IL98234A - Purified yeast-derived epidermal growth factor/urogastrone-like protein and medicinal compositions comprising said protein - Google Patents

Description

ΊΠΊΝ o !?>D)cin D>>ii£nnii o>-i ¾nm o>-m¾ra aian PURIFIED YEAST-DERIVED EPIDERMAL GROWTH FACTOR/UROGASTRONE-LIKE PROTEIN AND MEDICINAL COMPOSITIONS COMPRISING SAID PROTEIN BACKGROUND OF THE INVENTION Field of the Invention The present invention relates to medicinal compositions commonly known as Epidermal Growth Factor (EGF) /Urogastrone, and more particularly refers to related forms of these compositions derived from yeasts and yeast extracts.

Description of the prior art Naturally produced Epidermal Growth Factor was discovered a number of years ago. Relatively large amounts were found stored in mouse salivary glands. The material was given the name murine Epidermal Growth Factor (m-EGF) when it was discovered that newborn mice treated with the material opened their eyes earlier than did control mice. It was subsequently discovered that Epidermal Growth Factor was also involved in the control of gastric acid, which finding resulted from the discovery that Urogastrone v/as purified from the urine of pregnant women, and it was shown that this material was identical to human Epidermal Growth Factor.

Epidermal Growth Factor has been found to be very effective in the healing of wounds, and particularly wounds resulting from corneal transplants and cataract surgery. The same material, also designated as Urogastrone has been found useful in the treatment of gastric ulcers.

Because naturally occurring Epidermal Growth Factor and Urogastrone arc available in only minute quantities naturally, synthetic methods, including genetic engineering methods are now utilized to produce these materials. Such methods are disclosed in many patents, including the following: U.S. Patent Nos . 3,003,497, 3,940,875, 4,032,633, 4,035,405, 4,731,357, 4,743,679, 4,760,023, 4,764,593, and 4,820,690. However, these methods of producing the materials are not entirely satisfactory, as they are quite time-consuming and expensive.

SUMMARY OF THE INVENTION According to the invention there is provided an isolated water-soluble yeast protein which is naturally present in yeast, which is free of water-insoluble yeast components: which is insoluble in trichloroacetic acid, chloroform, absolute methanol, 95% ethanol, acetone, hexanes, petroleum ether, and 50% methanol-25% ether; which is soluble in acidic solutions containing 50% methanol, ethanol or acetone; which stimulates the growth and respiration of A431 cells in a culture without serum supplements; which has a murine Epidermal Growth Factor (mEGF) activity per milligram at least equivalent to 1000 nanograms of mEGF by Elisa Assay Titration, which EGF activity is provided by the presence therein of a water-soluble yeast protein having a molecular weight of about 4770 daltons and approximately the following amino acid composition: Asx (7) Thr (3) He (1) Glx (5) Ala (3) Leu (2) Ser (3) Pro (2) Phe (1) Gly (5) Tyr (1) Lys (4) His (1) Val (2) Trp (2), and Arg (1) Met (0) Cys (6) There is also provided a medicinal composition comprising, in admixture with a pharmaceutically acceptable carrier, a water-soluble yeast protein composition which is free of water-insoluble yeast components; which is insoluble in trichloroacetic acid, chloroform, absolute methanol, 95% ethanol, acetone, hexanes, petroleum ether, and 50% methanol-25% ether; which is soluble in acidic solutions containing 50% methanol, ethanol or acetone; which stimulates the growth and respiration of A431 cells in a culture without serum supplements; which has a murine Epidermal Growth Factor (mEGF) activity per milligram equivalent to at least 1000 nanograms of mEGF by Elisa Assay Titration, which EGF activity is provided by the presence therein of a water-soluble yeast protein having a molecular weight of about 4770 and approximately the following amino acid composition: ASX (7) Thr (3) He (1 ) Glx (5) Ala (3) Leu (2) Ser (3) Pro (2) Phe (1) Gly (5) Tyr (1) Lys (4) His (1 ) Val (2) Trp (2) Arg (1) Met (0) Cys (6) The yeast Epidermal Growth Factor may be prepared in various degrees of purity, including one composition which is substantially pure as determined by comparing its activity with that of murine Epidermal Growth Factor.

BRIEF DESCRIPTION OF THE DRAWINGS In the drawings: FIG. 1 is a graph showing the effect of yEGF on A431 cell growth.

FIG. 2 is a graph showing the results of the titration of mEGF (murine EGF) using A431 cellular respiration as an indicator of activity.

FIG. 3 is a graph showing the results of the titration of yEGF using A431 cellular respiration as an indicator of activity..

FIG. 4 is a graph showing the relative stability of mEGF and yEGF as measured by stimulation of A431 cellular respiration .

FIG. 5 is a graph showing . the effect of anti-mEGF on yEGF's stimulation of respiration.

FIG. 6 is a graph of titration of yEGF with 0.4 ug/ml anti-mEGF.

FIG. 7 is a graph showing percent stimulation of respiration by yEGF and mEGF mixtures.

FIG. 8 is a graph showing the results of the titration of yEGF activity with vaccinia virus infection.

FIG. 9 is a graph showing the results obtained from yEGF chromatography on a DEAE cellulose column FIG. 10 is a graph showing the results obtained from yEGF chromatography on a Waters Delta Pak RPC18 column resulting in yEGF in substantially pure form..

FIG. 11 is a graph showing the binding of yEGF to the EGF receptor of Λ431 cells, and FIG. 12 is a graph correlating the activity of substantially pure yEGF in Elisa units and Skin Respiratory Factor (SRF) units.

