Methods and means to promote gut absorption

Description of equivalent WO 2004001020 A2

METHODS AND MEANS TO PROMOTE GUT ABSORPTION

The present invention relates to epidermal growth factor (EGF) producing lactic acid bacteria and their use to increase intestinal villi height and to promote gut absorption. In particular, the invention relates to EGF producing Lactococcus lactis and Lactobacillus casei. Said organisms may be especially useful to treat short bowel syndrome.

The efficiency of gut absorption is essential for a good food conversion. Gut adsorption is largely determined by the gut surface, which is function, amongst others, of the length of the gut and the height of the villi. In cases where an operative removal of a part of the gut is necessary, as in the case of cancer or Crohn's disease, this may result in decreased gut adsorption, resulting in an unsufficient food conversion and a shortage of nutrients, dehydratation and even potentially lethal metabolic changes. These syndromes caused by the extensive resection of the small intestine are known as the Short Bowel Syndrome. Several methods have been proposed to improve the post operational adaptation of and to enhance the gut absorption in patients with the Short Bowel Syndrome. US5288703 discloses that both growth hormone and insulin like growth factor do have a positive effect on gut absorption in mammals. This positive effect can be enhanced by the administration of glutamine or glutamine equivalent. Administration of glutamine and growth hormone results in an increase of the villi length (Gu er a/., 2001 ; Zhou er a/., 2001). US5972887 demonstrated a reversal of the reduced intestinal mucosal mass and absorptive function in patients by the administration of low doses of exogenous Hepatocyte Growth Factor. Also the glucagon-like peptides GLP-1 and GLP-2 have been used with positive results. Studies on laboratory animals (Scott et al., 1998) as wall as on humans (Jeppesen er a/., 2001) showed a positive correlation between an increase in concentration of GLP-2 and an improvement of the intestinal adaptation. Short Bowel patients, of whom the ileum has been removed show a decrease in food-induced secretion of GLP-2 (Jeppesen et al. , 1999). Especially those patients can be treated successfully with GLP-2. It has been shown that also leptin has a positive effect on intestinal adaptation in a rat model (Pearson er a/., 2001 ). A lot of interest has been paid to the effect of Epidermal Growth Factor (EGF, urogastron). EGF is a relatively acid stable hormone that is produced in the salivary and the Brunner's glands. It is found in a wide variety of external secretions as well as in blood and amniotic fluid (Marti et al., 1989). The molecular weight of mature human EGF is 6.2 kDa (Carpenter et al., 1991). EGF is phylogenetically strongly conserved and is strongly cross-reactive between different species.

It is known that EGF increases the absorption of H₂O, Na⁺, Cl^" and glucose in a rabbit model (Opleta-Madsen er al. 1991). Moreover, EGF is stimulating the elongation of the villi. This results in an increase of the apical surface and a general increase in absorption of nutrients (Hardin et al., 1999). Absorption of carbohydrates is further facilitated by the EGF-stimulated secretion of pancreatic amylase (Piiper et al., 1994)

Several studies have shown a positive effect of the application of EGF in experimental animal models for Short bowel syndrome (Helmrath et al., 1988; Chaet et al., 1994; O'Loughlin et al., 1994; Swaniker ef al., 1996; Lukish etal., 1997 ; Dunn et al., 1997).

EGF-mediated effects after intestinal resection are strongly dose dependent: up to a certain limit, the adaptation increases with increasing doses. In intestinal studies, the normal dose is situated between 30 and 300μg/kg body weight/day. Systemical as well as enteral application seem effective. However, systemical application may be unwanted for possible side effects: several neoplasmas do have EGF receptors, and a general increase in EGF concentration in the blood might stimulate the formation of tumors. Enteral application of EGF, however, is less efficient as pepsin can process mature EGF into a truncated form that has only 25% of the initial biological activity (Playford et al., 1995).

Surprisingly, we were able to demonstrate that EGF can be delivered in situ by recombinant lactic acid bacteria producing EGF. Efficient production and secretion of EGF by lactic acid bacteria is not evident, and needs optimalization of the coding sequence. Moreover, it can not be forecasted that the lactic acid bacteria sufficiently survive the passage through the stomach to produce the appropriate amount of EGF to stimulate growth of the villi, to promote nutrient absorption and to treat the short bowel syndrome.

