WO1999000141A1

WO1999000141A1 - Use of bromelain in the manufacture of a medicament for enhancement of intestinal permeability

Info

Publication number: WO1999000141A1
Application number: PCT/GB1998/001895
Authority: WO
Inventors: Tracey Lehanne Mynott; Alessio Fasano
Original assignee: Provalis Uk Limited; The University Of Maryland At Baltimore
Priority date: 1997-06-27
Filing date: 1998-06-26
Publication date: 1999-01-07
Also published as: AU8225498A; EP0994720A1; GB9713667D0; JP2002511867A

Abstract

Bromelain is capable of enhancing the permeability of the intestine and therefore is able to increase the absorption of proteins such as insulin and other macromolecular biologically active agents.

Description

USE OF BROMELAIN IN THE MANUFACTURE OF A MEDICAMENT FOR ENHANCEMENT C<F INTESΗNAL PERMEABILITY

The present application relates to the enhancement of gut permeability, in particular to increase the absorption of macromolecules from the intestine.

Oral administration of pharmaceutical compounds is very often the preferred route of administration. Patients tend to prefer to take medication orally rather than by other routes, such as intravenous or intramuscular injection and thus the formulation of a medication for oral administration is generally acknowledged to lead to increased patient compliance.

However, problems arise when the agent to be administered is a macromolecule as such molecules are, in general, not easily absorbed from the intestine. As a result, many macromolecules, for example insulin, are currently administered by injection. This is not popular with patients who would greatly prefer oral formulations.

The intestinal epithelium represents the largest interface (more than 2,000,000 cm^) between the external environment and the internal host milieu and constitutes the major barrier through which molecules can either be absorbed or secreted. The intestinal epithelium acts as a barrier which prevents the free mixing of lumenal contents with underlying interstitial and vascular fluids. With the exception of molecules which are transported by active or facilitated transcellular mechanisms, the absorption of large and hydrophilic macromolecules is almost exclusively limited to the paracellular pathway (Madara and Trier, 1986). Under normal conditions, transport of molecules via the paracellular route is restricted to molecules having a molecular radius smaller than 11X (Bakker and Groot, 1989). The major barrier to paracellular transport is the tight junctions, or zonula occludens, which are narrow belt-like structures on the plasma membrane of epithelial cells that circumferentially wraps each cell and adjoin it with its neighbours (Madara, 1988). -The tight junctions have two known functions. First, they assist in the maintenance of surface polarity of the membrane which is required for vectorial transepithelial transport processes, such as salt absorption. Second, they act as a diffusion barrier.

In the past, attempts to increase paracellular transport by loosening tight junctions have been hampered because of unacceptable side effects induced by the absorption enhancing agents (Lee et al , 1991 ; Muranishi, 1990; Hochman and Artursson, 1994; Citi, 1992). In general, these agents fall into two classes, firstly, calcium chelators and, secondly, surfactants (Hochman and Artursson, 1994). Both types of agents have properties which limit their usefulness as agents to promote absorption of molecules. In the case of calcium chelators, Ca^+ depletion induces global changes in the cells, including disruption of actin filaments, disruption of adherent junctions, and diminished cell adhesion (Citi, 1992). In the case of surfactants, the potential lytic nature of these agents may cause exfoliation of the intestinal epithelium which irreversibly compromises its barrier functions (Hochman and Artursson, 1994).

