatase, an enzyme produced by human placenta during
pregnancy, to reduce blood glucose level in a mammal. Treatment
regimens provided by the invention maybe used to control Type 1
and Type 2 forms of diabetes in humans.
Significant recent changes in human behavior and lifestyle as well
as the human environment have resulted in the escalation of diabetes
during the last decades. Diabetes is a disease characterized by
elevated levels of blood plasma glucose, or hyperglycemia. Hyperglycemia,
if uncontrolled, can lead to other complications, such as blindness,
kidney disease, heart disease, stroke, nerve diseases, circulatory
disorders, and impotence in males. Diabetes is a chronic disease
with diverse pathologic manifestations, and is accompanied by lipid
metabolism and cardiovascular disorders as well as glycometabolism
disorders.
Diabetes mellitus is a heterogeneous group of disorders characterized
by high blood glucose (sugar) levels. There are two main types of
diabetes. Type 1, or insulin-dependent diabetes, results from a
deficiency of insulin due to autoimmunological destruction of the
insulin-producing pancreatic .beta.-cell islets [Bell, G. I. and
Polonsky, K. S., "Diabetes mellitus and genetically programmed
defects in .beta.-cell function." Nature 414, 788 791 (2001);
Mathis, D., Vence, L. and Benoist, C., ".beta.-cell death during
progression to diabetes." Nature 414, 792 798 (2001)]. People
with Type 1 diabetes must take exogenous insulin for survival to
prevent the development of ketoacidosis.
In Type 2 diabetes, or non-insulin-dependent diabetes mellitus
(NIDDM), muscle, fat, and liver cells are resistant to the actions
of insulin. Furthermore, compensatory mechanisms that are activated
in .beta.-cells to secrete more insulin to maintain blood glucose
levels within a normal physiological range fail to function properly.
Type 2 diabetes accounts for about 90% of all diabetes [Saltiel,
A. R., "New perspectives into the molecular pathogenesis and
treatment of Type 2 diabetes." Cell 104, 517 529 (2001)]. Type
2 diabetics are often prescribed blood glucose-lowering sulfonylurea-based
or -derived drugs, which are associated with the stimulation of
insulin production in the pancreatic .beta.-cells. Alternatively,
patients suffering from Type 2 diabetes may also be prescribed biguanide-based
or -derived drugs, which are associated with increasing a patient's
sensitivity to insulin.
Diabetes already afflicts an estimated 6% of the adult population
in Western society, and its worldwide frequency is projected to
grow by 6% per annum, potentially reaching a total of 200 300 million
cases in 2010 [Zimmet, P., Alberti, K. G. M. and Shaw, J., "Global
and societal implications of the diabetes epidemic." Nature
414, 782 787 (2001)]. The main forces driving this increasing incidence
are sedentary lifestyle and a staggering increase in obesity.
Diabetes is a potentially very dangerous disease because it is
associated with markedly increased incidence of coronary, cerebral,
and peripheral artery disease. As a result, patients with diabetes
have a much higher risk of myocardial infarction, stroke, limb amputation,
renal failure, or blindness. Atherosclerotic cardiovascular disease
is responsible for 80% of diabetic mortality and more than 75% of
all hospitalizations for diabetic complications [Moller, D. E.,
"New drug targets for Type 2 diabetes and the metabolic syndrome."
Nature 414, 821 827(2001)]. Recent evidence indicate that hyperglycemia
leads to overproduction of superoxide accounting for vascular damage,
which, in turn, underlies most diabetic complications [Brownlee,
M., "Biochemistry and molecular cell biology of diabetic complications."
Nature 414, 813 820 (2001); Ho, E. and Bray, T. M., "Antioxidants,
NF.kappa.B activation, and diabetogenesis." Proc. Soc. Exp.
Biol. Med. 222, 205 213 (1999)].
Despite large variations in carbohydrate intake with various meals,
blood glucose normally remains in a narrow range between 4 and 7
mM in non-diabetic individuals. Such tight control is regulated
by the balance among three major mechanisms, i.e. (i) glucose absorption
from the intestine, (ii) glucose production by the liver, and (ii)
uptake and metabolism of glucose by the peripheral tissues, mainly
the skeletal muscle and fat tissue. In skeletal muscle and fat tissue,
insulin increases the uptake of glucose, increases the conversion
of glucose to glycogen, and increases conversion of glucose to fat
(mainly triglycerides). In the liver, insulin inhibits the release
of glucose from glycogen. Insulin is the only known hormone which
can regulate all three mechanisms required to maintain the blood
glucose level in the normal range [Saltiel, A. R. and Kahn, C. R.,
"Insulin signaling and the regulation of glucose and lipid
metabolism." Nature 414, 799 806 (2001)].
At present, the only established available treatment for severe
Type 1 diabetes is daily (often multiple) insulin injection. Apart
from the inconvenience of the injection procedure, several adverse
effects may accompany insulin treatment, including occasionally
severe hypoglycemia (lower than normal blood glucose level) and
weight gain; unfortunately, this latter side effect can make the
target tissues even more resistant to the actions of insulin [U.K.
Prospective Diabetes Study Group, "U.K. Prospective Diabetes
Study 16. Overview of 6 years' therapy of Type 2 diabetes: A progressive
disease." Diabetes 44, 1249 1258 (1995)]. On an experimental
basis, clinical islet transplantation is also gaining acceptance,
particularly for patients with hypoglycemic awareness [Ryan, E.
A., Lakey, J. R. T., Paty, B. W., Imes, S., Korbutt, G. S., Kneteman,
N. M., Bigam, D., Rajotte, R. V. and Shapiro, A. M. J., "Successful
islet transplantation: Continued insulin reserve provides long-term
glycemic control." Diabetes 51, 2148 2157 (2002)]; however,
this method is still far from established. Apart from the problem
that it is very difficult to adjust insulin production to meet the
patient's needs, immuno-suppressants, which are used to prevent
rejection of the transplanted tissue, can exert potent and undesirable
side effects. It is obvious that even partial replacement of insulin
with a safer and effective agent that does not require a complicated
surgical procedure would greatly benefit Type 1 diabetic patients.
In Type 2 diabetes, an aggressive control of hyperglycemia by medication
is essential; otherwise this disease will progress into the even
more dangerous Type 1 diabetes. Several drugs in five major categories,
each acting by a different mechanism, are available for this purpose
[Moller, D. E., "New drug targets for Type 2 diabetes and the
metabolic syndrome." Nature 414, 821 827 (2001)]: (A) Insulin
secretogogues, including sulphonylureas (e.g., glipizide, glimepiride,
glyburide) and meglitinides (e.g., nateglidine and repaglinide),
enhance secretion of insulin by acting on the pancreatic .beta.-cells.
While this therapy can decrease blood glucose level, it has limited
efficacy and tolerability. In addition, it causes weight gain and
often induces hypoglycemia. Finally, patients often become refractory
to this treatment. (B) Biguanides (e.g., metformin) are thought
to act primarily by decreasing glucose production in the liver.
Biguanides often cause gastrointestinal disturbances and lactic
acidosis, which limits their use. (C) Inhibitors of .alpha.-glucosidase
(e.g., acarbose) decrease absorption of glucose from the intestine.
These agents also often cause gastrointestinal disturbances. (D)
Thiazolidinediones (e.g., pioglitazone, rosiglitazone) act on a
specific receptor (peroxisome proliferator-activated receptor-gamma
(PPAR.gamma.)) in the liver, muscle and fat tissues. They regulate
lipid metabolism and thus enhance the response of these tissues
to the actions of insulin. Frequent use of these drugs may lead
to weight gain and may induce edema and anemia. (E) Insulin is used
in more severe cases, either alone or in combination with the above
agents. All these medications are given to the patient daily, often
two or three times a day.
Because each agent that is being used in the medical treatment
of diabetes has either significant side effects or causes weight
gain (which, in turn, further impairs the actions of insulin), or
both, newer approaches to control Type 2 diabetes are desperately
needed. An effective new treatment would meet the following criteria:
(a) it would not have significant side effects including induction
of hypoglycemia; (b) it would not cause weight gain; (c) it would
at least partially replace insulin by acting via mechanism(s) that
are independent from the actions of insulin; (d) it would desirably
be metabolically stable to allow less frequent (e.g., once per week)
usage; and (e) it would be usable in combination with tolerable
amounts of any of the categories of drugs listed above.
Placental alkaline phosphatase (PALP) is a member of the alkaline
phosphatase group of enzymes that hydrolyze phosphate-containing
compounds at alkaline pH. Mature PALP is a dimer of two identical
glycosylated subunits. A primary source of PALP is human placenta,
which synthesizes this enzyme during pregnancy so that toward the
end of third term the enzyme's level in the placenta tissue and
maternal/fetal blood becomes very high. Therefore, it is very unlikely
that human PALP exerts toxic or pathological effects in human tissues.
Subunits of human placental alkane phosphatase have an approximate
molecular weight of 66 kDa, as determined by gel electrophoresis.
A determination of an in vivo half-life for human PALP was reported
in 1965 [Clubb, J. S., Neale, F. C. and Posen, S., "The behavior
of infused human placental alkaline phosphatase in human subjects."
J. Lab. & Clin. Med. 66, 493 507 (1965)]. In human subjects,
injected PALP is reported to remain remarkably stable in the circulation,
with an estimated biological half-life of about 7 days. In the reported
experiments, PALP was injected as a minor constituent in a mixture
of PALP and albumin obtained by extraction, without further purification.
The authors reported that PALP up to serum concentration of 975
"King-Armstrong" (KA) units appeared metabolically inert,
and hypothesized that PALP performs no measurable physiological
function in circulation.
The physiological function of PALP has been unknown until recently,
when Kiss and his co-workers discovered that, in human fetus and
mouse embryo fibroblasts, the enzyme functions both as a growth
factor and a promoter of survival in serum factor-deficient culture
medium [She, Q.-B., Mukherjee, J. J., Huang, J.-S., Crilly, K. S.
and Kiss, Z., "Growth factor-like effects of placental alkaline
phosphatase in human and mouse embryo fibroblasts." FEBS Lett.
469, 163 167 (2000); She, Q.-B., Mukherjee, J.-J., Chung, T. and
Kiss, Z., "Placental alkaline phosphatase, insulin, and adenine
nucleotides or adenosine synergistically promote long-term survival
of serum-starved mouse embryo and human fetus fibroblasts."
Cellular Signalling 12, 659 665 (2000)].
SUMMARY OF THE INVENTION
In one embodiment, the present invention provides a method of reducing
blood glucose level in a mammal, comprising the step of administering
a therapeutically effective amount of purified human placental alkaline
phosphatase, or an active derivative. The mammal may be a human.
The method may be effective to reduce a human's blood glucose level
to below about 10 mM, and preferably into the normal range of 4
mM to 7 mM. The method maybe used on a diabetic human, and may be
used in combination with administration of an anti-diabetic medicament.
In another embodiment, the invention provides a treatment regimen
for treating diabetes, comprising periodic administration to a diabetic
human of a therapeutically effective amount of purified human placental
alkaline phosphatase, or an active derivative. The treatment regimen
may be used in combination with administration of an anti-diabetic
medicament. The treatment regimen may be effective to maintain the
human's blood glucose level below about 10 mM, and preferably within
the normal range of 4 mM to 7 mM.
In another embodiment, the invention provides a preparation for
administration to a human, the preparation comprising homogeneous
purified human placental alkaline phosphatase in a physiologically
acceptable carrier.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows a picture of a gel separation, demonstrating that
homogeneous purified PALP used for the experiments described in
Examples 21, 22, and 24 does not contain any contaminating protein.
FIG. 2 demonstrates that in BDF1 male fasting mice, administration
of a preparation comprising commercial PALP prior to a glucose load
substantially reduced an increase in blood glucose level.