It is a first aspect of the invention to provide an EGF producing lactic acid bacterium. Preferably, said lactic acid bacterium is secreting the EGF produced in the growth environment. Preferably, said lactic acid bacterium is a Lactococcus lactis or a Lactobacillus casei. Even more preferably, said lactic acid bacterium comprises SEQ ID N° 1 and/or SEQ ID N° 3. A preferred embodiment is an EGF producing Lactococcus lactis comprising SEQ ID N° 3. Another preferred embodiment is an EGF producing Lactobacillus casei comprising SEQ ID N° 3.

Another aspect of the invention is the use of an EGF producing lactic acid bacterium according to the invention to promote gut absorption. Methods to measure gut absorption are know to the person skilled in the art. Still another aspect of the invention is the use of an EGF producing lactic acid bacterium according to the invention to treat the Short Bowel syndrome. Preferably, the lactic acid bacterium according to the invention is applied orally; it may be treated by any treatment know to the person skilled in the art to improve its survival during the passage of the intestinal system. As a non-limiting example, it may be freeze-dried or spray dried, and/or encapsulated in a suitable recipient so that the bacteria are only released in the small intestine. Encapsulation and treatments for delivery in the small intestine have been described, amongst others in US5972685, WO0018377 and WO0022909. The lactic acid bacterium, according to the invention, may be combined with other compounds, having a positive effect on gut absorption, and/or enhancing the positive effect of EGF. As a non-limited example, glutamine can be used in combination of the lactic acid bacterium according to the invention.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 : Outline of the construction of pTlhEGF. The construction of pTlmEGF is carried out in a similar way.

Figure 2: Expression of mEGF (A) and hEGF (B) in L. lactis and L. casei. Supernatant of the cultures as indicated is separated on a 20% polyacrylamide gel and the proteins are detected using a Western blot

Figure 3: Average villus length of the mice treated with either Lactococcus lactis or

Lactobacillus casei, transformed with the empty vector pTI X (pTI NX), with the vector pTl mEGF, expressing murine EGF (mEGF) or with the vector pTl hEGF expressing human

EGF (hEGF). Medium BM9 treated mice are used as additional negative control (BM9)

EXAMPLES Media and strains

M17 : - 5 g Bacto Tryptone

- 5 g Bacto Soytone

- 5 g Meat Digest

- 2,5 g Yeast Digest

- 0,5 g ascorbic acid

- 0,25 g MgSO₄

- 19 g disodium-β-glycerolphosphate in 1 I deionized H₂0

GM17 : M17 with 0.5% glucose

Recuperation medium: - 1 ml 2 x M17

- 0 .5 ml 2 M sucrose

- 50 μl 20% glucose

- 40 μl 1 M MgCI₂

- 4 μl 1 M CaCI₂

- 406 μl H₂0 Agar medium is obtained by adding 1.2 % agar

BM9 expression medium - 60 g Na₂HPO₄ - 30 g KH₂PO₄,

- 10 g NH₄CI

- 5 g NaCI .

- 50 Mm CO₃-buffer

- 2 mM MgSO₄

- 0,1 mM CaCI₂

- 0,5% casiton (Difco)

- 0,5% glucose in 1 liter H₂O

L lactis MG1363 is a plasmid and prophage free derivative of the L. lactis strain NCDO 712 (Gasson, 1983)

Example 1 : optimising the EGF coding sequence for expression in Lactococcus

Both the murine as well as the human are available in the public databases (http//:www.ncbi. nlm.nih.gov accession number X04571 for hEGF and NM 310113 for mEGF). The coding sequences were adapted to optimize the expression in Lactococcus. On the base of these sequences, primer sets were designed to assemble the optimized coding sequences of both hEGF and mEGF. At the 3' end of the coding sequence, a Spel restricition site was introduced. The primers are shown in table 1 (hEGF) and table 2 (m EGF).