To date, oral drug delivery via the paracellular route remains elusive due to a limited understanding of tight junction physiology and the lack of substances capable of increasing tight junction permeability without irreversibly compromising intestinal integrity and function (Lee et al , 1991; Muranishi, 1990; Hochman and Artursson, 1994; Citi, 1992). Recently however, Fasano and co-workers (1991, 1992) discovered a protein elaborated by the bacterium Vibrio cholerae called Zot (Zonula occluden toxin), which alters cytoskeletal organisation in epithelial cells leading to the opening of tight junctions. Zot's action is mediated by activation of a protein kinase C which leads to polymerisation of actin filaments and their rearrangement, thereby modulating tight junction and intestinal permeability (Fasano et al. , 1995). The action of Zot is reversible, time and dose-dependent, and is confined to the small intestine, since Zot does not affect colon permeability (Fasano, 1991; Uzzau, 1996). Since Zot modulates tight junction activity and the paracellular pathway, Zot may be of use to enhance intestinal absorption of orally administered macromolecules via the paracellular pathway. Indeed, in recent studies, co- administration of Zot with insulin (5733 da) or immunoglobulin G (140-160,000 da) resulted in enhanced absorption of these molecules. The orally administered insulin is biologically active with doses of insulin as low as 10 IU inducing a significant reduction in blood glucose concentration. Insulin administered without Zot had no effect on blood glucose levels. In diabetic rats, orally delivered insulin, when co- administered with Zot, was as efficient as the parentally administered hormone in controlling blood glucose levels (Fasano and Uzzau, 1997). No damage to the small intestine was observed by the Zot treatment as determined by histological examination.

We have now discovered another substance which modulates tight junction activity. We have shown that bromelain from pineapple stems, acts physiologically in a Zot- like manner. In experiments described below, bromelain causes a dose-dependent increase in intestinal permeability, which is readily reversible. Bromelain is therefore of use in a method for increasing the permeability of the intestine.

This effect of increasing the permeability of the intestine means that bromelain is useful in a method of increasing the absorption of large molecules from the intestine.

Therefore, in a first aspect of the invention, there is provided the use of bromelain in the preparation of an orally adminstrable agent for increasing the absorption of a macromolecular biologically active agent from the intestine. The invention is particularly advantageous as enables proteins and other macromolecular biologically active agents to be adminstered by the oral route. This greatly increases the likelihood of patient compliance. An additional advantage is that bromelain is a well known substance which is widely available at relatively low cost and, unlike Zot, is not derived from a pathogenic organism. Furthermore, since the action of bromelain on the tight junctions is reversible, any unwanted side effects can be rapidly and easily treated. Finally, it appears that bromelain does not, as might have been expected, have an adverse effect on nutrient influx.

Bromelain has been reported to increase the absorption of small molecules such as the antibiotics, tetracycline and penicillin (reviewed by Lotz-Winter 1989). The mechanism of action was unknown but it was thought that bromelain increased absorption by a non-specific mechanism such as damage to the intestinal lining. Such a mechanism would suggest that bromelain would not be a suitable agent for pharmaceutical use. Furthermore, since antibiotics are relatively small molecules, it would not have been predicted that bromelain would increase the absorption of macromolecules which are much larger than antibiotics.

The present inventors have made the surprising discovery that bromelain reversibly increases intestinal epithelial permeability without causing damage. It is thus able to increase the absorption of macromolecular biologically active agents as well as the absorption of smaller molecules. Furthermore, because the effect is reversible, it appears that the toxicity of bromelain is much lower than previously believed.

Bromelain is the collective name for the proteolytic enzymes found in the tissues of the plant Bromeliaceae . Bromelain is a mixture of various moieties derived from the stem of the pineapple {Ananas comosus). It contains at least five proteolytic enzymes but also non-proteolytic enzymes, including an acid phosphatase and a peroxidase; it may also contain amylase and cellulase activity. In addition, various other components are present, in particular, organically bound calcium. Bromelain is reviewed by Taussig and Batkin (J. Ethnopharmacol. , 22, 191-202 (1988)). It is available in various countries under the trade marks ANANASE, ANANASE FORTE, EXTRANASE, PORTEOLIS, RESOLVIT, ROGORIN, BROMASE and TRAUMANASE.

In the context of the present invention, the term "macromolecular biologically active agent" refers to any molecule having biological activity and which is a protein, glycoprotein, oligo- or polypeptide, polynucleotide, for example DNA or RNA, a polysaccharide or other large molecule having a molecular radius greater than 11X.

Specific examples of macromolecular biologically active agents which may be administered orally with bromelain in order to improve their absorption include proteins and peptides, for example Insulin glucagon, parathyroid hormone and its antagonists, calcitonin, vasopressin, renin, prolactin, growth hormones, thyroid stimulating hormone, carticitropin, follicle stimulating hormone, luteinising hormone, chorionic gonadotropin, interferon, tissue plasminogen activator, gamaglobulin and blood clotting factors such as Factor VIII.

