U.S. patent application number 14/656083 was filed with the patent office on 2015-07-02 for methods of treating anemia and red blood cell dysfunction with lecithin cholesterol acyltransferase.
The applicant listed for this patent is AlphaCore Pharma, LLC. Invention is credited to Bruce J. AUERBACH, Reynold HOMAN, Brian KRAUSE.
Application Number | 20150182600 14/656083 |
Document ID | / |
Family ID | 42676855 |
Filed Date | 2015-07-02 |
United States Patent
Application |
20150182600 |
Kind Code |
A1 |
AUERBACH; Bruce J. ; et
al. |
July 2, 2015 |
METHODS OF TREATING ANEMIA AND RED BLOOD CELL DYSFUNCTION WITH
LECITHIN CHOLESTEROL ACYLTRANSFERASE
Abstract
Disclosed are methods for treating conditions characterized by
anemia or red blood cells dysfunction by administering an agent
that increases the level of endogenous LCAT or LCAT activity.
Additionally disclosed are methods of treating conditions wherein
red blood cells have reduced function in relation to deformability,
oxygenation, increased adhesion and aggregability, reduced nitric
oxide function, or decreased life-span, increased free cholesterol,
or abnormal phospholipid content. Also disclosed are methods for
treating conditions characterized by an abnormal concentration of
free cholesterol in red blood cells and methods of normalizing the
free cholesterol content of red blood cells.
Inventors: |
AUERBACH; Bruce J.; (Ann
Arbor, MI) ; KRAUSE; Brian; (Ann Arbor, MI) ;
HOMAN; Reynold; (Ann Arbor, MI) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
AlphaCore Pharma, LLC |
Ann Arbor |
MI |
US |
|
|
Family ID: |
42676855 |
Appl. No.: |
14/656083 |
Filed: |
March 12, 2015 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
13946236 |
Jul 19, 2013 |
|
|
|
14656083 |
|
|
|
|
13377586 |
Jan 12, 2012 |
8492108 |
|
|
PCT/US10/38494 |
Jun 14, 2010 |
|
|
|
13946236 |
|
|
|
|
61241223 |
Sep 10, 2009 |
|
|
|
61186668 |
Jun 12, 2009 |
|
|
|
Current U.S.
Class: |
424/94.5 ;
514/44R |
Current CPC
Class: |
A61P 3/10 20180101; A61P
15/10 20180101; A61P 31/14 20180101; A61P 35/00 20180101; A61P 7/06
20180101; A61P 33/00 20180101; A61P 33/06 20180101; A61K 38/45
20130101; A61P 37/06 20180101; Y02A 50/409 20180101; A61P 31/04
20180101; A61P 33/02 20180101; A61P 25/08 20180101; A61P 9/00
20180101; A61P 25/28 20180101; Y02A 50/411 20180101; Y02A 50/415
20180101; A61P 19/02 20180101; Y02A 50/30 20180101; A61P 1/16
20180101; A61P 17/02 20180101; C12Y 203/01043 20130101; A61P 37/00
20180101; C12N 9/1029 20130101; A61P 29/00 20180101; A61K 48/00
20130101 |
International
Class: |
A61K 38/45 20060101
A61K038/45 |
Claims
1. A method of treating a patient having a condition characterized
by anemia or red blood cell dysfunction comprising administering to
the patient a therapeutically effective amount of an agent which
increases the activity of LCAT or increases the plasma level of
LCAT or both.
2. The method according to claim 1 wherein he agent is LCAT.
3. The method according to claim 1 wherein the agent is a drug that
increases the activity or level of endogenous LCAT.
4. The method according to claim 1 wherein the agent is a gene
therapy vector.
5. The method of claim 2 wherein the amount of LCAT is an amount
that increases LCAT concentration to above normal LCAT levels or
LCAT activity to above normal LCAT activity.
6. The method according to claim 5 wherein the condition is
characterized by red blood cells with reduced ability to deform,
reduced oxygenation, increased aggregation and adhesion, reduced
nitric oxide function, decreased life-span, or any combination
thereof.
7. The method according to claim 5 wherein the condition is
anemia.
8. The method of claim 5 wherein the condition is sickle cell
disease, diabetes, thalassemia, rheumatoid disease, autoimmune
disease, arthritis, liver disease, cirrhosis, hepatitis,
acanthosytosis, sepsis, dementia, anemia, or microvascular
disorders, inflammatory disorders, parasitic disease, erectile
dysfunction, cancer, pre-eclampsia, critical illness or trauma.
9. The method of claim 8 wherein the inflammatory disorder is
sepsis, rheumatoid disease, anemia of inflammation, or
post-surgical inflammation.
10. The method of claim 8 wherein the microvascular disorder is
dementia, or retinopathy.
11. The method of claim 8 wherein the condition is parasitic
disease and the parasitic disease is malaria, sleeping sickness,
filariasis, or leishmaniasis.
12. The method of claim 8 wherein the condition is an Alzheimer's
related dementia.
13. The method of claim 8 wherein the condition is sickle cell
disease.
14. The method of claim 8 wherein the condition is thalassemic
disease.
15. The method of claim 8 wherein the LCAT is a modified LCAT.
16. The method of claim 5 wherein the modified LCAT comprises an
amino acid substitution at position 31.
17. A method of improving a condition characterized by one or more
of the following: anemia, red blood cells with reduced ability to
deform, reduced RBC oxygenation, increased RBC aggregation and
adhesion, reduced nitric oxide function, decreased RBC life-span
comprising: obtaining a base-line measurement of one or more than
one of the following: hemoglobin level, hematocrit level, RBC
deformability, RBC oxygenation, RBC aggregation and adhesion, or
RBC life-span; a) administering to a patient in need thereof a
therapeutically effective amount of an agent which increases the
activity of LCAT or increases the plasma level of LCAT or both; b)
obtaining a post-treatment measurement of one or more of the
following: hemoglobin level, hematocrit level, RBC deformability,
RBC oxygenation, RBC aggregation and adhesion, or RBC life-span; c)
comparing the baseline measurement with the post-treatment
measurement wherein the occurrence of one or more of the following:
an increase in hemoglobin level, hematocrit level, an increase in
RBC deformability, an increase in RBC oxygenation, a decrease in
RBC aggregation and adhesion or an increase in RBC life-span,
indicates an improvement in the condition.
18. The method according to claim 17 wherein the agent is a drug
that increases the activity or level of endogenous LCAT.
19. The method according to claim 17 wherein the agent is a gene
therapy vector.
20. The method according to claim 17 wherein the therapeutically
effective amount of an agent is therapeutically effective amount of
LCAT.
21. The method of claim 20 wherein the RBC deformability is
increased following the administration of LCAT.
22. The method of claim 20 wherein RBC oxygenation is increased
following the administration of LCAT.
23. The method in claim 20 wherein the hematocrit level is
increased following the administration of LCAT.
24. The method in claim 20 wherein the hemoglobin level is
increased following the administration of LCAT.
25. The method in claim 20 wherein nitric oxide function is
increased following the administration of LCAT.
26. The method of claim 20 wherein RBC life-span is increased
following the administration of LCAT.
27. The method of claim 20 wherein RBC aggregation and adhesion is
decreased following the administration of LCAT.
28. The method according to any one of claims 1, 2, 5-16 and 20-27
wherein the therapeutically effective amount of LCAT is an amount
that raises the LCAT concentration or LCAT activity to above normal
LCAT levels.
29. The method according to any one of claims 1, 2, 5-16 and 20-28
wherein the administration of LCAT is by intravenous injection,
subcutaneous injection or intra-muscular injection.
30. The method according to any one of claims 1, 2, 5-16 and 20-29
and wherein the amount of LCAT administered is from about 10 mg to
about 5000 mg.
31. The method according to any one of claims 1, 2, 5-16 and 20-29
wherein the amount of LCAT administered is between 1-times and
1000-times the normal level of LCAT.