Table 1 : oligo's used for assembly of hEGF, and the amount available

Sense

HEGF01 AACTCAGATTCAGAATGTCCACTTTCACACGATGGTTACT 33,3 nmol

HEGF02 GTTTGCACGATGGTGTTTGTATGTACATCGAAGCTCTTGA 34,8 nmol

HEGF03 TAAATACGCTTGTAACTGTGTTGTTGGTTACATCGGTGAA 26,9 nmol

HEGF04 CGTTGTCAATACCGTGATTTGAAATGGTGGGAACTTCGTT 28,8 nmol

HEGF05 AACTAGTCTGCAGAATCTAG 29,7 nmol

Antisense

HEGF06 CTAGATTCTGCAGACTAGTTAACGAAGTTCCCACCATTTC 31,1 nmol

HEGF07 AAATCACGGTATTGACAACGTTCACCGATGTAACCAACAA 22,5 nmol

HEGF08 CACAGTTACAAGCGTATTTATCAAGAGCTTCGATGTACAT 23,6 nmol

HEGF09 ACAAACACCATCGTGCAAACAGTAACCATCGTGTGAAAGT 28,4 nmol

HEGF 10 GGACATTCTGAATCTGAGTT 37,8 nmol

Table 2 : oligo's used for assembly of mEGF, and the amount available

Sense

MEGF01 AACTCATACCCAGGTTGTCCATCATCATACGATGGTTACT 29,7 nmol

MEGF02 GTTTGAACGGTGGTGTTTGTATGCACATCGAATCACTTGA 28,0 nmol

MEGF03 TTCATACACTTGTAACTGTGTTATCGGTTACTCAGGTGAT 20,0 nmol

MEGF04 CGTTGTCAAACTCGTGATTTGCGTTGGTGGGAACTTCGTT 25,5 nmol

MEGF05 AACTAGTCTGCAGAATCTAG 29,7 nmol

Antisense

MEGF06 CTAGATTCTGCAGACTAGTTAACGAAGTTCCCACCAACGC 33,4 nmol

MEGF07 AAATCACGAGTTTGACAACGATCACCTGAGTAACCGATAA 30,2 nmol MEGF08 CACAGTTACAAGTGTATGAATCAAGTGATTCGATGTGCAT 27,3 nmol MEGF09 ACAAACACCACCGTTCAAACAGTAACCATCGTATGATGAT 26,2 nmol MEGF10 GGACAACCTGGGTATGAGTT 40,3 nmol

The oligonucleotides were dissolved in water at a concentration of 100 μM, and used in a 10 times diluted concentration.

1 μl of each oligonucleotide is added to 10 μl Taq buffer, 8 μl 2mM Mg²⁺, 2 μl 0.5mM XTP, 5u

Taq DNA polymerase (Boehringer, Mannheim, Germany) and 1 u Pfu DNA polymerase

(Promega, Madison, USA). The reaction mixture is added up to 100μl with water. The PCR reaction is carried out for 300 sec at 94°C, followed by 30 times the cycle of 45 sec at 94°C,

30 sec at 48°C and 30 sec at 72°C, with a final step of 10 sec at 15°C. After the assembly hEGF and mEGF are amplified in a PCR mixture containing 1 μl Vent DNA-polymerase (New

England Biolabs ; Beverly, USA), 10 μl Taq buffer, 4 μl 0.5mM XTP, 5 μl 0.5 μM of each primer, 1 μl template DNA, 1 μl 2 mM Mg₂SO₄ and 74 μl H₂O.

In case of hEGF, HEGF01 and HEGF06 were used as primer, for mEGF, MEGF01 and

MEGF06 were used. For hEGF, the same temperature schedule was used as for the first step.

In case of mEGF the hybridization step was carried out at 52°C in stead of 48°C.

After the assembly, the size of the optimized gene fragments was confirmed on a 2% agarose gel.