DESCRIPTION OF THE PREFERRED EMBODIMENTS A water soluble protein fraction that stimulates the growth and respiration of A431 cells in culture without serum supplements was isolated from several different yeast sources including bakers yeast, brewers yeast, torula yeast, and other specially engineered yeasts such as the new Gist-Brocades Bakers yeast, and also commercial extracts of yeast. Following purification (as described below) the substantially pure protein fraction contained all of the respiratory activity found in the initial extract and had many properties suggesting that it is an analogue of mammalian epidermal growth factors. This protein fraction is herein designated as Yeast Epidermal Growth Factor (yEGF) .

Example 1. Difco yeast extract (Baxter Scientific no. 0127-01-GB) was dissolved at 200mg /ml in water. An equal volume of absolute ethanol was . added and the solution was stirred. Insoluble material was removed by centrifugation at 10,000 x g for 10 minutes. The alcoholic supernatant was heated to 70°C in a water bath, until the volume had been reduced 75%. Alternatively, an equal weight of absolute ethanol may be added to the yeast cakes and the mixture extracted at room temperature overnight. The insoluble material was removed by centrifugation, and the ethanol was removed by distilling the supernatant at 70°C under vacuum until the volume had been reduced 50%. Alternatively, the yeast cakes were first autoclaved and then subjected to ethanol extraction. As an other alternative, commercially available yeast extracts were (LYCD, Live Yeast Cell Derivative) used directly. The resulting solutions had a characteristic yeast odor and brown color.

These extracts, designated as yEGF (Fraction I), can support the growth of Λ431 cells (ATCC no. CRL1555) in culture without serum. Cell numbers were measured by crystal violet staining according to the procedure of Braesaemle, 1988.

Referring to FIG. 1, a graph is shown demonstrating the results of a representative titration of a Difco yeast extract. A linear regression correlates yEGF (Fraction I) concentration with Λ431 growth from 10 to 100 micrograms/ml (Y = 8.76 mg/ml yEGF + 1.08). Y is ratio of the cell number for yEGF grown cells to control cell numbers, that are grown without yEGF.

In carrying out the experiment resulting in the data from which the graph of FIG. 1 was produced, 104 A431 cells were dispersed into each well of a 24 well plate in Dulbecco's Minimal Essential Media (DME) containing 0.1% bovine serum albumin. The cells were allowed to attach overnight and different concentrations of Difco yEGF (Fraction I) were added to each well. Each yEGF (Fraction I) concentration was prepared in quadruplicate. The first column of wells was used as a negative control. After 7 days, the wells were washed one time with phosphate buffered saline (PBS) containing 1ml ethylenediamine tetraacetic acid (EDTA) . The cells were fixed with absolute methanol for 15 minutes and stained with 0.1% crystal violet for 5 minutes. The excess stain was removed by inverting the plates, and the wells were washed twice with tap water. After the plates had air dried, 1ml of 2% sodium desoxycho,late was added to each well and the plates were incubated overnight on an orbital shaker. The quadruplicate samples were pooled, adjusted to 4 ml, where necessary, and their absorbance at 570nTn was recorded. Each of the samples is expressed relative to a yEGF-less control.

Example 2. Λ rapid assay for EGF activity was developed based on the observation that pure murine EGF ( EGF, Collaborative Research no. 40001) immediately stimulates A431 cellular respiration.

Referring to FIG. 2, a graph is shown demonstrating the results of a titration of mEGF using A431 respiration. The mEGF dose correlates with respiration from 5 to 250 ng/rol. The correlation at 98% confidence has been calculated to be: Z = 1.512 ug/ml EGF + 1.03. The respiratory assay was specific for EGF type growth factors, since fibroblast growth factors, insulin growth factors, interferons, interleukins, and platelet derived growth factors did not have any effect on the respiration assay.

In preparing to carry out the titration the results of which are shown in FIG. 2, 10 cm dishes of A431 cells were washed twice and incubated in 5 ml of DME containing 0.1% bovine serum albumin for 1 hour in a CO2 incubator. The cells were collected by trypsinization and resuspended at 2-3 x 106 cells per ml in DME containing 0.1% bovine serum albumin. One ml of the cell suspension was transferred to a glass tube that was maintained at 37°C with circulating water. The cells were allowed to equilibrate at temperature for 5 minutes, and a Clark oxygen electrode was inserted into the vessel. Potentiometric readings were continuously taken from the electrode using a YSI oxygraph that was attached to a recorder. After a stable respiration rate had been observed, 1 to 20 microliters of pure murine EGF was added to the reaction vessel. Respiration was allowed to continue until an accurate rate could be observed on the recorder. The rates of respiration were converted to microliters of oxygen per hour per 2 x 106 cells. The percent stimulation observed for any given mEGF dosage was calculated by comparing the respiration rate with EGF to the pre-established baseline for that sample.

Similarly, the yEGF fractions were titered by measuring their immediate effect on the consumption of oxygen by Λ431 cells.

Referring to FIG. 3, a graph is shown demonstrating the effects of yEGF(Fraction I) on A431 respiration. The results obtained with yEGF (Fraction I) prepared from the difco extract are summarized in the graph of FIG. 3. A linear regression relates yEGF(Fraction I) to A431 respiration in the range of 100 to 1000 micrograms per ml [Z = 0.465 mg/ml yEGF (Fraction I) + 1.01]. Z is the ratio of yEGF (Fraction I) respiration divided by the control respiration rate. A comparison of the regressions for yEGF (Fraction I) and mEGF indicate that 1 mg of yEGF (Fraction I) contains 329 ng of mEGF activity. An analysis of 10 independent preparations of yeast extract by the A431 respiratory assay indicated an average of 313ng of mEGF activity + 35ng.

