Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
W~ 93/19084 PCr/US93/02~57
, . .
2~1 09.~20
R~FOI.DXNG AND PllRIFI~ATION OF I~ E GROWT~ FAC~QR I
Field of the Invention
This invention relates to the field of biotechnology
processing, more particularly refolding and purification of
polypeptides, and even more particularly to t~e refolding and
purification of insulin-like growth factor I.
ackoround of_the Invention and In~ormation Disclosure Statement
The insulin gene family, comprised of insulin, relaxin,
: insulin-like growth factors I and II, and possibly the beta subunit
o~ 7S nerve growth factor, represents a group of structurally
related polypeptides whose biological functions have diverged as
~ reported in Dull, et al., ~ature 310:777-781 (1984~
: Insulin-like growth factor I tIGF-I), also known as
somatomedin C, is a protein of ~ppro~imately 7.8 kilodaltons~ IGF-
15~ I bi~ds to IGF rec:eptors. StructlLral similarity l~etween IGF-I and
IGF-II permits both to bind to IGF reoeptors. q~o IGF receptors
are kn~. to exist. IGF-I and IGF-II birld to the IGF type I
receptor, while insulin binds with less affinity to this receptor.
~ T$e type I receptor prefcre~ially binds ~F-I and is belie~ed o
: 20 transduce the mitogenic effects of IGF-I and IGF-II. IGF-II binds
to the type I rec~ptor with a ~0-fold lower affinity than IGF-I.
The second or t~pe II IGF receptor preferentially binds IGF-II.
Receptor binding is believed to be ne essary for the biolog~cal
activities of IGF-I and IGF-II.
: 25 In addition to specific cell surface receptors, there exist at
least six distinct IGF binding proteins (IGFBP-l through I~FBP-6)
WOg3/lgO84 PCT/US93/~2457
~lU~320
that circulate throughout the body. These proteins bind IGF I and
IGF-II. The ~inding of I~F-I and IGF-II to binding proteins
reduces the action of these I&Fs on cel}s by inhibiting IGF binding
to cell surface receptors. Oh et al., Endo~rinol. 132:1337-1344,
1993~ reports IGF-I and IGF-II are essentially equipotent in their
affinities for IGFBP-1. When IGF is bound to binding proteins, it
is u~able to bind to the IGF receptors and is therefore, no longer
active in the body.
IGF-I is mitogenic f~r a large number of cell types,
including fibroblasts, keratinocytes, endothelial cells and
osteoblasts (bone-forming cells). IÇF-I also stimulates
differentiation of many cell types, e.g., synthesis and secreti~n
~: of~ ~ollagens by osteoblasts. IGF-I exert5 its mitogenic and cell
dif~erentiating effects by binding to the specific IGF cell surfac~
` :`
15~ receptor~ IGF-I also has been shown to inhi~it protein catabolism
iD~ vi~o, to stimulate glucose upt~ke by cells and to promote
survival of isolated~neurons in culture~ These properties have led
to IGF-I being tested~as a therapeutic agent for ,a ~arie y of
: disease indica~io~s as reported in Froesch et al., Trends _in
20~ :EndocrinoloqY and~ :Metabolism, 254-260 (May/June 1990~ and
: Cotterill, Cli~ical En~aE~ gy, 37~ 16 (1992).
, The role of IGF-I as a useful therapeutic agent for ~everal
other disease onditions has also been suggested. For example,
~:: IGF-I ~as long been studied for its role in the growth of various
tissues. As xeported in Laron et al., Slinical EndocrinolooY, 35:
- ~ 145-~50 (1991), a marked rise in serum type III procollagen, a
.
WO93/19084 2 1 ~ ~ ~ 2 0 PCT/US93/02457
marker of bone formation, occurred after one week of administration
o~ recombinan~ly produced IGF-I to patients with dwarfism otherwise
non responsive to growth hormone. The effects of the infusion of
IGF-I in a child with Laron Dwarfism were described in Walker et
al., The New En~land Journal of Medicine, 324(21):1483-1488 (l991).
Increased weight gain t nitrogen retention and muscle protein
synthesis following treatment of diabetic rats with IGF-I or a
truncated form of IGF-I having a deletion of the first three amino
~ acids ordinarily found in IGF-I (referred to as ''(desl-3)IGF-II')
were demonstrated by Tomas et al., as reported in Biochem. _J.
~ 276:547-554 (1991). Growth restoration in insulin-deficient
; diabetic rats by administration of recombinantly produced human
IGF-I was reported in Schèiwiller et al., Nature, 323:169 (1986).
F-I and (desl~3)I~F I enh~nced growth in rats after gut
15~:~:reseotion~ as reported in Lemmey et al., Am. J. PhYsiol~ 260
; ~kndocr~inol. Metab. 23~)~ E213-E219 (1991). A combination of
platelet-derived growth:~a ~ or and insulin-like growth ~actors,
ind uding IGF-I, enhanoed periodontal regeneration in beagle dogs
as reported by: ~ynch et al., ~ L~L~ sie9~ 16:545-548
20~ (1989~. The~synergisti~c effects of platelet-derived growth factor
and IGF-I in wound healing were reported in L~nch et al., Proc.
~, Natl. Acad. Sci. ~4:7696-7700 ~1987). The effects of IGF~I and
:~ ~ g~owth ~ormone on longitudinal bone growth ~n ~itro were ~et forth
: in Scheven and ~amilton, Acta EndocrInoloqLica ~Copenhagen) 124:602-
~ 25 607 (199I). In viyo~ actions of IG~-I on bone formation and
:~; resorption in rats: were shown i~ Spencer et al., Bone 12:21-2
~; 3
WO93/19084 PCT/US93/0~457
210'9'g20
(1991). The use of IGF-I and IGF-II for e~hancing the survival of
non-mitotic, cholinergic neuronal cells in a mammal was described
in U.S. Pate~t 5,093,317 to Lewis et al. In addition, PCT
Application Publication No. W0 92111865 published on July 23, 1992,
5' describes the use of IGF-I for the treatm~nt of cardiac disorders.
Although it is possible to purify IGF-I from human plasma, it
is not co~mercially feasible to do so because o~ the significant
costs in~olved. Furthermore, proteins purified from human plasma
may be contaminated by pathogenic organisms such as viruses
: 10 includi.ng the hepatitis viruses and the AIDS ~irus. An alternati~e
method for producing large ~uantities of IGF-I cheaply is to
produce it by recombinant DM~ methods. With the~e methods, DNA
se~uences enooding IGF-I are cloned into a proka~yotic e~pression
vec~or, for e~mple, pT3XI 2 (described in W0 91/08285~, that is
:
15 ~capable of directing high l~vel expression of the recombinant
proteins in bacteria, particularly scherichia coli (~ col~). For
example, European Pate~t Application Publication No. 0130166
: ~
describes expression of IGF-I in ~ coli. Thi~ re~er-ence does not
~ teach purification nor how to render the protein biologically
::~ 2Q active~ :
-~ ~ As an alternative means for obtaining biol~gically active
recombinant IGF-I from bacteria, several groups have constructed
gene fusions in which DNA sequences encoding IGF-I are fused to
::~ other proteins, protective peptides! or a ~eries of charged amino
; ~ 25 acid residues. For example, in an attempt to obtain biologically
~ ~ active IGF-I from E. coli, DNA seguences encoding IGF-I were fused
'
~ 4
: ~
WO93/19084 PCT/US93/02457
2 1 Q ~ 0
to those encoding a hydrophobic ~'signal se~uence" from lamB or
ompF, which directs secretion of the fusi~n protein into the
periplasmic space of E. coli. Endogenous, membrane bound proteases
cleave ~he signal sequence ~rom the mature I~F-I protein. See
~uropean Patent Application Publication Mo. 0288451.