32. A method of treating a patient having a condition characterized
by a high PC/SM ratio in RBC membranes comprising administering to
the patient
33. The method according to claim 32 wherein the condition is
sickle cell disease, diabetes, thalassemia, rheumatoid disease,
autoimmune disease, arthritis, liver disease, cirrhosis, hepatitis,
acanthosytosis, sepsis, dementia, anemia, or microvascular
disorders, inflammatory disorders, parasitic disease, erectile
dysfunction, cancer, pre-eclampsia, critical illness or trauma.
34. The method according to claim 33 further comprising determining
a baseline ratio of PC to SM and determining the ratio of PC to SM
following administration of LCAT wherein a decrease in the ratio of
PC to SM indicates an improvement in the condition.
35. A method of treating a patient having a condition characterized
by a high Fe content comprising administering to the patient in
need thereof, a therapeutically effective amount of LCAT.
36. The method according to claim 35 wherein the condition is
sickle cell disease, diabetes, thalassemia, rheumatoid disease,
autoimmune disease, arthritis, liver disease, cirrhosis, hepatitis,
acanthosytosis, sepsis, dementia, anemia, or microvascular
disorders, inflammatory disorders, parasitic disease, erectile
dysfunction, cancer, pre-eclampsia, critical illness or trauma.
37. The method of claim 36 wherein the blood cell is a red blood
cell, monocyte, platelet, neutrophil or leukocyte.
38. The method of claim 37 wherein the blood cell is a red blood
cell.
39. A method of improving a condition characterized a high level of
FC in RBC membranes comprising: obtaining a base-line measurement
of the ratio of FC to PL; a) administering to a patient in need
thereof a therapeutically effective amount of LCAT; b) obtaining a
post-treatment measurement of the ratio of FC to PL; c) comparing
the baseline measurement with the post-treatment measurement
wherein a decrease in the ratio of FC to PL indicates an
improvement in the condition.
40. The method according to any of claims 32-39 wherein the LCAT is
administered by intravenous injection, subcutaneous injection or
intra-muscular injection.
41. The method according to any one of claims 32-40 wherein the
amount of LCAT administered is from about 10 mg to about 5000
mg.
42. The method according to any one of claims 32-40 wherein the
amount of LCAT administered is between 1-times and 1000-times the
normal level of LCAT.
43. The method according to any one of claims 32-42 wherein the
LCAT is a modified LCAT.
44. The method according to claim 43 wherein the modified LCAT
comprises an amino acid substitution at position 31.
45. A method of treating a patient having a condition characterized
by anemia or red blood cell dysfunction comprising administering to
a subject in need thereof, a therapeutically effective amount of
LCAT wherein the plasma HDL-C level in the subject is rapidly
increased after administration of the LCAT.
46. The method of claim 45 wherein the condition is sickle cell
disease, diabetes, thalassemia, rheumatoid disease, autoimmune
disease, arthritis, liver disease, cirrhosis, hepatitis,
acanthosytosis, sepsis, dementia, anemia, or microvascular
disorders, inflammatory disorders, parasitic disease, erectile
dysfunction, cancer, pre-eclampsia, critical illness or trauma.
47. The method of claim 46 wherein the inflammatory disorder is
sepsis, rheumatoid disease, anemia of inflammation, or
post-surgical inflammation.
48. The method of claim 46 wherein the microvascular disorder is
dementia, or retinopathy.
49. The method of claim 46 wherein the condition is parasitic
disease and the parasitic disease is malaria, sleeping sickness,
filariasis, or leishmaniasis.
50. The method of claim 46 wherein the condition is an Alzheimer's
related dementia.
51. The method of claim 46 wherein the condition is sickle cell
disease.
52. The method of claim 46 wherein the condition is thallasemic
disease.
53. The method according to any one of claims 44-52 wherein the
plasma HDL-C level in the subject 4 hours post administration of
the LCAT is increased by at least 50% of the plasma HDL-C level
prior to LCAT administration.
54. The method according to claim 44-52 wherein the plasma HDL-C
level in the subject 24 hours post administration of the LCAT is
increased by at least 100% of the plasma HDL-C level prior to LCAT
administration.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional
Application Ser. No. 61/186,668 filed Jun. 12, 2009, and U.S.
Provisional Application Ser. No. 61/241,223 filed Sep. 9, 2010. The
entire content of U.S. Provisional Application Ser. No. 61/186,668
and U.S. Provisional Application Ser. No. 61/241,223 is
incorporated herein by reference.
FIELD OF INVENTION
[0002] This invention relates generally to the field of medicine,
and in particular, the treatment of diseases characterized by
anemia and/or red blood cells that have abnormal function in terms
of deformability, oxygenation, aggregation, nitric oxide
metabolism, or life-span.
BACKGROUND
[0003] The quality and quantity of red blood cells (RBC) in the
blood stream is often degraded during periods of increased physical
stress, resulting in anemia and enhanced risks of morbidity and
mortality. Physical stresses that have been linked to the
development of anemia include autoimmune diseases, major surgery,
trauma, infectious diseases, cancer, critical illness, diabetes,
liver diseases, kidney disease, heart failure, and parasitic
diseases. Systemic inflammation is a characteristic common to all
these situations as evidenced by the presence of increased levels
of inflammatory cytokines in the circulation. Even in persons
predisposed to anemia because of a hemoglobinopathy, for example
sickle cell disease or thalassemia, inflammatory cytokine levels
are frequently elevated and can exacerbate the disease symptoms,
particularly during crisis episodes.
[0004] One consequence of elevated inflammatory cytokine levels is
a reduction in the hepatic production of the enzyme
lecithin:cholesterol acyltransferase (LCAT). Normally, LCAT is
released into the plasma from the liver to facilitate plasma lipid
turnover and maintain the balance of cholesterol and phospholipids
in the blood and the tissues perfused by blood. Excess cholesterol
is removed from tissues, such as arteries, and delivered to the
liver for excretion in bile by a process known as reverse
cholesterol transport (RCT). In the first step of RCT, cholesterol
passes from tissue cells to high-density lipoproteins (HDL) in the
circulation. In the second step, the enzyme LCAT enhances the
cholesterol-carrying capacity of HDL by catalyzing the
transesterification of a fatty acid from phosphatidylcholine (PC)
(also known as lecithin), to cholesterol to form cholesteryl ester
(CE). The CE product accumulates in the HDL interior until it is
removed at HDL-receptors in the liver. The CE delivered to the
liver by HDL is converted to cholesterol and bile acids that are
excreted in the bile.
[0005] The health consequences of diminished plasma LCAT activity
are most evident in persons with Familial LCAT Deficiency (FLD), a
rare genetic disease in which plasma LCAT activity is absent. The
absence of LCAT activity results in greatly diminished levels of
plasma CE, reflected in decreased HDL and low-density lipoprotein,
and in the accumulation of excess LCAT substrate in plasma. The
major health consequences of FLD are reduced vision resulting from
a diffuse build-up of lipid in the corneas, eventual kidney failure
due to renal lipid accumulation (glomerulosclerosis), and hemolytic
anemia.
[0006] Distortions in the plasma lipoprotein lipid compositions due
to lipid metabolic disorders such as those resulting from low LCAT
activity have been associated with changes in the lipid content of
RBC. A shift in RBC lipids in response to plasma lipid changes can
alter RBC performance and survival since these properties are
dependent on cellular lipid content. The types of RBC lipid changes
that can occur are evident in FLD subjects where the RBC are
enriched in cholesterol and PC and diminished in sphingomyelin (SM)
content. Evidence that these RBC lipid abnormalities depend on
disturbances in plasma lipoprotein lipids as a result of LCAT
deficiency was obtained in an experiment were a temporary
normalization of RBC cholesterol content occurred following
infusion of normal plasma into an FLD subject (Muryama et al. Am.
J. Hematol, 16:129-137, 1984). This temporary normalization of the
RBC lipids could be due to the replenishment of LCAT, HDL,
apolipoprotein A-I or other plasma factors that are absent or
greatly reduced in patients with FLD.
