WO2011005756A1 - Delivery of agents targeted to microbiota niches - Google Patents

Abstract

Methods for delivery of therapeutic, prophylactic, and diagnostic agents to an animal host, wherein the delivery of the agent is targeted at a bacterial niche present in the host, have been developed. In particular, delivery systems capable of selectively targeting or selectively avoiding bacterial niches, wherein the bacterial niches are identified by the functional role they serve in the host environment, such as metabolism of sugars, amino acids, or xenobiotics, or by their colonization of a given anatomical location of the host, such as the oral cavity, the gut, or the skin, are described. Use of the compositions results in an alteration of a host's microbial composition associated with a condition, a disease state, or a higher risk for a condition or disease.

Description

DELIVERY OF AGENTS TARGETED TO MICROBIOTA NICHES FIELD OF THE INVENTION

The present invention is generally in the field of drug delivery and relates in particular to methods and compositions for delivery of molecules and organisms to specific bacterial niches with the purpose of modulating or diagnosing a subject's microbiota.

BACKGROUND OF THE INVENTION

Animals, including humans, host multitude of microbes (collectively referred to as the host's microbiota) in anatomical locations including the mouth, esophagus, stomach, small intestine, large intestine, caecum, colon, rectum, vagina, skin, nasal cavities, ear, eyes, and lungs. These locations offer environments with varying conditions of pH, redox potential, presence of host secretions, and contact with the immune system, among other factors, where intense competition among bacteria leads to specialization in certain functional roles. Furthermore, the host exerts selective pressure for functional redundancy to prevent against the loss of key functions. As a result, groups of bacterial commensals that share a specialization or function are established. These can be generally referred to as functional niches.

Elucidation of the functional roles of such niches has been the focus of recent research, which has established that, collectively, the human microbiota is responsible for a multitude of critical processes, including metabolism of carbohydrates and proteins, maturation of the immune system, formation and regeneration of the epithelium, fat storage, production of hormones, metabolism of xenobiotics, production of vitamins, and protection from pathogen infections, among others (Hooper et al. Science. 2001 ;292: 1115; Rakoff-Nahoum et al. Cell. 2004 118:229; Backhed et al. Proc. Natl. Acad. Sci. U.S.A. 2004;101: 15718; Stappenbeck et al. Proc. Natl. Acad. Sci.

U.S.A. 2002;99: 15451;! Sonnenburg et al. Nat. Immunol. 2004;5:569;

Hooper, et al. Science. 2001;291:881)

Despite the key importance of the microbiota, there is a lack of approaches to modulate it, and the few approaches available are inadequate. Interventions known to modulate the microbiota are limited to antibiotics, prebiotics, probiotics, and synbiotics. Antibiotics generally eradicate the microbiota indiscriminately as a byproduct of targeting an infectious pathogen. In contrast, nutritional approaches involving live organisms (probiotics), non-digestible food ingredients that stimulate the growth or activity of bacteria (prebiotics), or combinations of both (synbiotics), are more benign but exert a moderate beneficial effect on the host. Antibiotics have been formulated for oral delivery with a focus on promoting rapid release in the small intestine, where most absorption occurs, or with a focus on extending retention in the gut, by using a number of delivery carriers. In either case, altering the microbiota has not been a goal, but rather an undesired consequence of the use of antibiotics, and no delivery systems have been developed to selectively target microbiota functional niches. Prebiotics and probiotics have been formulated in a number of delivery carriers, the main goal generally being ensuring stability to temperature and humidity during storage and to pH and proteolysis during passage through the stomach and intestine. None of the delivery systems developed has contemplated the possibility of targeting the cargo to a specific functional niche, and they have been relatively crude at ensuring delivery to the desired anatomical region. Other therapeutic modalities that could be envisioned as microbiota modulators, such as non-antibiotic small molecule modulators, biologies, DNA or RNA-based agents, etc., have not been developed, partly due to the lack of delivery systems for targeting the interventions to a desired bacterial niche.

It is therefore an object of the present invention to provide formulations suitable for specific targeting of interventions and diagnostic agents to relevant bacterial niches in a host, which enable efficient modulation of microbial imbalances and characterization of conditions associated with imbalances, respectively.

SUMMARY OF THE INVENTION

Methods and compositions for delivering interventions and diagnostic agents to bacterial niches of a host while avoiding other bacterial populations have been developed. A general method of delivering a pharmaceutical formulation to a bacterial niche of an animal host includes the steps of: first selecting a feature that differentiates the bacterial constituents of the niche from the rest of bacterial residents that coexist in the same anatomical location (such as the ability to perform a certain chemical reaction or the presence of differentially expressed surface molecules), and second, providing a delivery system that exploits the feature to release a pharmaceutical formulation preferentially in the vicinity of the constituents of a bacterial niche.

In one aspect, the delivery systems are tailored to deliver a range of active ingredients, including small molecules, proteins, nucleic acid, carbohydrates, lipids, and full organisms. In another aspect, the delivery systems target anatomical locations of the host which have widely dissimilar microbiota compositions, such as different sections of the gut, the oral cavity, the skin, the vagina, the nasal cavities, the ear, and the lungs. In another aspect, the delivery systems target regions and structures within the anatomical locations that have dissimilar local microbiota compositions (such as the hair follicle, the sweat glands, and the sebaceous glands in the case of skin).

In another aspect, the delivery systems target bacterial niches based on the functions that such niches perform. Target niches may be specialized in a number of functions, including metabolism of carbohydrates, metabolism of proteins, metabolism of xenobiotics, production of vitamins, or maturation of the immune system.

In another aspect, the delivery systems are triggered by differential properties of a bacterial niche, which cause the preferential release of a therapeutic, prophylactic, or diagnostic agent in the vicinity of the niche. For example, the delivery systems may be triggered by hydrolytic and reductive reactions that are preferentially performed by certain bacterial niches, such as nitro- and azo- group reductions, azo bond cleavage, sulfoxide reduction, reductive deamination, dehydroxylation, and hydrolysis reactions. The compositions may include synthetic and natural scaffolds that can be selectively degraded by the action of enzymes performing the reactions listed above, thus triggering the release of a cargo. The delivery systems may enable targeting to relevant members of a bacterial niche, including phyla relevant in the human microbiota, such as the Bacteroidetes, and the

Firmicutes, genus such as Bacteroides, Bifidobacterium, and Lactobacillus, and species, such as Bacteroides thetaiotaomicron or Clostridium

perfringens. In another aspect, the delivery systems are triggered by local changes in pH_s by pressure, or after certain lag times.

In another aspect, the invention relates to characterizing the bacterial populations of a host in order to identify differential properties and signatures of a bacterial niche that can be exploited to target the delivery of a drug to the niches. Delivery systems are then designed that can be triggered to release a drug upon exposure to the differential property. The differential properties are identified using methods to profile the microbiota including (i) screening of 16srRNA genes by PCR; and (ϋ) high-throughput

"metagenomic" sequencing methods such as pyrosequencing, which identify genes that are either over-represented or under-represented in the bacterial population.

In another aspect, the targeted delivery of a microbiota modulator to a human in need of a microbiota-modulating therapy (i) modulates a pathway involved in functions involved in metabolism of carbohydrates and proteins, maturation of the immune system, formation and regeneration of the epithelium, fat storage, production of hormones, metabolism of xenobiotics, production of vitamins, and protection from pathogen infections, among others (ii) promotes or prevents the growth or activity of a bacteria under- represented in a disease or over-represented in a disease, respectively

In yet another aspect, the general method delivers a pharmaceutical formulation away from a bacterial niche. The first step is to select a feature substantially lacking in the bacterial constituents of the niche compared to other bacterial residents that coexist in the same anatomical location (such as the ability to perform a certain chemical reaction or the presence of differentially expressed surface antigens). Second, a delivery system is provided that exploits the lack of such feature to release a pharmaceutical formulation preferentially in the vicinity of the constituents of a bacterial niche.

In yet another aspect, a method increases or decreases the

bioavailability of a drug metabolite generated by a microbial niche by selectively delivering the drug precursor to a certain niche. Delivery systems are presented that exploit a differentiating feature of a niche (e.g. the ability to perform a chemical reaction or the presence of certain surface antigens) to target a drug to a niche that then converts this drug to an active metabolite. Delivery systems are also presented that exploit the substantial lack of a feature to target a drug away from a certain niche, thus avoiding the formation of an undesired metabolite, such as a toxic bacterial metabolite.

DETAILED DESCRIPTION

I. Definitions:

The term "microbiota" refers, collectively, to the entirety of microbes found in association with a higher organism, such as a human.

Organisms belonging to a human's microbiota may generally be categorized as bacteria, archaea, yeasts, and single-celled eukaryotes, as wells as various parasites such as Helminths.

The term "commensal" refers to organisms that are normally harmless to a host, and can also establish mutualistic relations with the host. The human body contains about 100 trillion commensal organisms, which have been suggested to outnumber human cells by a factor to 10.

The term "anatomical niche" describes a group of organisms, such as microbes, that populate a region of a host, such as the gut, the oral cavity, the vagina, the skin, the nasal cavities, the ear, or the lungs. The term may also refer to a structure or sub-region within any of these regions, such as a hair follicle or a sebaceous gland in the skin.

The term "functional niche" describes a group of organisms, such as microbes, that specialize in a certain function, such as carbohydrate metabolism or xenobiotϊc metabolism.

The term "modulating" as used in the phrase "modulating a microbial niche" is to be construed in its broadest interpretation to mean a change in the representation of microbes in a bacterial niche of a subject. The change may be an increase or a decrease in the presence of a particular species, genus, family, order, class, or phylum. The change may also be an increase or a decrease in the activity of an organism or a component of an organism, such as a bacterial enzyme, a bacterial antigen, a bacterial signaling molecule, or a bacterial metabolite.

The term "metagenomics" refers to genomic techniques for the study of communities of microbial organisms directly in their natural

environments, without requiring isolation and lab cultivation of individual species.

The term "probiotic" refers to a dietary supplement of live microorganisms thought to be healthy for the host organism

The term "prebiotic" refers to non-digestible dietary components that can help foster the growth of microorganisms, which may lead to better health

The term "synbiøtic" refers to a supplement that contains both a prebiotic and a probiotic .. -.

The terms "targeting" or "targeted", as used in the phrase "targeted delivery" refers to methods of delivery of a cargo, such as a drug, to a patient in a manner that increases the concentration of the cargo in certain zones of the body relative to others

The terms "pharmaceutical formulation" and "pharmaceutical composition" refer to any composition which is suitable for administration to a human or other mammal, which comprises at least one bioactive ingredient (such as a drug, a probiotic, a prebiotic, a nutritional supplement, a botanical, etc.). Additionally, the formulation may comprise non-bioactive ingredients, such as excipients, fillers, and carrier materials. II. Method of Targeting Delivery System

A. Microbial Niches to be Targeted

Functional Niches to be Targeted and Factors Differentiating the Niches

The functional niches targeted by the delivery systems may be specialized in a number of functions, including metabolism of carbohydrates and proteins, maturation of the immune system, formation and regeneration of the epithelium, fat storage, production of hormones, metabolism of xenobiotics, production of vitamins, and protection from pathogen infections, among others

Features that allow specific targeting

Characteristics that differentiate a functional niche from the rest of commensals that may coexist in the same anatomical location may include the presence and concentration of certain microbial surface molecules. These molecules may be proteins, carbohydrates ,and lipids, and may function as receptors, adhesins, transporters, complex cell surface structures, and virulence factors. In a preferred embodiment, the surface molecule that differentiates the functional niche is expressed in high concentrations by members of the niche, and is lacking or substantially under-represented in other members of the microbiota. In one embodiment, the surface molecule is an antigen against which the host has developed antibodies, such as a flagellin molecule expressed by a microbe belonging to the Clostridium genus, a type of molecule known to be a dominant antigen in the microbiota of Crohn's disease patients. In another embodiment, the surface molecule is a lipopolysaccharide.