In one preferred embodiment the macromolecular biologically active agent is insulin.

Other categories of active molecule which may be administered with bromelain are physiologically active enzymes such as transferases, hydrolases, isomerases, proteases, ligases and oxidoreductases such as esterases, phosphatases, glycosidases and peptidases and enzyme inhibitors such as leupeptin, chymostatin and pepstatin and growth factors such as tumour antiogenesis factor , epidermal growth factor, nerve growth factor and insulin-like growth factors. In addition, the macromolecule may be an antibody or a vaccine which Ωiay be a proteinaceous vaccine or even an attenuated organism.

The bromelain and the macromolecular biologically active agent may be administered together or separately and therefore in a further aspect of the invention, there is provided a product comprising bromelain and a macromolecular biologically active agent as a combined oral preparation for simultaneous, separate or sequential use in the treatment of a condition for which the macromolecular biologically active agent is a therapeutic agent.

The invention also provides a pharmaceutical composition for oral administration comprising bromelain together with a macromolecular biologically active agent and a pharmaceutically acceptable excipient or carrier.

The bromelain may be provided either together with or separately from the biologically active agent in any formulation which is suitable for oral administration.

Such formulations include syrups, elixirs, tablets and capsules and the preparation of all of these types of formulation is familiar to those skilled in the art of formulation.

If the biologically active material is included with the bromelain, the formulation may take the form of an emulsion, microemulsion, or micellar or liposomal solution.

In order to assist the survival of the various components of the bromelain mixture through the stomach, it may be advisable to formulate the bromelain in an enteric- protected preparation. Enteric-coated tablets of bromelain are available (for example under the trade mark ANANASE FORTE in the United Kingdom). Other orally adminstrable formulations include syrups, elixirs and hard and soft gelatin capsules, which may also be enteric coated. As already suggested above, this preparation may also include the biologically active agent or, alternatively, the biologically active agent may be administered separately or sequentially. Bromelain activity is stable over a wide pH range (pH 2-9). Therefore, it may not be necessary to enteric-protect (or enteric coat) the bromelain from the acid conditions of the stomach. It may, however, be necessary to protect the bromelain from digestion by acid proteases in the gut. Bromelain may therefore be administered with a buffering agent, for example bicarbonate.

Dosage of bromelain is conventionally measured in Rorer units, FIP units, BTU (bromelain tyrosine units), CDU (casein digestion units), GDU (gelatin digestion units) or MCU (milk clotting units). One Rorer unit of protease activity is defined as that amount of enzyme which hydrolyses a standardisation casein substrate at pH 7 and 25°C so as to cause an increase in absorbence of 0.00001 per minute at 280nm. One FIP unit of bromelain activity is contained in that amount of a standard preparation which hydrolyses a suitable preparation (FIP controlled) under the standard conditions at an initial rate such that there is liberated per minute an amount of peptide, not precipitated by a specific protein precipitation reagent, which gives the same absorbence as 1 :mol of tyrosine at 275nm. BTUs, CDUs, GDUs and MCUs are as defined in the literature as follows:

BTU

One bromelain tyrosine unit is that amount of enzyme which will liberate one micromole of tyrosine per minute under the conditions of the assay (for example, after digestion of an acid denatured haemoglobin substrate at pH 5 and 30°C).

CDU

That amount of enzyme which will liberate one microgram of tyrosine after one minute digestion at 37°C from a standard casein substrate at pH 7.0. GDU

The enzyme activity which liberates one milligram (10³g) of amino nitrogen from a standard gelatin solution after 20 minutes digestion at 45°C and at pH 4.5.

1100 BTU/g = 750 CDU/mg = 1200 GDU/g.

While the precise dosage will be under the control of the physician or clinician, it may be found that daily dosages of from 50 to 4000 GDU/day is appropriate, for example from 100 10 1000 GDU/day. The precise dosage pattern will be dependent upon the dosage pattern of the macromolecular biologically active agent since the bromelain will be administered at a time such that the permeability of the intestine will be increased at the appropriate time for the biologically active agent to be absorbed.