Example 2: construction of pTl EGF and pTlmEGF and transformation into Lactococcus lactis

Spel cut assembled EGF (both for hEGF and mEGF) is ligated into a Λ/ael and Spel digested pTI NX (Steidler er a/., 1995), resulting in pTl hEGF and pTl mEGF. A schematic overview of the construction of pTl hEGF is shown in figure 1. Plasmids are transformed into competent cells of L. lactis by electroporation. 50μl of cells are electroporated in a precooled cuvet of 2mm, at 25 μF, 2.5 kV and 400 Ω (Bio-Rad electroporator). L. lactis is made competent by growing a 1/100 dilution of a saturated culture, in 200 ml GM17 with 2.5% glycine, till an OD₆oo of 0.5 (Wells er a/. , 1993). After electroporation, 1 ml of recuperation medium is added, and the cells are incubated for 1.5 hour at 28°C. Cells are plated on GM17 solid medium, comprising 5 μg/ml erythromycin.

For the transformation of L. casei, plasmid is isolated from L. lactis by on a Qiagen-tip 100, according to the instructions of the manufacturerer. The DNA is transformed into competent L. casei cells. L. casei cells are made competent by growing an 1/50 dilution of an overnight culture in 50 ml MRS (Oxoid LTD., Basingstoke, Hampshire, England) with 1 % glycine at 37°C, till an OD₆₀o of 0.6. The cells are harvested and washed twice with 10 ml 5 mM Na₃PO₄ pH 7.4, 1 mM MgCI₂, and resuspended in 500 μl electroporation buffer (0.3 M sucrose, 5 mM Na₃PO₄ pH 7.4, 1 mM MgCI₂). 10 μl of DNA is added to 50 μl of competent cells and the electroporation is carried out in a BioRad electroporator. After electroporation, 450 μl MRS is added and the cells are incubated for 2 hours at 37°C. Cells are plated on MRS agar with 5 μg/ml erythromycin. The presence of the plasmid is confirmed using PCR.

Example 3: expression of EGF in L. lactis and L. casei

The transformed L lactis strains MG1363 [pTI NX], MG1363 [pTlmEGF] and MG1363 [pTlhEGF] are pitched in 5 ml GM17 comprising 5 μg/ml erythromycin, and grown overnight at 30°C. This preculture is dilute 1/100 in 5 ml GM17 with erythromycin, and incubated for 3 hours at 28°C. The culture is centrifuged and resuspended in BM9 expression medium, and incubated overnight at 28°C. The transformed L. casei strains are grown under similar conditions, but using MRS as preculture, and BM9 as expression medium. To the culture supernatant, 1/10 volume sodium desoxycholate is added, and the mixture is kept on ice for 10 minutes. 1/10 of volume 100% TCA is added and the mixture is incubated on ice for 15 minutes. After centrifugation, the pellet is dissolved in 50 μl H₂0 and 50 μl 1M Tris-HCI pH 9.5. The proteins are analyzed on a 20% Laemmli protein gel. Detection is carried out using a Western blot, with mouse polyclonal anti hEGF and rabbit anti mEGF as primary antibodies. Alkaline phosphatase labelled anti-mouse and anti-rabbit secondary antibodies were from Southern Biotechnology (Birmingham, USA). The results are summarized in figure 2.

Example 4: in vivo testing of mice, using the transformed lactic acid bacteria strains

In order to assess the effect of the transformed lactic acid bacteria and the growth of the villi and the gut adsorption, seven groups of Balb/c mice (IFFA CREDO CR Broekman/ Sulzfield) were treated either with a mEGF or hEGF expressing lactic acid bacterium strain. L lactis and L casei transformed with an empty vector pTINX, or with BM9 medium was given to mice as a negative control.

600 μl of L casei is pitched in 15 ml MRS with 10 μg/ml erythromycin. In the case of L. lactis, GM17 is used instead of MRS, and only 5 μg/ml erythromycin is used for selection. L. casei is incubated overnight at 37°C, for L. lactis, 30°C is used. The overnight culture is harvested by centrifugation, and the pellet is resuspended in 1.5 ml BM9 expression medium. 100 μl of this solution is supplied daily, for a period of four weeks. At the end of the experiment, the mice are sacrificed and the intestine is isolated. The tissue is fixated in buffered formaldehyde and thin sections are coloured using heamatoxylin and eosin G, for microsopic analyis of the villi. The length of the villi is measured at several points to obtain a representative average. All section were taken from the terminal ileum.

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