[0007] No link between anemia and LCAT activity is seen in less
severe cases of diminished plasma LCAT activity. For example,
patients with fish eye disease, a milder form of LCAT deficiency,
exhibit less than 30% of normal plasma LCAT activity but have
normal hemoglobin and hematocrit (Rousset et al. Curr. Opin.
Endocrinol. Diabetes Obes. 16:163-171, 2009). Similarly, studies in
subjects with liver disease found no correlation between lowered
LCAT activity and anemia (L W Powell et al. (1975) Aust. N.Z. J.
Med. 5:101-107), or between LCAT activity and RAC lipid
abnormalities. (R A Cooper et al. (1972) J. Clin. Invest.
51:3182-3192).
[0008] Although there is evidence of deleterious lipid alterations
in RBC in persons under physical stress that are similar to those
detected in FLD patients, there is no apparent relationship between
LCAT and RAC level or lipids. Examples of anomalous RBC lipid
composition include reports of increased PC/SM ratio in RBC from
persons with liver disease and in persons with dyslipidemia due to
lipoprotein lipase deficiency or Tangier Disease. We (FIG. 1) and
others have also found an increase in the PC/SM ratio in RAC from
sickle disease patients who are not in crisis. Furthermore, there
are reports of cholesterol enrichment in RBC from persons with
diabetes, heart disease (including acute coronary syndromes),
hypercholesterolemia, sickle cell anemia, and in persons after
space flight.
[0009] The consequences of modified RAC lipid composition are not
fully known but in the case of elevated RBC cholesterol there is
evidence that activities of membrane proteins become abnormal.
Cholesterol-enriched RBC from liver disease patients exhibit
reduced activities of Mg++-ATPase and acetylcholine esterase.
Cholesterol enrichment has been linked to enhanced transfer of
phosphatidylserine from the inner to the extracellular membrane
surface, which is a signal for enhanced clearance of RAC by the
reticulo-endothelial system. Increased RBC cholesterol can reduce
RBC deformability and induce abnormal RBC morphologies, both of
which can impair RBC transit through the capillaries. Transmembrane
gas exchange, an essential RBC function, is also impacted by
cholesterol elevation.
[0010] The current evidence suggests abnormal RAC lipid
compositions can have a deleterious effect on red blood cell
function and therefore there is a need for methods to normalize RAC
lipid composition and methods to treat red blood cell
dysfunction.
SUMMARY OF THE INVENTION
[0011] There is no consensus in the literature as to the
correlation between HDL-C and endogenous LCAT activity. We made the
surprising discovery that an increase in plasma LCAT levels by
injection of recombinant human LCAT rapidly results in a removal of
cholesterol from tissues. Additionally, HDL-C was rapidly
increased. Given the equilibrium that exists between HDL and RBCs,
these surprising results indicate that the infusion of LCAT could
also be used to rapidly correct blood cell lipid abnormalities and
improve blood cell function.
[0012] Plasma HDL-C levels are often reported to be reduced in
cases of physical stress, for example: autoimmune diseases, major
surgery, trauma, infectious diseases, cancer, critical illness,
diabetes, liver diseases, kidney disease, heart failure and
parasitic diseases, and may be an important factor in the
distortion of RBC lipid content, in light of the direct lipid
interchange between RBC and lipoproteins. Anemia is highly
prevalent in the cases where HDL is reduced.
[0013] The present disclosure relates to methods modulating the
lipid content of red blood cell membranes by increasing LCAT
concentration and/or activity above normal human LCAT concentration
and/or activity by administering a therapeutically effective dose
of LCAT.
[0014] One embodiment of the disclosure is a method of treating a
patient having a condition characterized by red blood cell
dysfunction comprising administering to the subject a
therapeutically effective amount of LCAT.
[0015] Another embodiment is a method of treating a patient having
a condition characterized by anemia or red blood cells with reduced
deformability, reduced oxygenation, reduced nitric oxide function,
increased adhesion and/or aggregation, or decreased life-span, or
any combination thereof comprising administering to the patient in
need thereof, a therapeutically effective amount of LCAT.
[0016] In another embodiment, the method of treating a patient
having a condition characterized by anemia or red blood cells with
reduced deformability, reduced oxygenation, reduced nitric oxide
function, increased adhesion and/or aggregation, or decreased
life-span, or any combination thereof comprises determining a
baseline RBC deformability or RBC oxygenation or RBC aggregation or
adhesion or RBC life-span; administering to the patient in need
thereof, a therapeutically effective amount of LCAT; and
determining the changes following LCAT administration wherein an
increase in RBC deformability or RBC oxygenation or decreased RBC
aggregation or adhesion or increased RBC life-span indicates an
improvement in the condition.
[0017] In some embodiments, the condition treated is sickle cell
disease, diabetes, thalassemia, rheumatoid disease, autoimmune
disease, arthritis, liver disease, cirrhosis, hepatitis,
acanthosytosis, sepsis, dementia, anemia, or microvascular
disorders, inflammatory disorders, parasitic disease, erectile
dysfunction, cancer, pre-eclampsia, critical illness or trauma.
[0018] Another embodiment is a method of treating a patient having
a condition characterized by a high level of FC in RBC membranes
comprising administering to subject, in need thereof, a
therapeutically effective amount of LCAT. Another embodiment is a
method of treating a patient having a condition characterized by a
high level of FC in RBC membranes comprising determining a baseline
ratio of FC to PL; administering to subject, in need thereof, a
therapeutically effective amount of LCAT; and determining the ratio
of FC to PL following administration of LCAT wherein a decrease in
the ratio of FC to PL indicates an improvement in the condition.
Another embodiment is a method of reducing the FC content of a
blood cell in a patient comprising administering to the patient a
therapeutically effective amount of LCAT.
[0019] Another embodiment is a method of treating a patient having
a condition characterized by an increased PC/SM ratio in RBC
membranes comprising administering to subject, in need thereof, a
therapeutically effective amount of LCAT. Another embodiment is a
method of treating a patient having a condition characterized by an
increased PC/SM ratio in RBC membranes comprising determining a
baseline ratio of PC to SM; administering to subject, in need
thereof, a therapeutically effective amount of LCAT; and
determining the ratio of PC to SM following administration of LCAT
wherein a decrease in the ratio of PC to SM indicates an
improvement in the condition. Another embodiment is a method of
reducing the PC/SM ratio of a blood cell in a patient comprising
administering to the patient a therapeutically effective amount of
LCAT.
BRIEF DESCRIPTION OF THE FIGURES
[0020] FIG. 1 shows phospholipid composition of RBC from normal
subjects and from subjects with Sickle Cell Disease.
[0021] FIG. 2 is a graph depicting the increase in plasma HDL-C in
human ApoA-I transgenic mice after injection with LCAT.
[0022] FIG. 3 depicts the cholesterol content of tissues from
LCAT-knockout/apolipoprotein A-I transgenic mice after injection
with recombinant human LCAT.
DETAILED DESCRIPTION
[0023] The term "therapeutically effective amount", as used herein,
means the amount of LCAT which will elicit the desired therapeutic
effect or response when administered in accordance with the desired
treatment regimen. A preferred therapeutically effective amount is
an amount of LCAT that increases the level of plasma LCAT to above
normal levels.
[0024] As used herein "LCAT level" refers to the plasma
concentration of LCAT.
[0025] As used herein, a "normal level" of LCAT means the plasma
concentration of LCAT that is present in average healthy untreated
subject not currently on any medication which might alter LCAT
levels. "Normal level" and "endogenous level" are used
interchangeably herein.
[0026] For the avoidance of doubt, references herein to "treatment"
or "treating" include curative, palliative and prophylactic
treatment.
[0027] "Subject" and "Patient" are used interchangeably.
[0028] Between as used herein with reference to effective amount or
unit dosage is inclusive, e.g., "between 1 mg and 5000 mg" includes
1 mg and 5000 mg.
[0029] "From" as used herein with reference to effective amount or
unit dosage is inclusive, e.g., "from 1 mg to 5000 mg" includes 1
mg and 5000 mg.