FIG. 3 demonstrates that in BDF1 male mice, intraperitoneal application
of streptozotocin increased blood glucose level about 9-fold in
three days (.box-solid.), and that prior administration of a preparation
comprising commercial PALP greatly reduced such increase in glucose
level (.diamond-solid.).
FIG. 4 demonstrates that in BDF1 male mice, intraperitoneal application
of streptozotocin considerably decreased body weight over an eight
day period (.box-solid.), and that prior administration of a preparation
comprising commercial PALP partially prevented the decrease in body
weight (.diamond-solid.).
FIG. 5 indicates that in differentiated L1 adipocyte cells, commercial
PALP stimulates cellular uptake of D-[.sup.14C]glucose at various
levels of glucose and serum in the incubation medium.
FIG. 6 demonstrates that in differentiated L1 adipocyte cells,
commercial PALP stimulates the cellular synthesis of glycogen from
D-[.sup.14C]glucose at various compositions of the incubation medium.
FIG. 7 demonstrates that in differentiated L1 adipocyte cells,
commercial PALP stimulates the cellular synthesis of lipids from
D-[.sup.14C]glucose at various compositions of the incubation medium.
FIG. 8 demonstrates that in differentiated L6 muscle cells, both
1 U/mL commercial PALP and 50 nM insulin enhance the uptake and
metabolism of D-[.sup.14C]glucose after treatments for 6 hours;
PALP and insulin in combination had approximately additive effects.
FIG. 9 shows a picture of a gel separation demonstrating that digestion
of human PALP with bromelain results in the formation of a major
fragment and several smaller fragments, concomitant with the disappearance
of native PALP enzyme.
FIG. 10 demonstrates that in differentiated L1 adipocyte cells,
a product of the digestion of human PALP by bromelain enhances the
synthesis of glycogen, but not lipids, from D-[.sup.14C]glucose,
relative to native PALP. Bromelain did not affect the total cellular
level of D-[.sup.14C]glucose, but slightly enhanced glycogen and
lipid synthesis.
FIG. 11 demonstrates that in differentiated L1 adipocyte cells,
treatment with either commercial PALP or insulin for 6 hours decreased
the amount D-[.sup.14C]glucose in the incubation medium, indicating
that both agents enhance cellular uptake of the radiolabeled glucose.
FIG. 12 indicates that in differentiated L1 adipocytes, 1 U/mL
commercial PALP is more effective than 10 nM insulin in enhancing
cellular uptake of D-[.sup.14C]glucose, as well as metabolism of
the radiolabeled glucose to form glycogen and lipids, at both 2
hours and 6 hours after treatment.
FIG. 13 demonstrates that in differentiated L1 adipocytes, 1 U/mL
commercial PALP is somewhat less effective than 50 nM (supraphysiological)
insulin in stimulating uptake and metabolism of D-[.sup.14C]glucose,
at both 2 hours and 6 hours after treatment.
FIG. 14 demonstrates that in mouse embryo NIH 3T3 fibroblasts,
commercial PALP is more effective than insulin in stimulating uptake
and metabolism of D-[.sup.14C]glucose, and that insulin does not
modify the effect of PALP when used in combination with PALP.
FIG. 15 indicates that in differentiated L1 adipocytes, preparations
made from different batches of commercial PALP (each preparation
containing 1.5 U/mL) have effects on the loss of D-[.sup.14C]glucose
from incubation medium that include some variability which appears
to be larger than the inter-experimental error. Each of the preparations
was somewhat less effective than 50 nM insulin.
FIG. 16 indicates that in differentiated L1 adipocytes, preparations
made from different batches of commercial PALP have effects on the
synthesis of glycogen and lipids from D-[.sup.14C]glucose that include
some variability.
FIG. 17 demonstrates that in differentiated L1 adipocytes, homogeneous
purified PALP is somewhat less effective than insulin in reducing
the amount of D-[.sup.14C]glucose in incubation medium.
FIG. 18 demonstrates that in differentiated L1 adipocytes, homogeneous
purified PALP is somewhat less effective than insulin in enhancing
uptake and metabolism of D-[.sup.14C]glucose.
FIG. 19 demonstrates that in differentiated L1 adipocytes, an antibody
against PALP is capable of inhibiting all effects of homogeneous
purified PALP on the uptake and metabolism of D-[.sup.14C]glucose.
DETAILED DESCRIPTION OF THE INVENTION
The observation of the stimulatory effects of PALP on cell proliferation,
described above, led to the experiments described in the Examples
herein. In a first set of experiments, the effects of PALP on blood
glucose level were determined by glucose tolerance tests in mice,
as well as in models for Type 1 and Type 2 diabetes. In a second
set of experiments, the effects of PALP on cellular glucose uptake
in appropriate model systems for fat tissue (differentiated L1 adipocytes)
and skeletal muscle (differentiated L6 muscle cells) were observed.
The results of the sets of experiments demonstrate that PALP has
powerful insulin-independent stimulatory effects on glucose uptake
and metabolism in vitro, and corresponding significant glucose lowering
effects in vivo, without pathological effects and without causing
weight gain. The data, combined with other observations including
a) that high levels of PALP appear to exert only positive physiological
effects in pregnant women, and b) that the enzyme is very stable
in the circulation, indicate that PALP is likely to provide effective,
safe, and sustained control of hyperglycemia in diabetic patients.
Accordingly, the invention includes the use of PALP as an effective
and safe blood glucose-reducing agent, and as an effective agent
in a treatment regimen for the treatment of diabetes.
Method for Reducing Blood Glucose Level
In one embodiment, the present invention provides a method of reducing
blood glucose level in a mammal, comprising the step of administering
a therapeutically effective amount of purified human placental alkaline
phosphatase, or an active derivative. The term "therapeutically
effective amount" in this specification and in the claims indicates
a dosage that is effective in, or is targeted to, either attaining
a desired level of blood glucose or maintaining a desired level
of blood glucose over an appropriate time window.
In the practice of the methods of this embodiment, the mammal may
be a human. The method may be effective to reduce a human's blood
glucose level to below about 10 mM, and preferably into the normal
range of about 4 mM to about 7 mM. The method maybe used on a diabetic
human, and maybe used in combination with administration of an anti-diabetic
medicament.
The method may also be used for preventing the onset of Type 2
diabetes, for a human that is at risk for the development of diabetes.
In particular, the method may be appropriate for a human that exhibits
a chronically elevated blood glucose level, but that has not yet
been diagnosed with diabetes. Furthermore, the method may be useful
for preventing a progression from Type 2 diabetes to Type 1 diabetes.
This is because the persistently high blood glucose levels associated
with Type 2 diabetes eventually leads to the destruction of insulin-producing,
.beta.-cells in the islets of Langerhans, resulting in an insulin
deficiency.
The Active Component
The active component in the methods and compositions of the present
invention is human PALP, or an active derivative thereof. As used
herein, the term "PALP" and the phrase "human PALP"
are used interchangeably to refer to human placental alkaline phosphatase.
As is demonstrated by the examples herein, whole PALP enzyme in
its native state is not required to achieve a glucose-reducing effect.
Active derivatives of PALP are therefore suitable for the practice
of the present invention. For example, digestion of PALP by a protease,
such as bromelain, may provide an active derivative. Likewise, one
who is skilled in the art may synthesize or develop an active derivative
that is a smaller fragment of a PALP sequence which demonstrates
efficacy similar to that of native PALP enzyme. By way of example,
modification of a PALP sequence, or a sequence of smaller PALP peptides,
by exchanging amino acids at critical sites to yield an active derivative
may improve the glucose-reducing effect disclosed herein. In the
practice of the present invention, it is envisioned that modified
PALP, smaller PALP-derived peptides, or modified PALP-derived peptides
maybe similarly effective or even more effective than the native
PALP enzyme, and are each considered to be active derivatives.
Human PALP in solid form is available commercially from Sigma chemical
(St. Louis, Mo.), for example (Sigma catalog number P3895; CAS Registry
Number 9001-78-9). Another commercial source of human PALP is Calbiochem
(San Diego, Calif.; catalog number 524604).
Human PALP may also be obtained by extraction from placental tissue.
For example, a partially purified preparation maybe obtained by
butanol extraction of homogenized placenta. Other methods of extraction
from placental tissue are also suitable.
Human PALP, or an active derivative, may also be obtained by chemical
synthesis using conventional methods. For example, solid phase synthesis
techniques may be used to obtain PALP or an active derivative. Recombinant
methods of obtaining quantities of PALP are also suitable, as is
known in the art.
The methods and treatment regimens described herein include administration
of purified human placental alkaline phosphatase, or an active derivative.
In the practice of the present invention, PALP or an active derivative
is generally administered in the form of a preparation comprising
the PALP enzyme or active derivative. The term "preparation"
as used herein means a composition comprising purified human PALP
or active derivative dissolved or dispersed in a carrier. In a preparation
for administration to a human, the carrier should be a physiologically
acceptable carrier.
The term "purified" is used herein to encompass compositions
that are obtained from a starting material by one or more purification
steps (such as solvent extraction, column separation, chromatographic
separation, etc.) that enhance the concentration of active agent
relative to the starting material. The term "purified"
also encompasses compositions that contain a significant quantity
of active agent in relation to impurities, whether obtained by a
purification process or not. The term "purified" should
not be construed to connote absolute purity.
The phrase "purified human placental alkaline phosphatase"
therefore includes compositions such as a partially purified human
PALP available from Sigma Chemical, which was used as a starting
material for the experiments described in the following Examples.
A purified human PALP composition may also be obtained, for example,
by a purification procedure described elsewhere [She, Q.-B., Mukherjee,
J. J., Huang, J.-S., Crilly, K. S. and Kiss, Z., "Growth factor-like
effects of placental alkaline phosphatase in human and mouse embryo
fibroblasts." FEBS Lett. 469, 163 167 (2000)].
The term "homogeneous" is used herein to indicate a composition
that yields a single protein band in an electrophoretic gel separation,
such as by the SDS-PAGE technique described in Example 1. The phrase
"homogeneous purified human placental alkaline phosphatase"
therefore includes compositions that yield a single band for PALP
enzyme in an electrophoretic separation. A homogeneous purified
human PALP may be obtained, for example, by the purification procedures
described in Example 1.
A separate consideration is the degree of purity that is required
for PALP material that is to be used in a preparation for administration
to a human. An advantage of using a preparation comprising highly
purified or homogeneous PALP in the methods and treatment regimens
of the present invention is that possible side effects caused by
contaminating proteins will not be an issue. However, less extensively
purified PALP preparations, such as that comprising the commercially
available human PALP from Sigma, may also be used so long as safety
can be demonstrated. Since each additional purification step results
in significant loss of the enzyme, using less purified PALP material
for PALP preparations would be more cost-effective.
Administration of PALP
Since human PALP is a relatively large protein, and its actions
appear to involve the adipose tissue and muscle, systemic administration
is an appropriate mode of administration.
By way of example, systemic administration may include administration
of a PALP-containing preparation. For administration to a human,
purified human PALP is dissolved or dispersed in physiological saline
or in another physiologically acceptable carrier, or enclosed in
liposomes such as immunoliposomes, or other delivery systems or
formulations as are known to the art, to produce a PALP-containing
preparation.
For example, one suitable PALP preparation for the practice of
the present invention comprises purified human PALP dissolved in
a 0.9 N physiological salt solution to yield a PALP concentration
of 10 mg/mL. Another suitable PALP preparation comprises purified
human PALP dissolved in a 0.9 N physiological salt solution to yield
a PALP concentration of 30 mg/mL.
A PALP preparation may be administered by injection, for example.
A PALP preparation may be administered via intravenous injection,
intraperitoneal injection, subcutaneous injection, intradermal injection,
intramuscular injection, or any other mode of delivery that ensures
appropriate distribution and relative stability of the enzyme in
the body.