European Patent Application Publication No. 0155655 describes
synthesis, bacterial expression and purification of IGF-I fused to
oth~r proteins. No data are presented in this ref~rence which
demonstrated the activity of this mol~cule. Similarly, European
Patent Application Publication No. 0128733 describes bacterial
production of IGF-I fused to other proteins. The fusion protein so
produced was cleaved with proteases to release IGF-I. Again, no
data was presented in this referen~e demonstrating the activity of
his;~protein. Fusion proteins yielding five incorrectly folded
biologically inactive forms of IGF-I w~re described in S. Hober et
:~ al., BiochemistrY 31:1749~1756 (1992). European Patent ~pplication
~: ~ l?ublicatiorl No. 0286345 describe~ production of human IGF-I in
: :~ bacteria using a vector in which exprassion was controlled by ~
: lambda phage promotor and a temperature s~nsitive r~pressor
prot~in. The biological activity of the material produced was not
demonstrated. : Not taught was h~w to purify ~he protein, nor
whether the ~N-terminal methionine was cleaved fr~m the protein.
IGF-I not having the ~-terminal methionine cleaved is refer-red to
as met-IGF~I. PCT Application Publication No. W091/02807 describes
synthesis, expression in bacteriai~ and refolding of IGF-I fused to
charged amino a~ids:at the N-terminus of the protein~ The charged
W~93/19084 PCT/US93/02457
21~9~2~
amino acids were added ~o ~acilitate refolding of IGF-I; refolding
of IGF-I was found to be less than optimal without the charged
amino acid~. The protein of this reference was refolded and
subsequently treated with proteases to remoYe the extra charged
amino acids. The charged amino acids were chosen so that they
would be recognized as cleavage sites by diaminopeptidase, beef
sp~een Cathepsin C.
On~ of the problems encountered in expres~ing eukaryotic
protein~, particularly those that contain disulfide bonds, in
bacteria is that the recombinant proteins are synthesiæed in an
inactive form by the bacteria. Typically, cysteine residues in
recombinant proteins expressed in ba~teria are improperly paired
with one another. The improperly disulfide-bonded proteins tend to
:: :
; ~ be insoluble. This problem has been encountered in pre~ious
~ 15 atte~pts to express acti~e IGF-I in bacteria. See PCT Patent
,
Application Publication No~ WO 39/03423~ IGF-I contains six
cys~eine residues. ~ All six cysteine residues participate in
f~rming~disulfide bonds. The cysteine residues must be ~orrectly
paired in order for the prote~n to assume its proper conformation
0 and to exhibit full ~iological activity. As noted ab~Ye~ attempts
: :
at converting the:inactive recombinant IGF-I protein int~ acti~e,
properly folded protein by following reactivation and refolding
pro~ocols have not been successful due to improper disulfide
pairing and low yields. Several groups have set forth Yarious
25~:strategies for refolding, renaturingt or reactivating proteins.
: These include, for example, To Kohno, et al., Methods in EnzymolO,
W093~19084 PCT/US93/~2457
2~09~20
185:187-195 (1990). U.S. Pa~ent 4,620,948 reports the refolding of
proteins from inclusion bodies of reduced material. U.S. Patent
4,620,948 is deficient in describing purification steps or
analyti~al methods used to isolate ~nd characterize correctly
refolded protein~ Neither of ~hese references describes refolding
of IGF-I to form a biologically active protein. The references
als~ do not describe purification techniques for isolating
correctly folded IGF-I from incorrectly folded IGF-I.
Summarv_of the.Invention
In order to o~ercome the problems discussed above, the present
inventors have developed a process for producing and purifying
large amounts of biologically active rec~mbinant IGF-I, ~xpressed
in prokaryotic cells, particularly in bacteria, and more
~ particularly, in E.~ç~li. The present inYention resides in the
refoldi~g of recombinan~ly produced IGF I expres~ed in bacteria, to
render biologically~aGtive IGF-I. The method of refolding of the
present invention does not require the construction of fusion
proteins or the us:e ~of~charged amino acids attached to the N-
erminus~of the protein:to produce biologically active IG~
: 2`0 ~ The pres~nt invention provides methods whereby inactive met-
IG~-I expressed in bacteria can be refolded into its proper
:
conformationO The proteins produced by ~he instant invention ar~
indicated as being co~rectly refcld~d by evidence of their
iological activity :when compared to a commercially a~ailable
,~ :
standard.
The present invention also pro~ides a method for purif~iny
W093/l9084 PCT/US93/02457
21û9820
correctly refolded IGF-I from incorr~ctly refolded IGF-I.
The present in~ention also provides a method for converting
met-IGF-I to IGF~
Also pro~ided in the present invention are pharmaceutical
compositions comprising IGF-I and methods of using the IGF-I to
treat a patient having or potenkially having an IGF associated
condition.
petailed Description o the Invention
Often the lack of biologica~ a~tivity in proteins e~pressed in
microorganisms is related to improper formation of intramolecular
disulfide bonds. In the present in~ention, recombinant IGF-I
p~oduced in E. coli may be refolded t~ at~ain the correct
.
configuration of intramolecular disulfide bonds and, therefore,
exhibi~ full biological acti~ity.
15Terms used throughout this specification are de~ined as
; :~ foll w s: :
The term "acceptable phaxmac~utical carrier~' referc to a
physiologically-compati~le, aqueous or non aqueous solv~nt.
The: term ~'IGF-I" refers to a protein ha~ing the same amino
29 acid sequence as naturally occurring IGF-I, or a protein having the
: :same amino aaid sequence as naturally occurring IGF-I with the
addition 9f an N-terminal methionine, unless otherwise specified.
The term "IGF associated condition" rPfer~ to an existing or
potential adverse physiological condition which r~ults from an
~25 over-production or underproduction of IGF, IGF binding protein or
IGF re~eptor, inappropriate or inadequa~e binding of IGF to ~inding
: 8
WO93/l90~ 2 ~ Q ~ 8 2 0 PCT/US93/02457
proteins or receptors and any disease in which IGF administration
alle~iates disease symptoms. An IGF associated condition also
refers to a condition in which administration of IGF to a normal
patient has a desired effect.
The term "patient" refers to any animal, including ~umans, in
need of treatment for an IGF associated condition.
The term "denaturing agent, or d~natuxant" refers to any
material which will cause a change in the conformation of a protein
that results in a loss of biological activity. Acceptable
denaturing agents include, but are not limited to, guanidine and
urea.
The term "oxidizing agent" refers to any material which is
capable of removi~g an ~lectron from the compound being oxidized~
;~: Acceptable oxidizing:agents incIude, but are not limited to,
15 ~ oxidizing agents which are capable of aiding in the foxmation of
miYed~:disulfide bond~s,~for example, oxidized glutathione and
cystine.~
The:~term "reducing agent' refers to any material which îs
capable of~add~ing an~:~el~ectron to a compound. Acceptable r~duci~g
2~0~:agents in~1ude any reducing agent capable of ~he disruption of the
moleaular disulfide bonds~. Acceptable r~ducing agents include, but
~: are not limited-to, ~dithiothreitol (DTT), 2-mercaptoethano~, and
dithioerythritol. ~ .
,, :
:The term "thiol-containing reducing reagent" ref~rs to a
25~ reducing agent which ~contains a sulfh~dryl group. Examples
: incl~e, but are not limited to, dithiothreitol (DTT), 2-
; : g
W~93/19~ PCT/~S~3/02457
210~82~
mercaptoethanol, dithioerythritol, cysteine, cystamine, and
reducing agents containing added disulfide co~taining compoundsl
such as sodium borohydride or any of the Group ~IA hydrides
containing added cystine, sxidized glutathione or any cysteine-
containing dipeptide.
The term "biologically active" refers to the ability to
stimulate proliferation of UMR106 rat ostesarcoma cell line, as
described in Example ~. The biological acti~ity of a aorrectly
refolded protein stimulates proliferation of the ~MR106 cell line
at an EDSo of about 1 - 30 ng/ml, preferably about 2 - 10 ng/ml and
more preferably at about 7 - 8 ng/ml. Incorrectly refolded I~F-I
~; stimulates proliferation of UMR106 rat ostesarcoma cell line with
: an ED~ ~reater than 30 ng/ml, which for purposes of the present
in~ention is ~onsidered "biologically inactiven.