[0030] "FC" is an abbreviation for free cholesterol and as used
herein means non-esterified cholesterol.
[0031] "Nitric oxide function" means RBC mediated processes which
are dependent on nitric oxide including nitric oxide production,
nitric oxide delivery to the microvasculature, inhibition of
platelet and leukocyte adhesion, vasodilation, RBC deformability
and RBC survival.
[0032] "PC" is an abbreviation for phosphatidylcholine.
[0033] "SM" is an abbreviation for sphingomyelin
[0034] "RBC deformability" means the ability of cells to adapt
their shape to the dynamically changing flow conditions in order to
minimize their resistance to flow, and to enable their passage
through small blood vessels. Reduced deformability equates with
increased rigidity.
[0035] A "gene therapy vector" is an agent which is used to
incorporate and actively express a gene of interest in chromosomes
in parenchymal tissue cells. For example an adenovirus engineered
to carry the human LCAT gene.
[0036] "Osmotic fragility" means a cell's sensitivity to rupture
due to changes in surrounding osmotic pressure.
[0037] "RBC aggregability" means the ability to form multicellular
aggregates, normally in a rouleaux shape, in the presence of plasma
proteins or other macromolecules.
[0038] "LCAT" is used interchangeably with "lecithin-cholesterol
acyltransferase"
[0039] "LCAT" or "LCAT polypeptide" when used herein encompass
native sequence LCAT, LCAT variants, modified LCAT, and chimeric
LCAT. In specifying amino acid positions in the LCAT sequence,
reference is made to SEQ ID NO: 1
TABLE-US-00001 Human LCAT SEQ ID NO: 1 (Genbank Accession No.
AAB34898) FWLLNVLFPP HTTPKAELSN HTRPVILVPG CLGNQLEAKL DKPDVVNWMC
YRKTEDFFTI WLDLNMFLCL GVDCWIDNTR VVYNRSSGLV SNAPGVQIRV PGFGKTYSVE
YLDSSKLAGY LHTLVQNLVN NGYVRDETVR AAPYDWRLEP GQQEEYYRKL AGLVEEMHAA
YGKPVFLIGH SLGCLHLLYF LLRQPQAWKD RFIDGFISLG APWGGSIKPM LVLASGDNQG
IPIMSSIKLK EEQRITTTSP WMFPSRMAWP EDHVFISTPS FNYTGRDFQR FFADLHFEEG
WYMWLQSRDL LAGLPAPGVE VYCLYGVGLP TPRTYIYDHG FPYTDPVGVL YEDGDDTVAT
RSTELCGLWQ GRQPQPVHLL PLHGIQHLNM VFSNLTLEHI NAILLGAYRQ GPPASPTASP
EPPPPE
[0040] Specific amino acids in the native human LCAT protein
sequence are described using single letter amino acid designation
followed by the position in the protein sequence, for example W2
indicates that position 2 is a tryptophan. To represent a
substitution at a particular position, the substituted amino acid
follows the position, for example W2Y indicates that the tryptophan
at position 2 is replaced with a tyrosine.
[0041] A "native sequence LCAT" comprises a polypeptide having the
same amino acid sequence as a LCAT derived from nature. Thus, a
native sequence LCAT specifically encompasses naturally occurring
truncated forms of LCAT, and naturally-occurring allelic variants
of LCAT, naturally-occurring variant forms (e.g., alternately
spliced forms). The preferred native sequence LCAT is a mature
native sequence LCAT.
[0042] "Modified LCAT" means a polypeptide wherein one or more
amino acids in the native LCAT polypeptide is substituted with
another amino acid, or one or mom amino acids is added to a portion
of the native polypeptide, including, but not limed to, the
N-terminal or C-terminal amino acid. For example and without
limitation the modified LCAT may be a modified LCAT protein as
described in U.S. patent application Ser. No. 12/179,815. In other
embodiments the one or more amino acid is substituted with a
conservative substitution. Non-limiting exemplary conservative
substitutions are provided in Table 2. In other embodiments, the
one or more amino acids is substituted with a non-naturally
occurring amino acid. In addition, modified LCAT polypeptides
include derivatives of LCAT or modified LCAT. These derivatives
may, for example, improve the solubility, absorption, biological
half life, of the polypeptides. Derivatives of polypeptides are
well known in the art. One of skill in the art would know how to
derivatize polypeptides to improve their pharmacologic
properties.
TABLE-US-00002 TABLE 2 Exemplary Conservative Original Residue
Substitutions A G, S R K N Q, H D E C S Q N E D G A, P H N, Q I L,
V L I, V K R, Q, E M L, Y, I F M, L, Y S T T S W Y Tyr W, F Val I,
L
[0043] The present disclosure is directed to methods of treating
patients having a condition characterized by anemia or red blood
cell dysfunction comprising administering to a patient in need
there of an agent which increases the activity of LCAT or increases
the plasma level of LCAT or both. The LCAT level and/or LCAT
activity can be increased by any means available. This includes,
without limitation, direct administration of LCAT, expression of
LCAT through gene therapy, and the up-regulation of endogenous LCAT
through the administration of drugs.
[0044] In one embodiment the level of LCAT level and/or activity is
increased by direct administration of LCAT. Preferably the LCAT
administered in the methods according to the disclosure is
recombinantly produced human LCAT (e.g., using animals, mammalian
cells, fungi, insect cells or plants as a recombinant protein
expression system). Methods of producing proteins recombinantly is
well known in the art. LCAT may also he obtained by any suitable
methods e.g., isolation from human plasma. LCAT for can be prepared
in stable bulk or unit dosage forms. In one embodiment the level of
LCAT activity is increased through the use of gene therapy. As used
herein, "gene therapy" refers to the transfer and, preferably,
stable integration of new genetic information into cells in a
subject. Methods of increasing LCAT activity levels by gene therapy
involves transfecting cells with a nucleic acid that comprises a
nucleic acid sequence coding for expression of LCAT. The
transfected cells express LCAT and secrete it into the plasma of
the subject. The cells are transfected in sufficient number or for
such high expression of LCAT that they increase the amount of LCAT
to a therapeutically effective level. Genes encoding LCAT may be
introduced into the subject by any suitable method. In one
embodiment, the genes are introduced into cells of the individual
in vivo by means of expression vectors. In another embodiment, the
genes are introduced into cells ex-vivo, and transfected cells that
express and secrete LCAT are administered to the subject. In the in
vivo approaches, liver cells are useful targets for transfection.
Liver cells produce LCAT, so they possess the processing machinery
for making the enzyme recombinantly. Furthermore, vectors injected
into the blood stream quickly pass through the liver, so liver
cells are quickly exposed to the vectors. Hematopoietic stem cells
also are useful targets for gene therapy because they multiply
rapidly, thereby creating more cells capable of producing LCAT.
Ex-vivo approaches also are attractive because they allow more
control over the transfection process. For example, transfected
cells can be tested and the ones which express LCAT in the highest
amounts can be selected. Hematopoietic stem cells can be taken from
the subject, transfected ex vivo and reintroduced into the subject.
Therefore, in one embodiment, the cells are cells from the subject.
Methods of transfecting genes into mammalian cells, either in vivo
and ex vivo, and obtaining their expression are well known to the
art.
[0045] The present disclosure relates to methods of modulating
lipid content of red blood cell membranes by increasing LCAT levels
and/or activity above normal human LCAT levels. One embodiment of
the disclosure is a method of treating a patient having a condition
characterized by red blood cells dysfunction comprising
administering to the subject a therapeutically effective amount of
LCAT. Another embodiment of the disclosure is a method of treating
a patient having a condition characterized by red blood cells
dysfunction comprising administering to the subject a
therapeutically effective amount of a drug which increases the
endogenous production of LCAT or increases LCAT activity. In
particular embodiments the drug is a small molecule therapeutic
agent.