In another embodiment, the feature that differentiates the functional niche is recognized by a delivery system consisting of a bacteriophage, wich enables intracellular delivery of a cargo. The differentiating feature may be the presence or concentration of a receptor on the surface of bacteria, including a lipopolysaccharide, a teichoic acid, a protein or a flagella. To enter a bacterial cell, the bacteriophage attaches to the specific feature. This specificity in feature recognition enables the bacteriophage delivery system to only infect certain bacteria bearing receptors that it can bind to, and can be exploited to target a delivery system to the intracellular contents of a bacterial niche.

Endogenous features that trigger release of a drug from a delivery system in the presence of a bacterial niche

The release of a cargo may occur upon triggering by an enzymatic reaction preferentially performed by a microbial niche, which causes preferential release in the vicinity of the niche.

Bacteria produce a wide spectrum of enzymes that are involved in many processes, such as carbohydrate and protein fermentation, bile acid and steroid transformation, metabolism of xenobiotic substances, as well as the activation and destruction of potential mutagenic metabolites. In the gut, most bacterial enzymes perform hydrolysis and reduction reactions. These reactions may include nitro- and azo- group reduction, azo bond cleavage, sulfoxide reduction, reductive deamination, dehydroxylation, and hydrolysis. Enzymes responsible for the breakdown of polysaccharides include, among others, β-D-focosidase, α-L-arabinofuranosidase, β-D-galactosϊdase, β-D- glucosidase, and β-xylosidase, with the last three enzymes being the most active. Enzymes involved in reduction reactions include, among others, nitroreductase, azoreductase, and sulfoxide reductase. Pro-drugs, like sulfasalazine, exist, that rely on the action of colonic bacteria to break down an inactive precursor and release the active drug moiety. However, such approaches described in the literature do not exploit the enzymatic reactions of the gut flora in a specific manner and cannot be used to target specific microbial niches.

In a preferred embodiment, the reaction that triggers the release of the drug in the vicinity of the functional niche is performed by a secreted enzyme preferentially expressed by members of the niche. In one

embodiment, the enzyme is lacking or substantially under-represented in other members of the microbiota. Hydrolytic reactions triggering release of a drug by may be performed by enzymes of bacterial origin including β-glucoronidase, β- galactosidase and β-glucosidase, fucosidases, and sialidases. They may also be performed by bacterial peptidases, preferentially in the large intestine.

Reductive reactions may be performed by azoreductases (azo group reduction), nitroreductases (nitro group reduction), and sulfoxide reductases (sulfoxide group reduction) of bacterial origin.

The release of the cargo (e.g., drug) from a delivery system may occur by natural diffusion. Targeting of the delivery system to a feature of a microbial niche such as a surface molecule may ensure that the majority of the cargo release by diffusion occurs in the vicinity of the desired niche.

The release of a cargo may also be triggered by by a change in pH. Polymeric delivery systems with a dissolution threshold in the range of 6.8 to 7.5 may be used to exploit the natural shift towards a more alkaline pH in the distal sections of the gut for colonic delivery.

The release of a cargo may be delayed for a few hours after administration, preferably 3 to 5 hours, to enable transit through the small intestine and targeting to the colon. Shells of hydrogel that hydrate and swell on contact with gastrointestinal fluids, thereby effecting drug release, may be used.

The release of a cargo may also be triggered by a change in pressure such as the change in pressure that occurs in distal sections of the gut as a result of the production of gases by bacterial fermentation. Pressure- controlled colon delivery capsules consisting of a drug dispersed in a suppository base, coated with a hydrophobic polymer such as ethylcellulose, may be used.

Exogenous features that trigger release of a drug from a delivery system in the presence of a bacterial niche

The release of a cargo may occur upon triggering by an external stimulus. In a preferred embodiment, in a first step, an inactive form of the active agent is delivered to the target bacterial niche, and accumulates in that niche by any of the targeting mechanisms previously discussed. In a second step, which may occur after a time period has lapsed (for example, 1 hour, 3 hours, 10 hours, one day, or one week) an external stimulus triggers the release of the active form of the agent. The external stimulus may be light, for example visible or infrared light. The external stimulus may also be ultrasound.

In a preferred embodiment, an agent conjugated to a photosensitizer (a chemical compound that can be excited by light of a specific wavelength) is orally administered and is targeted to a bacterial niche in the human gut; the agent may be an antibody or antibody fragment that recognizes a surface molecule characteristic of a certain bacterial niche. The antibody or antibody fragment can be chemically conjugated to the photosensitizer via a thiol bond. For example, antibody fragments conjugated to the photosensitizer chlorin e6 have been described in the literature (Fowers, et al., J. of Drug Targeting, 2001; 9(4), 281-294). A period of 3-5 hours is allowed to lapse to guarantee arrival of the agent-photosensitizer conjugate to the distal gut and accumulation in the bacterial niche. After this period, the photosensitizer is externally excited by light, which causes molecular oxygen in the vicinity of the conjugate to create an excited singlet state oxygen molecule. Singlet oxygen is a very aggressive chemical species and very rapidly reacts with any nearby biomolecules thereby destructing the cells of the targeted bacterial niche.

Anatomical Differentiating Factors

The delivery systems may target anatomical locations of the host which have widely dissimilar microbiota compositions, such as different sections of the gut, the oral cavity, the skin, the vagina, the nasal cavities, the ear, and the lungs.

The delivery systems may also target regions and structures within the anatomical locations that have dissimilar local microbiota compositions. Regions that can be targeted within the oral cavity include the subgingival sulcus, tongue, cheek, or tooth (supragingival plaque). Regions with dissimilar microbiota in the skin include hair follicles, sebaceous, eccrine, and apocrine glands. These structures constitute differentiated ecological niches with a broad range of pH, temperature, moisture, and sebum content, and they comprise subhabitats associated with their own unique microbiota (Giice 2008 Genome Res. 2008 JuI; 18(7): 1043-50; Marples, M. (1965) The ecology of the human skin (Charles C Thomas, Bannerslone House, Springfield, IL; Kearney et al. 1984 J Gen Microbiol 1984 Apr;130(4):797- 801).

The environmental conditions of each niche can be exploited to design adequate delivery systems. For example, the nasal cavities are highly hydrated, and may be ideally targeted with a mucoadhesive delivery system. In one embodiment, a nasal delivery system comprises a mucoadhesive polysaccharide that sticks strongly to the nasal cavities upon hydration. In a preferred embodiment, the polysaccharide is a prebiotϊc compound. In another embodiment, the bacterial niche targeted is located in the lungs, and the delivery system comprises a pharmaceutical formulation contained in a porous particle suitable for aerosolization in a dry powder inhaler, wherein the particle has a density less than about 0.4g/cm3 and wherein at least 50% of the particles have an aerodynamic diameter of less than 4 microns. The particle may contain a prebiotic. The particle may also be substantially made of the prebiotic carbohydrate, containing more than 5% of dry mass weight of prebiotic.

Diseases and conditions associated with altered microbial niches Disease states may exhibit either the presence of a novel microbe(s), absence of a normal microbe(s), or an alteration in the proportion of microbes.

Recent research has established that disruption of the normal equilibrium between a host and its microbiota, generally manifested as a microbial imbalance, is associated with, and may lead to, a number of conditions and diseases. These include Crohn's disease, ulcerative colitis, obesity, asthma, allergies, metabolic syndrome, diabetes, psoriasis, eczema, rosacea, atopic dermatitis, gastrointestinal reflux disease, cancers of the gastrointestinal tract, bacterial vaginosis, neurodevelopmental conditions such as autism spectrum disorders, and numerous infections, among others. For example, in Crohn's disease, concentrations of Bacteroides, Eubacteria and Peptostreptococcus are increased whereas Bifidobacteria numbers are reduced (Linskens et al., 2001 Scand J Gastroenterol Suppl. 234:29-40); in ulcerative colitis, the number of facultative anaerobes is increased. In these inflammatory bowel diseases, such microbial imbalances cause increased immune stimulation, and enhanced mucosal permeability (Sartor, 2008 Proc Natl Acad Sci U S A. 28;105(43):16413-4). In obese subjects, the relative proportion of Bacteroidetes has been shown to be decreased relative to lean people (Ley et al, 2006 Nature 21 ;444(7122): 1022-3), and possible links of microbial imbalances with the development of diabetes have also been discussed (Cani et al., 2008 Pathol Biol (Paris). 56(5):305-9). Segmented Filamentous Bacteria have been shown to play a critical role in prevention of infection and development of autoimmune diseases (Ivanov et al, Cell.

139(3):485-98 2009). In the skin, a role for the indigenous microbiota in health and disesase has been suggested in both infectious and noninfectious diseases and disorders, such as atopic dermatitis, eczema, rosacea, psoriasis, and acne (Holland et al. 1977 Br J Dermatol. 96(6):623-6; Thomsen et al. 1980 Arch Dermatol. 116:1031-1034; Till et al. 2000 Br. J. Dermatol. 142:885-892; Paulino et al. 2006 J. Clin. Microbiol 44:2933-2941).

Furthermore, the resident microbiota may also become pathogenic in response to an impaired skin barrier (Roth and James 1988 Annu. Rev.

Microbiol. 42:441-464). Bacterial vaginosis is caused by an imbalance of the naturally occurring vaginal microbiota. While the normal vaginal microbiota is dominated by Lactobacillus, in grade 2 (intermediate) bacterial vaginosis, Gardnerella and Mobiluncus spp. are also present, in addition to Lactobacilli. In grade 3 (bacterial vaginosis), Gardnerella and Mobiluncus spp. predominate, and Lactobacilli are few or absent (Hay et al., Br. Med. J., 308, 295-298, 1994)

Other conditions where a microbial link is suspected based on preliminary evidence include rheumatoid arthritis, multiple sclerosis, Parkinson's disease, Alzheimer's disease, and cystic fibrosis. Types of Organisms Present in Niches

The delivery systems may enable targeting to relevant members of a bacterial niche, including phyla relevant in the human microbiota, such as the Bacteroidetes, and the Firmicutes, genus such as Bacteroides,

Bifidobacterium, and Lactobacillus, and species, such as Bacteroides thetaiotaomicron or Faecalibacterim prausnitzii.