As already mentioned above, one of the particular advantages of the present invention is that the increased intestinal permeability induced by bromelain is easy to reverse. This may be brought about by the administration to the patient of an anti- bromelain antibody. This antibody will usually be of the IgG isotype and may be raised in a conventional manner, for example by injecting rabbits with bromelain and extracting the antibody from them at an appropriate time thereafter.

Thus, in a further aspect of the invention, there is provided the use of an anti- bromelain antibody in the preparation of an agent for reversing increased intestinal permeability induced by bromelain. Although the effectiveness of the invention is not dependent upon the correctness or otherwise of this theory, the mechanism of action of bromelain, is thought to be mediated by its ability to modulate the activity of intracellular signalling pathways. Physiological regulation of the tight junctions and barrier function are influenced by second messengers and signalling pathways that control the assembly and disassembly of the actin cytoskeleton. Members of the Rho GTPase family, tyrosine kinases, Ca²⁺, protein kinase C, adenosine 3 ' ,5 '-cyclic monophosphate (cAMP) and phospholipase C appear to be key players in controlling actin cytoskeleton organisation (Anderson and Van Itallie, 1995; Tapon and Hall, 1997). Earlier, we have shown that bromelain prevents the effects of the intracellular second messengers, cAMP, cGMP and Ca²⁺ (Mynott et al , 1997). Mynott and Engwerda (1996) have also shown that bromelain prevents activation of the MAP kinase pathway, which is activated by Rac and Cdc42, members of the Rho GTPase family, which as mentioned earlier, are among key molecules that control the organisation of the actin cytoskeleton.

The invention will be described in greater detail with reference to the following example and the drawings in which:

FIGURE 1 shows the effect of bromelain on rabbit ileal Isc. Bromelain ( ) (15

:g/ml), PBS (N) or bromelain pre-incubated with specific anti-bromelain antibodies

(♦) were added to the mucosal and serosal bathing solution at time zero. Symbols with bars represent the mean + SE of n=9 tissue pairs. P reveals significance as determined by paired t-test.

FIGURE 2 shows the effect of bromelain on rabbit ileal Rt. (A) Effect of dose of bromelain on Rt. Columns with bars represent the mean change in Rt + SE of n=4 tissue pairs, 60 min after addition of bromelain. P value reveals significance as determined by ANOVA. (B) Effect of neutralization of bromelain with anti- bromelain antibodies and removal of bromelain on Rt. Bromelain ( ) (15 :g/ml), PBS (N) or bromelain pre-incubated for 30 min with specific anti-bromelain antibodies (♦) were added to the mucosal and serosal bathing solution at time zero. Symbols with bars represent the mean + SE of n=4 tissue pairs. P reveals significance as determined by paired t-test.

EXAMPLE

MATERIALS AND METHODS Reagents.

leucine, lysine, glutamic acid, glycine- phenylalanine and ^H-labelled inulin were purchased from Amersham (Milan, Italy). Proteolytic activity is measured in Bromelain Tyrosine Units (BTUs); 1 BTU is the amount of enzyme that will liberate 1 micromole of tyrosine after one minute digestion at 37°C of a standard casein substrate at pH 7.0. Crude bromelain extract (proteolytic activity, 1877 BTU/gl) was obtained from Miles Scientific (Elkhart,

IN).

Ussing Chamber Experiments. Experiments were conducted as previously described (Field et al , 1971), with modifications described by Fasano et al. (1991). Approval for the study was granted by the University of Maryland at Baltimore

Institutional Animal Care and Use Committee. Male New Zealand White rabbits (2 to 3 kg) were obtained from Charles River Breeding Laboratories (Wilmington, MA). Animals were killed by cervical dislocation and a 15 cm segment of distal ileum was quickly excised and cut open along the mesenteric border. The ileum was rinsed clean of luminal contents and stripped of muscular and serosal layers by means of blunt dissection. Sections of mucosa, prepared from a single animal, were mounted in Lucite Ussing Chambers of aperture 1.12 cm^ (World Precision Instruments, Sarasota, FL). Each surface of the tissue was continually bathed (10 ml per surface) in freshly prepared Ringer's solution containing NaCl (53 mM), KC1 (5 mM), Na₂SO₄ (30.5 mM), mannitol (30.5 mM), Na₂HPO₄ (1.69 mM), NaH₂PO₄