In one embodiment of the method, purified human placental alkaline
phosphatase, or an active derivative, is administered to a human.
A common way to express a suitable dosage is grams of active agent
per square meter of body surface area for the subject. Several formulas
are known for estimating a human subject's body surface area, based
on the human's height (in cm) and mass (in kg). Table 1 lists a
variety of known formulas for estimating body surface area (BSA)
proposed by researchers. Other suitable formulas may likewise be
employed.
TABLE-US-00001 TABLE 1 Formulas for estimating body surface area
(BSA). Author(s) BSA formula Du Bois and Du Bois BSA (m.sup.2) =
Mass(kg).sup.0.425 .times. Height(cm).sup.0.725 .times. 0.007184
Gehan and George BSA (m.sup.2) = Mass(kg).sup.0.51456 .times. Height(cm).sup.0.42246
.times. 0.02350 Haycock BSA (m.sup.2) = Mass(kg).sup.0.5378 .times.
Height(cm).sup.0.3964 .times. 0.024265 Mosteller BSA (m.sup.2) =
Mass(kg).sup.0.5 .times. Height(cm).sup.0.5 .times. 0.016666
Preferably, a dosage of about 0.2 to about 3 g/m.sup.2 of purified
human PALP or active derivative is administered to the human; more
preferably, a dosage of about 0.2 to about 1.0 g/m.sup.2 is administered
to the human.
The human may have an elevated blood glucose level before administration
of purified human PALP. By "elevated," it is meant that
the human's blood glucose level is greater than the normal range
of about 4 to about 7 mM. Systemic administration of PALP preferably
reduces blood glucose level in a human to below about 10 mM, more
preferably to below about 8 mM, and most preferably to within the
normal range of about 4 to about 7 mM.
Because of the relatively large size of the PALP enzyme, its partition
into target tissues after administration is expected to be slow.
Preferably, PALP is administered several hours prior to an expected
glucose load; more preferably PALP is administered at least about
12 hours prior to the expected glucose load; most preferably, PALP
is administered at least about 24 hours prior to the expected glucose
load.
In another embodiment of the method, purified human PALP, or an
active derivative, is administered to a diabetic human. The diabetic
human maybe afflicted with either Type 1 or Type 2 diabetes.
In the treatment of a diabetic human, administration of purified
human PALP may be used in combination with insulin or any anti-diabetic
medicament (i.e. biguanides, insulin secretogogues such as sulphonylureas
or meglitinides, inhibitors of .alpha.-glucosidase, thiazolidinediones,
and others). In this embodiment, the anti-diabetic medicament is
administered as part of a planned course of treatment for diabetes,
in conjunction with purified human PALP. The anti-diabetic medicament
may be administered orally or by any other conventional method.
While some anti-diabetic medicaments may work in combination with
PALP more effectively than others, presently no contra-indication
for using any medicament in combination with PALP has been observed.
For fine-tuning of glucose level in PALP-treated diabetic patients,
a recently developed long-acting derivative of glucagon-like peptide-1
(GLP-1), NN2211, maybe especially useful as the anti-diabetic medicament.
NN2211 has the useful property that it enhances insulin secretion
by the islet only at higher than normal blood glucose levels [Rolin,
B., Larsen, M. O., Gotfredsen, C. F., Deacon, C. F., Carr, R. D.,
Wilken, M. and Knudsen, L. B., "The long-acting GLP-1 derivative
NN2211 ameliorates glycemia and increases .beta.-cell mass in diabetic
mice." Am. J. Physiol. Endocrinol. Metab. 283, E745 E752 (2002)].
In the combination of treatments, the patient's situation will
determine if the administration of PALP occurs prior to or after
administration of the anti-diabetic medicament. For example, if
glycemic control is required very rapidly, then insulin will be
administered first followed by administration of PALP; in this embodiment
administration of PALP will ensure long-lasting glycemic control.
Treatment Regimen for Treating Diabetes
In another embodiment, the invention provides a treatment regimen
for treating diabetes, comprising periodically administering to
a diabetic human a therapeutically effective amount of purified
human placental alkaline phosphatase, or an active derivative. The
method maybe used in combination with administration of an anti-diabetic
medicament. The method may be effective to maintain the human's
blood glucose level below about 10 mM, and preferably in the normal
range of 4 mM to 7 mM.
The treatment regimen employs the same active component, purified
human placental alkaline phosphatase or active derivative, described
above. The active component may be obtained and purified as described
above.
Administration of purified human PALP in the treatment regimen
may also be carried out as described above. PALP preparations described
herein are also suitable for use in the treatment regimens.
As used with respect to the treatment regimens described herein,
the term "periodically" refers to repeated administration
of purified human PALP targeted to maintaining blood glucose level
within a desired range over the time of treatment. The term "periodically"
includes repeated administration at fixed intervals, but also includes
repeated administration over irregular intervals as is required
by the patient's condition. Furthermore, in the treatment regimens
of this embodiment, the therapeutically effective amount of purified
human PALP that is administered does not need to be identical for
each separate administration. More or less purified human PALP maybe
administered in separate administrations, as the patient's needs
dictate.
As will be appreciated by those skilled in the art, the dosage
and the number of treatments will be dependent on the severity of
diabetes and the tolerance of the individual patient. Preferably,
about 0.2 to about 3 g/m.sup.2 is administered to the patient once
daily, more preferably, about 0.2 to about 1.0 g/m.sup.2 is administered
to the patient once daily, even more preferably, about 0.2 to about
3 g/m.sup.2 is administered to the patient once or twice weekly,
and most preferably about 0.2 to about 3 g/m.sup.2 is administered
to the patient once biweekly.
It is expected that in severe cases of both Type 1 and Type 2 diabetes,
once weekly, twice weekly, or daily administration of PALP or active
derivative will result in significant reduction of blood glucose
level. The treatment regimen maybe effective to maintain the human's
blood glucose level below about 10 mM, preferably below about 8
mM, and most preferably in the normal range of 4 mM to 7 mM.
Furthermore, administration of an anti-diabetic medicament in combination
with the PALP may help to fully normalize glucose levels. In the
treatment regimens of this embodiment, administration of purified
human PALP may be used in combination with insulin or any anti-diabetic
medicament (i.e. biguanides, insulin secretogogues such as sulphonylureas
or meglitinides, inhibitors of .alpha.-glucosidase, thiazolidinediones,
and others) to ensure fine-tuning of glycemic control as required.
The anti-diabetic medicament maybe administered orally or by any
other conventional method. A recently developed long-acting derivative
of glucagon-like peptide-1 (GLP-1), NN2211, maybe especially useful.
While some of the anti-diabetic medicaments may work in combination
with PALP more effectively than others, presently no contra-indication
for using any medicament in combination with PALP has been observed.
By treating a diabetic patient using the treatment regimens described
herein, control over diabetes-related conditions may be attained.
For example, treatment by the treatment regimens maybe effective
to reduce diabetes-associated weight loss for a patient that would
normally experience weight loss when treated by an alternative method.
Furthermore, the treatment regimens maybe effective in treating
other diabetes-related complications that have been linked to hyperglycemia.
Diabetes-related complications related to hyperglycemia include
blindness, renal failure, and various neuropathies [Brownlee, M.,
"Biochemistry and molecular cell biology of diabetic complications."
Nature 414, 813 (2001)].
Method for Inducing Weight Loss or Reducing Weight Gain
Another embodiment of the invention provides a method for treating
a human to induce weight loss or to reduce weight gain, comprising
regularly administering purified human placental alkaline phosphatase,
or an active derivative, to the human in an effective amount to
induce weight loss, to reduce an expected weight gain, or to maintain
a constant body weight for the human over time.
The method may be particularly suitable for treatment of a human
afflicted with Type 2 diabetes mellitus. The method may then be
effective to induce weight loss or to reduce an expected weight
gain preceding, caused by, or associated with diabetes.
The method may also be suitable to treat an obese person, whose
blood glucose level is not elevated, to induce weight loss. Furthermore,
the method may be suitable to treat a human who is obese and non-diabetic,
but who has a chronically elevated blood glucose level prior to
beginning treatment. In this embodiment, the treatment may be effective
in preventing the onset of diabetes.
As used herein, the term "obese" refers to a condition
where a person's body mass index (BMI) is about 30 kg/m.sup.2 or
greater. Body mass index is defined as a person's mass (in kilograms)
divided by the square of the person's height (in meters). Persons
who are in the obese category are known to be at greater risk for
developing increased blood glucose (or "impaired glucose tolerance"),
insulin insensitivity, and eventually Type 2 diabetes.
The method furthermore may be useful to induce weight loss or reduce
an expected weight gain preceding the onset of diabetes for a person
at risk for Type 2 diabetes. In this embodiment, the treatment may
be effective in preventing the onset of diabetes.
The method employs the same active component, purified human placental
alkaline phosphatase or active derivative, described above. The
active component may be obtained and purified as described above.
Administration of purified human PALP in the method may also be
carried out as described above. By way of example only, the step
of administering maybe performed by injection of a preparation comprising
a physiologically acceptable carrier and human placental alkaline
phosphatase or active derivative, dissolved or dispersed in the
carrier. The mode of injection may be intravenous, subcutaneous,
intraperitoneal, intramuscular, or intradermal.
PALP preparations described herein are suitable for use in the
methods. By way of example, one suitable preparation comprises homogeneous
purified human placental alkaline phosphatase.
As used with respect to the methods described herein, the phrase
"regularly administering" refers to repeated administration
of purified human PALP targeted to induce weight loss or reduce
an expected weight gain over the time of treatment. The expected
weight gain may be caused by or associated with the onset of diabetes,
for example. The term "regularly" includes repeated administration
at fixed intervals, but also includes repeated administration over
irregular intervals. Furthermore, in the methods of this embodiment,
the effective amount of purified human PALP that is administered
does not need to be identical for each separate administration.
More or less purified human PALP maybe administered in separate
administrations, as the patient's needs dictate.
The step of regular administration may include administration of
human placental alkaline phosphatase or active derivative about
once per two weeks in some embodiments. In other embodiments, the
step of regular administration may include administration of human
placental alkaline phosphatase or active derivative about once per
week, about twice per week, or even about once per day.
By way of example only, the effective amount may be in the range
of about 0.2 grams to about 3 grams per square meter of calculated
surface area for the human. In one embodiment, the effective amount
is in the range of about 0.2 grams to about 1 gram per square meter
of calculated surface area for the human. In another embodiment,
the effective amount is in the range of about 1 gram to about 3
grams per square meter of calculated surface area for the human.
Physiologically Acceptable Preparation of Homogeneous PALP
In another embodiment, the invention provides a preparation for
administration to a human, the preparation comprising homogeneous
purified human PALP in a physiologically acceptable carrier. Human
PALP may be obtained as described above, and may be purified to
homogeneity by the purification procedures described in Example
1. By way of example, the preparation may be a PALP-containing solution
in which homogeneous purified human PALP is dissolved or dispersed
in physiological saline. Alternatively, the preparation may comprise
homogeneous purified human PALP dissolved or dispersed in another
physiologically acceptable carrier, or enclosed in liposomes such
as immunoliposomes, or other delivery systems or formulations as
are known to the art.
For example, one suitable preparation comprises homogeneous purified
human PALP dissolved in a 0.9 N physiological salt solution to yield
a PALP concentration of 10 mg/mL. Another suitable PALP preparation
comprises homogeneous purified human PALP dissolved in a 0.9 N physiological
salt solution to yield a PALP concentration of 30 mg/mL.
EXAMPLES
Example 1
Purification and Spectrophotometric Assay of PALP
Human PALP (Type XXIV, 1020 units of total activity in a partially
purified form was obtained commercially from Sigma Chemical. A butanol
extraction of placental tissue, followed by one or more chromatographic
steps, was performed by Sigma Chemical to obtain the partially purified
material. Butanol extraction inactivates most of the other placental
proteins, including growth factors, but does not reduce either the
mitogenic or the enzymatic activity of PALP.
As determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE), the partially purified PALP obtained from Sigma ("commercial
PALP") was not homogeneous and contained other proteins. FIG.
1 shows a picture of a gel separation of a preparation comprising
commercial PALP without further purification, and other preparations
of PALP of increasing purity. Lane 2 represents a preparation comprising
commercial PALP, lanes 3 and 4 represent preparations comprising
commercial PALP material after further purification steps (described
below), and lane 5 represents a preparation of homogeneous purified
PALP obtained by the complete purification procedure described below.
Lane 1 contains various molecular mass standards for comparison.
As can be seen by reference to FIG. 1 at lane 2, the preparation
comprising commercial PALP contained proteins other than PALP, and
did not yield a homogeneous band in the electrophoretic separation.
The preparation comprising commercial PALP contains at least three
major proteins (one is PALP at approximately 66 kDa, while a band
at approximately 52 kDa is .alpha..sub.1-antitrypsin) and several
minor proteins. Referring to lane 5 of FIG. 1, the preparation comprising
homogeneous purified PALP (obtained by the complete purification
procedure described below) apparently contains only PALP.
A purification procedure consisting of several steps was performed
to further purify the commercially obtained PALP and to yield a
homogeneous band in electrophoretic separation. The same purification
procedure was followed that is described elsewhere [She, Q.-B.,
Mukherjee, J. J., Huang, J.-S., Crilly, K. S. and Kiss, Z., "Growth
factor-like effects of placental alkaline phosphatase in human and
mouse embryo fibroblasts." FEBS Lett. 469, 163 167 (2000)].
A preparation of partially purified PALP was prepared by dissolving
350 mg of commercial PALP into 10 mL of buffer A (0.1 M sodium acetate,
0.5 M NaCl, 1 mM MgCl.sub.2, 1 mM CaCl.sub.2, adjusted to pH 6.5).
This preparation was then further purified by successive Concanavalin
A-Sepharose and Q-Sepharose chromatography, essentially following
the procedure described elsewhere [Chang, T.-C., Huang, S.-M., Huang,
T.-M. and Chang, G.-G., "Human placenta alkaline phosphatase:
An improved purification procedure and kinetic studies." Eur.
J. Biochem. 209, 241 247 (1992)], as follows.
The preparation was run through a Concanavalin A-Sepharose column
using buffer A as solvent. For elution, buffer A included 50 mM
.alpha.-methyl-D-mannopyranoside. Active fractions collected from
the effluent were pooled and dialyzed against buffer B (50 mM Tris-HCl
at pH 7.7). SDS-PAGE separation of the collected and dialyzed fraction
is shown in FIG. 1 at lane 3.
The collected and dialyzed fraction from the previous step was
then passed through a Q-Sepharose column. The fraction of interest
was eluted with buffer B using a linear gradient of 0 250 mM potassium
phosphate at a pH of 7.5. The active fractions from the Q-Sepharose
column were pooled and dialyzed against phosphate-buffered saline
and concentrated by Amicon ultrafiltration. SDS-PAGE separation
of the collected and dialyzed fraction is shown in FIG. 1 at lane
4, which demonstrates that at least two major proteins are still
present in the fraction after dialysis.
Then, the collected and dialyzed fraction from the previous step
was purified to homogeneity by t-butyl hydrophobic interaction chromatography
(HIC). Prior to adding the fraction to a t-butyl HIC column, the
fraction was made 2 M in ammonium sulfate, and pH was adjusted to
6.8. The 5 mL bed volume t-butyl HIC cartridge (BIO-RADIATION, Hercules,
Calif.) was connected to a fast performance liquid chromatography
(FPLC) system from PHARMACIA (Peapack, N.J.). The fraction was introduced
to the HIC column, and the column was eluted with buffer C (100
mM sodium phosphate buffer, 2 M ammonium sulfate at pH 6.8). The
column was eluted with buffer C until a first protein-containing
fraction completely eluted, and then a negative gradient of 2 M-0
M ammonium sulfate in 100 mM sodium phosphate at pH 6.8 was passed
over the column. The negative linear gradient was used to elute
a second protein-containing fraction, which contained the enzymatically
active PALP protein.
The enzymatically active fraction from the HIC separation was dialyzed
against phosphate-buffered saline and concentrated by Amicon ultrafiltration.
Presence and purity of the PALP enzyme in the fraction was confirmed
by SDS-PAGE. After electrophoretic separation, the gel was stained
using coomassie blue or silver stain for visual observation of protein
bands. A single protein band was observed with an approximate molecular
weight of 66 kDa (FIG. 1, lane 5). Identification of the PALP band
by sequence analysis was performed by the Mayo Clinic Protein Core
Facility Rochester, Minn.).
PALP enzyme activity was assayed spectrophotometrically by monitoring
the hydrolysis of 4-nitrophenylphosphate (as an increase in absorbance
at 410 nm) at room temperature (22.degree. C. as described elsewhere
[Chang, G.-G., Shiao, M.-S., Lee, K.-R. and Wu, J.-J., "Modification
of human placental alkaline phosphatase by periodate-oxidized 1,N.sup.6-ethenoadenosine
monophosphate." Biochem. J. 272, 683 690 (1990)]. Activity
analysis of 5 10 .mu.g purified enzyme was performed in 1 mL incubation
volume containing 50 mM Na.sub.2CO.sub.3/NaHCO.sub.3, 10 mM MgCl.sub.2,
10 mM 4-nitrophenylphosphate at pH 9.8. The extinction coefficient
of 4-nitrophenol was taken as 1.62.times.10.sup.4 M.sup.-1 cm.sup.-1.
An enzyme activity of 1 U (unit) is defined as 1 .mu.mol substrate
hydrolyzed/min at 22.degree. C. at pH 9.8.
Examples 2 8
Effects of PALP on Blood Glucose Levels and Body Weight in Mice
Example 2
Determination of Blood Glucose Levels in Treated and Untreated
Healthy Mice in Glucose Tolerance Tests
Two preparations of commercial PALP, without further purification,
were prepared. A first preparation included 30 mg/mL commercial
PALP in a 0.9 N physiological salt solution. A second preparation
included 10 mg/mL commercial PALP in a 0.9 N physiological salt
solution.
First-generation hybrid BDF1 (C57B1/6 female.times.DBA/2 male)
adult (10 to 12 weeks old) male mice, weighing 22 23 g each, were
used in the experiment. These animals, developed in the National
Cancer Institute (Budapest, Hungary), are in the specified pathogen-free
(SPF) hygienic category. The subject animals were kept in macrolon
cages at 22 24.degree. C. and 50 60% humidity, with lighting regimen
of 12 hours light/12 hours dark. The animals had free access to
tap water and were fed a sterilized standard diet (Charles River
VRF1, autoclavable; Germany) according to the provided instructions.
The animals were cared for according to "Guiding Principles
for the Care and Use of Animals" based upon the Helsinki Declaration.
The mice were fasted for 16 hours before administration of glucose.
Four groups of mice were treated and observed in this experiment.
A first group was treated by intraperitoneal injection of 500 .mu.g
commercial PALP (50 .mu.L injection of the second preparation) exactly
16 hours prior to intraperitoneal administration of glucose (2 mg/mouse).
A second group was treated by intraperitoneal injection of 1500
.mu.g commercial PALP (50 .mu.L injection of the first preparation)
24 hours prior to intraperitoneal administration of glucose (2 mg/mouse).
A third group was treated by 0.5 I.U. insulin, 15 minutes prior
to administration of glucose (2 mg/mouse). A control group received
only intraperitoneal administration of glucose (2 mg/mouse).
Blood glucose concentration was monitored for mice of each group,
over a period of six hours after administration of glucose. Blood
samples were taken from the corner of the eyes (canthus), and glucose
concentrations in whole blood samples were immediately measured
with the fast Glucose C test (Wako Chemicals USA Inc., Richmond,
Va.). Data is shown in the graph of FIG. 2.
The data shown in FIG. 2 demonstrates that, for mice of the control
group (.circle-solid.), glucose blood levels increased 3.2 to 4.3-fold
within 0.5 to 6 hours after intraperitoneal glucose administration,
with a maximum increase at the 3-hour time point. For mice receiving
insulin treatment (.tangle-solidup.), insulin was effective to initially
(i.e., at 30 minutes) block a sharp increase in blood glucose level,
but at later time points it was progressively less effective, and
at the 6-hour time point it had no significant effect. For mice
receiving 500 .mu.g PALP (.box-solid.), an effect was observed similar
to that observed with insulin at the 1-, 3- and 5-hour time points.
Finally, for mice receiving 1500 .mu.g PALP (.diamond-solid.), the
PALP treatment resulted in greater inhibitory effects on glucose
levels than that was observed with insulin at the 1-, 3- and 6-hour
time points.
This glucose tolerance experiment clearly indicated that an appropriate
amount of PALP may have longer-term effects than insulin when used
at a concentration that is safe in animals. In other experiments
(not shown), it was determined that for maximal effects PALP needs
to be administered about 16 to about 24 hours prior to the glucose
load. It is thought that preadministration is more effective because
PALP is a relatively large molecule and its distribution in the
body takes time. This observation is also an indication that PALP
is metabolically stable in vivo. Indeed, others demonstrated that
in human subjects injected PALP remains remarkably stable in the
circulation with an estimated biological half-life time of 7 days
[Clubb, J. S., Neale, F. C. and Posen, S., "The behavior of
infused human placental alkaline phosphatase in human subjects."
J. Lab. & Clin. Med. 66, 493 507 (1965)]. It is expected that,
for the treatment regimens described herein, metabolic stability
will allow administration of PALP once every 5 14 days, in contrast
to practically all anti-diabetic drugs that are presently available,
which require daily (often multiple) doses.
Example 3
PALP Decreases Blood Glucose Level in Streptozotocin-Treated BDF1
Mice
BDF1 male mice, weighing 27 28 g each, were used. Four groups of
mice were treated and observed in this experiment. A first group
and a second group were initially treated by intraperitoneal injection
of 1500 .mu.g of commercial PALP (50 .mu.L injection of the first
preparation from Example 2). A third group and fourth group were
untreated.
Twenty-four hours later (day 0), the first group and third group
were treated once with streptozotocin (STZ) at a dosage of 200 mg/kg.
STZ is widely used to selectively destroy insulin-producing .beta.-cells
in the islet by an oxidative mechanism. The second group and fourth
group were untreated at this time. Mice in the first and second
groups were then repeatedly administered 1500 .mu.g commercial PALP
by intraperitoneal injection (50 .mu.L injection of the first preparation
from Example 2) on days 2, 3, 4, and 5.
Blood glucose concentration was monitored for five mice of each
group, over a period of six days after administration of STZ (day
0). Blood samples were taken from the eyes, and glucose concentrations
in whole blood samples were immediately measured by the Glucose
C test. Blood glucose concentration was determined on days 0, 3,
4, 5 and 6. Data is shown in the graph of FIG. 3
The data shown in FIG. 3 demonstrates that, for mice of the fourth
group (.circle-solid.) (untreated), the basal level of blood glucose
was stable over the 6-day observation period. For mice of the second
group (.tangle-solidup.), administration of PALP did not increase,
and, importantly, did not decrease the basal blood glucose level.
For mice of the third group (.box-solid.), treated with STZ in the
absence of PALP, blood glucose level increased 9-fold by the third
day and 10.9-fold by the sixth day. For the first group (.diamond-solid.),
treatment with PALP decreased the effect of STZ by 63 67%.
These experiments clearly indicate that repeated administration
of PALP is effective in reducing blood glucose levels for mice having
damaged .beta.-cells.