The procedure for measuring respiration for the titration carried out and shown in FIG. 3 is the same as that summarized in FIG. 2. The Experimental Over Control (EXP/CON) is the rate of respiration observed with yEGF (Fraction I) divided by the basal respiration rate for that sample.

Example 3. The biological activity οϊ yEGF(Fraction I) can be eliminated by treatment with proteases such as trypsin and proteinase K (See TABLE 1 below) . Treatment of yEGF (Fraction I) with DNase or RNase had no effect on the activity. The activity of yEGF(Fraction I) demonstrated the same degree of sensitivity as pure murine EGF, except that it could be boiled without loss of activity whereas the mEGF was denatured at the higher temperature. Dithiotrietol (DTT) enhanced the activity of both murine EGF and the yeast fraction yEGF(Fraction I). The stability of the mEGF and yEGF (Fraction I) were compared at 55°C.

Referring to FIG. 4, a graph is shown summarizing the results obtained for yEGF (Fraction I) and mEGF. The ability of mEGF to stimulate respiration is lost within 10 minutes at 55°C, whereas yEGF(Fraction 1) activity was actually enhanced by incubation at the elevated temperature.

In preparation for the titration the results of which are shown in FIG. 4, twenty microliter samples of either 100 ng of mEGF or 500 ug of yEGF(Fraction I) were incubated for various times at 55°C. The samples were removed and assayed for their ability to stimulate A431 cell respiration. The procedures for measuring respiration were the same as those which are summarized in relationship to the work leading to FIG. 2.

Example 4^. Anti-mEGF monospecific antisera (Collaborative Research no. 40019) inhibited both the murine EGF and the yEGF ( Fraction I) ability to stimulate A431 respiration (See TABLE 1 below) . The addition of non-immune IGGs did not affect the respiration assay.

Referring to FIG. 5, a graph is shown demonstrating the effect of anti-mEGF on the stimulation of respiration of yEGF (Fraction I). [yEGF (Fraction I)= 0.8 mg/ml]. The inhibition of respiration by anti-mEGF was dependent on the concentration of the antibody, as shown in FIG. 5. 210ng anti-mEGF inhibited the respiratory stimulation of 800 ug/ml yEGF(Fraction I) by 50%.

In preparation for the titration the results of which are shown in FIG. 5, eight micrograms of yEGF (Fraction I) were mixed with different concentrations of anti-mEGF antibody in a final volume of 10 microliters. Five microliters of each sample was assayed in the A431 cell respiration assay as outlined in the material related to FIG. 2. The percent stimulation is the respiration rate of the experimental sample divided by the basal respiration rate.

Anti- urine EGF antisera was also used in an Enzyme Linked Immuno-sorbent Assay (Elisa test) to titer the amount of EGF epitope in partially purified yeast extracts (Engvall, 1971). One ml of a 100 mg/ml sample of a yeast extract yEGF ( Fraction I) was diluted to 10% with trichloroacetic acid (Fisher Scientific no. A322-100) and allowed to stand for 30 minutes on ice. Precipitated material was collected on a centrifuge at 10,000 x g for 10 minutes, and washed twice with 95% ethanol. The insoluble material was dissolved in 1 ml of water and dispersed in a 96 well plate by serial dilutions. Unbound antigen was removed after 3 hours at 37°C and the plate was incubated with monospecific anti-mEGF antibody overnight at 4°C. Excess antibody was removed by washing the plates and a second antibody conjugated to alkaline phosphatase was added to each well. After 3 hours at 37°C, the excess second antibody was removed and the amount of alkaline phosphatase per well was determined. The Elisa test was dependent on the amount of y-EGF present.

As shown in the graph of FIG. 6, yEGF is recognized by the anti-mEGF antibody. The reaction was 50% maximal at the 5th dilution. Each dilution is 20%. A comparison to mEGF titered under identical conditions indicated that yEGF (Fraction I) contains 375ng of mEGF units.

In preparation of the materials for the titration shown in the graph of FIG. 6, one hundred microliters of PBS was added to each well of a 96 well microtiter plate. Twenty five microliters of 10 g/ml yEGF was added to the second well of each row. After mixing, 25 microliters was transferred to the third well in the row. The process was repeated until all the wells had dilutions of yEGF. The antigen was allowed to bind at 37°C for 3 hours and the plate was washed 3 times with PBS containing 0.05% Tween-20 (TPBS). One-hundred microliters of goat anti- urine EGF which had been diluted 1/2500 in TPBS was'added to each well and the plate was incubated overnight at 4°C. The wells were washed 3 times with TPBS, and 100 ul of 1:4000 sheep anti-goat F(ab) which was conjugated to alkaline phosphatase was added to each well. The second antibody was incubated for 3 hours at 37°C and the plates were washed 3 times with TPBS.

The ΛΡ color reaction was developed according to the procedure of Blake (1984).

Example 5. The ability of alcohol extracts of yeast to stimulate growth and respiration of A431 cells may be explained entirely by their EGF activity. Subsaturating amounts of yEGF and mEGF were mixed together and assayed by Λ431 respiration. The percent stimulation of the observed combined reaction was compared to the theoretical amount of respiratory stimulation.

Referring to FIG. 7, the graph showing percent stimulation of respiration by yEGF (Fraction I) and mEGF mixtures indicate that a greater than 95% correlation resulted between the theory and the experimental.

