Note: Descriptions are shown in the official language in which they were submitted.
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-- 1 --
Obesity Protein Formulations
This application claims the benefit of U.S.
Provisional Application No. 60/010,229, filed January 19,
t1996
The present invention is in the field of human
medicine, particularly in the treatment of obesity and
disorders associated with obesity. More specifically, the
present invention relates to formulations of an obesity
protein.
1~ Obesity, and especially upper body obesity, is a
common and very serious public health problem in the United
States and throughout the world. According to recent
stat stics, more than 25% of the United States population and
27~ of the Canadian population are overweight. Kuczmarski,
Amer. J. of Clin. Mutr. 55: 495S - 502S (1992); Reeder et
al., Can. Med. ASs. J., 23: 226-233 (1992). Upper body
obesity is the strongest risk factor known for type II
diabetes mellitus, and is a strong risk factor for
cardiovascular disease and cancer as well. Recent estimates
for the medical cost of obesity are $150,000,~00,000 world
wide. The problem has become serious enough that the surgeon
general has begun an initiative to combat the ever increasing
adiposity rampant in American society.
Much of this obesity induced pathology can be
attributed to the strong association with dyslipidemia,
hyper~ension, and insulin resistance. Many studies have
demonstrated that reduction in obesity by diet and exercise
reduces these risk factors dramatically. Unfortunately,
treatments are largely unsuccessful with a failure rate
reaching 95%. This failure may be due to the fact that the
condi~ion is strongly associated with genetically inherited
factors that contribute to increased appetite, preference for
highly caloric foods, reduced physical activity, and
increased lipogenic metabolism. This indicates that people
~5 inheriting these genetic traits are prone to becoming obese
regardless of their effor~s to combat the condition.
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Therefore, a pharmacological agent that can correct this
adiposity handicap and allow the physician to success~ully
treat obese patients in spite of their genetic inheritance is
needed.
The ob/ob mouse is a model of obesity and diabetes
that is known to carry an autosomal recessive trait linked to
a mutation in the sixth chromosome. Recently, Yiying Zhang
and co-workers published the positional cloning of the mouse
gene linked with this condition. Yiying Zhang et al. Nature
~ 425-32 ~19~4). This report disclosed the murine and
human protein expressed in adipose tissue. Likewise,
Murakami et a~., in Biochemical and Bio~hvsical Research
Communications 209(3):944-52 (1995) report the cloning and
expression of the rat obese gene. The protein, which is
encoded by the ob gene, has demonstrated an ability to
effectively regulate adiposity in mice. Pelleymounter et
al., Science 269: 540-543 (1995).
A parenteral formulation containing insoluble
protein causes problems relating to inconsistency in the
dose-response as well as unpredictability. The
unpredictability is believed to be due to greater variability
in the pharamacokinetics in suspension ~ormulations. The
insoluble formulations must ~irst dissolve prior to
adsorption. It is hypothesized that this step has
significant variability in a subcutaneous depot.
Furthermore, non-native association and aggregation under
physiological conditions can lead to precipitation of the
protein at the site of injection, which could lead to
irritation or other immune response. For these reasons, a
formulation o~ human obesity protein that developed insoluble
protein particles would be unacceptable to patients seeking
its benefits and to regulatory agencies.
Unfortunately, the naturally occurring obesity
proteins demonstrate a propensity to aggregate making the
preparation of a soluble, pharmaceutically acceptable
parente~al ~ormulation exceedingly di~icult. The molecular
interactions amongst the preservative, bu~fer, ionic
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:
-- 3 --
strength, pH, temperature, and any additional excipients such
as a surfactant, or sugar are hi~hly unpredictable in view of
~ the propensity for the obesity protein to ag~regate and
precipitate from the formulation.
The present invention provides conditions under
which the obesity protein is soluble and commercially viable
as a multi-use pharmaceutical product. Most unexpectedly,
the physical stability of the ~ormulation is greatly enhanced
in the presence o~ methylparaben, propylparaben,
butylparaben, chlorobutanol or a mixture thereof. That is,
when formulated under the conditions described herein, the
obesity protein remains soluble at much higher concentrations
and at a pH range acceptable for a soluble, parenteral
~ormulation. Accordingly, the present invention provides a
soluble, parenteral formulations of an obesity protein.
This invention provides a soluble parenteral
formulation, comprising an obesity protein and a preservative
selected from the group consisting of alkylparaben,
chlorobutanol, or a mixture thereof.
The invention further provides a process for
preparing a soluble parenteral formulation, which comprises
mixing an obesity protein and a preservative selected ~rom
the group consisting of alkylparaben, chlorobutanol, or a
mixture thereof.
Additionally, the invention provides a method of
treating obesity in a mammal in need thereof, which comprises
administering to said mammal a soluble parenteral formulation
of the present invention.
For purposes of the present invention, as disclosed
and -laimed herein, the following terms and abbreviations are
de~ined as follows:
Alkylparaben -- refers to a Cl to C4 alkylparaben.
Preferably, alkylparaben is methylparaben, ethylparaben,
~ prop~ylparaben, or butylparaben.
Base pair (bp) -- refers to DNA or RMA. The
abbreviations A,C,G, and T correspond to the 5'-monophosphate
forms of the nucleotides (deoxy)adenine, (deoxy)cytidine,
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~deo~)guanine, and (deoxy)thymine, respectively, when they
occur in DNA molecules. The abbreviations U,C,G, and T
correspond to the 5'-monophosphate forms of the nucleosides
uracil, cytidine, guanine, and thymine, respectively when
they occur in RNA molecules. In double stranded DNA, base
pair may refer to a partnership of A with T or C with G. In
a DNA/RNA heteroduplex, base pair may refer to a partnership
of T with A or ~ with G.
Obesity protein -- refers to the native m~mm~l ian
protein produced from the native ob gene following
transcription and deletions of introns, translation to a
protein and processing to the mature protein with secretory
signal peptide removed, e.g. from the N-terminal valine-
proline to the C-terminal cysteine of the mature protein.
The human obesity protein is published in Zhang et al.
Nature 372: 425-32 (1994). The rat obesity protein is
published in Murakami et al., Biochemical and Bio~h~sical
Research Comm. 209(3): 944-52 (1995). Native porcine and
bovine Ob proteins are disclosed in a U.S. patent application
by Hansen M. Hsiung and Dennis P. Smith filed May 19, 1995,
Serial number 08/445,305 (EP0 743 321). Other mammalian Ob
proteins are disclosed in U.S. patent application serial
number 08f452,228, filed May 26, 1995 (EP0 744 408), U.S.
provisional application, 60/003935, filed September 19, 1995
(EP ). Obesity protein includes those proteins having a
leader sequence. A leader sequence is one or more amino
acids on the M-terminus to aid in production or puriflcation
of the protein. A preferred leader sequence is Met-Rl-
wherein Rl is absent or any amino acid except Pro.