Example 4
PALP Decreases Blood Glucose Levels Even After Pre-Treatment with
STZ
BDF1 male mice were initially treated with STZ (200 mg/kg). Two
weeks after the initial treatments, the mice were treated with 1500
.mu.g commercial PALP (50 .mu.L injection of the first preparation
from Example 2) once daily for 5 days, and blood glucose was determined
as before on the sixth day. PALP treatment reduced the average blood
glucose level from 22.5 mM (n=5) for a control group to 11.5 mM
(n=5) for the PALP-treated group.
This experiment strongly indicates that the effect of PALP on blood
glucose levels is independent of insulin, because in STZ-treated
animals practically no glycemic control remains (as shown by the
.about.10-times increase in glucose level compared to the fourth
group of Example 3), which is consistent with the massive death
of .beta.-cells and the strong reduction or absence of insulin in
the circulation. No attempt was made to determine the potentially
greater effects of higher concentrations of PALP.
Example 5
PALP Decreases the Loss of Body Weight Induced by Treatment with
STZ, and Increases Life Span of STZ-Treated Animals
In the same experiment described in Example 3, the weight of each
of the mice was measured on days 0, 2, 4, 6, and 8. Data is plotted
in FIG. 4, which shows that mice from the untreated fourth group
(.circle-solid.) and PALP-treated animals of the second group (.tangle-solidup.)
gained little, if any, weight during the 8-day observation period.
Mice of the third group (.box-solid.) treated with STZ in the absence
of PALP lost approximately 5 grams. Weight loss for mice of the
first group (.diamond-solid.) treated with both PALP and STZ was
inhibited about 50%.
It is known that in the absence of insulin, protein degradation
in muscles is enhanced leading to weight loss (wasting). In STZ-treated
animals, this mechanism almost certainly plays a key role in weight
loss. In unpublished work, Kiss and co-workers found that in differentiated
L6 muscle cells, administration of PALP increases protein synthesis
from various amino acids (alanine, proline, leucine, glutamine,
methionine). Thus, stimulation of protein synthesis in muscle is
likely to explain, at least in part, the ability of PALP to partially
prevent weight loss in STZ-treated annals.
PALP, administered either subcutaneously or intraperitoneally,
had a profound effect on the survival of STZ-treated animals. Of
the 15 animals treated with STZ alone, only 6 survived up to 6 months.
On the other hand, all 23 animals treated with STZ (one time) plus
1.5 mg PALP/mouse (for at least five consecutive days) survived
for at least 8 months. Because conventionally a high survival rate
of STZ-treated animals can be ensured only with daily injection
of insulin, the positive effects of PALP on animal survival further
indicate that PALP has insulin-like, but insulin-independent, physiological
effects, including reduction of blood glucose level. Reduction in
blood glucose level and increased protein synthesis together may
explain the ability of PALP to promote survival of these animals.
Example 6
Determination of Blood Glucose Levels in Untreated and PALP-Treated
Non-Obese Diabetic Mice
In this set of experiments, the effect of PALP on blood glucose
levels was determined in non-obese diabetic adult female mice. Data
for the experiments is given in Table 2.
Non-obese (NOD) diabetic inbred adult (140 150 days old) female
mice weighing 22 26 g each were obtained from Charles River Italia
S.P.A. (Italy). The NOD mouse strain is a spontaneous animal model
of human Type 1 diabetes isolated from the cataract-prone CTS subline
of outbred ICR mouse. The mice are also in the specified pathogen-free
(SPF) category. These mice are characterized by a cumulative incidence
of diabetes, reaching 60 80% in females and 10 20% in males at an
age of 150 200 days. In these mice, spontaneously autoreactive T-cells
destroy the insulin-producing islet .beta.-cells in the pancreas.
The following references provide a summary of the current understanding
on how diabetes develops in NOD mice: Lyons, P. A., Armitage, N.,
Lord, C. J., Phillips, M. S., Todd, J. A., Peterson, L. B. and Wicker,
L. S., "Mapping by Genetic Interaction: High resolution congenic
mapping of the type 1 diabetes loci Idd10 and Idd18 in the NOD mouse."
Diabetes 50, 2633 2637 (2002); Kaufman, D. L., Tisch, R., Sarvetnick
N., Chatenoud, L., Harrison, L. C., Haskins, K., Quinn, A, Sercarz,
E., Herrath, M., Wegmann, D., Wen, L. and Zekzer, D., "Report
from the 1.sup.st international NOD mouse T-cell workshop and the
follow-up mini-workshop." Diabetes 50, 2459 2463 (2001); Bonifacio,
E., Atkinson, M., Eisenbarth, G., Serreze, D., Kay, T. W. H, Lee-Chan,
E. and Singh, B., "International Workshop on lessons from animal
models for human type 1 diabetes: Identification of insulin but
not glutamic acid decarboxylase or IA-2 as specific autoantigens
of humoral autoimmunity in nonobese diabetic mice." Diabetes
50, 2451 2458 (2001).
The subject animals were kept in macrolon cages at 22 24.degree.
C. and 50 60% humidity, with lighting regimen of 12 hours light/12
hours dark. The animals had free access to tap water and were fed
a sterilized standard diet (Charles River VRF1, autoclavable; Germany)
according to the provided instructions. The animals were cared for
according to "Guiding Principles for the Care and Use of Animals"
based upon the Helsinki Declaration.
For treated and control NOD animals (180 days old), blood samples
were taken from the eye corner, and initial blood glucose levels
(day 1) were determined by the Fast Glucose C test. Over an eleven-day
period, a solution of commercial PALP (30 mg/mL in physiological
saline) was injected into four treated animals (1.5 mg/mouse) subcutaneously
(s.c.) or intraperitonially (i.p.) at four times. Injections were
given on days 1, 3, 5 and 8. On day 11, blood samples were taken
from the eye corner for both treated and control animals, and blood
glucose levels were determined by the Fast Glucose C test.
As indicated by the data in Table 2, for the three untreated NOD
mice, blood glucose concentrations increased by 7.2 8.4 mM during
the 11-day period. In contrast, in the four animals treated with
PALP subcutaneously, the increases in blood glucose levels were
considerably lower (in the range of 0.6 3.1 ml). For animals treated
with PALP intraperitonially, there was a similar reduction in the
increase (in the range of 0.7 3.4 mM) in blood glucose levels (data
not given in Table 2).
TABLE-US-00002 TABLE 2 Changes in blood glucose levels observed
for untreated and PALP-treated NOD mice. PALP- Untreated Mice Treated
Mice (subcutaneous) 1 2 3 1 2 3 4 Subject Blood glucose level (mM)
Blood glucose level (mM) Day 1 12.7 8.2 12.9 7.9 12.9 8.1 13.3 Day
11 20.8 15.4 21.3 8.5 16.0 8.7 16.2 Increase 8.1 7.2 8.4 0.6 3.1
0.6 2.9 Survival 12 22 16 <150 25 <150 27 (days)
Also, it was observed that the untreated mice survived for not
longer than 22 days; in contrast, PALP-treated mice survived for
at least 25 days, and up to over 150 days in some cases.
In this set of experiments, no attempt was made to examine the
potentially stronger effects of administering a greater quantity
of PALP. The results clearly indicate that administration of PALP
markedly reduces the rise in blood glucose levels in the non-obese
Type 1 diabetic model.
Example 7
Determination of Blood Glucose Levels in Untreated and PALP-Treated
Obese Diabetic Mice
In this set of experiments, the effect of PALP on blood glucose
levels was determined in obese diabetic adult female mice. Data
for the experiments is given in Table 3.
Obese diabetic inbred adult (six weeks old) female C57BI/6J Bom-ob
mice weighing 33 37 g were obtained from Charles River Laboratories,
Germany. These animals are in the specified pathogen-free (SPF)
hygienic category. A mutation in the leptin gene, responsible for
obesity, was propagated in the C57B1/6J (B1/6) inbred strain. These
homozygous obese (ob/ob) animals developed hyperglycemia, hyperinsulinemia
and obesity by six weeks of age. Food intake is greatly increased
by that time.
The subject animals were kept in macrolon cages at 22 24.degree.
C. and 50 60% humidity, with lighting regimen of 12 hours light/12
hours dark. The animals had free access to tap water and were fed
with a sterilized standard diet (Charles River VRF1, autoclavable)
according to the prescribed standard. The animals were cared for
according to "Guiding Principles for the Care and Use of Animals"
as stated in the Helsinki Declaration.
On day 43 (Day 1 in Table 3), the animals were divided into five
groups (I-V) each consisting of four animals, and the treatments
started as specified in Table 3. Animals with similarly high blood
glucose levels were selected for each of groups I-V. In short, the
treatments were: untreated (Group I); treated with commercial PALP
for 28 days, 5 times/week intraperitonially (Group II); treated
with commercial PALP for 28 days, 3 times/week, intraperitonially
(Group III); treated with commercial PALP for 28 days, 5 times/week
subcutaneously (Group IV); treated with commercial PALP for 28 days,
3 times/week, subcutaneously (Group V). Initially, each treatment
consisted of 1.5 mg commercial PALP/mouse in physiological saline;
then on day 15 (Group II and IV) or day 16 (Group II and V) the
dosage of commercial PALP was increased to 3 mg/mouse. Blood samples
were analyzed for glucose on the days indicated in the table. Blood
glucose level (BGL) data are the mean.+-.S.E. of single simultaneous
determinations from four animals of the group.
Data is tabulated in Table 3. For the untreated animals of Group
I, blood glucose levels increased steadily over the 28 days period.
However, for both intraperitoneal (Groups II and III) and subcutaneous
administration (Groups IV and V) of PALP, a steady decrease in blood
glucose was observed for the subject animals. The data further indicate
that the 1.5 mg PALP/mouse dosage was effective in decreasing the
blood glucose level, while the 3 mg/mouse dosage was slightly more
effective.
It should be noted that intraperitoneal and subcutaneous treatments
were similarly effective, with intraperitoneal administration providing
somewhat better results. It is also noteworthy that treatment 5
times/week was only slightly more effective than treatment 3 times/week
for either intraperitoneal or subcutaneous administration. The data
suggests that, in humans, daily administration may not be required
in order to effectively control hyperglycemia
TABLE-US-00003 TABLE 3 Changes in blood glucose levels observed
for untreated and PALP-treated obese diabetic mice. (BGL = Blood
Glucose Level (mM)) Group II Group III Group IV Group V Group I
intraperitoneal intraperitoneal subcutaneous subcutaneous Day BGL
.+-. S. E. BGL .+-. S. E. PALP BGL .+-. S. E. PALP BGL .+-. S. E.
PALP BGL .+-. S. E. PALP 1 5.6 .+-. 0.7 7.2 .+-. 0.2 1.5 mg 7.6
.+-. 0.9 7.1 .+-. 0.5 1.5 mg 7.3 .+-. 0.5 2 -- 7.3 .+-. 0.2 1.5
mg 7.6 .+-. 0.8 1.5 mg 7.1 .+-. 0.4 1.5 mg 7.3 .+-. 0.6 1.5 mg 3
5.8 .+-. 0.9 6.6 .+-. 0.2 1.5 mg 7.1 .+-. 0.6 6.7 .+-. 0.2 1.5 mg
7.3 .+-. 0.6 4 -- 6.4 .+-. 0.3 1.5 mg 6.8 .+-. 0.2 1.5 mg 6.8 .+-.
0.1 1.5 mg 6.8 .+-. 0.4 1.5 mg 5 6.2 .+-. 0.6 5.4 .+-. 0.3 1.5 mg
6.4 .+-. 0.5 6.5 .+-. 0.1 1.5 mg 6.4 .+-. 0.3 7 -- 5.1 .+-. 0.2
1.5 mg 6.1 .+-. 0.7 1.5 mg 6.3 .+-. 0.2 1.5 mg 6.2 .+-. 0.5 1.5
mg 8 7.6 .+-. 1.2 5.0 .+-. 0.5 1.5 mg 5.5 .+-. 0.7 6.1 .+-. 0.1
1.5 mg 5.8 .+-. 0.3 9 -- 5.2 .+-. 0.2 1.5 mg 5.7 .+-. 0.6 1.5 mg
6.1 .+-. 0.2 1.5 mg 5.7 .+-. 0.5 1.5 mg 10 7.9 .+-. 0.2 4.5 .+-.