The term l'ED~o'~ refers to the concentration which causes one-
half maximal ~ incorporation into the DN~ of cells.
~ The recombinant proteins of the present inYention ~were
; refold~d, purified and subse~ue.ntly treated with an aminopeptidase
.
: ~ to remove the extra N-tenmi~al ~e~hionine. Aminopeptidas~s useful
: 20 for this pu ~ ose include, but are not limited to, diaminopeptidase,
:: ~
from beef spleen Cathepsin C, and aminopeptidase from Aeromonas
P~
The examples below set forth the proced~res used to construct
~he IGF-I gene, which was done by forming a gene fusion with a
secretory leader sequence for E. coli. From this, a second J
construct was formed to e~press met-IGF-I without the secretory
WO93/19084 PCT/US93/02457
2109~20
leader sequence. The plasmid thus created was used to transform E.
coli to express the met-I~F-I. The yield of met-IÇF-I expressed
exceeds 10% of total cell protein. The protein thus expressed was
purified after disrupting the ~. coli cells. The insoluble
biologically inactive met-IGF-I was rendered solu~le and
biologically active by use of a re~olding procedure. Properly
refolded IGF-I was isolated from improperly refolded IGF-I by use
of several column chromatography procedures.
The instant invention resides in the refolding and
purification of the resultant recombinant protein to render
biologically acti~e IGF-I. In the present invention, recombinant
: ~ IGF-I may be r~folded by using the following ~teps:
(1) Any intramolecular or intermolecular disulfîde bonds
::~
~ andlor.any noncovalent interactîons ~hich ha~e occurred involvîng
:
: 15 :~he ~ature IGF-I produced in a mîcrooxganîsm are fîrst dîærupted.
In ord-r to do~ this,~ the protein is exposed to suffîcîent
enaturant (for example, guanidine hydrochlorîde or urea) and
~: sufficient~ reducing agent tfor~ example, ~eta-mercapto~thanol,
dithio~hreitol, or:~cysteine) to denature the proteîn, dîsrupt
~: 20 :nonco~alent interactions, and reduce disulfîde bonds.
,
(2):: After the; mature IGF-I has been denatured and reduced,
the free thiols present in the reduced protein are oxidîzed by
addltîon of a large excess of dîs~lfide-contaîning oxîdîzing agent
(for ex~mple, oxidized glutathîone:~or cystîne). This reaction
produces mixed disulfide bonds in~whîch each cysteine resîdue in
: ~ :
the mature IGF-I forms a disulfide bo~d with the monomeric form of
:
11
:
W093/19084 PCT/US93tO24~7
21~9820
the oxidizing agent. This step helps to prevent the formation of
inco~rect intramolecular disulfide bonds in the IGF~I during
subsequent processing.
(3~ The denaturant and oxidizing agent are then diluted to a
defined concentration and a then second reducing agent, also known
as a thiol-containing reducing reagent, is added to catalyze
disulfide interchange. The objective is to produce an environment
in which the denaturant is sufficiently reduced to allow the I~F-I
to assume various 3-dimensional configurations and in which the
oxidization/reduction potential is adjusted to allow the ~ormation
and breaking of disulfide bonds. It is believed that the proper 3-
dimensional structure and disulfide bonding pattern of the mature
:: :
IGF;I is energetically more stable than other possible
con~ormatiohs. Therefore, under conditions in which the IGF-I is
15 allowed ~o assume a variety of 3-dimensional co~foxmations and
: intramolecular disul ide~bond patterns, a sîgni~icant proportion of
the I~F-I will form the correct intramolecular disulfide bonding
pattern ~assuming :rthe correct 3-dimensional ~tructure, and~
therefore, b~come:biologically active.
These procedures :are mild and should not result in the
chemical mGdification~; of the IGF-I. If urea is used as ~
denaturant, any inter~ering cyanate that may form can be remoYed by
passing the urea solution oYer an anion exchange co~umn, such as
: DOWEX l-X8(Bio~ad). Cyanate can modi~y amino ~roups in the protein
tStark, ethods in~Enzvmolo~y 11:125 1967).
The optimal concentration and hoice of denaturant, oxidizing
WO93/19084 PCTJUS93/02457
2109~2()
agent, thiol-containing reducing reagent and their concentrations
in the final refolding solution are determined experimenta~ly by
mo~itoring the proportion of I~F-I properly refolded and
biologically active. The objective in the final refolding ~olutio.n
is to provîde a controlled environment in which disulfide
interchange and conformational changes ~an occur in the IG~-I un~il
the favored conformation and disulfide bonding pattern is achieved.
In an embodiment of the present in~enti~n, the IGF-I is
~ubs~antially purified from soluble pxoteins prior to refolding.
An alternative embodiment is contemplated whereby the IGF-~ is
substantially purified from soluble and insoluble proteins prior to
: r folding. Substantially purified in this context means the
:solution is substantially free of host cell proteins that interfere
:` :
with the rate or ef~iciency of:IGF-I ref~lding.
15~ : ~The correctly refolded IGF-I is separated from ~he incorrectly
refolded ~ IGF I by means of various column chroma~o~raphy
techniques, including, f~or ex~mpleO the technique5 described below
~ xample 4~ In ~ne::embodiment of the ~eparation me~hod, the
first:step~;is:dialysis to decrease the amount and concentration of
:20 redu~ing agent and;denaturing agent u~ed n th refolding process.
The second: step utilizes an ion exchange column which separates
~:, pr~tein i~mers according to charge. Although Example 4 teaches
:~ the use of an S-sepharose column for thi~ purpose, tho~e skilled in
;: ~ the~art can readily determine other cation exchange columns that
c~uld ~e used fo~ this:purpoæe. This:S~sepharose chromato~raphy
procedure of Example ~ yi~lded two major peaks. The peak
13
WO93/19084 PCT/US93/02457
21~820
corresponding to correctly folded protein was identified by
comparison to a commercial standard. The ED50 f this peak (Peak B)
was 7-8 ng/ml when measured by the UMRlO6 cell assay described in
Example 6. The E~so of the other peak (Peak A) was 30 - 40 ng/ml
when measured by the same assay. Peak A was determined to be
incorrectly folded protein. The final step for separating
correctly folded protein from incorrectly folded protein can be
reverse phase HPLC, whi~h is particularly useful ~or small scale
e~periments. Alternati~ely, a hydrophobic interacti~n column can
be used in the final step. The hydrophobic interaction column
separates the proteins base~ on their hydr~phobicity. Any
hydrophobic interaction chromatography column, such as, for
Pxample, Toyopearl Butyl-650S, can be used for ~his purpose. The
olated, correctly folded protein can then be analyzed using
lS re~erse phase HPLC, if desired.
Although IGF-I ha~ing an Nterminal me~hionione (met-IGF~I~
: exhibi~s biological activity comparable to IGF-I as demon~trated in
; the bioassays described below (Example 6), it may be-desirable to
cleave: ~he N-terminal methionine from the IGF-I. Sin~e ~aturally
: 20 oceurring IGF-I h~s no N-terminal methionine, met-IGF-I may gi~e
,
rise to an immune response in so~e circu~stances. For that reason,
:the present invention also provides a method for converting met-
: :I~F-I to IGF-I. This is accomplished by reacting the met-IG~-I
: :
~ wi~h an aminopeptidase, for example, an aminop~ptida~e from
: 2~ a5~a~a~ proteolY~i~a, to cleave the N-terminal methionine. The
reaction is stopped by lowering the pH of the solu~ion to ~elow pH
14
WO93/19084 PCT/US93/02457
2 1 U9 82 0
5. This can be acro~plished by the addition of any of several
acids. Suitable acids for this purpose include, but are not
limit~d to, trifluroacetic acid (TFA), acetic a~id, and
hydrochloric acid. This reaction can also be stopped by lowering
the tempera~ure to below 4C.