Plasmid -- an extrachromosomal self-replicating
genetic element.
Reading frame -- the nucleotide sequence from which
translation occurs "readl' in triplets by the translational
apparatus of tRNA, ribosomes and associated factors, each
triplet corresponding to a particular amino acid. Because
each triplet is distinct and of the same length, the coding
sequence must be a multiple of three. A base pair insertion
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-- 5 --
or deletion (termed a frameshift mutation) may result in two
di~ferent proteins being coded for by the same DNA segment.
To insure against this, the triplet codons corresponding to
the desired polypeptide must be aligned in multiples of three
from the initiation codon, i.e. the correct ~Ireading frame"
must be maintained. In the creation of fusion proteins
containing a chelating peptide, the reading frame of the DNA
sequence encoding the structural protein must be maintained
in the DNA sequence encoding the chelating peptide.
Recombinant DNA Cloning Vector -- any autonomously
replicating agent including, but not limited to, plasmids and
phages, comprising a DNA molecule to which one or more
additional DNA segments can or have been added.
Recombinant DNA Expression Vector -- any
recombinant DNA cloning vector in which a promoter has been
incorporated.
Replicon -- A DNA sequence that controls and allows
for autonomous replication of a plasmid or other vector.
Transcription -- the process whereby information
contained in a nucleotide sequence of DNA is transferred to a
complementary RNA sequence.
Translation -- the process whereby the genetic
information of messenger RNA is used to specify and direct
the synthesis of a polypeptide chain.
Vector -- a replicon used ~or the transformation of
cells in gene manipulation bearing polynucleotide sequences
corresponding to appropriate protein molecules which, when
combined with appropriate control sequences, confer specific
properties on the host cell to be transformed. Plasmids,
virus~s, and bacteriophage are suitable vectors, since they
are replicons in their own right. Artificial vectors are
constructed by cutting and joining DNA molecules from
different sources using restriction enzymes and ligases.
Vectors include Recombinant DNA cloning vectors and
Recombinant DNA expression vectors.
Treating -- as used herein, describes the
management and care of a patient for the purpose of combating
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.
-- 6
the disease, condition, or disorder and includes the
administration of a protein of present invention to prevent
the onset of the symptoms or complications, alleviating the
symptoms or complications, or eliminating the disease,
condition, or disorder. Treating as used herein includes the
administration of the protein for cosmetic purposes. A
cosmetic purpose seeks to control the weight of a mammal to
improve bodily appearance.
Isotonicity agent -- isotonicity agent refers to an
agent that is physiologically tolerated and embarks a
suitable tonicity to the ~ormulation to prevent the net flow
of water across the cell membrane. Compounds, such as
glycerin, are commonly used ~or such purposes at known
concentrations. Other possible isotonicity agents include
salts, e.g., MaCl, dextrose, mannitol, and lactose.
Physiologically tolerated buffer -- a
physiologically tolerated bu~er is known in the art. A
physiologically tolerated buf~er is preferably a phosphate
buffer, like sodium phosphate. Other physiologically
tolerated buffers include TRIS, sodium acetate, or sodium
citrate. The selection and concentration of buffer is known
in the art.
The nucleotide and amino acid abbreviations are
accepted by the United States Patent and Trademark Office as
set forth in 37 C.F.R. ~ 1.822 (b)(2) (1993). Unless
otherwise indicated the amino acids are in the L
configuration.
As noted above, the invention provides a soluble
parenteral formulation, comprising human obesity protein and
a preservative selected ~rom the group consisting of an
alkylparaben or chlorobutanol. In the presence of these
preservatives, the obesity proteins r~m~i n~ in solution
making a soluble, parenteral ~ormulation possible.
A parenteral formulation must meet guidelines ~or
preservative effectiveness to be a commercially viable
product. Pharmaceutical preservatives known in the art as
being acceptable in parenteral ~ormulations include: phenol,
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m-cresol, benzyl alcohol, methylparaben, chlorobutanol, p-
cresol, phenylmercuric nitrate, thimerosal and various
mixtllres thereof. See, e.g., WALLHAUSER, K.--H., DEVE:LoP. BIOL.
STANDARD. 24, pp. 9-28 (sasel~ s. Krager, 1974).
~ 5 Most unexpectedly, a select number of preservatives
have been identified which provide good ~ormulation stability
These select preservatives are an alkylparaben or
chlorobutanol. Most preferrably, the preservative is
meth~lparaben, propylparaben, or butylparaben. Most
unexpectedly, the obesity protein does not aggregate in the
presence of these preservatives at the conditions necessary
to formulate and particularly conditions at 37~C.
The concentration of obesity protein in the
formulation is about 1.0 mg/mL to about 100 mg/mL; preferably
about 5.0 mg/mL to about 50.0 mg/mL; most preferably, about
10.0 mg/mL. The concentration of preservative required is
the concentration necessary to maintain preservative
effectiveness. The relative amounts of preservative
necessary to maintain preservative effectiveness varies with
the preservative used. Generally, the amount necessary can
be found in WALLHAUSER, K.--H., DEVELOP. BIoL. STANDARD~ 24, pp.
9-28 (Basel, S. Krager, 1974), herein incorporated by
reference. The optimal concentration of the preservative
depends on the preservative, its solubility, and the pH of
the formulation.
An isotonicity agent, preferably glycerin, may be
additionally added to the formulation. The concentration of
the isotonicity agent is in the range known in the art for
parenteral formulations, preferably about 1 to 20 mg/mL, more
preferably about 8 to 16 mg/mL, and still more preferably
about 16 mg/mL. The pH of the formulation may also be
~ buffered with a physiologically tolerated buffer, preferably
a phosphate buffer, like sodium phosphate. Other acceptable
physiologically tolerated buffers include TRIS, sodium
acetate, or sodium citrate. The selection and concentration
of buffer is known in the art; however, the formulations of
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.
-- 8
the present invention are preferably prepared the mln;m~lly
accpetable concentration of buffer.
Other additives, such as a pharmaceutically
acceptable solubilizers like Tween 2~ (polyoxyethylene (20)
sorbitan monolaurate), Tween 40 (polyoxyethylene (20)
sorbitan monopalmitate), Tween 80 (polyoxyethylene (20)
sorbitan monooleate), Pluronic F68 (polyo ~ ethylene
polyoxypropylene block copolymers), BRIJ 35 (polyoxyethylene
(23) lauryl ether), and PEG (polyethylene glycol) may
optionally be added to the formulation to reduce aggregation.