(0.3 mM), CaCl₂ (1.25 mM), MgCl₂ (1.1 mM) and NaHCO₃ (25 mM). The

bathing solution was maintained at 37°C with water-jacketed reservoirs connected to a constant-temperature circulating pump and gassed with 95 % O₂/5 % CO₂. The potential difference (difference in voltage between the mucosal and serosal sides of the tissue, PD) was then measured under open-circuited conditions. The increase in voltage resulting from the passage of 100 :A current was used to calculate the short circuit current (amount of current needed to nullify the PD, I_sc) and tissue resistance

(Rt) from Ohm's Law (I_SC = PD/R_t) (Field et al. , 1971). Before the tissue was mounted, the fluid resistance was determined and incorporated into the calculations. After mounting the tissue and prior to experimentation, the I_sc was determined at approximately 10 min intervals, until a steady state was reached. Four or eight sections of tissue from each animal were mounted simultaneously and used for each experiment. To test for minute perforations of the tissue prior to experimentation, 0.01 M glucose and 0.01 M mannitol, diluted in Ringer's solution, were added to the serosal and mucosal sides, respectively. Tissues were discarded if they showed an increase in I_sc, reflective of Na-glucose co-transport, because serosally added glucose diffused via perforations to the mucosal surface. All experiments were conducted on tissues of similar resistance (_+ 10%).

Effect of Bromelain on Ileal I_sc and R_j When tissue reached a steady state, bromelain (15 μg/ml) diluted in phosphate-buffered saline (0.1 M, pH 7.4, PBS) or PBS alone as a control, was added to both the mucosal and serosal surfaces of mounted tissues. Variations in I_sc, PD and R_t were then recorded every 5 to 10 min for 2 h. Neutralization of Bromelain Activity. Antibody (immunoglobulin [Ig] G) raised against bromelain was used to block its inhibitory effect on I_sc in Ussing chamber experiments. Rabbits were given three injections of 60 :g of bromelain subcutaneously at intervals of four weeks. For all immunizations, bromelain was emulsified with Freund's incomplete adjuvant. The animals were bled two weeks after the final immunization. The IgG fraction was prepared by protein A affinity chromatography with protein A-Sepharose CL-4B (Pharmacia) as specified by the manufacturer. Anti-bromelain IgG was diluted 1: 100 in PBS and pre-incubated with an equal volume of bromelain for 30 min at 37°C prior to treatment of rabbit ileum in Ussing chambers. Control chambers were incubated with antibody alone.

Influx Experiments. Influx experiments were conducted as previously described (Guandalini et al , 1988). Male New Zealand White rabbits (2 to 3 kg) were killed by cervical dislocation. A 25 cm segment of distal ileum was excised, opened along the mesenteric border and rinsed free of intestinal contents with cold Ringer solution. Two 10 cm segments of intestine were rapidly mounted in Lucite influx chambers, where four adjacent portions of the mucosal side (surface area of 0.28 cm^) were exposed to the pre-incubation solution for 30 min at 37°C and gassed with 95 % O₂/5% CO₂. The pre-incubation solutions contained bromelain at 1 mg/ml for studies observing glucose influx, and 15 μg/ml for amino acid and dipeptide influx. Control tissues were incubated with Ringer solution alone. Studies commenced when the pre-incubation solution was replaced with Ringer's containing ^ -labelled nutrient and - Inulin as a marker of the extracellular space. Incubation in this solution was for 45-50 sec and stopped by quickly removing the solutions containing label and adding cold 0.3 M mannitol. Each piece of exposed tissue was then excised, gently blotted on filter paper, homogenized in 10% trichloroacetic acid and centrifuged to sediment particulate matter. Aliquots of the supernatants were assayed for radioactivity with Hpb Beckman scintillation fluid (Milan, Italy), in a Beckman LS 7500 Beta-counter. Calculations were conducted as previously described (Rubino et al. , 1971).