The present invention further provide5 a pharmaceutica~
composition containing IGF-I in a pharmaceutically acceptable
: carrier. One carxier is physiological saline solution, but it is
contemplated that other pharmaceutically acceptable carriers may
also be used. In one embodiment it is envisioned that the carrier
and the IGF-I constitute a physiologically-co~patible, slow-release
formulation. The primary solvent in suc~. a carrier may be either
~queous or non-aqueous in nature. In addition, the carrier may
contain other pharmacologically-acceptable excipaents for modifying
or main~aining the pH, os~Qlarity, ~iscosity, clarity, color,
.
: : starility, stability, rate o~ dissolutionl or odor o~ the
:ormul~ation. Similarly, the carrier may contain still other
pharmacologically-acceptable excipie~ts ~or modifying or
maintaining~ the ~stability, rate of dissolution, release/ or
20~ absorption of~th : ~GF-I> Such excipients axe those substances
usually ~and customarily employed to formulate dosages for
administration in either unit dose or ~ulti-dose form.
Once the pharmaceutical composition has been formulated, it
may be ~ored in sterile ~ial~ as a solution, su~pension, gel,
: : 25 emulsion, solid, or dehydrated or ~yophilized powdex. Such
formulations may be stored either in a ready to use form or
,,,~ . . ,....-.-
W093/19084 ~ PCT/US93/02457
2~9820
re~uiring reconstitution immediately prior to administration~ Thestorage of such formulations ~an ~e at temperatures at least as low
as 4C and preferably at -70C. Formulations containing IGF-I can
also be stored and admini~tered at or near physiological pH. It is
S presently believed that storage and administration in a formulation
at a high pH (i.e. greater than g) or at a low pH ~i.e. less than
2) is undesirable~
The pharmaceutical coLposition of the present invention an be
used to treat a patient having or potentially having an IGF
associated condition. Some of these conditions may include, for
example, dwarfism, diabetes; cachexia, peripheral neuropathy, renal
~: disease, impaired wound healing, amyotrophic lateral sclerosis
ALS), stroke, periodontal disease and osteoporosis. The
pharmaceutical composition of the present invention can also be
15 :used to treat a ~condition in which administration of IGF to a
normal patient has a~desired effect; for example, using IGF-I to
enhance growth of a~patient of normal stature.
The manner of administ~ring the formulations containing IGF-I
can be: via ~n: intraarticular, subcutanec)us, intramuscular or
20~: intra~renous injection ~ or~ infusion, suppositories, enema, irlhaled
aerosol, or oral or~topical routes. To achieve and maintain the
desired effective ~ dose~ of IGF-I, repeated subcutane~us or
intramuscular injections may be administered. Both of these
me~hods:are intended to~create a presele~ted concentration ra~ge of
IGF~ in the patient's blood :stream.~ It is believed that the
maintenance of circulating concentrations of I~F-I of less than
16
WO93J19084 PCT/US93/02457
2103~%0
0.0l ng per ml of plasma may not be effective, while the prolonged
maintenance of circulating levels in excess of l00 ~g per ml may be
undesirable. The fre~uen~y of dosing will depend on
pharmacokinetic parameters of ~he IGF-I in the formulation used.
5The following examples are intended to illustrate but not
limit the present invention.
~I,~ 1
IGF-I
A. Construction of the IGF-I qene
10The IGF-I gene was assembled in two stages. Initially, the
DNA seguence enc~ding ~he mat~re IGF-I protein was joined to DNA
: seguences encoding the secretory leader sequence of the E. coli
OmpA protein (ompAL). This gene fusion was constructed in order to
determine whether IGF-I a~uld be ef~iciently seGreted ~rom E. coli.
~ 15 A ~econd ~onstruct, in which IGF-I is expressed as an intracellular
:~ protein, was ~reated by deleting DNA sequences en~oding ~he OmpA
leader~se~uence and repla¢ing ~ em with appropriate DNA sequ~e~ces
for~intracel1ular expr~ssion of IGF-I.
Construction of the Om~A,-IGF-I qene fusion
;: 20 ~ The four synthetic~oligonucleotides labeled OmpAlU (SEQ ID
~: :
NO:l~, OmpA2U (SEQ ID NO:2), OmpAlL (SEQ ID NO:3) and OmpA2L (SEQ
ID NO:4~, were annealed pairwise (lU ~ lL ~nd 2U ~ 2L3 and ~he
~: pairs ligated together. All four o~ t~ese oligonucleotides were
: ~ synthesized using ~NA synthesizers purchased from Applied
::
Biosystems ~Models 39~ and 380A). The ligation mixture was then
: d.gested with the restriction enzyme HaeIII. The resulting
17
WO93/19~84 PCT/US93/02457
~l~)s~2n
BamHI/HaeIII restriction fragment coding for a translational start
signal and the first 21 ~mino acids of the ompA signal se~uence was
purified. This DNA fragment was mixed with ~ I + PstI-digested
pUC18 DNA (Boehringer Mannhein Biochemicals, Indianapolis, IN) and
the two synthetic oligonucleotides [IGF-I (1 14) U + ~ tSEQ I~
N0:5 and SEQ I~ No:6) were ligated together. The ligation mixture
was transformed into E. g~li strain JM109 tNew England Biolabs,
Beverly, M~) and indi~idual colonies isolated. These plasmids
: (OmpALIGF-IpUC18) have a translational start signal followed by DNA
saquenc~s encoding the OmpA signal sequence and the first 14 amino
acids of I~F-I.
An N13 phage containing DNA ~equences encoding ami~o acids 15
t~rough 70 of IGF-I was created by ligating together the two
; complementary pairs of oligonucleotides (IGFlU ~ lL and IGF2~ ~ 2L)
~15~ SEQ~ ID;~NO:7 and SEQ ID NO:8) and cloning the DN~ ~ragment into
HindIII-aigested Iql3 mpl9 DNA (New England Biola~s, ~evl2rly,
MA).~ Double-stranded~DNA wa~ purified from a phage clone and the
III fragmeDt encoding amino acids 15-70 of the IGF-I
.
pr~tein~:were isolated.~m is DNA fragment was ligated together with
20~: PstI~ +~indIII-digested plasmid OmpALI~F-IpUC18 DNA and used to
transform E. coli;strain~JM107 (GIBC0 BRL,~Gaithersburg, ~D). The
, B~mEI/~ III fragment containing the IGFeI gene fused to the OmpAL
se~uence wa~ isolated~and cloned into the ~3~HI ~ HindIII generated
site~of plasmid pT3XI-2. ~he completed plasmid containing the
: 25 OmpAL-IGF-I gene ~usion is called pT3XI-2 ~lOc(TC3)ompALIGF-I.
`~ :
c. con;struction of the MethionYl-IGF-I sYn~
18
WOg3J19084 PCT/US93/0~457
2 1 ~J () ~ '~ O
The Ba~HI/HindIII fragment containing the OmpAL-IGF-I gene
described abo~e was purified from plasmid pT3XI-2~10c(TC3)0mpALIGF-I
and digested with HinfI. The approximate 200 bp Hin~ a~III DNA
fragment was mixed with the annealed, comple~entary synthetic
oligonucleotides (Me~IGFlU + lL) ~SEQ ID N0:9 and SEQ ID N0:10) and
liga~ed with BamHI + ~n~III-digested plasmid pT3XI2 DNA, and used
to transform E. coli JM107. The ~ompleted plasmid construct is
called ~10c(TC3)IGF-IpT3XI-2 and contains an extra alanine residue
at the beginning of the IGF-I sequence. The BamHI/~a~III fragment
containi~g the mutant IGF-I gene was isolated and ligated into the
BamHI + ~indIII generated site of plasmid pTST (described in
y~, Vol. 343, No. 6256:, pp. 341-346). The ligation mixture was
used to trans~orm ~ col B~21/DE3 (US Patent 4,952,496) and
individual co~oni~s isolated. Thi~ construct was named
lS:: ~lO~TC3)IGF-IpT5T.