These additives are particularly useful if a pump or plastic
container is used to administer the formulation. The
presence of pharmaceutically acceptable surfactant mitigates
the propensity for the protein to aggregate.
Also included as optional embodiments are agents
known to be synergistic with the preservative to provide
enhanced antimicrobial effect. Such agents are recognized in
the art and include for example ethylene diaminetetraacetic
acid (EDTA), 1,2-di(2-aminoethoxy)ethane-N,N,M'N'-tetraacetic
acid (EGTA), citrate, and caprylic acid. The concentration
of these agents varies with the desired preservation effect.
A preferred agent is EDTA, particularly in conjunction with
an alkylparaben at a concentration of about 0.025% to 0.4%.
Notably, in preparations including EDTA or EGTA, the buffer
concentration is reduced to minim; ze ionic strength.
The present formulations may optionally contain a
physiologically tolerated solvent such as glycerol, propylene
glycol, phenoxy ethanol, phenyl ethyl alcohol. Such solvents
are generally added to enhance the solubility of the protein
in the preservation effectiveness of the formulation.
The parenteral fo~mulations of the present
invention can be prepared using conventional dissolution and
mixing procedures. To prepare a suitable formulation, for
example, a measured amount of human obesity protein in water
3~ is combined with the desired preservatlve in water in
quantities sufficient to provide the protein and preservative
at the desired concentration. The formulation is generally
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sterile filtered prior to administration. Variations of this
process would be recognized by one of ordinary skill in the
art. For example, the order the components are added, the
surfactant used, the temperature and pH at which the
formulation is prepared may be optimized for the
concentration and means of administration used.
The unexpected preservative e~fect on formulation
stability was demonstrated by preparing formulations
comprising methylparaben, propylparaben, butylparaben,
chlorobutanol, cresol, phenol, benzyl alcohol, or a mixture
thereof and comparing the amount o~ protein remaining in
solution after 3 days at 4~C and 37~C. The data in Table 1
demonstrate that the stability and solubility of human
obesity protein is enhanced in the presence o~ an
alkylparaben or chlorobutanol. The formulations used to
generate the data of Table 1 were prepared in a manner
analogous to Examples 1 and 2.
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Table 1. Recovery of protein as a function of preservative
and conditions. Values are the calculated least
square means and standard errors determined from
fitting the raw data to a factorial model of the
second degree containing the e~ects of
preservative, acid excursion, buffer, conditions,
and all two factor interactions. (R2 = 0.94, (Prob
> F) = 1.96e~39, total observations = 147). Percent
of protein in solution is calculated using a
theoretical target o~ 1.6 mg protein/mL.
Protein in Solution
(% of theoretical)
PreservativePreservative 3 days ~ 3 days @
Concentration 4~C 37~C
( )
methylparaben0.17 = 0.02 ~7." = . . = .
propyl~araben0.16 0.02 9. =
butylparaben 0.~15 ,7. = ~
chlorobutanol 0.5 87., = ~-. 8r. = ~.
methylparaben ~0.18 + 0.02 76.8 = ~. 6 .
propy~paraben0.017 + 0.002
~enzy alcohol 1.0 79.8 + 3.6 31.6 + 4.2
methy_paraben +0.16 + 0.02 62.8 + 4.3 16.7 + 4.3
propylparaben +0.02 + 0.002
benzyl :lcohol 0.~
m-creso~ 0. ~ .~- = - ~- - --'
p-creso_ 0._ : = , ~. = ~.-
phenol 0. J
The stabilizing ef~ect by the alkylparabens is most
unexpected in view of the structural similarities to other
preser~atives. The data clearly show that the alkylparabens
and chlorobutanol are superior at 37~C, which is the
temperature re~uired for in-use physical stability testing.
Preferably, the pH of the present formulations is
about pH 7.0 to about 8.0 and, most preferably 7.6 to 8Ø
The formulations are preferably prepared under basic
conditions by mixing the obesity protein and preservative at
a pH greater than pH 7Ø Preferably, the pH is about 7.6 to
8.0, and most pre~erably about pH 7.8. Ideally, a
preservative and water solution is mixed at pH 7.6 to 8Ø
Added to this solution is obesity protein in water. The pH
is adjusted, as necessary, to about pH 7.6 to 8Ø The
solution is then held until the components are dissolved,
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approximately 20 to 40 minutes, preferably about 30 minutes.
The base used to adjust the pH of the formulation may be one
or more pharmaceutically acceptable bases such as sodium
hydroxide and potassium hydroxide. The preferred base is
sodium hydroxide.
The formulations prepared in accordance with the
present invention may be used in a syringe, injector, pumps
or any other device recognized in the art for parenteral
administration.
Preferably, the obesity protein employed in the
formulations of the present invention is human obesity
prot~in of SEQ ID NO:1.
lo 15
Val Pl~c Ile Gln Lys Val Gln Asp Asp Thr Lys Thr Leu Ile Lys Thr
2c 25 30
Ile Val Thr Arg Ile Asn Asp Ile Ser His Thr Gln Ser Val Ser Ser
Lys Gln Lys Val Thr Gly Leu Asp Phe Ile Pro Gly ~eu His Pro Ile
S0 55 60
Leu Thr Leu Ser Lys Met Asp Gln Thr Leu Ala Val Tyr Gln Gln Ile
65 70 75 80
Leu Thr Ser Met Pro Ser Arg Asn Val Ile Gln Ile Ser Asn Asp Leu
85 90 95
Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser Cys
100 105 110
Hi~ L~u Pro Trp Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly
115 120 125
3 5 Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg
130 135 140
Leu Gln Gly Ser Leu Gln Asp Met Leu Trp Gln Leu Asp Leu Ser Pro
145
Gly C~s (SEQ ID NO:1)
The obesity protein employed in the formulations of
the present invention can be prepared by any of a variety of
reco~-nized peptide synthesis techniques including classical
~solution) methods, solid phase methods, semi synthetic
methods, and more recent recombinant DNA methods. The
preparation of the human obesity protein is known and
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.
- 12 -
disclosed, for example, in Halaas Jeffrey L. et al., Science
269 ~1995). The preparation of other ~mm~l ian obesity
proteins are described in U.S. appLication serial number
08/445,305, filed May 19, 1995 (EP0 743 321); U.S. patent
application serial number 08/452,~28, filed May 26, 1995 (EP0
744 408); and provisional application, 60,003935, filed
September 19, 1995, (EP ); all of which are herein
incorporated by reference.