[0046] Some embodiments are directed to methods of normalizing FC
content of RBC cellular membranes by increasing LCAT levels or
increasing LCAT activity in a subject in need thereof. One
embodiment according to the present disclosure is a method of
treating a patient having a condition which is characterized by
having RBC with increased FC content by administering a
therapeutically effective dose of LCAT to a patient in need
thereof. Increasing LCAT levels rapidly cause the net transfer of
FC from RBC to HDL, thus changing the composition of the RBC
membrane to a more fluid state. This action increases the
oxygenation of the RBC, improves the rheology (increase
deformability, flow, decrease phosphatidylserine externalization,
decrease the propensity for adhesion and aggregation) decrease
anemia (decrease the mechanical stress and destruction associated
with decreased deformability, increasing the life of the RBC), and
increase the ability of the RBC to oxygenate tissue, especially
peripheral tissues. In some embodiments erythropoiesis is increased
following the administration of a therapeutically effective amount
of LCAT. In some embodiments nitric oxide function is increased
following the administration of a therapeutically effective amount
of LCAT. There are many conditions in which the cell membranes of
the RBC have increased levels of FC in relation to phospholipid
levels. Increased FC content in blood cell membranes is present in
a number of disease states including, but not limited to, sickle
cell disease, diabetes, thalassemia, rheumatoid disease, autoimmune
disease, arthritis, liver disease, cirrhosis, hepatitis,
acanthocytosis, sepsis, dementia, anemia, or microvascular
disorders, inflammatory disorders, parasitic disease, erectile
dysfunction, cancer, pre-eclampsia, critical illness or trauma.
[0047] Although not a primary pathology in these diseases, the
change in RBC composition and function leads to exacerbation of the
morbidity of the underlying disorders. Thus, one embodiment of
present disclosure is a method of treating a patient with sickle
cell disease, diabetes, thalassemia, rheumatoid disease, autoimmune
disease, arthritis, liver disease, cirrhosis, hepatitis,
acanthosytosis, sepsis, dementia, anemia, or microvascular
disorders, inflammatory disorders, parasitic disease, erectile
dysfunction, cancer, pre-eclampsia, critical illness or trauma by
administering a therapeutically effective dose of LCAT to a patient
in need thereof.
[0048] Hemoglobin gene mutations such as in sickle cell disease
(SCD), thalassemias and hemoglobin E (HbE) can result in a variety
of pathologies which decrease RBC deformability and ability to
carry/deliver oxygen. As an example, SCD is an inherited disorder,
caused by a single amino acid replacement in the beta-globulin
subunit of hemoglobin (HbS). Under low oxygen conditions, HbS
polymerizes (aggregates), leading to changes in the shape of the
RBCs from normal concave to "sickle-shaped". The formation of rigid
HbS polymers decreases RBC elasticity or deformability, which is
detrimental to their function, since they have to be able to
repeatedly pass through capillaries four times smaller than their
own size to oxygenate tissues. Therefore, sickling leads to
vasoocclusive disease due to occlusion of postcapillary venules of
all sizes and increased RBC fragility, leading to lysis and
hemolytic anemia. Although sickling under low oxygen conditions
causes acute crises and the major problems associated with the
disease, the RBCs from patients that are not sickled, under normal
oxygen conditions, have more rigid membranes with decreased
deformability and increased aggregability. Chemical analysis of
erythrocyte membranes from SCD also demonstrates increased FC
content. Additionally, these patients usually have low HDL with a
decreased CE content, inferring a decreased LCAT activity or
functional LCAT deficiency. In fact, in one study, LCAT activity
was shown to be decreased by 30% in patients with SCD. Accordingly,
one embodiment of the current disclosure is a method of treating a
patient having sickle cell disease by administering to a patient in
need thereof, a therapeutically effective amount of LCAT.
[0049] Injecting high levels of LCAT, for example, an amount that
results in a doubling of the endogenous activity to 1000-times the
endogenous activity of LCAT in SCD patients, would force a movement
of FC from RBC and concomitantly increasing plasma HDL-C levels. A
reduction in the FC content of the RAC would lead to an increase in
the ability of the RBC to deform and improve the rate of O.sub.2
exchange. The improved function of the RBC may lessen the occlusive
events due to both improved flow properties of blood and decreased
rate of sickling (due to better re-oxygenation of the RBC). In
another embodiment the administration of a therapeutically
effective amount of LCAT to a patient in need thereof, increases
RBC deformability and RBC oxygenation. In some embodiments the RBC
life-span is increased following the administration of LCAT.
[0050] In liver disease RBC cholesterol is increased and anemia
often occurs. LCAT therapy will normalize the RBC cholesterol,
restore normal shape and function of the effected RBCs, decreasing
RBC destruction, increasing life-span thus reducing the propensity
for anemia. Therefore another embodiment is a method of treating a
patient having anemia by administering to a patient, a
therapeutically effective amount of LCAT.
[0051] Target cell and spur cell anemia (Acanthocytosis): Target
and spur cells have an increased FC content leading to decreased
function and increased hemolysis and anemia.
[0052] In conditions such as sepsis, rheumatic diseases and
inflammatory disorders (including anemia of inflammation) there are
myriad pathologies such as decreased RBC deformability and abnormal
rheology which lead to further complications. Damage to tissues and
organ systems due to decreased oxygenation and increased RBC
aggregation leads to increased morbidity and mortality from the
initial inflammatory insult. Thus another embodiment is a method
reducing RBC aggregability by administering, to a patient in need
thereof, a therapeutically effective amount of LCAT. LCAT can also
act upon oxidized phospholipids generated during inflammation. The
oxidized lipids are very reactive, and can increase damage to cells
and organ systems. Normalizing RBC membrane lipids would improve
flow and tissue oxygenation, and decrease the concentration of
reactive oxidized lipids. This will be useful post-surgery, where
occult infections can decrease RBC function, increasing
wound-healing time.
[0053] Microvascular disorders may occur when there is an increase
in RBC FC, thereby causing rigidity, increased adhesion and
aggregability of the RBCs. These changes are magnified in the low
flow (or low pressure) found in capillaries and venules. When RBCs
are unable to deform properly, their transit is slowed to a greater
extent in these small vessels. With the increased propensity for
aggregation and adhesion, there is a greater chance of blockages in
the peripheral vessels. In organs where the microvasculature is
critical for normal function (e.g., eyes, ears, brain, kidney,
penis, lungs), repeated ischemic events in these vessels could lead
to loss of function (e.g., blindness, hearing loss, kidney failure,
ischemic microvascular brain disease (e.g., dementia, Alzheimer's),
erectile dysfunction). LCAT treatment would decrease RBC FC
improving RBC rheology, decreasing risk of further blockages and
end organ damage.
[0054] As demonstrated in Example 1 of the present disclosure, mice
with approximately 30-fold the normal level of LCAT activity had
increased RBC mass as compared to normal mice, demonstrating that
LCAT activity is a major factor in regulating RBC mass, and can be
rate-limiting in this regard.
[0055] Thus, administering a high dose of LCAT, for example, from
1-times to 1000-times the endogenous level of LCAT or from 1-times
to 500-times the endogenous level of LCAT, or from 1-times to
100-times the endogenous level of LCAT to a patient having a
condition characterized by abnormal rheology (anemia, decreased
deformability, increased aggregation, decreased flow, decreased RBC
life-span) would result in an improvement of the condition.
[0056] Thus, another embodiment is a method of treating a patient
with sickle cell disease, diabetes, thalassemia, rheumatoid
disease, autoimmune disease, arthritis, liver disease, cirrhosis,
hepatitis, acanthosytosis, sepsis, dementia, anemia, or
microvascular disorders, inflammatory disorders, parasitic disease,
erectile dysfunction, cancer, pre-eclampsia, critical illness or
trauma by administering a therapeutically effective dose of LCAT.
Yet another embodiment of the present disclosure is a method of
mating a patient having sickle cell disease, diabetes, thalassemia,
rheumatoid disease, autoimmune disease, arthritis, liver disease,
cirrhosis, hepatitis, acanthosytosis, sepsis, dementia, anemia, or
microvascular disorders, inflammatory disorders, parasitic disease,
erectile dysfunction, cancer, pre-eclampsia, critical illness or
trauma by administration of a drug that increases LCAT activity or
LCAT level. In preferred embodiments the drug is a small molecule
therapeutic. In another embodiment the LCAT level and/or LCAT
activity is increased using gene therapy.