In preparation for the experimental work resulting in the study shown in FIG. 7, 25 ng of mEGF was mixed with 10-40 ug of yEGF (Fraction I) , and each of the mixtures was assayed with the A431 cell respiration assay as outlined in description in relation to FIG. 2. The percent stimulation for each mixture was calculated from the experimental data (Observed) . The theoretical percent .stimulation was calculated from the standard curves for yEGF and mEGF in Figures 2 and 3 assuming that the expected EGF units from yEGF or mEGF would be additive.

Example 6. Epstein et al (1985) reported that Vaccinia virus uses the EGF receptor to gain entry into fibroblasts and purified 60 ng/ml EGF blocks virus infectivity by 50%. A comparable experiment has been performed with yEGF.

Referring to FIG. 9, a graph shows the results of yEGF(Fraction I) pretreatment of the A431 cells for 24 hours prior to infection with vaccinia virus. As summarized therein, a 50% reduction of infectivity was observed at 175 ug/ml yEGF ( Fraction I) . An extrapolation indicates that 1 mg of yEGF(Fraction I) contains 343 ng of antiviral EGF units.

Subconfluent plates of either BSC-40 cells or A431 cells were incubated with varying concentrations of yEGF(Fraction I) for 24 hours. The yEGF(Fraction I) was . removed from the plates by washing and the cells were infected with vaccinia virus R at a multiplicity of infection equal to 5. Virus infection was allowed to continue to 48 hours and the infected cells were collected, hypotonically shocked and frozen. Aliquots of the samples were defrosted and the amount of virus was titered by plaque assay. The percent controls are the number of virus plaques in the yEGF (Fraction I) treated samples divided by the number of plaques in cells that had not been pretreated with yEGF.

Example 7. Active fractions of yeast extracts, i.e. yEGF (Fraction I), are insoluble in trichloroacetic acid, chloroform, absolute methanol, 95% ethanol, acetone, hexanes, petroleum ether and 50% methanol-25% ether. However the active fractions are soluble in acidic solutions containing 50% methanol, ethanol, or acetone. 50% alcoholic extracts from bakers yeast were precipitated with 10% trichloroacetic acid, neutralized and washed with ethanol. The material was dissolved and analyzed by electrophoresis on 10% polyacrylamide gels according to the procedure of Laemmli (1970) . One major protein peak was detected with a molecular weight between 4000 and 14,000. An identical gel was transferred to nitrocellulose paper by the procedure of Towbin (1979, 1984) and the paper was immunostained with anti-murine EGF or anti-human recombinant EGF using the procedures of Blake (1984) . A single immunopositive band was identified at a molecular weight of 4000-14,000. No staining was observed with other anti-murine antibodies or with second antibody alone.

Example 8. yEGF(Fraction I) was further purified by methanol to 75% (v/v) or by adding methanol to 50% and ether to 25% of the final volume. A precipitate formed and was collected by centrifuging at 10,000 x g for 10 minutes. The precipitate was dissolved in 0.1 of its initial volume in water to form yEGF (Fraction II) . The pH was adjusted to 1 to 2 with concentrated HC1 and the resulting precipitate was removed by centrif gation at 10,000 x g for 10 minutes. The supernatant was adjusted to pH 7 with 1M Tris base to form yEGF(Fraction III). 1.0 mg of Fraction III was loaded onto a 0.39x15 cm Waters Delta Pak RPC18 column. Proteins were eluted from the column with a linear gradient, starting with 0.94% trifluoroacetic acid (TFA) containing 0.6% acetonitrile (ACN) and ending with 0.02% TFA containing 76% ACN. The HPLC chromatographic separation is summarized in Figure 9. All of the immune activity and Skin Respiratory Factor were recovered in Peak-3 , producing yEGF (Fraction V) , a substantially pure form of yEGF.

Table 2 below summarizes the results of titrating each of the yEGF fractions with anti-murine EGF. Gel electrophoresis and western blotting, as outlined in Example 7, indicated that a single protein band contained all of the EGF activity. Alternatively, yeast cakes were washed with 4 grams of methanol per gram of yeast until they had been decolorized. An equal weight of IN HCl was added to the yeast solids and the mixture was extracted for 30 minutes. The yeast residue was removed and the supernatant was neutralized with IN NaOH. The solution was concentrated by pressure filtration to obtain yEGF(Fraction Ilia) Fraction III of the yeast extract was alternatively purified by DEAE - cellulose, . diethylaminoethyl cellulose chromatography (Cohen, 1977) , yielding a fraction designated as yEGF(Fraction IV) . A single activity peak was eluted from the DEAE column with 0. IN HCl in 50% ethanol.

Referring to FIG. 10, a graph is shown demonstrating the results of yEGF ( Fraction III) chromatography on a DEAE cellulose column. In the experiment lg yEGF (Fraction III) was loaded onto a 25 x 55mm column that had been equilibrated in lOmM sodium acetate (NaOAc) , pH 6.0. VJhile carrying out the study, fractions from the active peak were pooled, concentrated and analyzed on polyacr lamide gels by the Lae mli procedure. This fraction is designated as yEGF(Fraction IV). Λ single protein peak was detected with Coomasie Blue or silver staining at a molecular weight of 4,000 - 14,000. A companion acrylamide gel was transferred to i mobilon paper by the Towbin procedure and stained with anti-mEGF. A single immunopositive band was detected at the same position as the silver staining.

Alternatively, the yEGF(Fraction I) is completely purified by the procedure described for the preparation of murine EGF in U.S. Patent No.3 , 948, 875, which is herein incorporated by reference to produce substantially pure yEGF, yEGF(Fraction V).