The obesity proteins described herein may also be
produced either ~y recombinant DNA technology or well known
chemical procedures, such as solution or solid-phase peptide
synthesis, or semi-synthesis in solution beginning with
protein fragments coupled through conventional solution
methods. Recombinant methods are preferred if a high yield
is desired. The basic steps in the recombinant production of
protein include:
a) construction of a synthetic or semi-synthetic
(or isolation from natural sources) DNA
encoding the obesity protein,
b) integrating the coding se~uence into an
expression vector in a manner suitable for the
expression of the protein either alone or as a
fusion protein,
c) transforming an appropriate eukaryotic or
prokaryotic host cell with the expression
vector, and
d) recovering and purifying the recombinantly
produced protein.
Synthetic genes, the n vitro or n vivo transcription and
translation o~ which will result in the production of the
protein may be constructed by techniques well known in the
art. Owing to the natural degeneracy of the genetic code,
the skilled artisan will recognize that a sizable yet
definite number of DNA sequences may be constructed which
encode the desired proteins. In the preferred practice of
the invention, synthesis is achieved by recombinant DNA
technology.
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- 13 -
Methodology of synthetic gene construction is well
known in the art. For example, see Brown, et al. (1979)
Methods in Enzymology, Academic Press, N.Y., Vol. 68, pgs
109-151. ~he DNA sequence corresponding to the synthetic
claimed protein gene may be generated using conventional DNA
synthesizing apparatus such as the Applied Biosystems Model
380A or 380B DNA synthesizers (commercially available from
Applied Biosystems, Inc., 850 Lincoln Center Drive, Foster
City, CA 94404). It may desirable in some applications to
modify the coding se~uence of the obesity protein so as to
incorporate a convenient protease sensitive cleavage site,
e.g., between the signal peptide and the structural protein
facilitating the controlled excision of the signal peptide
from the ~usion protein construct
The gene encoding the obesity protein may also be
created ~y using polymerase chain reaction (PCR). The
template can be a cDNA library (commercially available from
CLOMETECH or STRATAGENE) or mRNA isolated from the desired
arrival adipose tissue. Such methodologies are well known in
the ~rt Maniatis, et al. Molecular Clonina: A Laboratorv
Manual, Cold Spring Harbor Press, Cold Spring Harbor
Laboratory, Cold ~pring Harbor, New York (1989).
The constructed or isolated DNA sequences are
usef-,ll for expressing the obesity protein either by direct
expression or as fusion protein. When the sequences are used
in a fusion gene, the resulting product will require
enzymatic or chemical cleavage. A variety of peptidases
which cleave a polypeptide at specific sites or digest the
peptides from the amino or carboxy termini (e.g.
diaminopeptidase) of the peptide chain are known.
Furthermore, particular chemicals (e.g. cyanogen ~romide)
- will cleave a polypeptide chain at specific sites. The
skille~ artisan will appreciate the modifications necessary
- to tl1e amino acid sequence (and synthetic or semi-synthetic
coding sequence if recombinant means are employed) to
incorporate site-specific internal cleavage sites. See U.S.
Patent No. 5,126,249; Carter P., Site Specific Proteolysis
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.
~ 14 -
of Fusion Proteins, Ch. 13 in Protein Purification: From
Molecular Mechanisms to Larae Scale Processes, American
Chemical Soc., Washlngton, D.C. (1990).
Construction of suitable vectors containing the
desired coding and control sequences employ standard ligation
techniques. Isolated plasmids or DMA fragments are cleaved,
tailored, and religated in the form desired to form the
plasmids required.
To effect the translation of the desired protein,
one inserts the engineered synthetic DNA sequence in any o~ a
plethora o~ appropriate recombinant DNA expression vectors
through the use of appropriate restriction erldonucleases. A
synthetic coding sequence may be designed to possess
restriction endonuclease cleavage sites at either end of the
transcript to facilitate isolation from and integration into
these expression and amplification and expression plasmids.
The isola~ed cDNA coding sequence may be readily modified by
the use of synthetic linkers to facilitate the incorporation
of this sequence into the desired cloning vectors ~y
techni~ues well known in the art. The particular
endonucleases employed will be dictated by the restriction
endonuclease cleavage pattern of the parent expression vector
to be employed. The restriction sites are chosen so as to
properly orien~ the coding sequence with control sequences to
achieve proper in-frame reading and expression of the
protein.
In general, plasmid vectors containing promoters
and control sequences which are derived from species
compatible with the host cell are used with these hosts. The
vector ordinarily carries a replication origin as well as
marker sequences which are capable of providing phenotypic
selection in transformed cells. For example, E. coli is
typically transformed using pBR322, a plasmid derived from an
~ coli species (Bolivar, et al., Gene 2: 95 (~977)).
Plasmid pBR322 contains genes for ampicillin and tetracycline
resistance and thus provides easy means for identifying
transformed cells. The pBR322 plasmid, or other microbial
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plasmid must also contain or be modified to contain promoters
and other control elements commonly used in recombinant DMA
technology.
The desired coding sequence is inserted into an
- 5 expression vector in the proper orientation to be transcribed
~rom a promoter and ribosome binding site, both of which
should be ~unctional in the host cell in which the protein is
to b~ expressed. An example o~ such an expression vector is
a plasmid described in Belagaje et al., U.S. patent No.
5,304,493, the teachings o~ which are herein incorporated by
re~erence. The gene encoding A-C-s proinsulin described in
U.S. patent No. 5,304,493 can be removed from the plasmid
pRB182 with restriction enzymes ~I and 3amHI. The isolated
DNA ~equences can be inserted into the plasmid backbone on a
NdeI/BamHI restriction ~ragment cassette.
In general, procaryotes are used for cloning o~ D~A
sequences in constructing the vectors use~ul in the
invention. For example, E. coli K12 strain 294 (ATCC No.
31446) is particularly use~ul. Other microbial strains which
may be used include E. CQli B and E. 5~,~ X1776 (ATCC No.
31537). These examples are illustrative rather than limiting.
Procaryotes also are used for expression. The
a~orementioned strains, as well as E. coli W3110
(prototrophic, ATCC No. 27325), bacilli such as Bacillus
subtilis, and other enterobacteriaceae such as ~almonella
tv~himurium or Serratia marcescans, and various pseudomonas
species may be used. Promoters suitable for use with
prokaryotic hosts include the ~-lactamase ~vector pGX2907
[ATCC 39344] contains the replicon and ~-lactamase gene) and
lactose promoter systems (Chang et al., Nature, 275:615
(1978); and Goeddel et al., Nature 281:544 (1979)), alkaline
phosphatase, the tryptophan (trp) promoter system (vector
pATHl [ATCC 37695] is designed to ~acilitate expression o~ an
open reading frame as a trpE ~usion protein under control o~
the trp promoter) and hybrid promoters such as the tac
promoter (isolatable ~rom plasmid pDR54Q ATCC-37282).