The extra aIanine codon was removed by ~ vitro mutagenesis.
Plasmid~:~lOc(T~3)IGF-IpT3~XI-2 was digest~d with ~am~ ia~III and
t~e~ -200 bp~ DNA fragment; ~ontai~ing the ~mutant IGF-I gene was
: puri~ied:~and ~loned~:into:the Bam~I an~ ites of plasmid Ml3
20~ mpl9.~ ~B Yi~ mutagenesis was performed; usi~g a Muta Gene kit
Bio-Rad~Laboratories,~Richmond, ~A). Th~ procedure foll~wed was
described in the instruction that accompany the kit. Uracil-
; containing :singIe-stranded~ template DNA was prepared ~ollowing
propagation of ~he~ phage in E. coIi:strain CJ236 (supplied with
Muta-Gene Kik, Bio-Rad Laboratories, Richmond, CA). The
oligonucleotide used for mutagenesis had th~ sequence: 5'
: :
~ 19
W093/19~84 PCT/US93~02457
2109820
GATGATTAAATGGGTCCGGAGACT - 3' (SEQ ID N0:11). The mutagenesis
reaction product w~s transformed into E. coli strain JM109 and
individual plaques picked. Double~stranded replicative form phage
DNA was is~lated, digested with BamHI ~ HindIII and the -200 bp
fragment containing the IGF-I gene purified. The purified DNA was
cloned into the Bam~I + HindIII generated site of plasmid pT5T and
used to transform E. ~1~ strain BL21/DE3. One bacterial ~olony
with the correct plasmid was named ~lO(TC3)mutIGF-IpT5T. Several
isolates were seguenced, and all were cnrrec~
D. Expression of Met-IGF-I in bacteria
For small-scale experiments, an overnight culture of E. coli
strain containing ~lO(TC3)mutIG~-IpT5~ was diluted 1:100 into 800
ml of ~uria ~roth (10 g/liter tryptone, 5 g/liter yeast extract and
10 g/liter NaCl, p~ 7.5) medium containing 15 ~g/ml tetracycline
and grown at 37 until ~ e optical density at 600 nm was 0.7-0~9.
IPTG (isopropyl-~-D-thioga~actopyranoside, Si~ma Chemical Company,
: St. Louis, ~0~ was added to a final çoncentration of 1 mM and the
culture grown for an.additional 2.5-3.~ hours at 37C~ At the end
o~ the induc~ion: pe~iod, the cells we~e harvested by
¢entri~ugation. The cell pellet was washed once with ice-cold
buffer A (50 mM Tris-HCl pH 7.5/ 25 mM NaCl~l mM DTT) and stored at
-70C or resuspended in ~uffer A and used immediately.
For large-~cale e~periments, E. co~i strai~ ~lO(TC3)mu~IGF-
IpT5T was growr in a 10 1 fermenter at 37C in complex m~dia (40
g/l N~ amîne HD, 2 g/l KX2P04, 1 g~l MgS04 7~2~ 1 g/l Na2S04, 1 g/l
Na3 citrate 2H20, 50 g/l glycerol, 0.1 ml/l Macol l9::GE60, ~ ml/l
WO93/190g4 21 ~ 9 8 2 ~ PCTJUS93/02457
trace minerals, 20 mg/l thiamine HCl, and 15 mg/l tetracycline HCl,
pH 7). When the optical density at 600 nm of the culture reached
approximately 10, IPTG was added to a final concentration of 0.1
m~. Bacteria were grown for an addi~ional 2-8 hours, harvested by
centrifugation and the cell pellet stored at o70C until use.
E~ANP~ 2
Purification of Net-IGF-I
E. oli cells were suspended in Buffer A (50 mM Tris, pH 7.5,
20 mM NaCl and 1 mM DTT~, and were disrupted at 1800 psi using a
~rench pressure cell. The suspension was ~entrifuged at 20,0~0 x
g for 30 minutes, and aliquots of the pellet and the supernatant
were analyzed by SDS-PAGE. A major band corre~pondiny to met-IGF-I
~ was~present in the pellet, but not the supernatant. The pellet was
; ~ ~ resusp~nded in ~u~fer A (40 ml/10 g cell paste), and re-centrifuged
at ~OpO00 x g for 30 min. This wash procedure was repeated 2
ti~esO The final pellet containing met-IGF-I was resuspended in 6
M guanidine, 50 mM Tris, ~H 7~5, 6 mN DTT ~25 ml/10 g cell paste)
using a ground glass homogenizer, and the suspension was incubated
~: ~ at room temperature for 15 minutes. The undissolved protein was
removed by centrifugation at 20,000 x g for 30 minute~. SDS-PAGE
::: analysis of the pellet ~nd super~atant showed that met-IGF~I was
prese~t in the upernatant only.
E~aNp~E 3
Refoldinq of ~et-IGF-I
The reduced met-IGFI from Example 2 was subjected to a
three-step refolding protocol.
21
WO 93/19084 P~/US93/0~457
2~a9~20
1) The oxidizing agent, oxidized glutathione (GSSG~, was
added to the supernatant from Example 2 to a final concentration of
2 5 ~I, and incubated at room temperature f or 15 minutes .
2) The solution was th~n diluted 10 fold gradually with 50
S ~ Tris, pH 9 . 7 to a f inal ~::oncentration of 100 - 2û0 ,ug/ml .
Cys~eine was added to a f inal concentration of 5 m~ to aid in
disul f ide exchange .
3) Tlhe solution from step (2) was incubated ~verniyht at 4~C
to allow complPtion of dis3llfide exchange, and then centrifuged at
lO 20,000 x g for 15 minutes. SDS-PAGE analysis of the pellet and the
supernatant s~owed that the supernatant was composed of relatively
homogalleous met-IGF-I.
Aliquots (50 ~Ll) of 'che supernatant were diluted to 1 ml with
0~05% TFAo inj~cted onto a reverse phase c:olu~n ~ 4, 1 x 250 mm,
l S Synt::hrom), and eluted with Buf f er B ( 8 û~6 acetonitri}~ in water ,
~; ~ 0.:04~:~% TFA) using a linear gradient (increase of 1% Buffer B/min~
at a ~low ra~e of 0.1 ml/minute.
~: :TWO major peaks were resolved: Peak I at 56.5 .minutes, and
; Peak II at 58.2 minutes. ~ ~In addition, a minor peak was present at
:20 60 minutes, and a broad peak at 75 79 minute~ containing impropPrly
refolded met-IGF-I speci~s. Based on the integration of the HPLC
chxomatogram, Peak I and Peak II repr~sented approximately 25~6 and
30% of the c:rude met-IGF-I pro ein loaded onto the reverse- phase
columr~ spectively. N-terminal ~;equenae analysis OI Peak I and
25 Peak II gave the sequence ~!letGlyProGluTfflI,ell. O . (SEQ ~D N0: 12),
whic:h matches the N-termlnal amino ac:id ~equ~nce of human IG~I
22
WO93~19084 PCT/US93/~2457
2l~ss2a
except for the extra methionine residue at the N-terminus.
Recombinant human met-IGF-I ~purchased from Bachem, Torrance, CA)
eluted ~t a retention time identical to Peak II. Therefore, Peak
II represents correctly rafolded met-IGF-I, as evidenc4.d by
~5 retention time identical to ~he purchased ~tandard as well as
biological activity identical to the purchased 5~andard. IGFoI
which has not been correctly refolded exhibits reduced or no
biolog~cal activity. Correctly refolded IGF-I is e~ide~ced by ED50
of 10 ng/ml or less when tested on the UMR106 cell line. This
assay is described in Example 6.