Example 9. The amino acid composition of yEGF Fraction V was determined by the Pico-Tag methods of Waters Associates (Milford, MA) using either hydrolysates prepared in constant boiling HCl containing 1% phenol or 4M methanesulfonic acid containing 0.2% 3 (2-aminoethyl) indole. The results are summarized below in Table 3. Referring to Table 3, the composition of yEGF(Fraction V) accounts for its observed molecular weights by electrophoretic techniques. Moreover the amino acid of composition of yEGF (Fraction V) more closely resembles that of human recombinant EGF than the murine EGF. yEGF(Fraction V) was also tested for its ability to bind to the human EGF receptor expressed on the surface of A431 cells. The binding constant (Kn) for yEGF was determined to be 1.61 to 2.92 x 10 M. The binding constants for EGF and hrEGF were determined in the same experiment to be 2.42 and 1.12 x 10~9M.

Referring to Figure 11, Tissue culture wells containing 1-2 x 10^ cells were washed free of medium and incubated with 1 ng of 125I-mEGF (Collaborative Research) and various amounts of Fraction V yEGF. After 30 minutes, the wells were washed four times with PBS and the cells were solubilized in NTS (Amersham Radiochemicals) according to the manufacturers protocol. The amount of radioactivity was determined by liquid scintillation.

Example 10. The purity of Fraction V yEGF was further evaluated by comparing its respiratory activity and immune activity to pure EGFs. One ug/ l yEGF(Fraction V) was determined to equal 0.0124 Respiratory stimulation units (SRF) - 0.0245. The regression was significant at <0.05 and had a standard error of 0.0025. yEGF (Fraction V) samples were converted to ug / ml EGF from their SRF values. A second linear regression was calculated from the experimental data obtained from titering pure mEGF in the Elisa assay. One ug of mEGF equaled 0.0518(ABS) - 0.0223. The regression was significant at <0.1 and had a standard error of 0.019. The absorbance readings for an Elisa titration of yEGF(Fraction V) were then converted to ug/ml mEGF using the regression. The results are summarized in Figure 12.

In sum, Fraction V yEGF (1) has been shown to be electrophoretically pure, that is, substantially pure, and migrates at the same apparent molecular weight as mEGF and hrEGF; (2) has an amino acid composition which is similar to mEGF and hrEGF; (3) binds to the human A431 cellular EGF receptor; (4) shares epitopes with mEGF and hrEGF as evidenced by its reactivity with monospecific antisera to either mEGF or hrEGF; and (6) stimulates cellular respiration as well as mEGF and hrEGF per nanogram of sample.

It should be noted that initial crude isolates of EGF/Urogastrone from animal tissues and fluids contain a plurality of other protein molecules. By comparison, the initial yeast Epidermal Growth Factor isolates, yEGF (Fraction I), and consequently Fractions II, III, Ilia, IV, and V contain substantially one protein species, yEGF, and are thus suitable for use in pharmaceutical product formulations based on EGF bio-assay content. Further, the other yeast extract components (which are non-toxic lecithins, carbohydrates, nucleic acids, etc.) serve to stabilize the low concentrations of yEGF employed medicinal preparations.

TABLE 1. Properties of yEGF (Fraction I) or mEGF (Stimulation of A431 Respiration) (mEGF) [yEGF (Fraction I) ] A431 Assay Additions % Stimulation % Stimulation 0.5 ug/ml mEGF 17.8 1.0 mg/ l yEGF(Fraction I) 19.3 Pretreatments 30 in 2ug/ml trypsin 1 2.3 Boiling 2.1 22.3 30 min 10 ug/ml DNasel 18.2 19.1 30 min 2 ug/ml RNase A 17.6 20.4 30 min 2 ug/ml ProK 1.0 2.6 O.lmM DTT 44.8 60.4 6.5 ug/ml a-mEGF 1.5 -2.5 10 ug/ml murine IGG 19.8 19.9 TABLE 2 ; Activities of various yEGF Fractions (ELISA ASSAY TITRATIONS) yEGF Specific Activity (micrograms mEGF/mg yEGF) Fraction I - 50% ethanol soluble Bakers Yeast 0. 366 Torula Yeast Extract 0. 405 Autoclaved Bakers Yeast 0. 412 Fraction II - 80% methanol insol 3. 453 Fraction Ila - 10% TCA insoluble 3. 126 Fraction III - pH 1 soluble 114 .652 Fraction Ilia - methanol/acid extract 117 .654 Fraction IV - DEAE Peak 3 214 .654 Fraction V- HPLC Peak 3 943 .560 The yEGF, at the initial Fraction I purity is useful in topical medicinal products in the range of one to 100,000 microgra s/kg/day . Fractions II, III, Ilia, IV, and V are also useful, although their concentrations in the formulations should be reduced commensurate with their activities as assayed in SRF units.

TABLE 3: Amino Acid Composition of Substantially Pure vEGF (Fraction V) yEGF hrEGF mEGF asx 7 7 7 glx 5 5 3 ser 3 3 6 gly 5 4 6 his 1 2 1 arg 1 3 4 thr 3 0 2 ala 3 2 0 pro 2 1 2 tyr 1 5 5 val 2 3 2 met 0 1 1 iso 1 2 2 leu 2 5 4 phe 1 0 0 lys 4 2 0 tryp 2 2 2 cys 6 6 6 #AA 52 53 53 Mol Wt = 4770 5261 5331 Table 4; Comparison EGF and yEGF (Fraction V) Mouse yEGF Human EGF (Fraction V) EGF . Acidic amino acids 10 12 12 . Basic amino acids 5 6 7 . Non-polar amino acids 17 15 11 . Aromatic amino acids 7 4 7 . Neutral amino acids 8 9 10 6. Cysteine 6 6 6 As seen in Table 4 above, the cysteine values for all three EGF compositions are the same, supporting the claim that the compositions are related.