However, other ~unctional bacterial promoters, whose
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- 16 -
nucleotide sequences are ~enerally known, enable one of skill
in the art to ligate them to DNA encoding the protein using
linkers or adaptors to supply any required restriction sites.
Promoters for use in bacterial systems also will contain a
Shine-Dalgarno sequence operably linked to the DNA encoding
protein.
The DNA molecules may also be recom~inantly
produced in eukaryotic expression systems. Preferred
promoters controlling transcription in mammalian host cells
may be obtained ~rom various sources, for example, the
genomes of viruses such as: polyoma, Simian Virus 40 (SV40),
adenovirus, retroviruses, hepatitis-B virus and most
preferably cytomegalovirus, or from heterologous mammalian
promoters, e.g. ~-actin promoter. The early and late
promoters of the SV40 virus are conveniently obtained as an
SV40 restriction fragment which also contains the SV40 viral
origin of replication. Fiers, et al., Nature, 273:113
(1978). The entire SV40 genome may be obtained from plasmid
pBRSV, ATCC 45019. The immediate early promoter of the human
cytomegalovirus may be obtained from plasmid pCMBb (ATCC
77177). Of course, promoters from the host cell or related
species also are useful herein.
Transcription of the DNA by higher eucaryotes is
increased by inserting an enhancer sequence into the vector.
Enhancers are cis-acting elements of DNA, usually about 10-
300 bp, that act on a promoter to increase its transcription.
Enhancers are relatively oriented and positioned
independently and have been found 5' (T,~;mln~, L. et ~l.,
PNAS 78:993 (1981)) and 3' (Lusky/ M. L., et al., Mol. Cell
Bio. 3:1108 (1983)) to the transcription unit, within an
intron (Banerji, J. L. et al., Cell 33:729 (1983)) as well as
within the coding sequence itself (Osborne, T. F., et al.,
Mol. Cell Bio. 4:1293 (1984)). Many enhancer sequences are
now known from m~mm~lian gene-s (globin, RSV, SV40, EMC,
elastase, albumin, alpha-fetoprotein and insulin).
Typically, however, one will use an enhancer from a
eukaryotic cell virus. Examples include the SV40 late
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enhancer, the cytomegalovirus early promoter enhancer, the
polyoma enhancer on the late side of the replication origin,
and adenovirus enhancers.
Expression vectors used in eukaryotic host cells
(yeast, ~ungi, insect, plant, animal, human or nucleated
cells from other multicellular organisms) will also contain
sequences necessary for the termination of transcription
which may a~fect mRNA expression. These regions are
transcribed as polyadenylated segments in the untranslated
portion of the mRNA encoding protein. The 3~ untranslated
regions also include transcription termination sites.
Expression vectors may contain a selection gene,
also termed a selecta~le marker. Examples of suitable
selectable markers for mammalian cells are dihydrofolate
reductase (DHFR, which may be derived from the BalII/HindIII
restriction fragment of pJOD-10 [ATCC 68815]), thymidine
kinase (herpes simplex virus thymidine kinase is contained on
the E~mHI fragment of vP-5 clone [ATCC 2028]) or neomycin
(G418) resistance genes (obtainable ~rom pNN414 yeast
artificial chromosome vector [ATCC 37682]). When such
selectable markers are successfully transferred into a
m~mm~l ian host cell, the transfected mammalian host cell can
survive i~ placed under selective pressure. There are two
widely used distinct categories of selective regimes. The
25 first category is based on a cell's metabolism and the use of
a mutant cell line which lacks the ability to grow without a
supplemented media. Two examples are: CHO DHFR- cells (ATCC
CRL-9096) and mouse LTK cells (L-M(TK-) ATCC CCL-2.3).
These cells lack the ability to grow without the addition of
30 such nutrients as thymidine or hypoxanthine. Because these
cells lack certain genes necessary for a complete nucleotide
synthesis pathway, they cannot survive unless the missing
nucleotides are provided in a supplemented media. An
r alternative to supplementing the media is to introduce an
35 intact DHFR or TK gene into cells lacking the respective
genes, thus altering their growth requirements. Individual
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cells which were not transformed with the DHFR or TK gene
will not be capable of survival in nonsupplemented media.
The second category is dominant selection which
refers to a selection scheme used in any cell type and does
not require the use of a mutant cell line. These schemes
typically use a drug to arrest growth of a host cell. Those
cells which have a novel gene would express a protein
conveying drug resistance and would survive the selection.
Examples of such domin~nt selection use the drugs neomycin,
Southern P. and Berg, P., J. Molec. A~l. Genet. 1: 327
(1982), mycophenolic acid, Mulligan, R. C. and Berg, P.
Science 209:1422 (1980), or hygromycin, Sugden, B. et al.,
Mol Cell. Biol. 5:410-413 (1985). The three examples given
above employ bacterial genes under eukaryotic control to
convey resistance to the appropriate drug G418 or neomycin
(geneticin), xgpt (mycophenolic acid) or hygromycin,
respectively.
A preferred vector for eucaryotic expression is
pRc/CMV. pRc/CMV is commercially available from Invitrogen
Corporation, 3985 Sorrento Valley Blvd., San Diego, CA
92121. To confirm correct sequences in plasmids constructed,
the ligation mixtures are used to transform E. coli K12
strain DHlOB (ATCC 31446) and successful transformants
selected by antibiotic resistance where appropriate.
Plasmids from the transformants are prepared, analyzed by
restriction and/or sequence by the method of Messing, et al.,
Nucleic Acids Res. 9:309 (1981).
Host cells may be transformed with the expression
vectors of this invention and cultured in conventional
nutrient media modified as is appropriate for inducing
promoters, selecting transformants or amplifying genes. The
culture conditions, such as temperature, pH and the like, are
those previously used with the host cell selected for
expression, and will be apparent to the ordinarily skilled
artisan. The techniques of transforming cells with the
aforementioned vectors are well known in the art and may be
found in such general references as ManiatiS, ~t al.,
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Molecular Clonina: A La~oratory Manual, Cold Spring Harbor
Press, Cold Spring Harbor Laboratory, Cold Spring Harbor, New
York (1989), or Cl]rrent Protocols in Molecular Bioloov
(1989) and supplements.