E~A~P~B 4
Isolation_ f ~orrectlY Refolded IGF-I
~: The following is a description of the preparation of IGF-I from
: 305 g of cell paste. The upernatant from th~ refolding procedure
~6700 ml~ was aoncentrated lO-fold and dialyzed to ~mpletion
against 20 mM ~EPES, p~ 7.5. The dialyzed sample was centri~uged
20,000 X g for 15 minutes to remove precipitat~d proteins, passed
, f~
through a 0.2 ~ filter (Corning, Corning, NY) and loaded ~nto an
S-Sepharo~e column tS.0 X 40 ~m, Pharmacia LKB, Piscataway, NJ)
previously equilibrated with the same buffer, at a flow rate of 40
: mlfminute.: The bound IGF-I was eluted with a 5000 ml linear
; gxadient to 0.5 M NaCl at a flow rate of 40 ml/minute. 25 ml
fractions were collecte~. Two symmetrical peaks were xesolved:
:~ Peak A eluting at 0.12 M NaCl, ~nd Peak B eluting at a. 1~ M NaCl.
: 25 SDS-PA~E analysis of aliguo~s of Peaks A and B showed th~t they
contained relatively homogeneous IGF-I (> 90% homogeneous);
~: 23
WO93/19Q84 PCT/US93/02457
~1 09~0
however, several high molecular weight E. coli proteins were still
present. The S-Sepharose fractions corresponding to Peaks A and B
were pooled separately. HPLC analysis (RP 4, 1 X 250 mm~ of the S~
Sepharose pools showed that Pools A and B were composed of major
peaks elutiny at 56.5 minutes and 58.2 minutes, respectively, as
well as se~eral minor peaks. The major RP--4 pe~k of the S-
Sepharose pool B eluted with the same retention time~ as
commercially purchased recombinant human met-IGF-I (Bachem,
Torrance CA).
The S-Sepharose pool B was made to 2 M NaClt 20 mN HEPES, pH
7.5, and loaded at a fl~w rate of 30 ml/mi~ute onto a Toyopearl
Butyl-6505 (Supelco, Bellefonte, PA) hydrophobic interaction column
previously eguilibr~ted with 20 m~ ~EPES, pH 7.5, 2N NaCl. The
bound protein was eluted with:a 1250 ml linear gradient to 20 m~
H~PES,:pH 7.5, 20 ~ ethanol at a flow rate of 40 ml/minute. 25 ml
ractions were collected. A:major peak eluted at approximately
17.5 % ethanol, as~well: as a minor p~ak at 13-15 % ethanol.
, f ~
Aliquots (50~ul) o~ the frac:tions were diluted to 200~1 with Buffer
C ~ (0.05% ~rFA) ~ injected: onto a reverse phase ::olumn (RP-4, 1 x
20 ~250mm, ~Sync~om~ ,- and eluted with 80% aoetorlitrile, 0.042% TFA
Buffer D) using a linear gradient (in~rease of 1~ Buffer Dlminute~
at a flow rate of 0 .1 mI/minute~. The major pealc ~lu~ing at 17 . 5%
ethanol contained homogeneous, correctly ref olded IG~-I . This
major peak was detenmlned to contain correctly xefolded IGF-I
~25~ because it eluted with ~he~ same retention time as commercial}y
purchased natural ~human met-IGF-I. (Bachem, Torrance, CA)
24
:
W093/l9084 P~T/US~3/02457
21 0~20
Fractions containing this peak were pooled, c~ncentrated to 2
mg/ml, dialyzed against 100 mM HEPES, 44 mM sodium phosphate/ pH
6.0, and stored at -70~C.
2~A~P~ 5
conversion of Met-IGF-I to IGF-I
In ordPr to conver~ recombinant met-IGF-I to natural human
~GF-I, an aminopeptidase, isolated fr~m Aeromonas proteolYtica
using a modification of a previously described method (Lorandl L~,
1976, Meth. Enzymol. 45:530-543), incorporated herein by reference,
was used to remove the N terminal methionine. Recombinant met-IGF-
I was incubated in the presenae of the purified aminopeptidase in
a 100 ~1 reaction~mixture containing 120 ~g met-IGF-I, 20 mM
: ~ricine, pH 8.0, and 1 ~g aminopeptidase fox 30 minutes at 2~C.
:
: ~ ~he reaction was stopped by the addition o~ 1 ml 0005% TFA in
water. Aliquots of ~he ~a~pl~s were analyzed on a rever~e phase
~; column, and the prDtein peak~ collected and eub; ected to ~equenc~
analysis. met-IGF-I eluted at 5802 minutes; whereas, the material
:
reaeted wi~h the ami~opeptidase comigrated with n~tural human IGF-I
: (purchased from Bachem, ~orrence, ~A) at 56 minu~es. The following
is a s~mmary of the:~pmoles of each residue recov~red at each
~: sequence cycle, no~malized for lOO:pmoles of starting material:
~ .
:~
WO 93/1908~ PCI/US93/1)2457
21G9~20
TAB~13 1
Met-IGF-I + 1 ~ Amino~eptidase
~moles Recovered _
Cycle Net Gly Pro Glu Thr
1 2.32 ~6.8 2.4 1.78 1.4~i
2 0.00 23.3 105.6 3.1 0.8
3 0 . 18 13 . 823 . 4 128 . 6 1 . 4
4 0.00 9,.3 4.6 2~.1 51.5
Sequence obtained: Gly, Pro, Glu, Thr; approximately 296 of the
10 ~molecules did not ~ave N- erminal Met cleaved by amillopeptidase.
TA~LE 2
Met-IGF-I ~o ~m op~ti~lase
~oles Reco~rered_ e
~: Cycle Met Gly Pro Glu Thr
15~ 8 ~ 16 11 ~ 62 ~ 15 L o 62 0 ~ ~
2 : ~ 4.8 108.~ 2.31 4.1 2.0
3 ~ 0 . 5 2~5 . 8 ~8û . 72 7 . 6 ~, 3
:4: ; :: ~ ~ 0.0 ~.:15.~9~25.2: 71.6 0.. 9
Segu~nce~obtained: ~et:, Gly, Pro, ~lu
20~These~results ~show::that approxîmately g8% of met-IG~-I was
: conver~ed~to~na~ura;l~ IGF-I by the~aminop tida~e.
y ~ 6
: 2~ : ~ The~ in ~ :biological activities~of~ purified recombinant
met-IGF-I were tested in cell proliferation ~assay~; using mouse 3T3
26
~ '
W~3/19084 PCT/US93/0~457
210~820
fibro~lasts, and on rat osteosarcoma UMK106 cells. The cell
proliferation assay used is set forth below.
1. Effect~of Met IGF-I on Mouse 3~3 Fi~ro~lasts
A crystal violet dye as~ay was used to measure cell
proliferation. Assays were performed in 96 well gelatin-coated
plates. Balb/c 3T3 fibroblasts (available from Americ~n Type
Culture Collection, Rockville, MD~ Accession $CCL 163) were plated
at 25,000 cells/well in 200 ~l of serum-free DMEM (~ulbecco's
Modification of Eagle's Medium, Mediatech, Herndon, ~A) containing
0.03 M glycerol and 0-1,000 ng/ml met-IGF-I. Cells were incubated
for 72 houxs at ~7C. At this time, the media was replaced with
~ ~ 150 ~l of 0.2% crystal ~iolet, 10% formaldehyde, 10 mM potassium
:~ phosphate pH 7Ø After incubation at room temperature for ~0
~-; minutes, the wells were washed 3 times with PBS, and the cell-bouna
15~ dye was released by in~uba~ion with ~00 ~llwell of 50% e~hanol/O~M
s~di~ citrate, pH 4~2. Absorbance at 570 nm was read the next
day. The results show ~hat recombinant met-IGF-I stimulates
~prolieration o~ 3T3~fi~roblast cells in a dose-dependent ma~ner.