FORMULATION EXAMPLE8 Example 11. A capsule containing yEGF ( Fraction V) , substantially pure yEGF, for oral administration is prepared by formulating the following ingredients: - grams/lOOg Lactose 39.99 Crystalline cellulose 15 Calcium stearate 15 Talc 30 yEGF (Fraction V) 0.00025 Substantially pure yEGF, yEGF(Fraction V) , promotes the healing of damaged tissues of the alimentary tract such as, for example, ulcers of the mucosa and submucosa of the gastrointestinal tract when administered orally.

The partially purified yEGF fractions described in this application, such as Fraction I, Fraction II, and Fraction III are also useful when administered orally to treat duodenal and gastric ulcers. Separately, such oral yEGF formulations also provide amelioration of Inflammatory Bowel Disease.

The present invention relates in part to the discovery that yEGF in its various states of purity- will promote the healing of damaged tissues of the alimentary tract such as, for example, ulcers of the mucosa and submucosa of the gastrointestinal tract when administered orally.

For yEGF an optimal daily ingestion of 4000 to 20,000 SRF units requires .4g to 2.0 g of yEGF(Fraction I).

A preferable oral pharmaceutical composition is one suitable for administration in unit dosage form, for example in aqueous, elixir, or oily solutions or suspensions, or an emulsion containing yEGF at between 40ug to 200u of yEGF(Fraction V) per ml. Alternatively, a yEGF composition of unit dosage form, for example a tablet, capsule, lozenge, each containing between 5.5ug and lOOug, and preferably between 5 ug and lOOug of yEGF (Fraction V.

Example 12. A capsule containing yEGF is formulated as follows: parts/lOOg Lactose 40 Crystalline cellulose 15 Calcium Stearate 15 Talc 30 yEGF (Fraction V) 50ug 0 Example 13.

A tablet containing yEGF (Fraction I) is formulated as follows : parts/lOOg Lactose 40 3% HPC lactose 30 Crystalline cellulose 20 Potato starch 8 Talc stearate 2 (a 1:1 mixture of magnesium stearate and talc) yEGF (Fraction I) 100 g Purified water q.s.

The capsule or tablet formulations are taken one to four times per day, and are also suitable for overnight medication.

Example 14. Preparation of a sterile solution of yEGF(Fraction I). log to lOOg of yEGF (Fraction I) is dissolved in pyrogen-free water (1000ml) and the solution filtered through a sterilizing membrane filtration system, for example a 0.22 mille micron filter, into sterile vials or ampoules so that each container receives 100 ml and is sealed under sterile conditions. This product, which is stable for a year or longer at refrigerated temperatures is suitable for washing surgical wounds; and may also be used by parenteral injection to promote bone and/or tendon repair in a warm blooded animal.

Example 15. Lyophilized preparation of yEGF (Fraction III) : One gram of yEGF (Fraction III), prepared as previously described, is dissolved in pyrogen-free 5% w/v dextrose solution to give a final concentration of 1 mg/ml. This solution is dispensed into vials in aliguotes of 5.0ml each through a sterilizing membrane filtration system, for example, through a 0.22mmu filter. The contents of each vial are then lyophilized and the vials capped and sealed under sterile conditions. The vials, containing a sterile mixture of yEGF(Fraction III), and dextrose are stored at 4°C.

Example 16. A solution of yEGF(Fraction V) , suitable for topical application to skin or scalp -is formulated with the following ingredients: Alcohol 55.0% w/v Oleyl alcohol 5.0 w/v yEGF (Fraction V) 0.00590 w/v Propylene glycol to 100% Example 17. A cream formulation of yEGF (Fraction I) for topical application is prepared from the following ingredients in stated amounts per 100 grams: Dimethicone 5.0 g D-pantheol 4.0 g Benzalkonium chloride 0.1 g yEGF (Fraction I) 0.5 g A water washable cream base .. to 100 g.

Example 18. An ointment formulation of yEGF(Fraction I), for topical application is prepared from the following ingredients, stated in terms of parts per ingredient for each 100 parts of ointment: Beeswax 4.0 Lanolin 4.0 yEGF( Fraction I) 1.0 Methyl parabens 0.01 Petrolatum to 100 parts Example 19. Preparation of ophthalmic solution of yEGF(Fraction III) : One gram of yEGF (Fraction III) is dissolved in isotonic, pyrogen-free saline to give a concentration of 0.1% of yEGF. This solution is dispensed into vials in aliquotes of 1.0 ml each through a sterilizing membrane filtration system, for example a 0.22 micron filter. The contents of each vial are then lyophilized and the vials capped and sealed under sterile conditions. The vials containing a sterile mixture of yEGF (Fraction III) and isotonic saline are stored at refrigerator temperature.

The availability of yeast Epidermal Growth Factor as a readily available lower cost replacement for human recombinant EGF/Urogastrone permits the development of topical therapeutic products suitable for use in the following areas: a) burn/wound healing, including bed-sores, decubitus ulcers, diabetic ulcers, and other non-healing skin ulcers; b) skin protectant and skin wrinkle control, including photo-aging, acne, psoriasis, dermatoses, insect bites, and inflammation; c) soft tissue and bone repair in Orthopedic and periodontal surgery; d) control of post surgical adhesions in gastrointestinal, neurological, and cardiac surgery; e) in ophthalmology as an aid to corneal transplant surgery and cataract removal; and f) actinic keratosis. In addition, oral yEGF is an effective anti-ulcer agent.