In one embodiment, Bidifobacterium and Bacteroides spp. are targeted by a delivery system triggered by the action of a β-glucoronidase enzyme. The delivery system may be substantially comprised of

polysaccharides or mucopolysaccharides that are hydrolyzed by β- glucoronidase by breakage of a glycosidic bond. Additionally, the drug being delivered may be formulated into a prodrug precursor by a bond to a glucuronic acid molecule. This bond can be selectively cleaved by a β- glucoronidase. Since Bidifobacterium and Bacteroides spp account for 99.5% of β-glucoronidase activity in humans, the delivery systems described can be used to target niches rich in these species.

In another embodiment, lactic acid bacteria (Bidifobacterium and Lactobacillus spp.) are targeted by using carbohydrate-based delivery systems that can be degraded by β-D-galactosidase and β-D-glucosidase.

In another embodiment, putrefactive bacteria {Bacteroides,

Peptsostreptococcus, Clostridia) are targeted with a delivery system comprising a protein matrix that is decomposed by putrefaction reactions.

In another embodiment, a vitamin-producing bacterial niche or an isoprenoid-producing bacterial niche is targeted through use of a delivery system targeted to a cell surface component of the 2-rnethyl-d-erythritol 4- phosphate pathway, or triggered by an enzyme component of the pathway. The delivery system may target species responsible for the production of vitamin K, such as Bacteroides, Propionibacterium, and Veillonella spp. The delivery system may also target species responsible for the production of folic acid, such as Bifidobacterium bifidum, Bifidobacterium infantis,

Bifidobacterium breve, Bifidobacterium longum, Enterococcus faecalis, and Escherichia coll The delivery system may also target species responsible for the production of vitamin B 12, such as E. coli, Bifidobacterium spp., Klebsiella spp., Veillonella spp. , Fusobacterium spp., Eubacterium spp., and Clostridium spp.

In another embodiment, Clostridia spp. are targeted by use of a delivery system triggered by a reduction reaction consisting of

decomposition by loss of an imidazole ring. In a further embodiment, the species targeted is Clostridium perfringens, a microbe known to have a high capacity to conduct imidazole ring loss reactions

In another embodiment Bacteroides and Clostridium spp. are targeted by a delivery system triggered by an enzyme from the microbes that performs a thiazole ring-opening reaction. In a preferred embodiment, the Bacteroides is B. thetaiotaomicron,

In another embodiment Clostridium spp. are targeted by a delivery system triggered by a reaction with a Clostridial enzyme selected from a ferredoxin-reducing hydrogenase, a membrane-bound oxidoreductase, an enzyme performing an imidazole ring opening reaction, and an enzyme performing a thiazole ring-opening reaction.

In another embodiment, Bifidobacteria spp. are targeted by a delivery system containing substantial amounts of human milk oligosaccharides which can be degraded by β-galactosidases, α-fucosidases, and sialidases.

In another embodiment, E, coli, Lactobacillus spp., and Bacteroides spp. are targeted by a delivery system containing substantial amounts of lactulose, which delivers its cargo when triggered by a lactulose-hydrolyzing enzyme from the bacteria. This same delivery system can be used to target a cargo away from Clostridium spp., which substantially lack the ability to hydrolyze lactulose.

In another embodiment, Eubacterium ramulus (a bacterium that plays a dominant role in the transformation of flavonoids) and Enterococcus casseliflavus are targeted by a delivery system triggered by a deglycosylation reaction.

In another embodiment, bacteria belonging to the Enterobacteria family are targeted by a delivery system triggered by a reaction consisting of breaking a nucleotide from a nitrogen ring. The bacteria targeted may include Bacteroides eggerthii and Bacteroides vulgates.

In another embodiment, the delivery system targets a group of bacteria that populates the skin, selected from, Staphylococcus, Micrococcus, Corynebacterium, Brevibacteria, Propionibacteria, and Acinetobacter spp, or a selected species, such as Propionibacterium acnes

In another embodiment, the delivery system targets a niche containing Firmicutes by selectively recognizing one or more features from the cell surface of a Firmicute, selected from an amino acid transporter, a sugar transporter, an ATP-binding cassette (ABC) transporter, and a

Phosphotransferase system (PTS). Such transporters are known to be overrepresented in Firmicutes. In a preferred embodiment;, the Firmicute is selected from, F. prausnitzii E. rectale, and E. eligens,

In another embodiment, the delivery system targets a niche containing sulfate-reducing bacteria by being triggered by a disulfide exchange reaction.

In another embodiment, the delivery system targets a niche containing Segmented Filamentous Bacteria (SFB) by a delivery system triggered by a reaction with an SFB enzyme selected from a ferredoxin- reducing hydrogenase, a membrane-bound oxidoreductase, an enzyme performing an imidazole ring opening reaction, and an enzyme performing a thiazole ring-opening reaction.

Methods for identifying features from microbial niches that allow specific targeting or specific triggering of delivery systems

In one aspect, the invention relates to characterizing the bacterial populations of a host in order to identify differential properties and signatures of a bacterial niche that can be exploited to target the delivery of a drug to the niches. Delivery systems are then designed that can be triggered to release a drug upon exposure to the differential property. The differential properties may be identified using several methods to profile the microbiota.

In one embodiment, the method used involves screening of 16srRNA genes by PCR, which enables characterization of microorganism at the phylum, class, order, family, genus, and species level. The sequences of the lόsrRNA gene contain hypervariabie regions which can provide specific signature sequences useful for bacterial identification. (Schloss and

Handelsman, Microbiol. MoI. Biol. Rev., 2004, 68: 686-691).

In another embodiment, the method used involves screening of taxa, phylum, class, order, family, genus, and species level specific genes by RT- PCR, which enables the identification and quantification of microorganism at the desired taxonomical level.

In another embodiment, mass spectrometry or nuclear magnetic resonance are used to identify bacterial signatures such as nucleic acids, proteins glycans, lipids, and metabolites.

In another embodiment, a high-throughput "metagenomic" sequencing method is used, such as pyrosequencing. The feature that differentiates the bacterial niche is identified by isolating a sample from a bacterial niche, extracting the DNA of the bacterial fraction, cloning the DNA in a vector that replicates in a cultured organism, introducing the vectors in bacteria to create a metagenomic library, and identifying phylogenetic markers in the DNA sequences of the library that link the cloned sequences to the probable origin of the DNA and the probable functions encoded by such genes. The method identifies genes that are either over-represented or under-represented in the bacterial population.

Furthermore, the method enables the sequencing of genetic material from uncultured communities of microbial organisms directly in their natural environments, bypassing the need for isolation and lab cultivation of individual species (Handelsman et al. (1998). Chem. & Biol 5: 245-249)

In another embodiment, an immune response by the host can be used as a reporter to identify a key microbial protein. A microbial cell surface antigen characteristic of a certain niche can be detected by administering a strain to a host and isolating a serum antibody against the strain secreted by the host. For example, a lambda phage expression library of total cecal bacterial DNA can be constructed and then screened using serum IgG from a patient suffering from colitis. Positive clones can be collected and rescreened for verification. At the end of the process, the remaining clones can be sequenced. The sequences can be matched against clones in reference datasets, such as GenBank, and homology with existing bacterial proteins is established. Additionally, a recombinant version of the microbial antigen- binding antibodies identified, or relevant fragments of the antibody, or relevant epitope sequences introduced into a recombinant construct, may be expressed in a recombinant system (e.g. E. coli, yeast, or a Chinese Hamster Ovary cells), purified, and conjugated to a delivery system to enable specific targeting of the original microbial cell surface antigen.

In another embodiment, phage display technology is used to purify and characterize differentiating proteins from bacteria. In this method, bacterial proteins are displayed on the surface of the bacteriophage virion. Display is achieved by fusion of a bacterial protein or library of proteins of interest to any virion proteins such as the pill and pVIII proteins.

Filamentous phage virion proteins are secreted by translocation from the cytoplasm via the Sec-dependent pathway and anchored in the cytoplasmic membrane prior to assembly into the virion (Jankovic et al., Genome Biol. 2007; 8(12): R266). In this fashion, all types of bacterial secreted proteins, including receptors, adhesions, transporters, complex cell surface structures, secreted enzymes, toxins, and virulence factors, can be identified. In order to deduce whether a protein is likely to be secreted, several methods can be used, including SignalP 3.O₅ TMHMM 2.0, LipoPred, or PSORT (Bendtsen JD₅ Nielsen H₅ von Heijne G, Brunak S: J MoI Biol 2004, 340:783-795). These methods deduce secreted proteins from a completely sequenced genome by using a range of algorithms that identify signal sequences and transmembrane α-helices, which are characteristic of secreted proteins.

B. Delivery Systems

Therapeutic, Diagnostic or Prophylactic Agents to be Delivered

The agents delivered may include therapeutic, prophylactic, or diagnostic agents selected from small molecules, nucleic acids, proteins, polypeptides, carbohydrates, lipids, full organisms, a mixture of organisms, and combinations thereof.

In one embodiment, the agent delivered is a small molecule delivered that has low oral bioavailability and acts on a microbial niche of the host's gut. Low oral bioavailability is generally undesirable in drugs, since absorption through the intestine is an objective of most oral therapies.

However, in the context of modulating the intestinal flora, it may be a desirable attribute since the site of action is the contents of the gut. The permeability of the small molecule may be decreased by tethering the small molecule to a high molecular weight compound such as a polymer, thereby reducing the molecule's bioavailability. Highly hydrophilic or hydrophobic small molecules may be used, since both extremes are detrimental to oral bioavailability (highly hydrophilic molecules do not cross the epithelium, while highly hydrophobic molecules are not solubilized in aqueous media). In one embodiment, a prodrug is formed by chemically modifying a small molecule drug with highly charged groups that increase the hydrophilicity of the drug, thereby decreasing its oral bioavailability.

In another embodiment, the agent delivered is a prebiotic

carbohydrate selected from a fructooligosaccharϊde, a

galactooligosaccharide, inulin, a xylooligosaccharide, polydextrose, a manoolϊgosaccharide, and an arabinoxylan

In another embodiment, the agent delivered is a glycoprotein or a glycolipid.

In another embodiment, the agent delivered is a protein or peptide selected from an antibody, antibody fragment, a peptide, a minibody, a fibronectin domain, a multimer of an Ankyrin repeat fold, a protein A protein fold, a lipocalin protein fold, and a nucleic acid aptamer. In a related embodiment, the agent binds to a bacterial surface antigen selected from a receptor, and adhesion molecule, a transporter, a cell surface structure, a secreted enzyme, a toxin, and a virulence factor. In a related embodiment, the bacterial surface antigen targeted by the agent mediates an immune response of the host. In another embodiment, the protein delivered is a recombinant version of a protein naturally secreted by human commensals or by probiotic organisms. If the protein or peptide agent is targeted to a bacterial niche located in the gut, it may be protected from proteolysis reactions in the gastrointestinal tract by a number of techniques known in the art, including PEGylation, conjugation to other macromolecules, formation of covalent bonds that stabilize the polypeptide structure, such as disulfide bonds, or encapsulation within polymeric carrier systems, such as methacrylic acid, polyvinylalcohol, polyvinylpirrolidone, gelling

polysaccharides, polyethylene oxide, or polyethylene glycol.