Naxi~al pr~liferation oc~urred at met-IGF~I oncentration of about
20 ~30 ng/ml, The ED50 was approximately 2 ngtml.
The mitogen:ic~(grow*h stimulating) activity of the refolded
~ met-IGF-I was measured;~by the amo~nt~of ~-thymidine incorporated
: ; into rat osteosarcoma cells when the met-IGF-I was incubated with
these~ ce~ls under serum fre~ conditions. The rat osteosarcoma
:
: cells (the U~R106 cell line; American Type Culture Collection,
;~ 27
W093~190~ PCT~US93~02457
2109820
Accession No C~L-1661, Rockville, Maryland) were plated at 5-6 X 104
cells in 0.5 ml of ~am's F12 (Cat. #10-080-L~, Mediatech)
containing 7% fetal bovine serum, lO0 U/ml penicillin and 100 ~g/ml
streptomycin a~d 2 mM L-glutamine per well in 48-well tissue
culture plates (Costar, Cambridge, ~A). A*ter incubating for 72
hours at 3~C when the cell~ became confluent, the cells were washed
twice with PBS and pre~incubated in serum-free Ham's F12 medium
containing 100 U/ml penicillin and 100 ~glml streptomycin and 2 mM
L-glutamine for 24 hours. After the pre-incubation, 0.5 ml of F12
serum-~ree medium containing serial dilutions (1.0 - 1,000 ng/ml)
of met-IGF-I, were incubated for an additional 20-24 hours. Each
well was ~hen pulse labeled~with 0.5 uCi of 3H-thymidine (t::at. ~NET-
027Z, NEN Research Products, Du Pont Co., Boston, ~A~ for 4 hours,
~ ,
then washed with cold PBS three times and inc:orporated 3H-thymidine
15~: was precipi~ate~ wi~h cold 7% trichloroacetic a~id (Cat. #0414-01,
J.~:. Baker Inc., Phillipsburg, NJ). 3H-thymidine was ~uantitated by
liquid scintillation counting. All ass~ys were performed in
~ ~ ~ , f ~
triplicate. The results show that recombinant met-IGF-I stimulates
proli~eration of rat:U~R106 cells in a dose dependent m~n~er. The
ED~of re~olded met-IGF-I was 2 - 20 ny/ml.
B~ In viyo_activities of recom~inant Me~t-IGF-I
IGF-I possesses both growth-promoting and metabolic properties
~: similar to those of insulin tL. Rossetti. Diabetes 40:444-4489
:~ 1991). We have dem~nstrated in r~ts that met-IGF I posses~es both
: 25 growth-promoting and meta~olic e~fects and is therefore bioactive.
1. Growth of _h~ hysectomized _rats _i~L_~romoted bY the
:
2 8
WOg3/19084 PCT~US93/~2~57
21 Q9820
_ub~utaneous iniectlon of Met-IGF-I
Hypophysectomized rats are deficient in both growth hormone
(G~) and IGF-I. GH i~ believed to stimulate growth indirectly by
inducing synthesis of I~F-I which then acts directly on tissues to
regulate growth. Although ~he hypophysectomized rat i~ stunte~,
~rowth can be stimulated by the administration of either IGF-I or
GH. Subcutaneous i~fusion of IGF-I purified from natural ~ources
~timulates an increase in body weight and tibial epiphyseal width
(Schoenle, E. Nature 296:252-253, 1982).
Male Sprague Dawley rats which were surgically
hypophysectomized at 120-130 grams of body weight were obtained
from a commercial sourc2 (Charles River, Wilmin~ton, M~), The body
weights of these rats were monitor~d for three w~eks before the
~ ~ beginning of ~he experiment in order to Yerify c~mpleteness of the
:~ 15 hypop~ys~ctomy. Rats gaining mor~ ~ an 2 grams per we~k were
: excluded from the study.
,
The rats were di~ide~ into ~wo groups containing f~ur rats in
each. One group was inject~d:subcutaneou~Iy at the nape of the
~eck with recombinant met-IG~-I produced as set forth above (80
~g/r~t/in~ection~ twice a day at 9:00 a.m. and 8:00 p.m. for nine
: :consecutive days. The other group of four rats was injected with
an equal volume of vehicle (O.2 ml). Body weights were measured
daily at the time of the morning injection. Twelve hours after the
last:iniection, the rats;were killed, and the right and left tibias
w re removed. The formalin-fixed tibias were split at the prvximal
end in a sagittal plane and stai~ed with sil~er nitxate (Greenspan,
WO93/19084 PCT/US93/02457
~10~8~0
F.S., Endocrinology 45:455-463, 194g). ~he calcified tissue was
stain d dark brown and the proliferating zone of cartilage appeared
as a clearly defined white band. The cartilaginous epiphyseal
plate was measured with a stereomicroscope with a calîbrated
micrometer eyepiece. Approximately ten individual readings were
made acro~s the width of ~he epiphysis. The mean of the combined
~eadings from the right and left tibias was ~alculated for each
rat.
A significant change in body weight occurred o~er a nine day
period in four met-IGF-I;;injected rats, but not in four Yehicle-
injected rats. ~he ~irst injections were given on day zero. The
change in body weigh~ was calculated as the differe~ce between the
: body weigh~ at ach su~sequent day of injection and that on day
~:: ; zero. The increase in body weight in the met-IGF-I-treated rats
was significantly greater tha~ the ~hange in weight in the vehicle-
treated rats on days 3-9. Overall during the nine day period, the
met-IGF-I-treated ~ats gained an average o~ 8.3 + 0.5 grams of body
, f ~
: :weight per rat; whereas, the ~ody w~ights of ~he vehicle-treat2d
rats remained stabilized~ith a chanye of only 1.0 ~ 1.2 grams on
average per rat. ~he difference ~etween ~he two group is
tatistically significant (p<0.05 using an unpaired t test).
he width of the epiphyseal cartilage of the met-IGF-I treated
~: rat~ w~s greater than :~hat of the ~ehicl~ treated rats. The
epiphyseal widths were 0.20 ~ 0.01 mm in ~et-IGF-I-treated rats and
0.14 ~ 0.01 in vehicle-treated ratC. The difference between the
two groups is statis~icaIly significant (p<0.00~ using an unpaired
WO93t19084 PCT/US93/024~7
210~
t ~st).
2. HvPoqlycemia is induced by the intravenous in~ection Qf
recombinant Met-IGF-I
The intravenous, bolus injection of IGF-I purified from
natural sources provokes a rapid fall in blood sugar (Zapf, ~.J.,
Clin. Invest. 77:1768-1775, 1986). In order to determine the
effe~t of intravenously injected met-I~F-I produced in accordance
with the present invention on blood glucose levels,
hypophysectomized ra~s weighing between 129 and 140 grams were
lV tranquilized ~y the subcutaneous administration of diazepam (11
mg/kg). At time zero, blood samples were obtained from the tail
~: vein ~or dete~mination of the pre-injecti~n blood glucose lPvels.
: Intravenous injections of either me~-XGF-I at 5, 12.5, and 17.5
g/rat, or vehicle, were administered through the tail ~ein. There
were three rats per dosage group of met-IGF-I. Four rats were
~: injected with vehicle. Blood samples were obtained at 15 minute
:
~: intervals during:the 120 minute period ~hereafter. One drop of
blood (about 50 ~l).was collected by ~equential nick$ of the rat's
tail at a single site ~;uperficial to the lateral tail vein. Blood
~:~ 20 glucose levels were measured immediat~ly on an Ames glucometer
Miles Inc ., Elkhart , IN) .
Blood glucose levels were not signif ica~tly lowered in rats
,
injected with either vehicle or 5 ~g met-IGF-I per rat. In fact,
these rats were probably undergoing a stress-induced hyperglycemia
due to experimental manipulations. However, rats injected with
12.5 and 17.5 ~g met IGF~I experienced a marked ~rop in blood
~: 31
WO93/19084 PCT~US93/02457
2109~2~
glucose levels. Glucose values decreased by 40% of the time zero
values.