Various combinations of yEGF formulations at representative concentrations of EGF activity are thus made available for a whole range of medicinal applications in warm blooded animals. Combinations with other lymphokine/cytokine growth factors, and other compatible topical medicinal agents are also contemplated.

The biological activity of yEGF is enhanced and/or stabilized by limited proteolysis, reduction of disulfide bonds, glycosylation, myristilation, palmitation, pegylation, and phosphorylation.

In summary, the isolation and characterization of yeast Epidermal Growth Factor as a new Epidermal Growth Factor/Urogastrone-like protein has been effected using five distinct assays to correlate and quantify yEGF and murine EGF activity: Λ431 cell growth.

A431 cell respiration.

Anti-murine EGF Elisa.

Anti-murine EGF Western Blot.

Anti-vaccinia virus growth in cultured cells.

The substantially pure yEGF (Fraction V) protein has a molecular weight of 4,000-14,000. It is insoluble in methanol, ethanol, hexanes, petroleum ether and chloroform. Substantially pure yEGF is soluble in acidic solutions of 50% ethanol or methanol.

Sequencing of the yEGF protein and identification of its encoding gene(s) is accomplished by current classical techniques. Alteration of the yEGF protein by recombinant genetic methodology in yeasts, bacteria or viruses, including fusion to other proteins can result in products which retain or enhance the biological activities measured by the assays described herein.

References: Blake, M.S. et al (1984) Analytical Biochemistry 136: 175-184.

Brasae le, D.L. & A.D. Attie (1988) BioTechniques 6: 418-419.

Cohen, S & R Savage (1977) US Patent 3,948,875 Engvall, E. & P. Perlmann (1971) Immunochemistry 8: 871-879.

Epstein, D.A. et al (1985) Nature 318: 663-665.

Laemmli, E.K. (1970) Nature 227: 680-685. - 21a - Towbin, H., Staehelin, T. & J. Gordon (1979) Proc Nat Acad Sci (USA) 76: 4350-4354.

Bentley et al., Arch. Surg. vol. 125, May 1990, discloses similar compositions treated with activated charcoal to purify them. The Applicants' compound would have been removed by such a procedure and therefore Bentley et al could not prepare the Applicants' compound.

Urdea et al., Proc. Natl. Acad. Sci. USA, vol. 80, pp. 7461-7465, 1983, synthesize human Epidermal Growth Factor by the recombinant DNA method. In doing so they first form DNA fragments and fuse them to form a plasmid or vector coding for human Epidermal Growth Factor. They then express the plasmid in a eukaryotic medium, yeast, which results in the synthesis of the human Epidermal Growth Factor protein. As opposed to this, Applicants start with yeast or a yeast extract, forming a substantially pure compound which is not human Epidermal Growth Factor. 22 In summary, the present invention comprises a series of yeast-derived Epidermal Growth Factor-like materials which are derived by extraction from various sources of yeast, and exhibit many of the properties exhibited by Epidermal Growth Factors obtained from murine species and those obtained by genetic engineering methods. The yeast-derived Epidermal Growth Factor-like materials obtained herein by extraction from yeast are designated yEGF. The first fraction obtained by extraction with alcohol is designated as yEGF (Fraction I). This fraction exhibits an equivalency of 300 to over 400 nanograms, or .3 to over .4 micrograms of mEGF activity per milligram of yEGF (Fraction I). Although this activity is sufficient for some purposes, a higher equivalency is desirable. yEGF Fraction II is prepared by further extracting yEGF (Fraction I) in acidic solutions containing 50% methanol, ethanol or acetone, as demonstrated in Example 6. The resulting yEGF (Fraction II) has an equivalency of 1000 to over 3400 nanograms, or 1 microgram to over 3.4 micrograms of mEGF activity per milligram of yEGF (Fraction II) . Other extraction methods yield yEGF(Fraction III) having an equivalency of 114000 to over 117000 nanograms, or 114 to over 117 micrograms, of mEGF activity per milligram of yEGF(Fraction III) . Further, by HPLC chromatography extraction of yEGF (Fraction III), yEGF (Fraction V) is obtained having an equivalency of over 943 micrograms mEGF activity per milligram of yEGF. yEGF(Fraction V) is considered to be homogeneous, since the amino acid composition accounts for the observed electrophoretic molecular weight. yEGF ( (Fraction V) co-migrates in a single band with pure mEGF and hrEGF. yEGF (Fraction V) contains all of the respiratory activity in the extract. yEGF (Fractions II, III, IV and V are also useful in many medical applications in which mEGF and genetically engineered hrEGF are presently used. 23 Although the invention has been described in connection with specific embod ments thereof, it is -evident that many alternatives, modifications, and variations will be apparent to those skilled in the art in the light of the foregoing description. Accordingly, it is intended to embrace all such alternatives, modifications and variations within the spirit and scope of the invention as defined by the appended claims.

The invention relates only to what is claimed. Other disclosed matter does not constitute part of this invention.