[0057] Another embodiment is a method of treating a patient with an
Alzheimer's related dementia by administering a therapeutically
effective dose of LCAT. Another embodiment is a method of treating
a patient with sickle cell disease, diabetes, thalassemia,
rheumatoid disease, autoimmune disease, arthritis, liver disease,
cirrhosis, hepatitis, acanthosytosis, sepsis, dementia, anemia, or
microvascular disorders, inflammatory disorders, parasitic disease,
erectile dysfunction, cancer, pre-eclampsia, critical illness or
trauma by administering a therapeutically effective dose of a
modified LCAT. In some embodiments the modified LCAT comprises a
conservative amino acid substitution. In one embodiment the
modified LCAT comprises a substitution at position F1, L3, L4, N5,
L7, C31, N384 or E416. In various embodiments the modified LCAT
comprises an amino acid substitution at position 31. In other
embodiments the modified LCAT comprises a C31Y substitution and a
substitution at one or more of amino acid residues F1, L4, L32, or
N34. In another embodiment the modified LCAT comprises a C31Y
substitution and one or more of the following substitutions: F1S,
F1W, L4M, L4K, N34S, L32F, or L32H. In various embodiments the
modified LCAT comprises one or more of the following substitutions:
F1A, F1G, F1I, F1L, F1M, F1P, F1V, F1Y, F1T, F1Q, F1N, F1H, F1D,
L3I, L3F, L3C, L3W, L3Y, L4A, L4I, L4M, L4F, L4V, L4W, L4Y, L4T,
L4Q, L4R, N5A, N5M, N5H, N5K, N5D, N5E, L7M, L7R, L7E, C31A, C31I,
C31M, C31F, C31V, C31W, C31Y, C31T, C31R, C31H, N384C, N384Q, or
E416C. In other embodiments the level of LCAT in the patient is
increased by using gene therapy techniques. In another embodiment
LCAT expression is upregulated through drug administration.
[0058] In the methods according to the present disclosure, the LCAT
is generally administered to the subject in a pharmaceutical
composition comprising a pharmaceutically acceptable carrier or
diluent. A pharmaceutical composition may be formulated in
accordance with routine procedures as a pharmaceutical composition
adapted to the chosen route of administration, i.e., orally,
parenterally, by intravenous, intramuscular or subcutaneous
routes.
[0059] Pharmaceutical compositions suitable for the delivery of
compounds of the present disclosure and methods for their
preparation will be readily apparent to those skilled in the art.
Such compositions and methods for their preparation may be found,
for example, in Remington's Pharmaceutical Sciences, 19th Edition
(Mack Publishing Company, 1995).
[0060] The compositions may take such forms as suspensions,
solutions, or emulsions in oily or aqueous vehicles, and may
contain formulatory agents such as suspending, stabilizing and/or
dispersing agents. Alternatively, tile active ingredient may be in
powder form for constitution with a suitable vehicle, e.g., sterile
pyrogen-free water, before use. Typically such compositions are
solutions in sterile isotonic aqueous buffer. The compositions may
he a hermetically sealed container such as an ampoule, syringe, or
vial with or without an added preservative.
[0061] A liquid carrier or vehicle can be a solvent or liquid
dispersion medium comprising, for example, water, ethanol, a polyol
(for example, glycerol, propylene glycol, liquid polyethylene
glycols, and the like), vegetable oils, nontoxic glyceryl esters,
and suitable mixtures thereof. The proper fluidity can be
maintained, for example, by the formation of liposomes, by the
maintenance of the required particle size in the case of
dispersions or by the use of surfactants. The prevention of the
action of microorganisms can be brought about by various
antibacterial and antifungal agents, for example, parabens,
chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In
many cases, it will be preferable to include isotonic agents, for
example, sugars, buffers or sodium chloride. Prolonged absorption
of the injectable compositions can be brought about by the use in
the compositions of agents delaying absorption, for example,
aluminum monostearate and gelatin.
[0062] According to the present disclosure, LCAT can be used alone
or in combination therapy with other drugs used to treat the
foregoing conditions. Such therapies include, but are not limited
to simultaneous or sequential administration of the drugs involved.
For example, LCAT formulations can be administered with drugs that
are commonly used as a standard of care for a particular condition.
For example LCAT can be administered in combination with
erythropoiesis stimulating agents (ESA) such as erythropoietin,
methoxypolyethylene-glycol-epoetin-beta, darbepoetin-.alpha.,
romiplostim, and epoetin-.alpha. for treatment of anemia. Or for
example LCAT can be administered in combination with hydroxyurea,
hydroxycarbamide, decitabine, or butyrate for treatment of sickle
cell disease.
[0063] In one embodiment the therapeutically effective amount of
LCAT is administered by subcutaneous injection. In another
embodiment the therapeutically effective amount of LCAT is
administered by intramuscular injection. In another embodiment the
therapeutically effective amount of LCAT is administered by
intravenous injection or infusion. In some embodiments the
therapeutically effective amount of LCAT is from 1 mg to 5000 mg,
or from 1 mg to 2000 mg, or from 10 mg to 5000 mg, or from 10 mg to
1000 mg, or from 10 mg to 500 mg or from 5 mg to 100 mg.
[0064] In some embodiments the therapeutically effective amount of
LCAT is from 1-times to 1000-times, from 25-times to 1000 times,
from 50-times to 1000-times, from 1-times to 100 times, from
50-times to 500-times, or from 1-times to 500-times the endogenous
level of LCAT.
[0065] The specific dosage used can vary. For example, the dosage
can depend on a number of factors including, but not limited to,
the dosing frequency, the specific activity of the recombinant LCAT
enzyme, the body weight of the patient, special requirements of the
patient, special conditions of the patient (e.g., abnormal kidney
or liver function), the condition being treated, etc. The dosing
frequency and amount may, at the physician's discretion, fall
outside of the typical range given herein. These dosages are based
on an average human subject having a weight of about 60 kg to 70
kg. Determination of optimum dosages for a particular patient is
well-known to those skilled in the art. The physician will readily
be able to determine doses for subjects whose weight falls outside
this range, such as infants and the elderly.
[0066] Depending on the disorder and the patient being treated, one
skilled in the art (i.e. a physician) could determine that an
initial dose which is higher than following doses is appropriate.
For example, a patient presenting with crisis state sickle cell
disease might he administered an initial dose of 30-times the
"normal" level. Once that patient's RBC oxygenation level reaches
the desired level the dose would be reduced for example to 3-times
the "normal" level.
[0067] The efficacy of a particular dose may be assessed by
reference to biomarkers or improvement in certain physiologic
parameters. Suitable biomarkers include, but are not limited to,
the ratio of FC to PL, FC to membrane protein, PC to SM or HDL-C
levels. Suitable physiologic parameters include, but are not
limited to, reduced anemia, improved rheology as measured by an
increase in RBCs, RBC deformability, blood flow, and/or RBC
aggregability, osmotic fragility, or RBC oxygenation level; an
increase in any one these parameters indicates improvement.
Measurement of biomarker levels and parameters described above may
be measured using methods that are well known in the art. For
example, reduced anemia can be measured by increased hematocrit or
hemoglobin or hemoglobin break-down products (e.g.,
unconjugated-bilirubin) measured with standard, well-established,
clinical techniques. Deformability can be measured by filtration,
viscometry, ektacytometry and by the use micropipettes. Aggregation
may be measured by a variety of instruments including
ektacytometers and aggregometers. RBC oxygenation can be measured
by standard pulse oximetry and blood gas analysis; tissue
oxygenation can be measured with direct probes with sialastic
tonometers with oxygen sensors. One of skill in the art would
understand the significance of the results and may choose to adjust
the dose based on assessments such as those described above.