Preferred suitable host cells for expressing the
vectors encoding the claimed proteins in higher eucaryotes
include: African green monkey kidney line cell line
transformed by SV40 (COS-7, ATCC CRL-1651); transformed human
primary embryonal kidney cell line 293,(Graham, F. L. ~ al.,
J. Gen Virol. 36:59-72 (1977), Viroloov 77:319-329, Virolo~Y
86:10-21); baby hamster kidney cells (BHK-21(C-13), ATCC CCL-
10, Viroloov 16:147 (1962)); Chinese hamster ovary cells CHO-
DHFR-- (ATCC CR~-9096), mouse Sertoli cells (TM4, ATCC CRL-
1715, Biol. Re~rod. 23:243-250 (1980)); African green monkey
kidLLey cells (VERO 76, ATCC CRL-1587); human cervical
epitheloid carcinoma cells (HeLa, ATCC CCL-2); canine kidney
cells (MDCK, ATCC CC~-34)i buffalo rat liver cells (BRL 3A,
ATCC CRL-1442); human diploid lung cells (WI-38, ATCC CCL-
75); human hepatocellular carcinoma cells (Hep G2, ATCC HB-
8065);and mouse m~mm~ry tumor cells (MMT 060562, ATCC CCL51).
In addition to prokaryotes, unicellular eukaryotes
such as yeast cultures may also be used. Saccharom~ces
cere~isiae, or common baker's yeast is the most commonly used
euka~yotic microorganism, although a number of other strains
are commorLly available. For expression in Saccharomyces, the
plasmid YRp7, for example, (ATCC-40053, Stinchcomb, et al.,
Nature 282:39 (1979); Kingsman et al., Gene 7:141 (1979);
Tschemper et al., Gene 10:157 (1980)) is commonly used. This
plasmid already contains the trp gene which provides a
selection marker for a mutant strain of yeast lacking the
ability to grow in tryptophan, for example ATCC no. 44076 or
PEP4-1 (Jones, Genetics 85:12 (1977)).
Suitable promoting sequences for use with yeast
hosts include the promoters for 3-phosphoglycerate kinase
(found on plasmid pAP12BD ATCC 53231 and described in U.S.
Patent No. 4,935,350, June 19, 1990) or other glycolytic
enzymes such as enolase ~found on plasmid pAC1 ATCC 39532),
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- 20 -
glyceraldehyde-3-phosphate dehydrogenase (derived from
plasmid pHcGAPC1 ATCC 57090, 57091), zymomonas mobilis
(United States Patent No. 5,000,000 issued March 19, 1991),
hexokinase, pyruvate decarboxylase, phosphofructokinase,
glucose-6-phosphate isomerase, 3-phosphoglycerate mutase,
pyruvate ~inase, triosephosphate isomerase, phosphoglucose
isomerase, and glucokinase.
Other yeast promoters, which contain inducible
promoters having the additional advantage of transcription
controlled by growth conditions, are the promoter regions for
alcohol dehydrogenase 2, isocytochrome C, acid phosphatase,
degradative enzymes associated with nitrogen metabolism,
metallothionein (contained on plasmid vector pCL28XhoLHBPV
ATCC 39475, United States Patent No. 4,840,896),
glyceraldehyde 3-phosphate dehydrogenase, and enzymes
responsible for maltose and galactose ~GAL1 found on plasmid
pRY121 ATCC 37658) utilization. Suitable vectors and
promoters for use in yeast expression are further described
in R. Hitzeman et al., European Patent Publication No.
73,657A. Yeast enhancers such as the UAS Gal from
Saccharomvces cerevisiae (found in conjunction with the CYC1
promoter on plasmid YEpsec--h~lbeta ATCC 67024), also are
advantageously used with yeast promoters.
The following examples will help describe how the
invention is practiced and will illustrate the invention.
The scope of the present invention is not to be construed as
merely consisting of the following examples.
Pre~aration 1
~orcine OB Gene and Gene Product
Total RNA was isolated from porcine fat tissue
obtained from Pel~Freez~, Pel-Freez Inc., and the cDNA was
cloned in accordance with the techniques described in Hsiung
et al., Neuro~e~tide 25: 1-10 (1994).
Primers were designed based on the published amino
acid sequence of the human o~ gene. The primers were
prepared for use in polymerase chain reaction (PCR)
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amplification methods using a Model 380A DNA synthesizers
(PE-Applied Biosystems, Inc., 850 Lincoln Center Drive,
Foster City, CA 94404). Primers PCROB-l (12504) (ATG CAT TGG
GGA MCC CTG TG), PCROB-2 (12505) (GG ATT CTT GTG GCT TTG GYC
~ 5 CTA TCT), PCROB-3 (12506) (TCA GCA CCC AGG GCT GAG GTC CA),
and PCRoB-4 (12507) (CAT GTC CTG CAG AGA CCC CTG CAG CCT GCT
CA) were prepared.
For cDNA synthesis, total RMA 1 ~L (1 ~g/~l)
isolated from porcine adipose tissue and 1 ~L Perkin Elmer
Random primers (50 ~M) in a total volume o~ 12 ~L were
annealed for 10 minutes at 70~C and then cooled on ice. The
following were then added to the annealed mixture: 4 ~L o~
BRL 5x H-reverse transcriptase (RT) reaction buffer (Gibco-
BRL CAT#28025-013), 2 ~L of 0.1 M DTT, 1 ~L of lQ mM dNTPs.
This annealed mixture was then incubated at 37~C for 2
minutes before adding 1 ~L BRL M-MLV-reverse transcriptase
(200 U/~L) (CAT#28025-013) and incubated at 37~C for
additional 1 hour. After incubation the mixture was heated
at 95~C for 5 minutes and then was cooled on ice.
For amplification of cD~A, the polymerase chain
reaction (PCR) was carried out in a reaction mixture (100 ~L)
containing the above cDNA reaction mixture (1 ~L), 2.5 units
of AmpliTaq DNA polymerase (Perkin-Elmer Corporation) 10 ~1
of 10x PCR reaction bu~er (Perkin-Elmer Corporation) and 50
pmol each of the sense (PCROB-l) and antisense (PCROB-3)
primers for porcine OB amplification. The condition ~or PCR
was 9~~C ~or 1 minute, 57~C ~or 1 minute and 72-~C for 1
minute ~or 30 cycles using a PCR DNA Thermal Cycler (Perkin-
Elmer Corporation). After PCR amplification, 5 ~L BRL T4 DNA
polymerase (5 U/~L), 2 ~L BRL T4 polynucleotide kinase (10
U/~L), and 5 ~L ATP (10 mM) were added to the PCR reaction
mixtu:-e (100 ~1) directly and incubated for 30 minutes at
37~C. After the incubation the reaction mixture was heated
at 95~C for 5 minutes and then was cooled on ice. The 500 bp
~ragment (~0.5 mg) was purified by agarose gel
electrophoresis and isolated by the freeze-squeeze method.