In another embodiment, the agent delivered is selected from one or more probiotic organisms, one or more commensal organisms, and combinations thereof.

In another embodiment, the agent delivered is an imaging agent Types of components of the delivery system that enable selective targeting of bacterial niches

The delivery systems may incorporate targeting moieties that confer selectivity towards a desired bacterial niche. The moiety may have selectivity towards a microbial surface protein, and may consist of a polypeptide selected from an antibody, an antibody fragment, a peptide, a minibody, a fibronectin domain, a multimer of an Ankyrin repeat fold, a protein A protein fold, a lipocalin protein fold, and a nucleic acid aptaraer. The moiety may also consist of a glycoprotein or a glycolipid. The moiety may have selectivity towards a sugar structure on the surface of a microbe, and may consist of a lectin (a sugar-binding protein which is highly specific for a sugar moiety). The moiety may have general selectivity towards bacteria with varying levels of surface charge, zeta potential, and hydrophobicity, and may consist of a polymer. The moiety may have selectivity towards a receptor on the surface of bacteria, including a lipopolysaccharide, a teichoic acid, a protein or a flagella, and may consist of a bacteriophage that attaches preferentially to a bacterial niche. The moiety may have selectivity towards an endogenous lectin expressed by a bacterial niche, and may consist of a carbohydrate such as mannose. The moiety may be selectively activated in the vicinity of an antibiotic-resistant bacteria, and may consist of an antibiotic conjugated to a delivery system. For example, a polymer conjugated to penicillin groups can be used. The penicillin groups can only be broken down by penicillin-resistant microbes that express a penicillinase enzyme, which enables specific targeting to penicillin-resistant microbes.

In one embodiment, a dextran-drug-antibiotic conjugate is formed by tethering both the desired drug as well as one antibiotic selective for a bacterial niche to a dextran backbone. Upon recognition of the antibiotic moiety by the targeted bacterial niche, degradation of the dextran structure by bacterial niche enzymes follows, thereby causing release of the drug entrapped in the dextran backbone. Methods of designing the necessary linker chemistry to form such conjugates have been described in the literature (Ying Chau, Frederick E. Tan, and Robert Langer, Bioconjugate Chem,, 2004, 15 (4). pp 931-941)

Types of components of the delivery system that can be triggered or degraded by microbial niche factors

The compositions of the delivery systems may include synthetic and natural scaffolds that can be selectively degraded by the action of enzymes performing the reactions listed above, thus triggering the release of a cargo. Natural GRAS (generally recognized as safe) compounds that may be used to target niches rich in carbohydrate-hydrolyzing enzymes include food components such as non-starch polysaccharides, amylose, xanthan gum, dextran, pectin, and galactomannan (L. Hovgaard, H. Brøndsted, Crit. Rev. Ther. Drug Carr. Syst. 13 (1996) 185-223; See also COLAL®, a delivery system consisting of non-starch polysaccharides that has undergone Phase II clinical trials to deliver prednisolone). These natural polysaccharides can be designed so that they remain intact in the stomach and small intestine (e.g. by chemical crosslinking or by addition of a protective coat) but once they enter the colon, they are degraded by hydrolytic enzymes, which release the drug into the colon.

In one embodiment, a drug containing a carboxylic acid moiety can be conjugated to a high molecular weight dextran through ester linkages in the dextran hydroxyl groups; the conjugate escapes absorption in the small intestine, but can be degraded by enzymes upon reaching the distal gut.

Compounds that may be degraded by reductive enzymes include azo- crosslinked polymers and azo-crosslinked hydrogels (See Saffran et al._Λ 1986, Science, 233, 1081). Drugs may be attached to a polymer via azo bonds, for selective release in the colon. These systems can be used to target anaerobic bacteria in the colon. In one embodiment, azo-crosslinked polymers or azo- crosslinked hydrogels are used to target a species with high azoreductase activity, selected from Eubacterium spp., C. clostridiiforme, Butyrivibrio spp., Bacteroides spp., C. papaputrificum, C. nexile. C. perfringens (the azoreductase activity of the listed species has been characterized by Rafii F, Franklin W, Cerniglia CΕ,Appl Environ, Microbiol. 1990, July, 56 (7): 2146-2151). In another embodiment, the same polymers are used to target a Staphylococcus aureus niche in the skin (Chen H, Hopper SL, Cerniglia CE, Microbiology, 2005, 151, 1433-1441 have shown that this bacterium produces NADPH-flavin azoreductase). In one embodiment, N-(2- hydroxypropyl)methacrylamide (HPMA) copolymers containing side chains conjugated to a drug are synthesized according to a method described in the literature (Pavla Kopeckova, Jindrich Kopecek , Macromolecular Chemistry and Physics, VoI 191 Issue 9, Pages 2037 - 2045). These polymers are used to target the drug to a bacterial niche rich in azoreductases. The

azoreductases cleave the HPMA backbone, thus releasing the drug in the bacterial niche.

The compositions of the delivery systems may also include prodrug constructs, consisting of a drug conjugated to a moiety that can be cleaved by a microbial enzyme. For example, drugs conjugated to nitro-, azo-, and sulfoxide groups can be cleaved by nitro-, azo-, and sulfoxide-reductase enzymes respectively. Drugs conjugated to glucuronic acid can be cleaved by glucuronidase enzymes. In one embodiment, a prodrug consisting of glucuronic acid conjugated to a drug via a glycosidic bond is degraded by β- glucoronidase from Bifidobacteria and Bacteroides spp. (which account for 99.5% of β-glucoronidase activity), thereby targeting a bacterial niche rich in the species.

Compounds that can be degraded by microbial peptidase activity include proteins and peptides. In one embodiment, a drug is entrapped inside a protein matrix that can be digested by peptidases.

Compounds that can be degraded by microbial sulfoxide reductases include materials that are crosslinked by disulfide bonds and can undergo a disulfide exchange reaction in the presence of a free thiol, hi one

embodiment, a sulfate-reducing bacterial niche is targeted by using a polymer crosslinked with disulfide bonds, which react in the presence of hydrogen sulfide produced by the bacterial niche.

Other possible conjugates that can be cleaved by bacterial enzymes may be appreciated by one skilled in the art, and may include cyclodextrin conjugates, glycoside conjugates, polypeptide conjugates, and polymeric prodrugs,, among others.

General strategies to attain niche selectivity

Targeted delivery to a bacterial niche can be accomplished by use of a delivery system that includes combinations of any of the targeting and triggering moieties described above. The targeting efficacy of these delivery systems will vary depending on the expression profiles of the niche differentiating factors as well as the niche enzymes used to trigger delivery.

Targeted delivery with a high selectivity to a bacterial niche can also be accomplished by designing an antibody against a bacterial niche antigen, and chemically linking the antibody to an enzyme (methods of creating recombinant fusions of enzymes with antibodies have been described in the art (Bagshawe KD. Expert Rev Anticancer Ther. 2006; 6(10): 1421-1431), resulting in selective binding of the enzyme to the bacterial niche. When discrimination between the bacterial niche and the surrounding microbiota is sufficient (e.g. after a period of time), a prodrug is administrated which is converted to an active drug (e.g. an antibiotic) by the enzyme, only within the bacterial niche. Selectivity is achieved by the bacterial niche specificity of the antibody and by delaying prodrug administration until there is a large differential of enzyme-antibody conjugate between the bacterial niche and the microbiota surrounding it For example, a penicillinase or β-lactamase enzyme can be recombinantly linked to an antibody recognizing an antigen differentially expressed by a bacterial niche detrimental to the host. After administration of the antibody-enzyme construct, a penicillin- or β-lactam prodrug construct can be administered to specifically eradicate the detrimental bacterial niche. Optionally, the antibody-enzyme construct may be encapsulated in a proteolysis-resistant delivery system (as described elsewhere in this invention), which may enable delivery to the distal portions of the gastrointestinal tract.

Alternative variations of this concept may involve constructs that substitute the antibody for a lectin, a virus, a bacteriophage, or a polymer. In the first case, selectivity is conferred by a lectin. A lectin-enzyme conjugate is designed, whereby the lectin group recognizes with high affinity a glycan structure differentially expressed in the surface of a bacterial niche. In the second and third cases, a gene expressing a desired enzyme is cloned into a virus or bacteriophage vector capable of preferentially infecting abacterial niche. Following infection of the bacterial niche by the virus or

bacteriophage, intracellular expression of the gene delivered takes place, leading to formation of functional enzyme. Following this, a prodrug that can be cleaved by the enzyme (e.g. an antibiotic prodrug) can be administered, thereby preferentially eradicating the targeted bacterial niche. In the last case, a polymer-enzyme conjugate is synthesized. The polymer preferentially accumulates in a bacterial niche characterized by a given combination of properties such as surface charge, zeta potential, or hydrophobicity.

General strategies for delivery to the colon

Numerous methods have been described in the art to enable general delivery of drugs to the colon, and any of them can be combined with the novel features of this invention to improve delivery to a niche in the colon. Such methods include pH-sensitive formulations (e.g. formulations coated with enteric polymers that release drug when the pH move towards a more alkaline range, after passage through the stomach), formulations that delay the release of the drug for a lag time of 3-5 hours, roughly equivalent to small intestinal transit time, thereby securing delivery to the colon, drugs coated with bioadhesive polymers that selectively provide adhesion to the colonic mucosa (e.g. see US Patent 6,368,586), and delivery systems that incorporate protease inhibitors to prevent proteolytic activity in the gastrointestinal tract from degrading biologic drug agents.

General strategies for vaginal, skin, nasal, ear, ocular and lung delivery

Drugs targeting bacterial niches in the vagina may be ideally delivered by rectal administration in the form of suppositories or enemas.

Drugs targeting bacterial niches in the skin may be ideally delivered by formulation into creams, gels, lotions, skin patches, and skin microneedle systems, all of which have been extensively described in the art.

Drugs targeting bacterial niches in the nasal cavity may be ideally delivered by formulation into aerosols. In one embodiment, the

pharmaceutical formulation is contained in a particle comprising a carbohydrate that strongly sticks to the nasal epithelia. In a preferred embodiment, the pharmaceutical agent contains a probiotic or a commensal, and the carbohydrate is a prebiotic that serves the dual function of improving stability and survival of the probiotic cargo as well as securing attachment to the nasal cavity.

Drugs targeting bacterial niches in the ear may be ideally delivered by formulation into drops.

Drugs targeting bacterial niches in the eye may ideally be delivered by formulation into eye drops and ophthalmic inserts such as drug-coated lens.