0.3 1.5 mg 5.5 .+-. 0.6 6.1 .+-. 0.3 1.5 mg 5.5 .+-. 0.6 11 -- 4.4
.+-. 0.3 1.5 mg 5.2 .+-. 0.4 1.5 mg 5.9 .+-. 0.3 1.5 mg 5.3 .+-.
0.4 1.5 mg 14 8.6 .+-. 1.3 4.6 .+-. 0.2 1.5 mg 5.1 .+-. 0.5 1.5
mg 5.7 .+-. 0.3 1.5 mg 5.3 .+-. 0.4 1.5 mg 15 -- 4.4 .+-. 0.1 3
mg 4.8 .+-. 0.4 5.2 .+-. 0.3 3 mg 5.0 .+-. 0.4 16 8.8 .+-. 0.4 3.5
.+-. 0.3 3 mg 4.4 .+-. 0.4 3 mg 4.1 .+-. 0.2 3 mg 4.2 .+-. 0.2 3
mg 17 -- 2.9 .+-. 0.2 3 mg 3.4 .+-. 0.2 3.7 .+-. 0.2 3 mg 3.6 .+-.
0.2 18 8.0 .+-. 0.2 2.7 .+-. 0.2 3 mg 3.1 .+-. 0.2 3 mg 3.5 .+-.
0.1 3 mg 3.4 .+-. 0.3 3 mg 21 -- 2.5 .+-. 0.2 3 mg 2.9 .+-. 0.3
3 mg 3.4 .+-. 0.3 3 mg 3.3 .+-. 0.2 3 mg 22 8.6 .+-. 0.5 2.3 .+-.
0.2 3 mg 2.7 .+-. 0.2 3.6 .+-. 0.2 3 mg 3.0 .+-. 0.4 23 -- 2.4 .+-.
0.3 3 mg 2.5 .+-. 0.1 3 mg 3.1 .+-. 0.1 3 mg 2.6 .+-. 0.3 3 mg 24
8.8 .+-. 0.5 2.3 .+-. 0.1 3 mg 2.2 .+-. 0.2 2.9 .+-. 0.2 3 mg 2.6
.+-. 0.3 25 -- 2.0 .+-. 0.2 3 mg 2.1 .+-. 0.3 3 mg 2.8 .+-. 0.2
3 mg 2.5 .+-. 0.3 3 mg 28 8.5 .+-. 0.4 1.5 .+-. 0.1 1.6 .+-. 0.1
2.3 .+-. 0.3 2.3 .+-. 0.2
Example 8
Effect of PALP Treatment on Body Weight for Obese Diabetic Mice
Obesity is characterized by an increase in adipose tissue and body
weight to a level that produces adverse health effects, including
increased risk for development of diabetes. White adipose tissue
secretes several factors that regulate energy balance and whole-body
homeostasis; one of the most important such factors is the hormone
leptin [F. M Gregoire, "Adipocyte differentiation: From fibroblast
to endocrine cell." Exp. Biol. Med. 226, 997 2001 (2001)].
The gene responsible for leptin production is called the ob gene.
In ob/ob animals, the leptin secreted from the adipose tissue is
truncated and thereby inactivated [B. M. Spiegelman and J. S. Flier,
"Obesity and the regulation of energy balance." Cell 104,
531 543 (2001)]. Due to absence of leptin-mediated regulation, ob/ob
animal gain substantial weight, leading dually and inevitably to
decreased insulin sensitivity in the peripheral tissues such as
muscle, adipose tissue, and liver. As a consequence, in ob/ob animals
the blood glucose level is high due to impaired uptake and metabolism
of glucose by the peripheral tissues, and to increased production
of glucose by the liver, which is normally inhibited by insulin.
The obesity syndrome in ob/ob mice can be corrected by administration
of leptin [B. M. Spiegelman and J. S. Flier, "Obesity and the
regulation of energy balance." Cell 104, 531 543 (2001)].
Importantly, high glucose levels that are induced by physiological
events other than obesity do not lead to obesity. Also, lowering
of glucose level does not necessarily lead to weight loss; i.e.,
high glucose level is not a risk factor for obesity. For example,
some of the most important agents that are used to treat diabetes
by lowering blood glucose level, including insulin, sulphonylureas
and thiazolidinediones, can cause significant weight gain [see Table
1 in D. E. Moller, "New drug targets for type 2 diabetes and
the metabolic syndrome." Nature 414, 821 827 (2001)], while
biguanides (e.g., metformin) can cause moderate weight gain [U.K.
Prospective Diabetes Study Group, "U.K. Prospective Diabetes
Study 16. Overview of 6 years' therapy of Type 2 diabetes: A progressive
disease." Diabetes 44, 1249 1258 (1995)]. None of the presently
used glucose-lowering agents are known to cause weight loss.
In view of the differential regulation of adipose tissue weight
and blood glucose level, it was of interest to determine whether
administration of PALP also modifies body weight that primarily
results from increased weight of adipose tissue in ob/ob mice. Accordingly,
in the experiment described in Example 7, the body weight of untreated
and PALP-treated obese diabetic mice was also regularly measured.
The schedule of treatments and the results are given in Table 4.
TABLE-US-00004 TABLE 4 Effect of administration of PALP on body
weight of obese diabetic mice. Group II Group III Group IV Group
V Group I intraperitoneal intraperitoneal subcutaneous subcutaneous
Body Body Body Body Body weight weight weight weight weight Day
(g) .+-. S. E. (g) .+-. S. E. PALP (g) .+-. S. E. PALP (g) .+-.
S. E. PALP (g) .+-. S. E. PALP 1 35.5 .+-. 0.9 37.2 .+-. 1.3 1.5
mg 33.3 .+-. 1.7 33.5 .+-. 0.6 1.5 mg 33.0 .+-. 0.7 2 34.4 .+-.
1.0 36.7 .+-. 0.6 1.5 mg 31.7 .+-. 1.6 1.5 mg 32.1 .+-. 1.0 1.5
mg 31.6 .+-. 1.1 1.5 mg 3 35.5 .+-. 0.8 36.3 .+-. 0.5 1.5 mg 32.2
.+-. 1.4 31.7 .+-. 1.0 1.5 mg 32.7 .+-. 0.8 4 35.6 .+-. 0.9 35.8
.+-. 0.7 1.5 mg 31.7 .+-. 1.9 1.5 mg 31.5 .+-. 0.9 1.5 mg 31.6 .+-.
0.5 1.5 mg 5 36.0 .+-. 1.1 35.3 .+-. 0.7 1.5 mg 31.8 .+-. 1.5 32.1
.+-. 1.2 1.5 mg 31.7 .+-. 0.5 7 37.3 .+-. 0.9 -- 1.5 mg 32.3 .+-.
1.3 1.5 mg 32.9 .+-. 0.9 1.5 mg 33.2 .+-. 0.3 1.5 mg 8 38.5 .+-.
1.3 35.9 .+-. 0.6 1.5 mg 32.2 .+-. 2.1 33.4 .+-. 0.6 1.5 mg 32.4
.+-. 1.1 9 38.0 .+-. 1.8 35.4 .+-. 0.8 1.5 mg 32.2 .+-. 1.6 1.5
mg 33.7 .+-. 0.9 1.5 mg 32.8 .+-. 0.6 1.5 mg 10 37.9 .+-. 1.0 35.3
.+-. 0.5 1.5 mg 32.3 .+-. 1.6 33.7 .+-. 1.6 1.5 mg 32.2 .+-. 0.5
11 37.9 .+-. 1.3 35.0 .+-. 0.4 1.5 mg 32.3 .+-. 1.9 1.5 mg 33.8
.+-. 1.9 1.5 mg 32.9 .+-. 0.4 1.5 mg 14 39.7 .+-. 1.2 33.9 .+-.
1.0 1.5 mg 33.0 .+-. 1.2 1.5 mg 34.8 .+-. 2.3 1.5 mg 34.2 .+-. 1.0
1.5 mg 15 39.7 .+-. 1.0 34.0 .+-. 1.9 3 mg 33.0 .+-. 1.9 36.0 .+-.
1.0 3 mg 33.7 .+-. 0.5 16 39.9 .+-. 1.4 33.8 .+-. 0.8 3 mg 32.9
.+-. 2.1 3 mg 34.7 .+-. 2.1 3 mg 33.9 .+-. 0.5 3 mg 17 39.8 .+-.
1.5 33.5 .+-. 1.1 3 mg 32.6 .+-. 2.2 34.8 .+-. 2.2 3 mg 33.8 .+-.
0.4 18 40.1 .+-. 1.3 33.3 .+-. 1.0 3 mg 32.8 .+-. 2.3 3 mg 35.2
.+-. 1.6 3 mg 34.1 .+-. 0.4 3 mg 21 41.4 .+-. 1.7 33.6 .+-. 1.2
3 mg 33.7 .+-. 2.4 3 mg 36.2 .+-. 1.7 3 mg 36.2 .+-. 0.7 3 mg 22
41.7 .+-. 1.6 33.1 .+-. 1.8 3 mg 33.5 .+-. 1.8 35.9 .+-. 1.7 3 mg
36.0 .+-. 1.0 23 41.6 .+-. 2.8 -- 3 mg 33.4 .+-. 2.3 3 mg 35.6 .+-.
1.7 3 mg -- 3 mg 24 41.8 .+-. 2.0 -- 3 mg 33.2 .+-. 1.5 35.8 .+-.
1.6 3 mg -- 25 41.6 .+-. 1.4 32.2 .+-. 1.3 3 mg 32.6 .+-. 1.4 3
mg 36.1 .+-. 2.1 3 mg 37.1 .+-. 0.8 3 mg 28 43.7 .+-. 2.7 32.8 .+-.
0.8 32.9 .+-. 1.4 37.4 .+-. 1.6 37.4 .+-. 1.0 % 23.1% -11.8% -1.2%
11.6% 13.3% Gain
Untreated animals of Group I steadily gained weight over the 28-day
observation period, with body weight increasing approximately 23%
overall. In contrast, animals treated with PALP administered intraperitoneally
five times per week (Group II) actually decreased in weight by approximately
12% overall, while animals treated with PALP administered intraperitoneally
three times per week essentially maintained their weight (Group
III). Animals treated with PALP five times per week (Group IV) administered
subcutaneously, or three times per week (Group V) administered subcutaneously
gained about half as much weight as the control group.
Because intraperitoneal and subcutaneous treatments had similar
effects on the blood glucose level (Table 3), the clearly established
ability to achieve weight control by intraperitoneally administering
PALP may involve some mechanism in addition to the control of glucose
level. In fact, considering that for the ob/ob model obesity leads
to insulin insensitivity and higher blood glucose levels, but not
vice-versa, it is likely that in this model a primary mode of action
of PALP was weight control, and that the reduction in blood glucose
level was a secondary effect. The mechanism by which PALP achieves
weight control is not known. However, since the absence of functional
leptin hormone is responsible for obesity in ob/ob animals, it is
reasonable to assume that PALP acts as a substitute for the most
important weight control-related functions of leptin.
Examples 9 12
Effects of PALP on Glucose Uptake and Metabolism in Differentiated
3T3-L1 Adipocytes and Rat L6 Cells at Different Levels of Glucose
and Serum in the Medium
The above-described experiments suggested that PALP decreased the
blood glucose level in treated mice by stimulating glucose uptake
and metabolism in peripheral tissues, which explains the relatively
long time required for body distribution and action. This hypothesis
was examined for differentiated adipocytes and muscle cells.