The 500 bp fragment (_0.2 ~g) was then ligated into SmaI
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- 22 -
linearized pUC18 plasmid (~1 ~g) and the ligation mixture was
used to transform DH5a (BRL) competent cells. The
transformation mixture was plated on 0.02% X-Gal TY broth
plates containing ampicillin (Amp) (100 ~g/mL) and was then
incubated overnight at 37~C. White clones were picked and
were grown at 37~C overnight in TY broth containing Amp (lQ0
~g/mL). The plasmid was isolated using a Wizard Miniprep DNA
purification system (Promega) and submitted for DNA
seqeuncing on a Applied Biosystem 370 DNA sequencer.
Pre~aration 2
Bovine OB Gene and Gene Product
The DNA sequence o~ the bovine OB gene was obtained
by techniques analogous to Example 1, except sense (PCROB-2)
and antisense (PCRoB-3) primers were used for bovine OB cDNA
amplification.
Pre~aration 3
Vector Construction
A plasmid containing the DNA sequence encoding the
obesity protein is constructed to include NdeI and ~HI
restriction sites. The plasmid carrying the cloned PCR
product is digested with ~I and BamHI restriction enzymes.
The small - 450bp fragment is gel-purified and ligated into
the vector pRB182 from which the coding sequence for A-C-B
proinsulin is deleted. The ligation products are transformed
into F.. coli DH10B (commercially available from GIBCO-BRL)
and colonies growing on tryptone-yeast (DIFCO) plates
supplemented with 10 mg/mL of tetracycline are analyzed.
Plasmid DNA is isolated, digested with NdeI and BamHI and the
resulting fragments are separated by agarose gel
electrophoresis. Plasmids containing the expected - 450bp
NdeI to ~mHI fragment are kept. E. coli K12 RV308
(available from the NRRL under deposit number B-15624) are
transformed with this second plasmid, resulting in a culture
suitable for expressing the protein.
The techniques of transforming cells with the
aforementioned vectors are well known in the art and may be
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- 23 -
found in such general references as Maniatis, et al. (1988)
Molecular Clonin~: A Laboratorv Manual, Cold Spring ~Iarbor
Press, Cold Spring Harbor Laboratory, Cold Spring Harbor, New
York or Cllrrent Protocols in Molecular Biolo~v ~1989) and
supplements. The techniques involved in the trans~ormation
of E. coli cells used in the preferred practice of the
invention as exemplified herein are well known in the art.
The precise conditions under wh-ich the transformed ~ coli
ce~ls are cultured is dependent on the nature of the ~ coli
host cell line and the expression or cloning vectors
employed. For example, vectors which incorporate
thermoinducible promoter-operator regions, such as the c1857
thermoinducible lambda-phage promoter-operator region,
require a temperature shift from about 30 to about 40 degrees
C. in the culture conditions so as to induce protein
synthesis.
In the preferred embodiment of the invention, ~.
SQ~i K12 RV308 cells are employed as host cells but numerous
other cell lines are available such as, but not limited to,
F 5~L~ K12 L201, L687, L693, L5~7, L640, L641, L695, L814
(E cQli B). The transformed host cells are then plated on
appropriate media under the selective pressure of the
an~ibiotic corresponding to the resistance gene present on
the expression plasmid. The cultures are then incubated for
2S a t:ime and temperature appropriate to the host cell line
emE)loyed .
Proteins that are expressed in high-level bacterial
expression systems characteristically aggregate in granules
or inclusion bodies which contain high levels of the
overexpressed protein. Kreuger et al., in Protein Foldin~,
Gierasch and King, eds., pgs 136-142 (1990), American
Association for the Advancement of Science Publication No.
89--18S, Washington, D.C. Such protein aggregates must be
solubilized to provide further purification and isolation of
the desired protein product. Id. A variety of techni~ues
us~ng strongly denaturing solutions such as guanidinium-HCl
and/or weakly denaturing solutions such as dithiothreitol
5~ TE SHEET(RULE 2~)
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.
- 24 -
(DTT) are used to solubilize the proteins. Gradual removal
of the denaturing agents (often by dialysis) in a solution
allows the denatured protein to assume its native
conformation. The particular conditions for denaturation and
folding are determined by the particular protein expression
system and/or the protein in question.
Preferably, the DNA sequences are expressed with a
dipeptide leader sequence encoding Met-Arg or Met-Tyr as
described in U.S. Patent No. 5,126,249, herein incorporated
by reference. This approach facilitates the efficie~t
expression of proteins and enables rapid conversion to the
active protein form with Cathepsin C or other
dipeptidylpeptidases. The purification of proteins is by
techniques known in the art and includes reverse phase
chromatography, affinity chromatography, and size exclusion.
The following examples and preparations are
provided merely to further illustrate the preparation of the
formuations of the invention The scope of the inve~tion is
not construed as merely consisting of the following examples.
Example 1
Human Obesitv Protein Formulation
To generate a solution of Human Obesity Protein
(hOb), the lyophilized solid material was first dissolved in
water to generate a stock solution (stock 1). The
concentration of hOb in stock 1 was verified by W /Vis
Spectrophometry using the known extinction coe~ficient for
hOb at the maximum spectral absorbance (279nm or 280nm),
measuring the maximum spectral absorbance (279nm or 280nm),
and utilizing the dilution factor. A stock preservative
solution containing methylparaben was prepared by dissolving
the solid in water (stock 2). A solution of hOb at 1.6 mg/mL
was prepared by addition of an aliquot of stock 1 to a
container which held an aliquot of stock 2 along with the
required quantity of water, at an alkaline pH (7.8~0.3)i
adjusted if necessary with HCl or NaOH. After an appropriate
incubation period at room temperature (30 minutes), the
CA 02243229 1998-07-1~
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solution pH was examined and adjusted if necessary with trace
~L ~uantities of HCl or NaOH, to yield an hOb solution at 1.6
mg/mL with 0.17% methylparaben pH 7.8+0.1. The solution was
then hand-filtered using a glass syringe with an attached
0.22 ~m syringe filter into a glass vial.