Drugs targeting bacterial niches in the lung may be ideally delivered by formulation into aerosols. Aerosols for the delivery of therapeutic agents to the respiratory tract have been described, for example, Adjei, A. and Garren, J. Pharm. Res., 7: 565-569 (1990); and Zanen, P. and Lamm, J.-WJ. Int. J. Pharm., 114: 111-115 (1995. Inhaled aerosols may be used for the treatment of microbiota imbalances associated with asthma and cystic fibrosis. Dry powder formulations with large particle size and improved flowability characteristics, such as less aggregation have been described, (Visser, J., Powder Technology 58: 1-10 (1989)). A novel feature of this invention involves the use of dry powder particulate aerosols containing prebiotics and probiotic organisms in large carrier particles that display minimum aggregation (thus avoiding particle-particle interactions, such as hydrophobic, electrostatic, and capillary interactions, which are detrimental to lung delivery). In a preferred embodiment, the pharmaceutical formulation is contained in a porous particle suitable for aerosolization in a dry powder inhaler, wherein the particle has a density less than about 0.4g/cm3 and wherein at least 50% of the particles have an aerodynamic diameter of less than 4 microns.

General strategies for targeting a cargo away from a bacterial niche

The general principles of the strategies described for targeting bacterial niches can be exploited to deliver drugs away from these same niches. Delivery systems to target drugs away from a niche can be constructed by: (i) selecting at least one feature that is lacking in the bacterial constituents of the niche in a given anatomical location and present in other bacterial residents of that same anatomical location, and; (ii) providing a delivery system that releases a pharmaceutical formulation preferentially away from the vicinity of the constituents of a bacterial niche.

General strategies for increasing or decreasing the bioavailability of a bacterial drug metabolite

A method of increasing or decreasing the bioavailability of an active drug metabolite generated by a bacterial niche includes the steps of (i) providing a drug known to have an active bacterial metabolite, (ii) selecting at least one feature that differentiates or is substantially lacking in the bacterial constituents of the niche that generate the active drug metabolite in a given anatomical location from the rest of bacterial residents of that same anatomical location, and (iii) providing a delivery system that releases the drug in the vicinity or away from the constituents of a bacterial niche; wherein release of the drug is triggered by the differentiating feature of the bacterial niche.

There exists a growing list of drugs that have toxic or active drug metabolites, and the microbiota is now widely regarded as an organ with metabolic potential equal to the liver. Over 30 drugs have been identified as substrates for colonic bacteria (omeprazoles, digoxin, nizatinide, and nitrazepam, among others) with one of them, sorivudine, having caused a notorious incident involving its toxic bacterial drug metabolite, which caused numerous deaths in Japan in 1993. Methods to avoid or increase the metabolism of drugs that have active bacterial metabolites are therefore needed.

Formulations

Formulations are prepared using a pharmaceutically acceptable "carrier" composed of materials that are considered safe and effective and may be administered to an individual without causing undesirable biological side effects or unwanted interactions. The "carrier" is all components present in the pharmaceutical formulation other than the active ingredient or ingredients. The term "carrier" includes but is not limited to diluents, binders, lubricants, desintegrators, fillers, and coating compositions.

"Carrier" also includes all components of the coating composition which may include plasticizers, pigments, colorants, stabilizing agents, and glidants. The delayed release dosage formulations may be prepared as described in references such as "Pharmaceutical dosage form tablets", eds. Liberman et. al. (New York, Marcel Dekker, Inc., 1989), "Remington - The science and practice of pharmacy", 20th ed., Lippincott Williams & Wilkins, Baltimore, MD, 2000, and "Pharmaceutical dosage forms and drug delivery systems", 6th Edition, Ansel etal, (Media, PA: Williams and Wilkins, 1995) which provides information on carriers, materials, equipment and process for preparing tablets and capsules and delayed release dosage forms of tablets, capsules, and granules.

Examples of suitable coating materials include, but are not limited to, cellulose polymers such as cellulose acetate phthalate, hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate and hydroxypropyl methylcellulose acetate succinate; polyvinyl acetate phthalate, acrylic acid polymers and copolymers, and methacrylic resins that are commercially available under the trade name Eudragit® (Roth Pharma, Westerstadt, Germany), Zein, shellac, and polysaccharides.

Additionally, the coating material may contain conventional carriers such as plasticizers, pigments, colorants, glidants, stabilization agents, pore formers and surfactants.

Optional pharmaceutically acceptable excipients present in the drug- containing tablets, beads, granules or particles include, but are not limited to, diluents, binders, lubricants, disintegrants, colorants, stabilizers, and surfactants. Diluents, also termed "fillers," are typically necessary to increase the bulk of a solid dosage form so that a practical size is provided for compression of tablets or formation of beads and granules. Suitable diluents include, but are not limited to, , dicalcium phosphate dihydrate, calcium sulfate, lactose, sucrose, mannitol, sorbitol, cellulose,

microcrystallϊne cellulose, kaolin, sodium chloride, dry starch, hydrolyzed starches, pregelatinized starch, silicone dioxide, titanium oxide, magnesium aluminum silicate and powder sugar.

Binders are used to impart cohesive qualities to a solid dosage formulation, and thus ensure that a tablet or bead or granule remains intact after the formation of the dosage forms. Suitable binder materials include, but are not limited to, starch, pregelatinized starch, gelatin, sugars (including sucrose, glucose, dextrose, lactose and sorbitol), polyethylene glycol, waxes, natural and synthetic gums such as acacia, tragacanth, sodium alginate, cellulose,including hydorxypropylmethylcellulose, hydroxypropylcellulose, ethylcellulose, and veegum, and synthetic polymers such as acrylic acid and methacrylic acid copolymers, methacrylic acid copolymers, methyl methacrylate copolymers, aminoalkyl methacrylate copolymers, polyacrylic acid/polymethacrylic acid and polyvinylpyrrolidone.

Lubricants are used to facilitate tablet manufacture. Examples of suitable lubricants include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, glycerol behenate, polyethylene glycol, talc, and mineral oil.

Disintegrants are used to facilitate dosage form disintegration or "breakup" after administration, and generally include, but are not limited to, starch, sodium starch glycolate, sodium carboxymethyl starch, sodium carboxymethylcellulose, hydroxypropyl cellulose, pregelatinized starch, clays, cellulose, alginine, gums or cross linked polymers, such as cross- linked PVP (Polyplasdone XL from GAF Chemical Corp).

Stabilizers are used to inhibit or retard drug decomposition reactions which include, by way of example, oxidative reactions.

Surfactants may be anionic, cationic, amphoteric or nonionic surface active agents. Suitable anionic surfactants include, but are not limited to, those containing carboxylate, sulfonate and sulfate ions. Examples of anionic surfactants include sodium, potassium, ammonium of long chain alkyl sulfonates and alkyl aryl sulfonates such as sodium dodecylbenzene sulfonate; dialkyl sodium sulfosuccinates, such as sodium dodecylbenzene sulfonate; dialkyl sodium sulfosuccinates, such as sodium bis-(2- ethylthioxyl)-sulfosuccinate; and alkyl sulfates such as sodium lauryl sulfate. Cationic surfactants include, but are not limited to, quaternary ammonium compounds such as benzalkonium chloride, benzethonium chloride, cetrimonium bromide, stearyl dimethylbenzyl ammonium chloride, polyoxyethylene and coconut amine. Examples of nonionic surfactants include ethylene glycol monostearate, propylene glycol myristate, glyceryl monostearate, glyceryl stearate, polyglyceryl-4-oleate, sorbitan acylate, sucrose acylate, PEG- 150 laurate, PEG-400 monolaurate, polyoxyethylene monolaurate, polysorbates, polyoxyethylene octylphenylether, PEG-1000 cetyl ether, polyoxyethylene tridecyl ether, polypropylene glycol butyl ether, Poloxamer® 401, stearoyl monoisopropanolamide, and polyoxyethylene hydrogenated tallow amide. Examples of amphoteric surfactants include sodium N-dodecykbeta.-alanine, sodium N-lauryl-.beta.-iminodipropionate₅ myristoamphoacetate, lauryl betaine and lauryl sulfobetaine. If desired, the tablets, beads granules or particles may also contain minor amount of nontoxic auxiliary substances such as wetting or emulsifying agents, dyes, pH buffering agents, and preservatives.

As will be appreciated by those skilled in the art and as described in the pertinent texts and literature, a number of methods are available for preparing drug-containing tablets, beads, granules or particles that provide a variety of drug release profiles. Such methods include, but are not limited to, the following: coating a drug or drug-containing composition with an appropriate coating material, typically although not necessarily incorporating a polymeric material, increasing drug particle size, placing the drug within a matrix, and forming complexes of the drug with a suitable complexing agent.

The delayed release dosage units may be coated with the delayed release polymer coating using conventional techniques, e.g., using a conventional coating pan, an airless spray technique, fluidized bed coating equipment (with or without a Wurster insert), or the like. For detailed information concerning materials, equipment and processes for preparing tablets and delayed release dosage forms, see Pharmaceutical Dosage Forms: Tablets, eds. Lieberman et al. (New York: Marcel Dekker, Inc., 1989), and Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, ό.sup.th Ed. (Media, PA: Williams & Wilkins, 1995).

A preferred method for preparing extended release tablets is by compressing a drag-containing blend, e.g., blend of granules, prepared using a direct blend, wet-granulation, or dry-granulation process. Extended release tablets may also be molded rather than compressed, starting with a moist material containing a suitable water-soluble lubricant. However, tablets are preferably manufactured using compression rather than molding. A preferred method for forming extended release drug-containing blend is to mix drug particles directly with one or more excipients such as diluents (or fillers), binders, disintegrants, lubricants, gUdants, and colorants. As an alternative to direct blending, a drug-containing blend may be prepared by using wet-granulation or dry-granulation processes. Beads containing the active agent may also be prepared by any one of a number of conventional techniques, typically starting from a fluid dispersion. For example, a typical method for preparing drug-containing beads involves dispersing or dissolving the active agent in a coating suspension or solution containing pharmaceutical excipients such as polyvinylpyrrolidone, methylcellulose, talc, metallic stearates, silicone dioxide, plasticizers or the like. The admixture is used to coat a bead core such as a sugar sphere (or so-called "non-pareil") having a size of approximately 60 to 20 mesh.

An alternative procedure for preparing drug beads is by blending drug with one or more pharmaceutically acceptable excipients, such as microcrystalline cellulose, lactose, cellulose, polyvinyl pyrrolidone, talc, magnesium stearate, a disintegrant, etc., extruding the blend, spheronizing the extrudate, drying and optionally coating to form the immediate release beads.

Extended release dosage forms

The extended release formulations are generally prepared as diffusion or osmotic systems, for example, as described in "Remington - The science and practice of pharmacy" (20th ed., Lippincott Williams & Wilkins, Baltimore, MD, 2000). A diffusion system typically consists of two types of devices, reservoir and matrix, and is well known and described in the art. The matrix devices are generally prepared by compressing the drug with a slowly dissolving polymer carrier into a tablet form. The three major types of materials used in the preparation of matrix devices are insoluble plastics, hydrophilic polymers, and fatty compounds. Plastic matrices include, but not limited to, methyl acrylate-methyl methacrylate, polyvinyl chloride, and polyethylene. Hydrophilic polymers include, but are not limited to, methylcellulose, hydroxypropylcelluiose, hydroxypropylmemylcelluiose, sodium carboxymethylcellulose, and carbopol 934, polyethylene oxides. Fatty compounds include, but are not limited to, various waxes such as carnauba wax and glyceryl tristearate.