Mouse 3T3-L1 pre-adipocyte cells and rat L6 skeletal muscle cells
were obtained from the American Type Culture Collection (Rockville,
Md.). 3T3-L1 cells were grown in high-glucose Dulbecco's modified
Eagle medium (DMEM) supplemented with 10% (v/v) fetal bovine serum
(FBS) and 1% antibiotic/antimycotic (GIBCO, Grand Island, N.Y.)
composed of 10,000 units/mL penicillin G, 10 mg/mL streptomycin,
and 25 .mu.g/mL amphotericin B.
To induce cell differentiation of the two cell lines, conventional
methods were used, as follows. 3T3-L1 cells were maintained in a
state of confluency for 2 days, followed by treatments for 3 days
with 400 nM insulin (Boehringer Mannheim, Indianapolis, Ind.), 250
nM dexamethasone (Sigma-Aldrich, Inc., St. Louis, Mo.) and 0.5 mM
isobutylmethylxanthine (Sigma-Aldrich, Inc.). The medium was replaced
with fresh 10% serum-containing medium, and the medium was changed
twice a week. The differentiated cells were used 14 days after removing
the differentiation-inducing agents.
The L6 cells in monolayer culture were induced to differentiate
in minimal essential medium (MEM) containing 2% (v/v) FBS and 1%
(v/v) antibiotic/antimycotic solution. The cells were fed fresh
medium every 48 hours. Myoblast differentiation, which occurred
by about the seventh day, was monitored by phase contrast microscopy.
To examine cellular glucose uptake and metabolism, differentiated
(attached) cells in 12-well plates were incubated with D-[.sup.14C]glucose
at 37.degree. C. in a CO.sub.2 incubator (95% air: 5% CO.sub.2)
in 2% serum- or 10% serum-containing (as specified in the specific
examples) glucose-free or 5 mM D-glucose-containing medium for 2
6 hours. In the various experiments, the quantity of other reagents
(such as PALP or insulin) in the incubation medium was varied, as
specified.
At the termination of incubation, the medium was removed; an aliquot
of the medium was taken to determine the loss of D-[.sup.14C]glucose
from the medium, which corresponds to glucose uptake by cells. Cells
were washed twice with 3--3 mL medium to remove traces of medium
containing unincorporated radioactivity. Then ice-cold 99.8% methanol/0.2%
water (v/v) mixture (1 ml) was added to the monolayers, and cells
were extracted for 2 hours at -20.degree. C. This treatment resulted
in the precipitation of glycogen and the solubilization of cellular
free glucose and lipids.
Precipitated [.sup.14C]glycogen was suspended in 0.75 mL of 1 M
NaOH and transferred to scintillation vials; this procedure was
repeated with another 0.75 mL aliquot of NaOH. 10 mM HCl (.about.150
.mu.L) was added to the vials to neutralize the suspension, followed
by the addition of 8.5 mL ECOLUME (ICN Pharmaceuticals, Inc., Costa
Mesa, Calif.). While the 1 M NaOH suspension contained some particulate
material (mostly protein which was not labeled with radiolabeled
glucose), after transfer to ECOLUME no precipitate remained. This
procedure resulted in quantitative removal of precipitated glycogen;
no additional radioactivity could be removed from the well by 30%
KOH (which was neutralized with HCl prior to determination of radioactivity.
The methanol mixture containing cellular free glucose and lipids
was treated as follows. The methanol phase (1 ml) and a following
1 mL methanol wash were added to 2 mL chloroform, followed by the
addition of 3 mL water. Separation of this organic-aqueous mixture
was facilitated by brief centrifugation. Of the resulting two phases,
the lower one contained the total radiolabeled lipids, while the
upper one contained radiolabeled glucose. Aliquots of the upper
and lower phases were transferred to scintillation vials to determine
the amounts of radiolabeled glucose and total lipids, respectively,
by liquid scintillation counting.
In some experiments, a published procedure [Huang, D., Cheung,
A. T., Parsons, J. T. and Bryer-Ash, M., "Focal adhesion kinase
(FAK) regulates insulin-stimulated glycogen synthesis in hepatocytes."
J. Biol. Chem. 277, 18151 18160 (2002)] parallel to the above method
was used to determine glucose incorporation into glycogen. After
incubations with D-[.sup.14C]glucose, cells were solubilized with
20% KOH for 2 hours. Lysates were extracted with 8% tricarboxylic
acid, neutralized with 2.0 M HCl, then boiled for 5 minutes. Total
glycogen was precipitated by the addition of 80% ethanol (final
concentration) for 2 hours at -20.degree. C. followed by centrifugation
at 1100.times.g for 10 minutes. Precipitation and centrifugation
was repeated. The precipitate was then re-dissolved in distilled
water, and the solids were precipitated again as before. This method
yielded essentially the same results as the methanol precipitation
method described above. Thus, in all subsequent experiments we used
the methanol precipitation method because this permitted simultaneous
analysis of cellular glucose, glycogen and lipids for an individual
sample.
Example 9
Stimulatory Effect of PALP on Glucose Uptake in Differentiated
L1 Cells
Three groups of differentiated L1 cells (used 14 days after the
differentiation treatment described above) in 12-well plates were
treated as follows: a first control group was untreated; a second
group was treated with 0.75 mL of a preparation comprising 0.5 U/mL
commercial PALP in serum-free and glucose-free DMEM for 30 minutes,
and a third group was treated with 0.75 mL of a preparation comprising
1.0 U/mL commercial PALP in serum-free and glucose-free DMEM for
30 minutes. The following experiment was performed in triplicate
for each group.
Each group was then incubated as described above for 5 hours following
addition of D-[.sup.14C]glucose, under four different conditions
as follows: (1) the incubation medium contained no serum or unlabeled
D-glucose; (2) the incubation medium contained 2% fetal bovine serum
(FBS) but no unlabeled D-glucose; (3) the incubation medium contained
5 mM unlabeled D-glucose but no serum; and (4) the incubation medium
contained 5 mM unlabeled D-glucose+2% FBS.
For each condition for each group, the amount of radiolabeled glucose
was determined by liquid scintillation counting. Results are charted
in FIG. 5. The data are the mean.+-.S.D. of three determinations
for each group under each condition. Under each of the four conditions,
treatment with PALP at both 0.5 U/mL and 1.0 U/mL (.box-solid.)
concentrations enhanced the cellular content of D-[.sup.14C]glucose
approximately two-fold or more, relative to the untreated control
group (.quadrature.). In the presence of 5 mM unlabeled D-glucose
(conditions 3 and 4), L1 cells obviously incorporated less D-[.sup.14C]glucose
(because of greatly decreased specific activity); at the same time
PALP had somewhat greater stimulatory effects on D-[.sup.14C]glucose
uptake compared to its effect in glucose-free medium. The experiment
was repeated for each group at least once, with similar results.
Example 10
Stimulatory Effect of PALP on Cellular Synthesis of [.sup.14C]Glycogen
from D-[.sup.14C]Glucose
The same conditions as described in Example 9 were used, except
the amount of radiolabeled glycogen was determined by liquid scintillation
counting. Results are charted in FIG. 6. Treatments included PALP
at 0.5 U/mL and 1.0 U/mL (.box-solid.), and the untreated control
group (.quadrature.). Again, while in the presence of 5 mM unlabeled
D-glucose L1 cells obviously incorporated less D-[.sup.14C]glucose
into glycogen, in the presence of 5 mM D-glucose PALP had slightly
greater (.about.2 to .about.3-fold) stimulatory effects on the synthesis
of [.sup.14C]glycogen.
Example 11
Stimulatory Effect of PALP on the Incorporation of D-[.sup.14C]Glucose
into the Total Lipid Fraction
The same conditions as described in Example 9 were used, except
the amount of total radiolabeled lipids was determined by liquid
scintillation counting. Results are charted in FIG. 7. Treatments
included PALP at 0.5 U/mL E and 1.0 U/mL (.box-solid.), and the
untreated control group (.quadrature.). Although in the presence
of 5 mM unlabeled D-glucose cells incorporated less D-[.sup.14C]glucose
into lipids, as expected, PALP had slightly greater (.about.2 to
.about.2.5-fold) stimulatory effects on [.sup.14C]lipid synthesis
compared to its effects in the absence of D-glucose.
Overall the experiments described in Examples 9 11 indicate that,
in differentiated L1 cells, PALP clearly stimulates cellular uptake
and metabolism of D-glucose, that PALP is more effective at 1 U/mL
than at 0.5 U/mL concentration, and that the effects of PALP do
not strongly depend on the concentration of glucose in the medium.
These experiments strongly suggest that a primary target of PALP
in mice, and by extension in humans, is fat tissue.
Example 12
Stimulatory Effect of PALP on Glucose Metabolism in Differentiated
L6 Cells
Another potential target of PALP may be the skeletal muscle. The
effects of PALP on glucose uptake/metabolism were determined in
differentiated L6 cells, a widely used model for skeletal muscle.
Differentiated L6 cells in 12-well plates were incubated in 2%
FBS-containing MEM for 6 hours with 1 .mu.Ci/mL of D-[.sup.14C]glucose
in the absence (.quadrature.) or presence of 1 U/mL commercial PALP
, 50 nM insulin ("Ins") , or 1 U/mL commercial PALP+50
nM Ins (.box-solid.). The data, shown in FIG. 8, are the mean.+-.S.D.
of three determinations in one experiment (the experiment was repeated
once with similar results).
PALP clearly stimulated, although only slightly, both the cellular
uptake of D-[.sup.14C]glucose and its metabolism to [.sup.14C]glycogen
and [.sup.14C]total lipid for the differentiated L6 cells. Insulin
was at least 50% more effective than PALP in stimulating all three
mechanisms, and in each case the effects of PALP plus insulin were
roughly additive or less than additive.
Examples 13 and 14
Digestion with Protease does not Reduce the Effects of PALP on
Glucose Metabolism
Example 13
Digestion of PALP by Bromelain
Bromelain (BRL) is a protease which was previously shown to effectively
digest PALP leading to the formation of fragments of lower molecular
mass [Kottel, R. H. and Hanford, W. C., "Differential release
of membrane-bound alkaline phosphatase isoenzymes from tumor cells
bybromelain." Biochem. Biophys. Methods 2, 325 330 (1980)].
Based on this observation, bromelain was used to digest commercial
PALP to determine if digestion by a protease can generate a smaller
PALP fragment which is able to stimulate glucose metabolism.
A preparation comprising 20 U/mL commercial PALP and 0.01 mg/mL
of BRL (Sigma-Aldrich) in 1 mL of 25 mM Tris-HCl (pH 7.4) buffer
was incubated at 37.degree. C. for 2 hours. FIG. 9 shows a gel picture
(obtained by SDS-PAGE using 7.5% polyacrylamide) with undigested
commercial PALP in lane 1 and BRL-digested PALP in lane 2. It is
clear that, after digestion with 0.01 mg/mL of BRL, no detectable
amount of the original PALP molecule remains.
Example 14
Effect of Bromelain Digestion Product of PALP on Glucose Metabolism
Differentiated L1 cells were preincubated in serum-free and glucose-free
DMEM for two hours under the following conditions: (a) untreated;
(b) in the presence of 1 U/mL commercial PALP; (c) in the presence
of 0.01 mg/mL of BRL; and (d) in the presence of 1 U/mL commercial
PALP+0.01 mg/mL BRL. Each group was then incubated for 5 hours in
the presence of D-[.sup.14C]glucose. Total radiolabeled lipids,
radiolabeled glucose, and radiolabeled glycogen were determined
by liquid scintillation counting, as described above.