Example 2
Human Obesitv Protein Solution
To generate a solution of Human Obesity Protein
(hOb~, the lyophilized solid material was first dissolved in
water to generate a stock solution (stock 1). The
concentration of hOb in stock 1 was verified by W/Vis
Spectrophometry using the known extinction coefficient for
hOb at the maximum spectral absorbance (279nm or 280nm),
measuring the maximum spectral absorbance (279nm or 280nm),
and utilizing the dilution factor. A stock preservative
solution containing chlorobutanol, for example, was prepared
by dissolving the solid in water (stock 2). A solution of
hOb at 1.6 mg/mL was prepared by addition of an ali~uot of
stock 1 to a container which held an aliquot of stock 2 along
with the required quantity o~ water, at an alkaline pH
(7.8+0.3); adjusted if necessary with HCl or NaOH. After an
appropriate incubation period at room temperature (30
minutes), the solution pH was examined and adjusted if
necessary with trace ~L quantities of HCl or NaOH, to yield
an hOb solution at 1.6 mg/mL with 0.50% chlorobutanol pH
7.8+0.1. The solution was then hand-filtered using a glass
syringe with an attached 0.22 ~m syringe filter into a glass
vial.
Example 3
Human Obesitv Protein Solution
To generate a solution of Human Obesity Protein
(hOb), the solid bulk material, lyophilized from a neutral
water solution, was redissolved in water to generate a stock
solution (stock 1). The concentration of hOb in stock 1 was
verified by W /Vis Spectrophometry by multiplying the maximum
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- 26 -
spectral absorbance (279nm or 280nm) by the dilution factor
divided by the known extinction coef~icient for hOb. A stock
preservative solution containing methylparaben was prepared
by dissolving the solid in water (stock 2). A stock of an
isotonicity agent, such as glycerin was prepared by
dissolving the neat liquid in water (stock 3). A stock of a
physiologically-tolerated buffer, such as sodium phosphate
was prepared by dissolving the solid in water (stock 4). A
solution of hOb at 1.6 mg/mL was prepared by addition of an
aliquot of stock 1 to a container which held an aliquot of
stock 2 along with an aliquot of stock 3 along with the
required quantity of water, adjusted if necessary with HCl or
NaOH to an alkaline pH (7.8+0.3). After an approp~iate
incubation period at room temperature (30 minutes) an aliquot
of stock 4 was added. The solution pH was then readjusted if
necessary with trace ~ quantities of HCl or NaO~, to yield
an hOb solution at 1.6 mg/mL with 0.17% methylparaben, 16
mg/mL glycerin, and 14mM sodium phosphate at pH 7.8~0.1. The
solution was then hand-filtered using a glass syringe with an
attached 0.22~m syringe filter into a glass vial.
Example 4
Hllm~n Obesitv Protein Solution
To generate a solution of Human Obesity Protein
(hOb), the solid bulk material, lyophilized from a neutral
water solution, was redissolved in water to generate a stock
solution (stock 1). The concentration of hOb in stock 1 was
verified by UV/ViS Spectrophometry by multiplying the maximum
spectral absorbance (279nm or 280nm) by the dilution factor
divided by the known extinction coefficient for hOb. A stock
preservative solution containing chlorobutanol was prepared
by dissolving the solid in water (stock 2). A stock of an
isotonicity agent, such as glycerin, was prepared by
dissolving the nea~ liquid in water (stock 3). A stock of a
physiologically-tolerated buffer, such as sodium phosphate
was prepared by dissolving the solid in water (stock 4). A
solution of hOb at 1.6 mg/mL was prepared by addition of an
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- 27 -
aliquot of stock 1 to a container which held an aliquot of
stock 2 along with an aliquot of stock 3 along with the
required ~uantity of water, adjusted if necessary with HCl or
NaOH to an alkaline pH (7.8+0.3). After an appropriate
incubation period at room temperature (30 minutes) an aliquot
of stock 4 was added. The solution pH was then readjusted if
necessary with trace ~L quantities of HCl or NaOH, to yield
an hOb solution,.for example, at 1.6 mg/mL with 0.5%
chlorobutanol, 16 mg/mL glycerin, and 14mM sodium phosphate
(if required by the formulation) at pH 7.8+0.1. The solution
was then hand-filtered using a glass syringe with an attached
0.22~m syringe filter into a glass vial.
Example 5
Pre~aration of Human Obesitv Protein Solution
To generate a preserved solution of human obesity
protein (hOb), the individual solution components were added
in succession to a neutral water solution, allowing each
component to dissolve prior to addition of the next
component. To the container were added: approximately 5-6
mL of sterile water (~inal solution volume = 10.00 mL)
followed by preservative solids (1.51 mg of butylparaben;
final concentration = 0.015~). The solution was mixed at
medium speed with a Te~lon stir bar and heat was applied to
promote dissolution o~ the solids. Care was exercised to
dissolve the preservative solids, but not to overheat or boil
the solution. A~ter dissolution was achieved (approximately
1-1.5 hr.), the solution was allowed to cool to room
temperature while maintaining stirring. Glycerin (160.0 mg)
was then added to the solution and stirred until dissolution
was achieved. The final glycerin concentration was targeted
to be 16 mg/mL. A 10.0 mg quantity o~ solid ethylenediamine
tetra_etate (EDTA) was added to the solution and stirred to
dissolve (final concentration = 0.10%). Dibasic sodium
phosphate crystals (17 0 mg) were then added and allowed to
dissolve by stirring. The ~inal phosphate concentration was
targe~ed at 6.3 mM. Sucrose (600.0 mg) was then added and
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- 28 -
allowed to stir until dissolved (final concentration of 60
mg/mL). Bulk lyophilized hOb (115.4 mg) was added
incrementally to the stirring solution, allowing each aliquot
to dissolve prior to the addition of successive portions.
The quantity of hOb added to the solution yielded a final
concentration of 10.2 mg/mL. The amount added was determined
by using the "as is" weight ~, determined for the specific
hOb lot used by W /Vis spectrophotometry. Following the
addition of all bulk ingredients, the pH of the solution was
adjusted to 8.5 using approximately 11 ~L of 10~ NaOH to
promote dissolution of the solids. After stirring the
solution for a 10-15 minute period, the pH was decreased to
7.8 using approximately 4-5 ~L of 10% HCl and the solution
was allowed to stir for an additional 10 minutes. The
solution was then transferred to a 10 mL volumetric flask,
and brought to final volume with sterile water. The ~lask
was then inverted 20x to thoroughly mix the contents and
transferred to a glass sample vial where a recheck of the pH
yielded 7.78. A trace quantity of 10% NaOH (approximately 1
~L) was added to yield a final pH of 7.83. The solution was
then hand-filtered, using a 5 mL glass syringe with an
attached 0.2~m syringe filter into a 20 mL glass vial, capped
and stored refrigerated until needed.
The principles, preferred embodiments and modes of
operation of the present invention have been described in the
foregoing specification. ~he invention which is intended to
be protected herein, however, is not to be construed as
limited to the particular forms disclosed, sinc~ they are to
be regarded as illustrative rather than restrictive.
Variations and changes may be made by those skilled in the
art without departing from the spirit of the invention.