Claims (26)

98234/2 - 24 - C L A I M S

1. An isolated water-soluble yeast protein which is naturally present in yeast, which is free of water-insoluble yeast components: which is insoluble in trichloroacetic acid, chloroform, absolute methanol, 95% ethanol, acetone, hexanes, petroleum ether, and 50% methanol-25% ether; which is soluble in acidic solutions containing 50% methanol, ethanol or acetone; which stimulates the growth and respiration of A431 cells in a culture without serum supplements; which has a murine Epidermal Growth Factor (mEGF) activity per milligram at least equivalent to 1000 nanograms of mEGF by Elisa Assay Titration, which EGF activity is provided by the presence therein of a water-soluble yeast protein having a molecular weight of about 4770 daltons and approximately the following amino acid composition: Asx (7) Thr (3) lie (1 ) Glx (5) Ala (3) Leu (2) Ser (3) Pro (2) Phe (1 ) Gly (5) Tyr (1 ) Lys (4) His (1 ) Val (2) Trp ( 2 ) , and Arg (1 ) Met (0) Cys (6)

2. A yeast protein according to claim 1 having a per milligram mEGF activity at least equivalent to 3000 nanograms of mEGF.

3. A yeast protein according to claim 1 having a per milligram mEGF activity at least equivalent to 114,000 nanograms of mEGF.

4. A yeast protein according to claim 1 having a per milligram mEGF activity at least equivalent to 214,000 nanograms of mEGF. 98234/2 - 25 -

5. A yeast protein according to claim 1 having a per milligram mEGF activity at least equivalent to 943,000 nanograms of mEGF.

6. A process for the isolation of a water-soluble yeast protein which is naturally preent in yeast, which process comprises extracting the water-soluble proteins from yeast, separating water insoluble yeast components therefrom to obtain a first water-soluble fraction (Fraction I), and separating from Fraction I a water-soluble protein fraction in the 4000-14,000 molecular weight range which is free of water-insoluble yeast components, which has an mEGF activity per milligram equivalent to at least 1000 nanograms of mEGF, which mEGF activity is provided by the presence therein of a water-soluble yeast protein which is insoluble in trichloroacetic acid, chloroform, absolute methanol, 95% ethanol, acetone, hexanes, petroleum ether, and 50% methanol-25% ether, which is soluble in acidic solutions containing 50% methanol, ethanol or acetone; which stimulates the growth and respiration of A431 cells in a culture without serum supplements; which has a murine Epidermal Growth Factor (mEGF) activity per milligram at least equivalent to 1000 nanograms of mEGF by Elisa Assay Titration, which EGF activity is provided by the presence of said water-soluble protein fraction which has a molecular weight of about 4770 daltons, and which has approximately the following amino acid composition: Asx (7) Thr (3) He (1) Glx (5) Ala (3) Leu (2) Ser (3) Pro (2) Phe ( 1 ) Gly (5) Tyr- ( 1 ) Lys (4) His (1) Val (2) Trp (2), and Arg ( 1 ) Met (0) Cys (6) 98234/2 - 26 -

7. A process according to claim 6, wherein said protein fraction isolated from Fraction 1 has a mEGF activity per milligram equivalent to at least 3000 nanograms of mEGF.

8. A process according to claim 6, wherein said protein fraction isolated from Fraction I has a mEGF activity per milligram equivalent to at least 114,000 nanograms of mEGF .

9. A process according to claim 6, wherein said protein fraction isolated from Fraction I has a mEGF activity per milligram equivalent to ast least 214,000 nanograms of mEGF .

10. A process according to claim 6, wherein the protein fraction isolated from Fraction I is the protein therein with mEGF activity and a molecular weight of about 4770, in substantially pure form, with mEGF activity per milligram equivalent to at least 943,000 nanograms of mEGF.

11. A medicinal composition comprising, in admixture with a pharmaceutically acceptable carrier, a water-soluble yeast protein composition which is free of water-insoluble yeast components; which is insoluble in trichloroacetic acid, chloroform, absolute methanol, 95% ethanol, acetone, hexanes, petroleum ether, and 50% methanol-25% ether; which is soluble in acidic solutions containing 50% methanol, ethanol or acetone; which stimulates the growth and respiration of A431 cells in a culture without serum supplements; which has a murine Epidermal Growth Factor (mEGF) activity per milligram equivalent to at least 1000 nanograms of mEGF by Elisa Assay Titration, which EGF activity is provided by the presence therein of a water-soluble yeast protein having a molecular weight of about 4770 and approximately the following amino acid composition : 98234/2 - 27 Asx (7) Thr (3) He (1 ) Glx (5) Ala (3) Leu (2) Ser (3) Pro (2) Phe (1 ) Gly (5) Tyr (1 ) Lys (4) His (1 ) Val (2) Trp (2) Arg (1 ) Met (0) Cys (6)

12. A medicinal composition according to claim 11 in oral dosage form.

13. A medicinal composition according to claim 11, wherein said yeast protein composition has a per milligram mEGF activity at least equivalent to 3000 nanograms of mEGF .

14. A medicinal composition according to claim 13 in oral dosage form.

15. A medicinal composition according to claim 11, wherein the yeast protein composition has a per milligram mEGF activity at least equivalent to 114,000 nanograms of mEGF .

16. A medicinal composition according to claim 15 in oral dosage form.

17. A medicinal composition according to claim 11, wherein the yeast proteic composition has a per milligram mEGF activity at least equivalent to 214,000 nanograms of mEGF.

18. A medicinal composition according to claim 17 in oral dosage form.

19. A medicinal composition according to claim 11, wherein the yeast protein composition has a per milligram mEGF activity at least equivalent to 943,000 nanograms of mEGF. 98234/1 - 28 -

20. A medicinal composition according to claim 19 in oral dosage form.

21. A topical medicinal composition according to claim 20.

22. An opthalmic medicinal composition according to claim 20.

23. A parenteral medicinal composition according to claim 20.

24. A topical medicinal composition according to claim 11.

25. An opthalmic medicinal composition according to claim 11.

26. A parenteral medicinal composition according to claim 11. AGENT FOR APPLICANT