[0068] As described in Example 4 and shown in FIG. 2, following
administration of LCAT to human apolipoprotein A-I transgenic mice
plasma HDL-C levels increased. The increase was surprisingly rapid;
plasma HDL-C levels increased by about 70% of control at 4 hours
and by about 120% by 24 hours. Thus, another embodiment of the
disclosure is a method of treating a patient having a condition
characterized by anemia or red blood cell dysfunction comprising
administering to a subject in need thereof, a therapeutically
effective amount of LCAT wherein the plasma HDL-C level in the
subject is rapidly increased after administration of the LCAT. In a
particular embodiment the plasma HDL-C level in the subject 4 hours
post administration of the LCAT is increased by at least 30% or by
at least 40% or by at least 50%, or by at least 70% or by at least
80% of the plasma HDL-C level prior to LCAT administration. In yet
another embodiment the plasma HDL-C level in the subject 12 hours
post administration of the LCAT is increased by at least 40%, or by
at least 50% or by at least 60% or by at least 70% or by at least
80%, or by at least 90% or by at least 100%, or by at least 110%,
or by at least 120% of the plasma HDL-C level prior to LCAT
administration. In still another embodiment the plasma HDL-C level
in the subject 24 hours post administration of the LCAT is
increased by at least 40%, or by at least 50% or by at least 60% or
by at least 70% or by at least 80%, or by at least 90% or by at
least 100%, or by at least 110%, or by at least 120%, or by at
least 130%, or by at least 140%, or by at least 150% of the plasma
HDL-C level prior to LCAT administration.
[0069] As described in example 5, and shown in FIG. 3,
administration of LCAT to LCAT-knockout/apolipoprotein A-I
transgenic mice resulted in an increase in tissue cholesterol,
(aorta and liver) an increase in plasma cholesterol levels. The
combined data from examples 4 and 5 demonstrate that the injection
of LCAT rapidly redistributes lipids from tissues to plasma HDL.
Given the effect of LCAT injection on the cholesterol content of
liver and aorta it would be expected that a similar change would be
rapidly be observed in red blood cells.
[0070] A transfer of FC from RBC to HDL, should change the
composition of RBC membrane to a more normal state. This action
will increase the oxygenation of the RBC, improve the rheology
(increase deformability, flow, decrease the propensity for adhesion
and aggregation) decrease anemia (decrease the mechanical stress
and destruction associated with decreased deformability, increasing
the life of the RBC), and increase the ability of the RBC to
oxygenate tissue, especially peripheral tissues.
[0071] In some embodiments the LCAT is self-administered by the
patient either by subcutaneous or intramuscular injection.
Self-administration is a preferred embodiment for chronic
treatment, including, but not limited to, of patients suffering
with sickle cell disease, diabetes, rheumatoid disease, or
hepatitis.
Example 1
Effect of LCAT Level on Hematocrit in Mice
[0072] Blood was sampled from 3 groups of mice: LCAT deficient
(LCAT-KO), LCAT over-expressing transgenic (.about.30.times. normal
LCAT activity), and control C57/b6 mice. RBC membranes were
isolated from the blood sample, and choline containing
phospholipids were measured (Wako Phospholipids B, Richmond) as a
surrogate for RBC mass or hematocrit. RBC mass was significantly
lower in the LCAT deficient mice as compared to normal mice
(402.+-.22.0 .mu.g/ml whole blood vs. 486.+-.25.7 .mu.g/ml whole
blood, respectively). The anemia in the LCAT deficient mice
demonstrated here is similar to the extent of anemia observed in
FLD patients. Surprisingly RBC mass was significantly elevated in
LCAT over-expressing transgenic mice as compared to mice with
normal LCAT activity (556.+-.20.1 .mu.g/ml whole blood vs.
486.+-.25.7 .mu.g/ml whole blood, respectively). These results show
that there is a positive relationship between LCAT levels and
hematocrit. Additionally, and most importantly, supra-normal levels
of LCAT can increase hematocrit in animals not considered anemic.
These studies show that increasing levels is a viable therapeutic
option for patients with anemia due a variety of causes, even in
patients with normal LCAT activity.
Example 2
Phospholipid Composition of RBC Ghost Membranes Prepared from
Normal and SCD Subjects (not in Crisis)
[0073] Samples of washed RBC in phosphate buffered saline were
prepared from fresh blood collected from normal subjects (n=7) and
SCD patients (n=6). Fifty microliter aliquots of packed RBC were
suspended in 0.95 ml phosphate-buffered saline. Lipids were
extracted by combining 0.4 ml aliquots of each RBC suspension with
20 .mu.l of a 1 mg/ml solution of 1-eicosanol in ethyl
acetate:acetone (2:1) (internal standard) and 2 ml of ethyl
acetate:acetone:methanol (6:3:1) in glass tubes. The capped tubes
were shaken for 2 minutes and then centrifuged at 2000 rpm for 5
min. The upper organic phase was transferred to 12-32 min HPLC
vials. Solvent was evaporated from the vials under a stream of N2
followed by at least 1 hr of high vacuum. The dried lipids were
reconstituted in 200 .mu.l
trimethylpentane:methanol:tetrahydrofuran (95:5:2). Membrane lipids
were chromatographed by high-performance liquid chromatography on a
silica column. Phosphatidylcholine (PC) and sphingomyelin (SM) were
detected and quantitated with an evaporative light-scattering
detector. The results show that the RBC lipids in SCD patients are
enriched in PC and diminished in SM content, compared to normal
subjects (FIG. 1), resulting in an increase in the PC/SM ratio from
0.67 to 0.98 for control and SCD, respectively. The SCD RBC
analyzed in this study exhibit a phospholipid composition pattern
that is distinct from normal RAC. The SCD RAC lipid composition is
analogous to that reported for RAC in other cases of low plasma
LCAT activity.
Example 3
Preparation of Recombinant Human LCAT
[0074] The plasmid pCMV6-XL4/LCAT encoding human LCAT protein was
purchased from Origene Technologies (Rockville, Md.) and ligated
into pcDNA3.1/Hygro (Invitrogen, Carlsbad, Calif.). The pcDNA3.1
vector was transfected into HEK293f cells. Stably-transfected cells
were selected with 200 .mu.g/ml hygromycin B and grown in Freestyle
293 serum-free medium (Invitrogen) in 10 L shake flasks for 4 days.
The rhLCAT was isolated from the culture medium by precipitation
with zinc chloride followed by batch capture and elution with
phenylsepharose.
Example 4
HDL Cholesterol Increase in Human Apolipoprotein A-I Transgenic
Mice Injected with LCAT
[0075] Male transgenic mice expressing the human apolipoprotein A-I
gene (Jackson Laboratory) were maintained on a normal chow diet, ad
libitum. The mice were given a single intravenous injection of
saline or recombinant human LCAT in saline (4 mg/kg) via the
retro-orbital sinus. Blood was collected at the orbital plexus in
isoflurane-anaesthetized animals at 0, 1, 4, 24, 48 and 72 hours
post-injection. Plasma cholesterol concentration was determined
with commercial enzymatic assay kits. The amount of cholesterol in
HDL (HDL-C) was determined by agarose gel electrophoresis with the
SPIFE system from Helena Labs. FIG. 2 shows that the mice that were
administered LCAT showed a significant increase in the levels of
plasma HDL-C by as much as 120% of control. The HDL-C level
remained at increased levels for the duration of the experiment (72
hours). The rise in plasma HDL was surprisingly rapid showing an
increase of about 70% of control at 4 hours and about 120% by 24
hours.
Example 5
Effect of LCAT Infection on Cholesterol Content in Tissues of
LCAT-Knockout/Apolipoprotein A-I Transgenic Mice
[0076] Transgenic mice expressing human apolipoprotein A-I (Jackson
Laboratory) were cross-bred with LCAT-KO mice to obtain
LCAT-KO/apoA-1-Tg mice. The LCAT-KO/apoA-I-Tg mice were maintained
on normal rodent chow, ad libitum. Intravenous (IV) injections of
saline or 0.4 mg LCAT were performed daily for 4 days via the
retro-orbital sinus. Animals were sacrificed on the fifth day.