Alternatively, extended release formulations can be prepared using osmotic systems or by applying a semi-permeable coating to the dosage form. In the latter case, the desired drug release profile can be achieved by combining low permeable and high permeable coating materials in suitable proportion.

The devices with different drug release mechanisms described above could be combined in a final dosage form comprising single or multiple units. Examples of multiple units include multilayer tablets, capsules containing tablets, beads, granules, etc.

An immediate release portion can be added to the extended release system by means of either applying an immediate release layer on top of the extended release core using coating or compression process or in a multiple unit system such as a capsule containing extended and immediate release beads.

Extended release tablets containing hydrophilic polymers are prepared by techniques commonly known in the art such as direct compression, wet granulation, or dry granulation processes. Their formulations usually incorporate polymers, diluents, binders, and lubricants as well as the active pharmaceutical ingredient. The usual diluents include inert powdered substances such as any of many different kinds of starch, powdered cellulose, especially crystalline and microcrystalline cellulose, sugars such as fructose, mannitol and sucrose, grain flours and similar edible powders. Typical diluents include, for example, various types of starch, lactose, mannitol, kaolin, calcium phosphate or sulfate, inorganic salts such as sodium chloride and powdered sugar. Powdered cellulose derivatives are also useful. Typical tablet binders include substances such as starch, gelatin and sugars such as lactose, fructose, and glucose. Natural and synthetic gums, including acacia, alginates, methylcellulose, and polyvinylpyrrolidine can also be used. Polyethylene glycol, hydrophilic polymers, ethylcellulose and waxes can also serve as binders. A lubricant is necessary in a tablet formulation to prevent the tablet and punches from sticking in the die. The lubricant is chosen from such slippery solids as talc, magnesium and calcium stearate, stearic acid and hydrogenated vegetable oils.

Extended release tablets containing wax materials are generally prepared using methods known in the art such as a direct blend method, a congealing method, and an aqueous dispersion method. In a congealing method, the drug is mixed with a wax material and either spray- congealed or congealed and screened and processed.

Delayed release dosage forms

Delayed release formulations are created by coating a solid dosage form with a film of a polymer which is insoluble in the acid environment of the stomach, and soluble in the neutral environment of small intestines.

The delayed release dosage units can be prepared, for example, by coating a drug or a drug-containing composition with a selected coating material. The drug-containing composition may be, e.g., a tablet for incorporation into a capsule, a tablet for use as an inner core in a "coated core" dosage form, or a plurality of drug-containing beads, particles or granules, for incorporation into either a tablet or capsule. Preferred coating materials include bioerodible, gradually hydrolyzable, gradually water- soluble, and/or enzymatϊcally degradable polymers, and may be conventional "enteric" polymers. Enteric polymers, as will be appreciated by those skilled in the art, become soluble in the higher pH environment of the lower gastrointestinal tract or slowly erode as the dosage form passes through the gastrointestinal tract, while enzymatically degradable polymers are degraded by bacterial enzymes present in the lower gastrointestinal tract, particularly in the colon. Suitable coating materials for effecting delayed release include, but are not limited to, cellulosic polymers such as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxymethyl cellulose, hydroxypropyl methyl cellulose, hydroxypropyl methyl cellulose acetate succinate,

hydroxypropylmethyl cellulose phthalate, methylcellulose, ethyl cellulose, cellulose acetate, cellulose acetate phthalate, cellulose acetate trimellitate and carboxymethylcellulose sodium; acrylic acid polymers and copolymers, preferably formed from acrylic acid, methacrylic acid, methyl acrylate, ethyl acrylate, methyl methacrylate and/or ethyl methacrylate, and other methacrylic resins that are commercially available under the tradename Eudragit®. (Rohm Pharma; Westerstadt, Germany), including Eudragit®. L30D-55 and L100-55 (soluble at pH 5.5 and above), Eudragit®. L-IOO (soluble at pH 6.0 and above), Eudragit®. S (soluble at pH 7.0 and above, as a result of a higher degree of esterification), and Eudragits®. NE, RL and RS (water-insoluble polymers having different degrees of permeability and expandability); vinyl polymers and copolymers such as polyvinyl pyrrolidone, vinyl acetate, vinylacetate phthalate, vinylacetate crotonic acid copolymer, and ethylene-vinyl acetate copolymer; enzymatically degradable polymers such as azo polymers, pectin, chitosan, amylose and guar gum; zein and shellac. Combinations of different coating materials may also be used. Multi-layer coatings using different polymers may also be applied.

The preferred coating weights for particular coating materials may be readily determined by those skilled in the art by evaluating individual release profiles for tablets, beads and granules prepared with different quantities of various coating materials. It is the combination of materials, method and form of application that produce the desired release characteristics, which one can determine only from the clinical studies.

The coating composition may include conventional additives, such as plasticizers, pigments, colorants, stabilizing agents, glidants, etc. A plasticizer is normally present to reduce the fragility of the coating, and will generally represent about 10 wt. % to 50 wt. % relative to the dry weight of the polymer. Examples of typical plasticizers include polyethylene glycol, propylene glycol, triacetin, dimethyl phthalate, diethyl phthalate, dibutyl phthalate, dibutyl sebacate, triethyl citrate, tributyl citrate, triethyl acetyl citrate, castor oil and acetylated monoglycerides. A stabilizing agent is preferably used to stabilize particles in the dispersion. Typical stabilizing agents are nonionic emulsifiers such as sorbitan esters, polysorbates and polyvinylpyrrolidone. Glidants are recommended to reduce sticking effects during film formation and drying, and will generally represent approximately 25 wt. % to 100 wt. % of the polymer weight in the coating solution. One effective gUdant is talc. Other glidants such as magnesium stearate and glycerol monostearates may also be used. Pigments such as titanium dioxide may also be used. Small quantities of an anti-foaming agent, such as a silicone (e.g., simethicone), may also be added to the coating composition. Particle materials

The particles can be prepared entirely from a therapeutic or diagnostic agent, or from a combination of the agent and a surfactant. The particles can be made of a variety of materials. Both inorganic and organic materials can be used. For example, ceramics may be used. Polymeric and non-polymeric materials, such as fatty acids, may be used to form

aerodynamically light particles. Other suitable materials include, but are not limited to, gelatin, polyethylene glycol, trehalose, and dextran. Particles with degradation and release times ranging from seconds to months can be designed and fabricated, based on factors such as the particle material.

Polymeric particles for lung delivery

Polymeric particles may be formed from any biocompatible,, and preferably biodegradable polymer, copolymer, or blend. Preferred polymers are those which are capable of forming aerodynamically light particles having a tap density less than about 0.4 g/cm3, a mean diameter between 5 μm and 30 μra and an aerodynamic diameter between approximately one and five microns, preferably between one and three microns. The polymers may be tailored to optimize different characteristics of the particle including: i) interactions between the agent to be delivered and the polymer to provide stabilization of the agent and retention of activity upon delivery; ii) rate of polymer degradation and, thereby, rate of drug release profiles; iii) surface characteristics and targeting capabilities via chemical modification; and iv) particle porosity.

Surface eroding polymers such as polyanhydrides may be used to form the particles. For example, polyanhydrides such as poly[(ρ- carboxyphenoxy)-hexane anhydride] (PCPH) may be used. Biodegradable polyanhydrides are described in U.S. Patent No. 4,857,311.

In another embodiment, bulk eroding polymers such as those based on polyesters including poly(hydroxy acids) can be used. For example, polyglycolic acid (PGA), polylactic acid (PLA)₅ or copolymers thereof may be used to form the particles. The polyester may also have a charged or functionalizable group, such as an amino acid. In a preferred embodiment, particles with controlled release properties can be formed of poly(D,L-lactic acid) and/or poly(D,L-lactic-co-glycolic acid) ("PLGA") which incorporate a surfactant such as DPPC.

Other polymers include polyamides, polycarbonates, polyalkylenes such as polyethylene, polypropylene, poly(ethylene glycol), poly(ethylene oxide), poly(ethylene terephthalate), poly vinyl compounds such as polyvinyl alcohols, polyvinyl ethers, and polyvinyl esters, polymers of acrylic and methacrylic acids, celluloses and other polysaccharides, and peptides or proteins, or copolymers or blends thereof. Polymers may be selected with or modified to have the appropriate stability and degradation rates in vivo for different controlled drug delivery applications.

In one embodiment, aerodynamically light particles are formed from functionalized polyester graft copolymers, as described in Hrkach et al., Macromolecules, 28:4736-4739 (1995); and Hrkach et al., "Poly(L-Lactic acid~co-amino acid) Graft Copolymers: A Class of Functional Biodegradable Biomaterials" in Hydrogels and Biodegradable Polymers for Bioapplications, ACS Symposium Series No. 627, Raphael M. Ottenbrite et al., Eds.,

American Chemical Society, Chapter 8, pp. 93-101, 1996.

Materials other than biodegradable polymers may be used to form the particles. Suitable materials include various non-biodegradable polymers and various excipients. The particles also may be formed of a therapeutic or diagnostic agent and surfactant alone. In one embodiment, the particles may be formed of the surfactant and include a therapeutic or diagnostic agent, to improve aerosolization efficiency due to reduced particle surface

interactions, and to potentially reduce loss of the agent due to phagocytosis by alveolar macrophages.

Excipients

In addition to a therapeutic or diagnostic agent (or possibly other desired molecules for delivery), the particles can include excipients such as a sugar, such as lactose, a protein, such as albumin, and/or a surfactant. Complex Forming Materials

If the agent to be delivered is negatively charged, protamine or other positively charged molecules can be added to provide a lipophilic complex which results in the sustained release of the negatively charged agent.

Negatively charged molecules can be used to render insoluble positively charged agents.

Materials Enhancing Sustained Release

If the molecules are hydrophilic and tend to solubilize readily in an aqueous environment, another method for achieving sustained release is to use cholesterol or very high surfactant concentration. This complexation methodology also applies to particles that are not aerodynamically light.

Formation of Particles

Formation of Polymeric Particles

Polymeric particles may be prepared using single and double emulsion solvent evaporation, spray drying, solvent extraction, solvent evaporation, phase separation, simple and complex coacervation, interfacial polymerization, and other methods well known to those of ordinary skill in the art. Particles may be made using methods for making microspheres or microcapsules known in the art, provided that the conditions are optimized for forming particles with the desired aerodynamic diameter, or additional steps are performed to select particles with the density and diameter sufficient to provide the particles with an aerodynamic diameter between one and five microns, preferably between one and three microns.

Methods developed for making microspheres for delivery of encapsulated agents are described in the literature, for example, as described in Doubrow, M., Ed., "Microcapsules and Nanoparticles in Medicine and Pharmacy," CRC Press, Boca Raton, 1992. Methods also are described in Mathiowitz and Langer, J. Controlled Release 5,13-22 (1987); Mathiowitz et al., Reactive Polymers 6, 275-283 (1987); and Mathiowitz et al., J. Appl. Polymer Sci. 35, 755-774 (1988). The selection of the method depends on the polymer selection, the size, external morphology, and crystallinity that is desired, as described, for example, by Mathiowitz et al., Scanning

Microscopy 4: 329-340 (1990); Mathiowitz et al., J. Appl. Polymer Sci. 45, 125-134 (1992); and Benita et al., J. Pharm. Sci. 73, 1721-1724 (1984).