Data Analysis. For each experiment, data from bromelain-treated tissue was compared with its paired PBS control. Paired t-tests (Student's t test; 2 way) were used to test for differences among means in I_sc, R_t and PD responses and for testing differences in nutrient uptake. Analysis of variance (ANOVA) was used when comparing the mean of more than one treatment against PBS controls. Numerical data showing I_sc and R_t values are expressed as mean + standard error. Because changes in PD followed changes in the I_sc, only changes in I_sc are shown.

RESULTS Effect of Bromelain on I_sc and R_t. Rabbit ileum mounted in Ussing chambers were treated on both the mucosal and serosal surface with bromelain (15 :g/ml). Bromelain addition caused a transient increase in I_sc which returned to approximate baseline values 30 min after its addition (Figure 1). The I_sc response could be prevented when bromelain was pre-incubated with anti-bromelain IgG. The ability of antibody to neutralise the effect of bromelain on I_sc would suggest that the

transient increase in I_sc was not a result of co-transport of Na⁺ and nutrients that may have been present in the crude bromelain extract.

Bromelain addition also caused a reduction in R_t in a dose-dependent manner (Figure 2A). The reduction in R_t was readily reversible; removal of bromelain was followed by an increase in R_t (Figure 2B) to baseline values. In control tissues, -or tissues incubated with bromelain plus anti-bromelain IgG, no change in R_t was observed.

Intestinal Influx of Glucose and Amino Acids. Because of the potential for damage to the small intestine from the proteolytic activity of bromelain, we investigated its effect on nutrient influx (initial rate of unidirectional flux of nutrient from the incubation medium into mucosal cells). We tested the influx of glucose, glutamic acid, lysine, leucine and the di-peptide, glycine-phenylalanine in the presence and absence of bromelain.

Influx studies indicated that there was no interference with glucose and amino acid influx, indicating no adverse effects on nutrient carriers important for nutrient uptake (see Table 1).

The affinity of glucose (K_t) for intestinal mucosa and the maximal influx (V_maχ)

(Rubino et al , 1971) of glucose into mucosal cells were unaffected by bromelain (Table 1). Although the difference between bromelain-treated and untreated tissues is not significant, it is noteworthy that there is a trend towards increased influx of nutrients in bromelain-treated tissues. Electron microscopy and histological examination of tissue treated with bromelain (15 :g/ml) revealed no evidence of tissue damage (Mynott, 1993).

Table 1. Influence of bromelain treatment of rabbit ileum on nutrient influx.

NOTE: Data (:mols/cm^.hr) are means ± SE for n tissue pairs. Difference between means is not significant (paired t-test).

^Concentration of bromelain used to treat cells for amino acid influx and glucose influx experiments was 15 :g/ml and 1 mg/ml respectively.

Gly-Phe = glycine-phenylalanine; Glut. Ac. = glutamic acid.

DISCUSSION

Bromelain treatment of intestinal epithelium increased intestinal permeability in a dose-dependent manner, which was reversed when bromelain was removed.

Variation in transepithelial resistance reflects modification of tissue permeability through the intercellular space, since plasma membrane resistances are relatively high (Madara, 1989). Since tight junctions represent the major barrier of the paracellular pathway, it is possible that bromelain is modulating tight junctions. This effect is intriguing and is reminiscent of a Zot-like activity (Fasano et al , 1991) whereby an increase in transepithelial permeability by Zot coincided with modification of the structure of intercellular tight junctions. The time course of Zot and bromelain effects are similar.

We also noted that bromelain caused a transient increase in I_sc in rabbit ileum, which could also be neutralised with specific antibodies. The effect of bromelain on tissue I_sc and R_t may be modulated by Ca^{2 +} . Agents such as thapsigargin, calcium ionophores and carbachol cause a transient increase in I_sc in tissues, which return to baseline values after 20 to 30 mins (Dharmsathaphorn and Pandol, 1986; Traynor- Kaplan et al , 1994). An increase in tissue permeability is also associated with Ca- agonist action. Increased intracellular concentrations of Ca^ + leads to increased intestinal permeability via rearrangement of actin filaments of the cytoskeleton and reassembly of tight junctions (Anderson and Van Itallie, 1995). The effect of bromelain may also be exerted by effects on MAP kinase. Earlier, Mynott and Engwerda (1996) showed that bromelain inhibits tyrosine phosphorylation and modulation of MAP kinase activity. MAP kinase is activated by members of the Rho GTPase family, which are among key molecules that control cytoskeleton arrangement. Therefore, it is possible that bromelain modulates tight junction activity via an effect on the MAP kinase pathway and/or by activating ^~a Ca^{2 +} signalling pathway.