Animals were anaesthetized and exsanguinated by perfusion with
heparinized saline. A liver lobe and the aorta were removed front
each animal and extracted with a chloroform and methanol solution.
The cholesterol in the lipids recovered from the extracted tissues
was measured with a commercial enzymatic assay kit.
[0077] FIG. 3 shows the cholesterol content of (A) liver, (B) aorta
and (C) plasma for mice injected with saline (Ctrl) or LCAT (Exp).
Treatment with LCAT significantly reduced the levels of cholesterol
in the liver and aorta and significantly raised the plasma
cholesterol level. The combined data from examples 4 and 5
demonstrate that the injection of LCAT rapidly redistributes lipids
from tissues to plasma HDL. Given the effect of LCAT injection on
the cholesterol content of liver and aorta it would be expected
that a similar change would be observed in red blood cells.
Example 6
[0078] A child (30 kg) in sickle cell crisis is admitted to the
hospital. Along with standard of care treatment, he is infused with
5 mg/kg of recombinant human LCAT (rhLCAT) over a 1-hour period in
a total of 100 ml saline. Following treatment, blood oxygen levels
are measured and have improved. As the crisis abates, red cell
morphology and physical characteristics (RBC deformability, RBC
aggregability, and osmotic fragility) are measured and the results
are compared to results from blood sample taken upon admittance.
Improvements in RBC physical characteristics and oxygenation are
maintained with weekly subcutaneous injections of rhLCAT at a dose
of 0.5 mg/kg.
Example 7
[0079] A 35 year old female (55 kg) presents with rheumatoid
arthritis has anemia with a hemoglobin level of 9 g/dl (normal
range 12-14 g/dl). A blood sample is taken, and demonstrates that
her red blood cells are less deformable and aggregate more easily
than normal red blood cells. The patient is prescribed weekly
injections of rhLCAT at a dose of 1 mg/kg to be administered
subcutaneously. Hematocrit and hemoglobin levels are measured after
6 weekly injections and are found to have increased 20%. After 6
months treatment, hemoglobin is 14 g/dl. The physician decides to
maintain the patient on rhLCAT at a dose of 1 mg/kg injected
hi-weekly.
Example 8
[0080] A 65 year old male (80 kg) is scheduled for quadruple bypass
surgery. The patient is advised to stop taking clopidogrel five
days prior to surgery to reduce the chance of post-operative
bleeding in order to reduce the risk of platelet activation,
thrombosis, or RBC aggregation, the patient is brought into the
doctor's office for an infusion of 1 mg/kg of rhLCAT five days
prior to surgery. The patient is infused with 1 mg/kg of rhLCAT
directly after surgery, 7 days post-surgery, and 14 days
post-surgery. After recovery (21 days post-surgery), the patient is
returned to chronic clopidogrel treatment.
Example 9
Gene Transfer for Hepatic Specific Over-Expression of LCAT
[0081] A patient presents with Rheumatoid arthritis accompanied by
chronic anemia. The patient is administered a dose of
4.times.10.sup.12 adenoviral particles (AdrLCAT)/kg by injection
through an intra-portal catheter. LCAT levels are monitored weekly
post-treatment. At four weeks post treatment the patients has an
LCAT levels of 10 mg/L, or approximately two-fold greater than the
concentration in a non-arthritic subject. After 8 weeks post
treatment the patient is monitor monthly. If the patient's LCAT
level drops below 5 mg/L the procedure is repeated.
Example 10
[0082] A child (30 kg) in sickle cell crisis is admitted to the
hospital. Along with standard of care treatment, he is infused with
5 mg/kg of recombinant human LCAT (rhLCAT) over a 1-hour period in
a total of 100 ml saline. Following treatment, blood oxygen levels
are measured and have improved. As the crisis abates, red cell
morphology and physical characteristics (RBC deformability, RBC
aggregability, and osmotic fragility) are measured and the results
are compared to results from blood sample taken upon admittance.
The patient then has a procedure in which a medical device is
placed under the skin. The medical device comprises mammalian cells
engineered to secrete active LCAT. Sufficient LCAT is released by
the cells to raise the endogenous LCAT activity by greater than
100% of normal LCAT levels.
[0083] It should be appreciated that the scope of this invention is
to be defined by the claims and is not to be limited by the
specifically described embodiments and examples herein.
Sequence CWU 1
1
11416PRTHuman 1Phe Trp Leu Leu Asn Val Leu Phe Pro Pro His Thr Thr
Pro Lys Ala 1 5 10 15 Glu Leu Ser Asn His Thr Arg Pro Val Ile Leu
Val Pro Gly Cys Leu 20 25 30 Gly Asn Gln Leu Glu Ala Lys Leu Asp
Lys Pro Asp Val Val Asn Trp 35 40 45 Met Cys Tyr Arg Lys Thr Glu
Asp Phe Phe Thr Ile Trp Leu Asp Leu 50 55 60 Asn Met Phe Leu Cys
Leu Gly Val Asp Cys Trp Ile Asp Asn Thr Arg 65 70 75 80 Val Val Tyr
Asn Arg Ser Ser Gly Leu Val Ser Asn Ala Pro Gly Val 85 90 95 Gln
Ile Arg Val Pro Gly Phe Gly Lys Thr Tyr Ser Val Glu Tyr Leu 100 105
110 Asp Ser Ser Lys Leu Ala Gly Tyr Leu His Thr Leu Val Gln Asn Leu
115 120 125 Val Asn Asn Gly Tyr Val Arg Asp Glu Thr Val Arg Ala Ala
Pro Tyr 130 135 140 Asp Trp Arg Leu Glu Pro Gly Gln Gln Glu Glu Tyr
Tyr Arg Lys Leu 145 150 155 160 Ala Gly Leu Val Glu Glu Met His Ala
Ala Tyr Gly Lys Pro Val Phe 165 170 175 Leu Ile Gly His Ser Leu Gly
Cys Leu His Leu Leu Tyr Phe Leu Leu 180 185 190 Arg Gln Pro Gln Ala
Trp Lys Asp Arg Phe Ile Asp Gly Phe Ile Ser 195 200 205 Leu Gly Ala
Pro Trp Gly Gly Ser Ile Lys Pro Met Leu Val Leu Ala 210 215 220 Ser
Gly Asp Asn Gln Gly Ile Pro Ile Met Ser Ser Ile Lys Leu Lys 225 230
235 240 Glu Glu Gln Arg Ile Thr Thr Thr Ser Pro Trp Met Phe Pro Ser
Arg 245 250 255 Met Ala Trp Pro Glu Asp His Val Phe Ile Ser Thr Pro
Ser Phe Asn 260 265 270 Tyr Thr Gly Arg Asp Phe Gln Arg Phe Phe Ala
Asp Leu His Phe Glu 275 280 285 Glu Gly Trp Tyr Met Trp Leu Gln Ser
Arg Asp Leu Leu Ala Gly Leu 290 295 300 Pro Ala Pro Gly Val Glu Val
Tyr Cys Leu Tyr Gly Val Gly Leu Pro 305 310 315 320 Thr Pro Arg Thr
Tyr Ile Tyr Asp His Gly Phe Pro Tyr Thr Asp Pro 325 330 335 Val Gly
Val Leu Tyr Glu Asp Gly Asp Asp Thr Val Ala Thr Arg Ser 340 345 350
Thr Glu Leu Cys Gly Leu Trp Gln Gly Arg Gln Pro Gln Pro Val His 355
360 365 Leu Leu Pro Leu His Gly Ile Gln His Leu Asn Met Val Phe Ser
Asn 370 375 380 Leu Thr Leu Glu His Ile Asn Ala Ile Leu Leu Gly Ala
Tyr Arg Gln 385 390 395 400 Gly Pro Pro Ala Ser Pro Thr Ala Ser Pro
Glu Pro Pro Pro Pro Glu 405 410 415
* * * * *