Data is shown in FIG. 10. Treatment by BRL alone slightly enhanced
both [.sup.14C]glycogen and [.sup.14C]lipid synthesis but had no
clear effect on the cellular content of D-[.sup.14C]glucose, relative
to untreated cells (.quadrature.). Treatment with the bromelain
digestion product of PALP (.box-solid.) enhanced glycogen synthesis,
lipid synthesis, and glucose uptake relative to untreated cells
(.quadrature.). Treatment with the bromelain digestion product of
PALP (.box-solid.) slightly enhanced glycogen synthesis but not
lipid synthesis, and had no clear effect on the cellular content
of D-[.sup.14C]glucose, relative to treatment by PALP .
This experiment indicated that at least one PALP fragment (i.e.
a sequence of PALP which is smaller than the full size PALP) at
least partially retained the ability of native PALP to enhance glucose
metabolism. Furthermore, the experiment also justifies the assumption
that a fragment of PALP may be even more active in stimulating glycogen
synthesis than native PALP. Potentially any sequence derived from
PALP could be active in promoting glucose metabolism.
Examples 15 17
Comparison of the Effects of PALP and Insulin on Glucose Metabolism
in Differentiated L1 Cells
During the course of the experiments, it was observed that L1 cells
respond better to PALP if the cells are used 8 to 11 days after
terminating treatment with differentiation-inducing agents, as opposed
to 14 days which was practiced in the previous experiments. Therefore,
in the subsequent experiments we used L1 cells within 9 to 11 days
after terminating treatment with differentiation-inducing agents.
Example 15
Effect of PALP and Ins on Glucose Content of Incubation Medium
in Differentiated L1 Cells
In this experiment, differentiated L1 cells in 12-well plates were
incubated (in 0.75 mL volume/well) in 2% BFS-containing glucose-free
DMEM for 6 hours with 1 .mu.Ci/mL of D-[.sup.14C]glucose in the
absence or presence of 1 U/mL commercial PALP or 50 nM Ins. At the
conclusion of the experiment, 0.5 mL aliquots of the incubation
medium were added to 9.5 mL ECOLUME, and the radioactivity was determined
by scintillation counting. The data indicating loss of D-[.sup.14C]glucose
from the medium are the means.+-.S.E.M. of three experiments each
performed in triplicate.
Data is graphed in FIG. 11. The experiment showed that treatment
with PALP and 50 nM Ins (.box-solid.) stimulated the loss of D-[.sup.14C]glucose
from the medium (i.e. uptake by the cells) by .about.86% and 77%,
respectively, relative to the untreated control (.quadrature.).
Thus, Ins, at the physiologically supra-maximal 50 nM concentration,
was more effective than PALP in increasing the uptake of D-[.sup.14C]glucose
by differentiated L1 cells.
Example 16
Effect of PALP and Ins on the Cellular Uptake and Metabolism of
Glucose in Differentiated L1 Cells
Differentiated L1 cells in 12-well plates were incubated in 2%
FBS-containing glucose-free DMEM with 1 .mu.Ci/mL of D-[.sup.14C]glucose
for 2 hours (FIG. 12, upper panel or 6 hours (FIG. 12, lower panel)
in the absence (.quadrature.) or presence of 0.25 U/mL commercial
PALP (), 1 U/mL commercial PALP , or 10 nM Ins (.box-solid.). The
data shown in FIG. 12 are the mean.+-.S.D. of three determinations
in one experiment (the experiment was repeated once with similar
results). Note that on this occasion, the concentration of Ins was
only 10 nM, which is closer (although still higher) to the physiological
concentration of Ins (around 1 nM or less).
After treatments for 2 hours, 1 U/mL PALP had somewhat greater
effects than Ins on the cellular level of D-[.sup.14C]glucose and
[.sup.14C]glycogen (upper panel). After incubations for 6 hours,
1 U/mL of PALP was more effective than 10 nM Ins in stimulating
uptake of D-[.sup.14C]glucose and its subsequent conversion into
both [.sup.14C]glycogen and [.sup.14C]total lipid (lower panel).
Example 17
Comparison of the Effects of PALP and a High Concentration of Ins
on the Cellular Uptake and Metabolism of Glucose in Differentiated
L1 Cells
Differentiated L1 cells in 12-well plates were incubated in 2%
FBS-containing glucose-free DMEM for 2 hours (FIG. 13, upper panel
or 6 hours (FIG. 13, lower panel) with 1 .mu.Ci/mL of D-[.sup.14C]glucose
in the absence (.quadrature.) or presence of 0.25 U/mL commercial
PALP , 1 U/mL commercial PALP , or 50 nM Ins (.box-solid.). The
data shown in FIG. 13 are the mean.+-.S.D. of three determinations
in one experiment (the experiment was repeated once with similar
results).
After incubations for either 2 hours (FIG. 13, upper panel) or
6 hours (FIG. 13, lower panel), 50 nM Ins was significantly more
effective than 1 U/mL of PALP on [.sup.14C]glycogen synthesis and
slightly more effective on [.sup.14C]total lipid synthesis and the
cellular content of D-[.sup.14C]glucose.
Example 18
Comparison of Stimulation of Glucose Uptake and Metabolism in Mouse
Embryo NIH 3T3 Fibroblasts by PALP and Ins
Data shown in FIG. 14 is for an experiment comparing the effects
of PALP and Ins on glucose metabolism in mouse embryo NIH 3T3 fibroblasts.
This cell line was chosen because it is relatively insensitive to
Ins, and, therefore, it appeared to be suitable to further verify
what had been suggested by animal experiments; namely, that the
effects of PALP occur independently of Ins. Since serum may contain
Ins, the fibroblasts were incubated in serum-free medium to further
exclude any contribution by Ins.
Accordingly, confluent NIH 3T3 fibroblast cultures in 12-well plates
were incubated for 5 hours in serum-free glucose-free DMEM with
1 .mu.Ci/mL of D-[.sup.14C]glucose in the absence (.quadrature.)
or presence of 1 U/mL commercial PALP (), 50 nM Ins (), or 1 U/mL
commercial PALP+50 nM Ins (.box-solid.). The data shown in FIG.
14 are the mean.+-.S.D. of three determinations in one experiment
(the experiment was repeated once with similar results).
PALP enhanced cellular uptake of D-[.sup.14C]glucose by 38% and
its metabolism to [.sup.14C]glycogen and [.sup.14C]total lipid by
77% and 55%, respectively. In each case, treatment with Ins alone
() was only about 50% as effective as PALP alone () and Ins did
not enhance any of the effects of PALP when used in combination
(.box-solid.), relative to treatment by PALP alone (). Thus, this
experiment demonstrates that PALP exerts an effect on glucose metabolism
without requiring the presence of insulin.
Examples 19 and 20
Effects of PALP Preparations on Glucose Metabolism in Differentiated
L1 Cells in the Presence of 10% Serum
In the presence of 10% FBS most cellular events, particularly cell
proliferation, are maximally or near maximally stimulated due to
the presence of optimum concentrations of growth factors. It is
difficult to assess which serum level (2% or 10% serum) used for
in vitro experiments corresponds better to the situation in vivo
(which, in turn, will also depend on the eating habit and the combination
of various physiological parameters of the individual). However,
as a conservative approach, it is generally accepted that if an
effect can be observed in vitro even in the presence of 10% serum,
then it is very likely that this particular phenomenon is physiologically
relevant. Therefore, next it was determined if PALP and Ins still
can enhance glucose metabolism in the presence of 10% serum, and
if the effects of preparations made from various batches of commercial
PALP were comparable.
Example 19
Effect of PALP Preparations and Ins on Glucose Content of Incubation
Medium for Differentiated L1 Cells
Data shown in FIG. 15 is for an experiment in which the effects
of four different preparations made from various batches of commercially
obtained PALP on the loss of D-[.sup.14C]glucose from the incubation
medium in differentiated L1 cell cultures were compared to the effect
of Ins.
Preparations were made from four separate batches of commercially
obtained PALP (preparations 1 through 4), the preparations comprising
1.5 U/mL PALP in serum-free glucose-free DMEM. Differentiated L1
cells were pretreated for 30 minutes with PALP-free DMEM (), with
preparation 1 (), 2 (), 3 (), or 4 (), or with 50 nM Ins in serum-free
glucose-free DMEM (.box-solid.), followed by the addition of 0.5
.mu.Ci of D-[.sup.14C]glucose for 10 hours. The data shown in FIG.
15 are [.sup.14C]glucose content of the incubation medium at the
end of incubation (time=10 hours), compared to [.sup.14C]glucose
content at the start of incubation (time=0 hours; .quadrature.),
and represent the mean.+-.S.D. of three determinations in one experiment
(the experiment was repeated once with similar results).
While all four PALP preparations clearly enhanced the loss of D-[.sup.14C]glucose
from the medium, there was some variability in their respective
effects which appeared to go beyond the experimental error. Again,
50 nM Ins was more effective than any of the commercial PALP preparations.
Example 20
Exploration of the Differences in Effect on Glucose Metabolism
for Various PALP Preparations
Data shown in FIG. 16 is for an experiment in which potential differences
in the effects on glucose metabolism in differentiated L1 cells
among the four commercial PALP preparations was further explored.
Differentiated L1 cells were pretreated (in 10% FBS-containing medium)
for 30 min with PALP-free DMEM (.quadrature.), or with preparation
1 (), 2 (), 3 (), or 4 () of commercial PALP (1.5 U/mL), or 50 nM
Ins in serum free glucose-free DMEM (.box-solid.), followed by the
addition of 0.5 .mu.Ci of D-[.sup.14C]glucose for 6 hours. The data
are the mean.+-.S.D. of three determinations in one experiment (the
experiment was repeated once with similar results).
While, again, each PALP preparation enhanced the synthesis of both
[.sup.14C]glycogen and [.sup.14C]total lipid, the preparation which
decreased the medium content of D-[.sup.14C]glucose the most (PALP
1 in FIG. 15) was also the most effective in stimulating the synthesis
of [.sup.14C]glycogen and [.sup.14C]total lipid (FIG. 16).
In addition to demonstrating variability in the actions of various
batches of commercially obtained PALP, the data in FIGS. 15 and
16 also indicate that PALP exerts considerable effects on glucose
metabolism in L1 cells even in the presence of 10% serum. Such effects
of PALP were observed in the presence of 10% serum only if cells
were used 8 12 days after terminating the differentiation treatment.
Examples 21 and 22
Effect of Homogeneous Purified PALP on Glucose Metabolism in Differentiated
L1 Cells
In the experiments described in Examples 21 and 22, homogeneous
purified PALP was used to demonstrate that the effects of partially
purified commercial PALP that were observed in preceding Examples
were indeed elicited by activity of the PALP enzyme and not by a
contaminating protein.
Homogeneous purified PALP was prepared by the complete purification
procedure described in Example 1. The homogeneous purified PALP
used in the experiments of Examples 21 and 22 produces a single
band in an electrophoretic separation, such as shown in lane 5 of
FIG. 1.
Example 21
Effect of Homogeneous Purified PALP on Glucose Content of Incubation
Medium in Differentiated L1 Cells
Data for this Example is shown in FIG. 17. Differentiated L1 cells
in 12-well plates were incubated in 10% serum-containing glucose-free
DMEM for 10 hours with 0.5 .mu.Ci of D-[.sup.14C]glucose in the
absence () or presence of 200 nM homogeneous purified PALP (), or
50 nM Ins (.box-solid.), followed by the determination of D-[.sup.14C]glucose
in the medium. The data shown in FIG. 17 are [.sup.14C]glucose content
of the incubation medium at the end of incubation (time=10 hours),
compared to [.sup.14C]glucose content at the start of incubation
(time=0 hours; .quadrature.), and represent the mean.+-.S.D. of
three determinations in one experiment.
Homogeneous purified PALP at 200 nM effectively reduced the medium
content of D-[.sup.14C]glucose, although its effect was somewhat
less than that of 50 nM insulin. The effects of purified PALP on
glucose uptake were comparable to the effects of the best comm |