Bromelain did not have an adverse effect on nutrient influx suggesting that the use of this substance is safe. Indeed, bromelain has been in clinical use for many years with indications that include uses as an anti-inflammatory agent and for the debridement of third-degree burns (reviewed by Taussig and Batkin, 1988).

The ability of bromelain to modulate intracellular signalling pathways linked to tight junctional activity and its ability to increase intestinal permeability suggests that bromelain will be a useful agent to enhance the absorption of macromolecules.

REFERENCES

1. Anderson JM, Van Itallie CM. Tight junctions and the molecular basis for regulation of paracellular permeability. Am J Physiol 1995;269 (Gastrointest Liver Physiol 32):G467-G475.

2. Bakker R, and Groot JA. Further evidence for the regulation of the tight junction ion selectivity by cAMP in goldfish intestinal mucosa. J Membr Biol

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3. Baudry B, Fasano A, Ketley JM, Kaper JB. Cloning of a gene (zot) encoding a new toxin produced by Vibrio cholerae. Infect Immun 1992;60:428-434.

4. Citi S. Protein kinase inhibitors prevent junction dissociation induced by low extracellular calcium in MDCK epithelial cells. J Cell Biol 1992; 117:169-178.

5. Dharmsathaphorn K, Pandol SJ. Mechanism of chloride secretion induced by carbachol in a colonic epithelial cell line. J Clin Invest 1986;77:348-354. 6. Fasano A, Baudry B, Pumplin DW, Wasserman SS, Tall BD, Ketley JM, Kaper JB. Vibrio cholerae produces a second enterotoxin, which affects intestinal tight junctions. Proc Natl Acad Sci USA 1991 ;88:5242-5246.

7. Fasano A, Fiorentini C, Donelli G, Uzzau S, Kaper JB, Margaretten K, Ding X,

Guandalini S, Comstock L, Goldblum SE. Zonula occludens toxin modulates tight junctions through protein kinase C-dependent actin reorganisation, in vitro. J Clin Invest 1995;96:710-720.

8. Fasano A, Uzzau S. Modulation of intestinal tight junctions by zonula occludens toxin permits enteral administration of insulin and other macromolecules in an animal model. J Clin Invest 1997 in press.

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10. Guandalini S, Fasano A, Albibi F, Marchesano G, Nocerino A, De Curtis M, Rubaltelli FF, Pettenazzo A, Rubino A. Unconjugated bilirubin and the bile from light exposed Gunn rats inhibit intestinal water and electrolyte absorption. Gut 1988;29:366-371.

11. Hochman J, Artursson P. Mechanisms of absorption enhancement and tight junction regulation. J Controlled Release 1994;29:253-267.

12. Lee VHL, Yamamoto A, Kompella VB. Mucosal penetration enhancers for facilitation of peptide and protein drug absorption. Crit Rev Ther Drug Carrier

Sys 1991 ;8:91-192.

13. Lotz- Winter H. On the pharmacology of bromelain: An update with special regard to animal studies on dose-dependent effects. Planta Med 1990:56;249- 253. 14. Madara JL. Tight junction dynamics: Is paracellular transport regulated? Cell 1988;53:497-498.

15. Madara JL. Loosening tight junctions: Lessons from the intestine. J Clin Invest

1989;83:1089-1094.

16. Madara JL, Trier JS. Functional morphology of the mucosa of the small intestine. In: Physiology of the Gastrointestinal Tract, L.R. Johnson (ed). Raven Press, New York. 1987; 1209-1250.

17. Muranishi S. Absorption enhancers. Crit Rev Ther Drug Carrier Sys 1990;7: 1- 33.

18. Mynott, TL. Protease and prevention of disease caused by enterotoxigenic Escherichia coli. Ph.D thesis, La Trobe University, Australia.