In solvent evaporation, described for example, in Mathiowitz et al., (1990), Benita; and U.S. Patent No. 4,272,398 to Jaffe, the polymer is dissolved in a volatile organic solvent, such as methylene chloride. Several different polymer concentrations can be used, for example, between 0.05 and 1.0 g/ml. The therapeutic or diagnostic agent, either in soluble form or dispersed as fine particles, is added to the polymer solution, and the mixture is suspended in an aqueous phase that contains a surface active agent such as polyvinyl alcohol). The aqueous phase may be, for example, a

concentration of 1% polyvinyl alcohol) w/v in distilled water. The resulting emulsion is stirred until most of the organic solvent evaporates, leaving solid microspheres, which may be washed with water and dried overnight in a lyophilizer. Microspheres with different sizes (between 1 and 1000 microns) and morphologies can be obtained by this method.

Solvent removal was primarily designed for use with less stable polymers, such as the polyanhydrides. In this method, the agent is dispersed or dissolved in a solution of a selected polymer in a volatile organic solvent like methylene chloride. The mixture is then suspended in oil, such as silicon oil, by stirring, to form an emulsion. Within 24 hours, the solvent diffuses into the oil phase and the emulsion droplets harden into solid polymer microspheres. Unlike the hot-melt microencapsulation method described for example in Mathiowitz et al., Reactive Polymers, 6:275 (1987), this method can be used to make microspheres from polymers with high melting points and a wide range of molecular weights. Microspheres having a diameter for example between one and 300 microns can be obtained with this procedure.

With some polymeric systems, polymeric particles prepared using a single or double emulsion technique vary in size depending on the size of the droplets. If droplets in water~in-oil emulsions are not of a suitably small size to form particles with the desired size range, smaller droplets can be prepard, for example, by sonication or homogenation of the emulsion, or by the addition of surfactants.

If the particles prepared by any of the above methods have a size range outside of the desired range, particles can be sized, for example, using a sieve, and further separated according to density using techniques known to those of skill in the art.

The polymeric particles are preferably prepared by spray drying. Prior methods of spray drying, such as that disclosed in PCT WO 96/09814 by Sutton and Johnson, disclose the preparation of smooth, spherical microparticles of a water-soluble material with at least 90% of the particles possessing a mean size between 1 and 10 μm. The method disclosed herein provides rough (non-smooth), non-spherical microparticles that include a water-soluble material combined with a water-insoluble material. At least 90% of the particles possess a mean size between 5 and 30 μm, and a low mass or tap density (less than 0.4 g/cc).

The particles can incorporate various complexes of therapeutic or diagnostic agents to be delivered with molecules of an opposite charge, or can include substances such as lipids which allow for the sustained release of small and large molecules. Addition of these complexes or substances is applicable to particles of any size and shape, and is especially useful for altering the rate of release of therapeutic agents from inhaled particles.

Aerodynamically Light Particles

Aerodynamically light particles, having a tap density less than about 0.4 g/cm3 and an aerodynamic diameter between one and five microns, preferably between one and three microns, may be fabricated using the methods disclosed herein.

Aerodynamically Light Particle Size

The aerodynamically light particles may be fabricated or separated, for example by filtration or centrifugation, to provide a particle sample with a preselected size distribution. For example, greater than 30%, 50%, 70%, or 80% of the particles in a sample can have a diameter within a selected range of at least 5 μm. The selected range within which a certain percentage of the particles must fall may be for example, between about 5 and 30 μm, or optionally between 5 and 15 μm. In one preferred embodiment, at least a portion of the particles have a diameter between about 9 and 11 μm.

Optionally, the particle sample also can be fabricated wherein at least 90%, or optionally 95% or 99%, have a diameter within the selected range. The presence of the higher proportion of the aerodynamically light, larger diameter (at least about 5 μm) particles in the particle sample enhances the delivery of therapeutic or diagnostic agents incorporated therein to the deep lung.

In one embodiment, in the particle sample, the interquartile range may be 2 μm, with a mean diameter for example, between about 7.5 and 13.5 μm. Thus, for example, between at least 30% and 40% of the particles may have diameters within the selected range. Preferably, the the percentages of particles have diameters within a 1 μm range, for example, between 6.0 and 7.0 μm, 10.0 and 11.0 μm or 13.0 andl4.0 μm.

The aerodynamically light particles, optionally incorporating a therapeutic or diagnostic agent, with a tap density less than about 0.4 g/cm3, mean diameters of at least about 5 μm, and an aerodynamic diameter of between one and five microns, preferably between one and three microns, are more capable of escaping inertial and gravitational deposition in the oropharyngeal region, and are targeted to the airways or the deep lung. The use of larger particles (mean diameter at least about 5 μm) is advantageous since they are able to aerosolize more efficiently than smaller, denser aerosol particles such as those currently used for inhalation therapies.

Enteric Coated Capsules

"Gastric resistant natural polymer", as used herein, refers to natural polymers or mixtures of natural polymers which are insoluble in the acidic pH of the stomach.

"Film-forming natural polymer", as used herein, refers to polymers useful for surface coatings that are applied by spraying, brushing, or various industrial processes, which undergo film formation. In most film-formation processes, a liquid coating of relatively low viscosity is applied to a solid substrate and is cured to a solid, high-molecular-weight, polymer-based adherent film possessing the properties desired by the user. For most common applications, this film has a thickness ranging from 0.5 to 500 micrometers (0.0005 to 0.5 millimeters, or 0.00002 to 0.02 inches).

"Gelling agent", as used herein, refers to substances that undergo a high degree of cross-linking or association when hydrated and dispersed in the dispersing medium, or when dissolved in the dispersing medium. This cross-linking or association of the dispersed phase alters the viscosity of the dispersing medium. The movement of the dispersing medium is restricted by the dispersed phase, and the viscosity is increased.

Composition

Gastric resistant film-forming compositions containing (1) a gastric resistant natural polymer; (2) a film-forming natural polymer; and optionally (3) a gelling agent, are described herein.

Gastric Resistant Natural Polymers

Exemplary gastric resistant natural polymers include, but are not limited to, pectin and pectin-like polymers which typically consist mainly of galacturonic acid and galacturonic acid methyl ester units forming linear polysaccharide chains. Typically these polysaccharides are rich in galacturonic acid, rhamnose, arabinose and galactose, for example the polygalacturonans, rhamnogalacturonans and some arabinans, galactans and arabinogalactans. These are normally classified according to the degree of esterification.

In high (methyl) ester ("HM") pectin, a relatively high portion of the carboxyl groups occur as methyl esters, and the remaining carboxylic acid groups are in the form of the free acid or as its ammonium, potassium, calcium or sodium salt. Useful properties may vary with the degree of esterification and with the degree of polymerization. Pectin, in which less than 50% of the carboxyl acid units occur as the methyl ester, is normally referred to as low (methyl) ester or LM-pectin. In general, low ester pectin is obtained from high ester pectin by treatment at mild acidic or alkaline conditions. Amidated pectin is obtained from high ester pectin when ammonia is used in the alkaline deesterification process. In this type of pectin some of the remaining carboxylic acid groups have been transformed into the acid amide. The useful properties of amidated pectin may vary with the proportion of ester and amide units and with the degree of

polymerization.

In one embodiment, the gastric resistant natural polymer is pectin. The gastric resistant natural polymer is present in an amount less than about 5% by weight of the composition, preferably from about 2 to about 4% by weight of the composition.

Film-Forming Natural Polymers

Exemplary film-forming natural polymers include,, but are not limited to, gelatin and gelatin-like polymers. In a preferred embodiment, the film- forming natural polymer is gelatin. A number of other gelatin-like polymers are available commercially. The film-forming natural polymer is present in an amount from about 20 to about 40% by weight of the composition, preferably from about 25 to about 40% by weight of the composition.

Gelling Agent

The compositions can optionally contain a gelling agent. Exemplary gelling agents include divalent cations such as Ca2+ and Mg2+. Sources of these ions include inorganic calcium and magnesium salts and calcium gelatin. The gelling agent is present in an amount less than about 2% by weight of the composition, preferably less than about 1% by weight of the composition.

Plasticizers

One or more plasticizers can be added to the composition to facilitate the film-forming process. Suitable plasticizers include glycerin, sorbitol, sorbitans, maltitol, glycerol, polyethylene glycol, polyalcohols with 3 to 6 carbon atoms, citric acid, citric acid esters, triethyl citrate and combinations thereof. The concentration of the one or more plasticizers is from about 8% to about 30% by weight of the composition. In one embodiment, the plasticizer is glycerin and/or sorbitol. Method of Making

The film-forming composition can be used to prepare soft or hard shell gelatin capsules which can encapsulate a liquid or semi-solid fill material or a solid tablet (Softlet®) containing an active agent and one or more pharmaceutically acceptable excipients. Alternatively, the composition can be administered as a liquid with an active agent dissolved or dispersed in the composition.

Capsules

Shell

The film-forming composition can be used to prepare soft or hard capsules using techniques well known in the art. For example., soft capsules are typically produced using a rotary die encapsulation process. Fill formulations are fed into the encapsulation machine by gravity.

The capsule shell can contain one or more plasticizers selected from the group consisting of glycerin, sorbitol, sorbitans, maltitol, glycerol, polyethylene glycol, polyalcohols with 3 to 6 carbon atoms, citric acid, citric acid esters, triethyl citrate and combinations thereof.

In addition to the plasticizer(s), the capsule shell can include other suitable shell additives such as opacifiers, colorants, humectants, preservatives, flavorings, and buffering salts and acids.

Opacifiers are used to opacify the capsule shell when the

encapsulated active agents are light sensitive. Suitable opacifiers include titanium dioxide, zinc oxide, calcium carbonate and combinations thereof.

Colorants can be used to for marketing and product identification/ differentiation purposes. Suitable colorants include synthetic and natural dyes and combinations thereof.

Humectants can be used to suppress the water activity of the softgel. Suitable humectants include glycerin and sorbitol, which are often components of the plasticizer composition. Due to the low water activity of dried, properly stored softgels, the greatest risk from microorganisms comes from molds and yeasts. For this reason, preservatives can be incorporated into the capsule shell. Suitable preservatives include alkyl esters of p- hydroxy benzoic acid such as methyl, ethyl, propyl, butyl and heptyl

(collectively known as "parabens") or combinations thereof.

Flavorings can be used to mask unpleasant odors and tastes of fill formulations. Suitable flavorings include synthetic and natural flavorings. The use of flavorings can be problematic due to the presence of aldehydes which can cross-link gelatin. As a result, buffering salts and acids can be used in conjunction with flavorings that contain aldehydes in order to inhibit cross-linking of the gelatin.

Fill Material

Soft or hard capsules can be used to deliver a wide variety of pharmaceutically active agents. Suitable agents include small molecules, proteins, nucleic acid, carbohydrates, lipids, and full organisms.