Although this invention has been described with respect to
specific embodiments, it is not intended to be limited thereto and
modifications made by those ~killed in the art are considered to
fall within the spirit and scope o~ the instant invention~
32
WO 93/1908~ PCr/lJS93/02457
21 0~820
( 1 ) t;hrNERAL INFORMATION
( i ) APPLICANT: SYNERGEN, INC .
(ii) TITLE: Refolding of Insulin-Like Growth Factor I
( iii ) ~VMBER OF SEQUE~CES: 12
( iv) CORRESPONDEMCE ADDRESS:
(A) ADDRESSEE: Synexgen, Inc.
(B~ STR~3ET: 1885 33rd Street
( C) CITY: Boulder
(D) STATE: Colorado
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(F) ZIP: 80301
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storage
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~vii) PRIOR APPLICATION DATA:
(A) APPLI~TION NI~ER: 07/858 ,161
(B) FILING D~TE: 24 ~CH 1992
(~iii) ATTORNEYJAGENT INFOR~TION:
~: 30 : (A3 NAME: Ruth }:ure
~: (B~ REGISTRATION N~ 35, 31
(C) REFERENCE/DOC~ET N~: S5rNE--240
( ix~ TEI ECONMUNICATION INFORMP.TION: .--
: (A) TELEP~ONE: (303) 441--5508
.: (B) TELEFAX: (303) 541--1370
( 2 ) INFORMATION FOR SEQ ID NO: l:
~i~ SEQUEN~E ~aRACTE~RISTXCS:
(A~) LENGT~I: 51 ba~es
t ~ TYPE: nucleic acid
( C) STR~EDNESS: ~ingle
(D) TOPOLOGY: linear
(xi~ SEQUENCE I)ESCRIPTION: SEQ ID NO:1:
GAT CCG ~TC GTG GA :; GAT TAA ATG AAA, AAG ACA GCT ~TC GC~: A~C ~ 8
GCA 51
.
33
WO 93/19084 PC~/US93/02~57
210g820
(3) INFORMATION FOR SEQ ID NO:2:
( i ~ SEQUENCE C~RP.CTERISTICS:
(P.) LENGTH: 51 ~ases
~B) TYPE: nucleic acid
( C~ STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi~ SEQU:ENCE I)ESCRIPTION: SEQ ID NO: 2:
GTG GCA CTG t;CT ~;GT TTC GCT ACC GTA GCG CA~; GCC GCT CCG TÇ;G CAG 48
TGC 51
: 15
( 4 ) INFO~$ATION F~ SEQ ID NO: 3:
(i) SEQIJENCE ~CTERISTICS:
(A) LENGTH: 56 ba33es
(B) TYP~: nucleic acid
(C) Sl~NDEDNESS: ~ingle
TOPOI~GY: linear
~: ~ (xi) SEQUENCI: DESCRIPTION: SEQ ID NO: 3 :
CAG $GC CAC T~i;C G~q? CGC GAT AGC: TGT CTT TTT ~AT TTA ATC CTC 48
QC GAT CG 5 6
3 0 ( 5 ) I~FOR~ION FOR SEQ ID NO: 4:
( i ) SEQ~ENClS CH~RACTERISTIC:S:
(A) ~ LENGTH: 42 bases
: ~ : (B) TYPE:~ nus~leic acid ._-~
35 ~ (C) :i ST~DNESS: sin~le
(I)) TOPOLOGY: linear
(xi) SEQU:~:NCE DESRIPTION: 5EQ ID NO: 4:
40: ~GCA CTG CCA CGG AGC GGC~ CTG CGC TAC GGT AGC ~A ACt:: AGC: 42
6 ) IMFORMATION FOR Sl :~ ID NO: 5:
( i3 SEQUENCE CHI~ACTERISTICS:
4 5 (A) L~GTH: 4 6 base~
B) TYPE: nuc:leic~ acid
( C) STF~DXDNESS: single
(D) ~OPC)LO~ linear:
(~i) SEQUENE DESCRIPTION: SEQ ID NO:5:
CC GGT CCG GAG ACT CTG TGC GGC GCA G~A CTG GTT GAC GCT CTG CA 4 6
34
.
WO 93/l9084 2 1 ~ ~ 8 2 1~ PCr/US93/02457
(7 ) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENOE CHARI~CTER~STICS:
(A) LENGTH: 42 bas~s
(B) TYP~: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOI.OGY: linear
(xi~ SEQUENCE DESCRIPTION: SEQ ID NO:6:
1~
GAG CGT CAA CCA G~T CTG CGC ACA G~G TCT CCG GA~ CGG 4 2
( 8 ) INFORMATION FOR SEQ ID NO: 7:
~i) SEQUENCE ~ACTE~ISTICS:
(A) LENG~I: 85 bas~s
(B~ TYPE: nucleic acid
(C) S~DEDNESS: single
(D~ TOPOLt)GY: linear
~xi) SEQUENCE DESCRIPTION: 5EQ :l:D NO: 7:
~:
GTTC GTA TGC GGC GAC CGT GGC TTC TAC TTC AAC AAA CCG ACT GGC Tl,C 49
~: 2S
: GGT: T~'C AGC TCT CGT GCA CCG CAG ACT GGT ATC 85
~: ( 9 ) IlRFORMA~ION FVR SEQ ID NO: 8:
: :~30 ~i) SEQUl~CE C:~ACTERISTICS:
(A) LENGTH: 98 bases
:: (B~ TYPE: Tlucleic acid
( C) S~DNE55: single
(D3 TOPOLOGY: lin~ar
~xi~ 5EQUENCE DESCRIPTION: SEQ ID NOo E~
T~C GTC AAC GAT ~C ~:GT CTG C:GG TGC ACG AGA GCT GGA ACC GT~ 4 8
~: GC AGT CGG TTT GTT GAA GTP. G~ GCC ACG GTC GCC t;CA TP.C ~A~ CTG 96
~: 40
C~ 9 8
~, (10) INFORMATION FOR SES~ ID NO:9:
4 5 ( i ) SE0UENCE CH~RACTERISTICS:
(A) LENGTH: 39 bases
~B) ; TYPE: nucleic acid
( C) SltRANDEDNESS: single
(D) TOPOLOGY: linear
(xi~ SEQUE~CE DESCRIPTIC)N: S:13Q TD NO. 9:
WO 93/19084 PCr/US93/02457
21D~20
GATCCGATCG TGGAGGATGA TTAAATGGCC GGTCCGGAG 3 9
~11) INFORMATION FOR SEQ ID NO:10:
S ( 1 ) SEQUENCE C~ACT33RISTICS:
~A) LENGTH: 38 bases
( 13 ) TYPE: nucl~ic aciâ
(C) STRANDEDNESS: single
( D ) TO}?OLOGY: linear
(xi~ SEQtJENCE DESCRIPTION: S~Q ID Nû: 10:
AGTCTCCG~:A CCGGCCATTT A~TCATCCTC CACGATCG 3 8
15 ( 12 ) INFORM~TION FOR SEQ ID NO: 11:
( i ) 5EQUENt::E CilARACTERISTICS:
(A) LEN~ 24 bases
(B) TYPE: nucleic acid
(C,~ STRANDEDNESS: single
(D) TOPOLOGY: linear
~xi) SEQUENCE DESe:RIPTION: SEQ XD NO:11:
G2LTGATT~AA TGGGTCCGGA GACT 24
(13) INFORMATION FOR SEQ ID NV: 12:
(i~ S~5QUENCE CH~ACTERISTICS:
~O (A) L~NGTH: 6 amino acids
(B) TYPE: amino acid
tc)~ TOPOLOGY: linear
(xi~ SEQUENCE DlESCRIPTIOM: SEQ ID NO:12
__
Me~ Gly Pro Glo r~ Leu
36