19. Mynott TL and Engwerda CR. Medical use of bromelain. International Patent Application PCT/GB95/01501

20. Mynott TL, Guandalini S, Raimondi F, Fasano A. Bromelain prevents secretion caused by Vibrio cholerae and Escherichia coli enterotoxins in rabbit ileum in vitro. Gastroenterol 1997 in press

21. Rubino A, Field M, Schwachmann H. Intestine transport of amino acid residues of dipeptides. I Influx of the glycine residues of Glcyl-L Proline across mucosal border. J Biol Chem 1971;246:3542-3548.

22. Tapon N, Hall A. Rho, Rac and Cdc42 GTPases regulate the organisation of the actin cytoskeleton. 1997;9:86-92. 23. Taussig SJ, Batkin S. Bromelain, the enzyme complex of pineapple-(^«a«^ comosus) and its clinical application. An update. J Ethnopharmacol 1988;22:191-203.

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Claims

1. The use of bromelain in the preparation of an orally adminstrable agent for increasing the absorption of a macromolecular biologically active agent from the intestine.

2. A product comprising bromelain and a macromolecular biologically active agent as a combined oral preparation for simultaneous, separate or sequential use in the treatment of a condition for which the macromolecular biologically active agent is a therapeutic agent.

3. A pharmaceutical composition for oral administration comprising bromelain together with a macromolecular biologically active agent and a pharmaceutically acceptable excipient or carrier.

4. A product as claimed in claim 2 or a composition as claimed in claim 3 which is enteric coated.

5. A product as claimed in claim 2 or a composition as claimed in claim 3 which is a syrup, elixir or a hard or soft gelatin capsule.

6. The use as claimed in claim 1 or a product or composition as claimed in any one of claims 2 to 5 wherein the daily dosage of bromelain is from 50 to 4000 GDU/day.

7. The use, product or composition as claimed in claim 6, wherein the daily dosage of bromelain is from 100 to 1000 GDU/day.

8. The use, product or composition as claimed in claim 7 wherein the macromolecular biologically active agent is insulin.

9. The use of an anti-bromelain antibody in the preparation of an agent for reversing increased intestinal permeability induced by bromelain.

AMENDED CLAIMS

[received by the International Bureau on 24 November 1998 (24.11.98); original claims 2 and 3 amended; remaining claims unchanged (2 pages)]

2. A product comprising bromelain and a macromolecular biologically active agent as a combined oral preparation for simultaneous, separate or sequential use in the treatment of a condition for which the macromolecular biologically active agent is a therapeutic agent; wherein the macromolecular biologically active agent is selected from:

insulin, glucagon, parathyroid hormone and its antagonists, calcitonin. vasopressin, rerun, prolactin, growth hormones, thyroid stimulating hormone, caiticitropin, follicle stimulating hormone, luteinising hormone, chorionic gonadotropin, interferon, tissue plasminogen activator, gamaglobulin and blood clotting factors such as Factor VIII; physiologically active enzymes selected from transferases, hydrolases, isomerases, ligases and oxidoreductases such as esterases, phosphatases, glycosidases and enzyme inhibitors such as leupeptin. chymostatin and pepstatin and growth factors such as tumour antiogenesis factor, epidermal growth factor, nerve growth factor and insulinlike growth factors: and antibodies.

3. A pharmaceutical composition for oral adπnnistration comprising bromelain together with a macromolecular biologically active agent and a pharmaceutically acceptable excipient or carrier, wherein the macromolecular biologically active agent is selected from: insulin, glucagon, parathyroid hormone and its antagonists, calcitonin, vasopressin, renin, prolactin, growth hormones, thyroid stimulating hormone, carticitropin, follicle stimulating hormone, luteinising hormone, chorionic gonadotropin, interferon, tissue plasminogen activator, gamaglobulin and blood clotting factors such as Factor VTII; physiologically active enzymes selected from transferases, hydrolases, isomerases, ligases and oxidoreductases such as esterases, phosphatases, glycosidases and enzyme inhibitors such as leupeptin, chymostatin and pepstatin and growth factors such as tumour antiogenesis factor, epidermal growth factor, nerve growth factor and insulinlike growth factors; and antibodies.