Fill formulations may be prepared using a pharmaceutically acceptable carrier composed of materials that are considered safe and effective and may be administered to an individual without causing undesirable biological side effects or unwanted interactions. The carrier is all components present in the pharmaceutical formulation other than the active ingredient or ingredients. As generally used herein "carrier" includes, but is not limited to surfactants, humectants, plasticizers, crystallization inhibitors, wetting agents, bulk filling agents, solubilizers, bioavailability enhancers, pH adjusting agents, and combinations thereof.

Alternatively, the composition can be administered as a liquid with an active agent dissolved (e.g. solution) or dispersed (e.g. suspension) in the composition. Suitable active agents are described above. The solution or suspension may be prepared using one or more pharmaceutically acceptable excipients. Suitable excipients include, but are not limited to, surfactants, humectants, plasticizers, crystallization inhibitors, wetting agents, bulk filling agents, solubilizers, bioavailability enhancers, pH adjusting agents, flavorants and combinations thereof

Muco adhesive Particles and methods of manufacturing

In general terms, adhesion of polymers to tissues may be achieved by (i) physical or mechanical bonds, (ii) primary or covalent chemical bonds, and/or (iii) secondary chemical bonds (i.e., ionic). Physical or mechanical bonds can result from deposition and inclusion of the adhesive material in the crevices of the mucus or the folds of the mucosa. Secondary chemical bonds, contributing to bioadhesive properties, consist of dispersive interactions (i.e., Van der Waals interactions) and stronger specific interactions, which include hydrogen bonds. The hydrophilic functional groups responsible for forming hydrogen bonds are the hydroxyl (-OH) and the carboxylic groups (-COOH).

Adhesive polymeric microspheres have been selected on the basis of the physical and chemical bonds formed as a function of chemical composition and physical characteristics, such as surface area, as described in detail below. These microspheres are characterized by adhesive forces to mucosa of greater than 11 mN/cm². The size of these microspheres range from between a nanoparticle to a millimeter in diameter. The adhesive force is a function of polymer composition, biological substrate, particle morphology, particle geometry (e.g., diameter) and surface modification."

Classes of Polymers Useful in Forming Bioadhesive

Microspheres.

Suitable polymers that can be used to form bioadhesive microspheres include soluble and insoluble, biodegradable and nonbiodegradable polymers. These can be hydrogels or thermoplastics, homopolymers, copolymers or blends, natural or synthetic. A key feature, however, is that the polymer must produce a bioadhesive interaction between 110 N/m² (11 mN/cm²) and 100,000 N/m² when applied to the mucosal surface of rat intestine.

The forces described herein refer to measurements made upon rat intestinal mucosa, unless otherwise stated. The same adhesive measurements made on a different species of animal will differ from those obtained using rats. This difference is attributed to both compositional and geometrical variations in the mucous layers of different animal species as well as cellular variations in the mucosal epithelium. However, the data shows that the same general trends prevail no matter what animal is studied {i.e., P(FA:SA) produces stronger adhesions than PLA in rats, sheep, pigs, etc.).

The mucous layer varies from species to species and even animal to animal due to differences arising from variations in diet, location, GI activity, sex and state of health. In general, GI mucus is made of 95% water and 5% electrolytes, lipids, proteins and glycoproteins, as described by Spiro, R.G., "Glycoproteins," Annual Review of Biochemistry, 39, 599-638, 1970; Labat-Robert, J. & Decaeus, C, "Glycoproteins du Mucus Gastrique: Structure, Function, et Pathologie," Pathologie et Biologie (Paris), 24, 241 1979. However, the composition of the latter fraction can vary greatly. Proteins, including the protein core of the glycoproteins, can made up anywhere from 60 to 80% of this fraction, Horowitz, M.I.,

"Mucopolysaccharides and Glycoproteins of the Alimentary Tract" in Alimentary Canal (eds. C. F. Code), pp. 1063-1085 (Washington: American Physiological Society, 1967). The glycoproteins typically have a molecular weight of approximately two million and consist of a protein core

(approximately 18.6-25.6% by weight) with covalently attached

carbohydrate side chains (approximately 81.4-74.4% by weight) terminating in either L-fucose or sialic acid residues. Spiro, R.G., "Glycoproteins," Annual Review of Biochemistry, 39, 599-638, 1970; Scawen, M. & Allen, A., "The Action of Proteolytic Enzymes on the Glycoprotein from Pig Gastric Mucus," Biochemical Journal, 163, 363-368, 1977; Horowitz, M.I. & Pigman, W., The Glycoconjugates, pp. 560 (New York: Academic Press, Inc., 1977); Pigman, W. & Gottschalk, A., "Submaxillary Gland

Glycoproteins" in Glycoproteins: Their Composition, Structure and Function (eds. A. Gottschalk), pp. 434-445 (Amsterdam: Elsevier Publishing

Company, Inc., 1966). Species and location differences in the composition of these glycoproteins have been reported by Horowitz, M.I.,

"Mucopolysaccharides and Glycoproteins of the Alimentary Tract" in Alimentary Canal (eds. C. F. Code), pp. 1063-1085 (Washington: American Physiological Society, 1967). In order for bioadhesive particles to embed themselves or become engulfed in the mucus lining the GI tract, the radius of the individual particles should be as thick as the thickness of the natural mucous layer. It has been shown that the gastric mucous layer thickness typically varies from 5 to 200 μ in the rat and 10 to 400 μ in man. Occasionally, however, it can reach thicknesses as great as 1000 μ in man, as described by Spiro, R. G., "Glycoproteins," Annual Review of Biochemistry, 39, 599-638, 1970; Labat- Robert, J. & Decaeus, C, "Glycoproteins du Mucus Gastrique: Structure, Fonction, et Pathologie," Pathologie et Biologie (Paris), 24, 241, 1979;

Allen, A., Hutton, D.A., Pearson, J.P., & Sellers, L.A., "Mucus Glycoprotein Structure, Gel Formation and Gastrointestinal Mucus Function" in Mucus and Mucosa, Ciba Foundation Symposium 109 (eds. J. Nugent & M.

O'Connor), pp. 137 (London: Pitman, 1984). Obvious physical differences in the mucus thickness were observed in the studies described herein. For example, the mucous layers in the rat and monkey were substantially thinner than those observed in the pig and sheep. Although the general order of adhesiveness was maintained throughout the studies, it must be noted that the tenacity of adhesion was dependent on the abundance of mucus.

In the past, two classes of polymers have appeared to show useful bioadhesive properties: hydrophilic polymers and hydrogels. In the large class of hydrophilic polymers, those containing carboxylic groups (e.g., poly[acrylic acid]) exhibit the best bioadhesive properties. One could infer that polymers with the highest concentrations of carboxylic groups should be the materials of choice for bioadhesion on soft tissues. In other studies, the most promising polymers were: sodium alginate, carboxymethylcellulose, hydroxymethylcellulose and methylcellulose. Some of these materials are water-soluble, while others are hydrogels.

Rapidly bioerodible polymers such as poly[lactide-co-glycolϊde], polyanhydrides, and polyorthoesters, whose carboxylic groups are exposed on the external surface as their smooth surface erodes, are excellent candidates for bioadhesive drug delivery systems. In addition, polymers containing labile bonds, such as polyanhydrides and polyesters, are well known for their hydrolytic reactivity. Their hydrolytic degradation rates can generally be altered by simple changes in the polymer backbone.

Representative natural polymers include proteins, such as zein, modified zein, casein, gelatin, gluten, serum albumin, or collagen, and polysaccharides, such as cellulose, dextrans, polyhyaluronic acid, polymers of acrylic and methacrylic esters and alginic acid. Representative synthetic polymers include polyphosphazines, poly(vinyl alcohols), polyamides, polycarbonates, polyalkylenes, polyacrylamldes, polyalkylene glycols, polyalkylene oxides, polyalkylene terephthalates, polyvinyl ethers, polyvinyl esters, polyvinyl halides, polyvinylpyrrolidone, polyglycolides,

polysiloxanes, polyurethanes and copolymers thereof. Synthetically modified natural polymers include alkyl celluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, and nitrocelluloses. Other polymers of interest include, but are not limited to, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxymethyl cellulose, cellulose triacetate, cellulose sulfate sodium salt, poly(methyl methacrylate), poly(ethyl methacrylate), poly(butyl methacrylate), poly(isobutyl methacrylate), poly(hexyl methacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), ρoly(ρhenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl acrylate) polyethylene, polypropylene, poly(ethylene glycol), poly(ethylene oxide), poly (ethylene terephthalate), poly(vinyl acetate), polyvinyl chloride, polystyrene, polyvinyl pyrrolidone, and polyvinylphenol. Representative bioerodible polymers include polylactides, polyglycolides and copolymers thereof, poly(ethylene terephthalate), poly(butic acid), poly(valeric acid), poly(lactide-co-caprolactone), poly[Iactide-co-glycolide], polyanhydrides, polyorthoesters, blends and copolymers thereof.

These polymers can be obtained from sources such as Sigma

Chemical Co., St. Louis, MO., Polysciences, Warrenton, PA, Aldrich, Milwaukee, WI, Fluka, Ronkonkoma, NY, and BioRad, Richmond, CA. or else synthesized from monomers obtained from these suppliers using standard techniques.

In the studies detailed below, a variety of polymer microspheres were compared for adhesive force to mucosa. Negatively charged hydrogels, such as alginate and carboxymethylcellulose, with exposed carboxylic groups on the surface, were tested, as well as some positively-charged hydrogels, such as chitosan. The rationale behind this choice is the fact that most cell membranes are actually negatively charged and there is still no definite conclusion as to what the most important property is in obtaining good bioadhesion to the wall of the gastrointestinal tract. Thermoplastic polymers, including (a) non-erodible, neutral polystyrene, and (b) sermcrystalHne bioerodible polymers that reveal or generate carboxylic groups as they degrade, polylactides and polyanhydrides, were also tested. Polyanhydrides are better candidates for bJoadJhesive delivery systems since, as hydrolysis proceeds, causing surface erosion, more and more carboxylic groups are exposed to the external surface. However, polylactides erode more slowly by bulk erosion. In designing bioadhesive polymeric systems based on polylactides, polymers that have high concentrations of carboxylic acid are preferred. This can be accomplished by using low molecular weight polymers (Mw 2000), since low molecular weight polymers contain high concentration of carboxylic acids at the end groups.

Modification of Polymers.

The polymers were selected from commercially available materials that could be fashioned into microsphere delivery devices or used to coat pre-existing microspheres. In some instances, the polymeric material could be modified to improve bioadhesion (to force values greater than 11 mN/cm2) either before or after the fabrication of microspheres.

For example, the polymers can be modified by increasing the number of carboxylic groups accessible during biodegradation, or on the polymer surface. The polymers can also be modified by binding amino groups to the polymer. The polymers can also be modified using any of a number of different coupling chemistries that covalently attach ligand molecules with bioadhesive properties to the surface-exposed molecules of the polymeric microspheres.

One useful protocol involves the "activation" of hydroxyl groups on polymer chains with the agent, carbonyldiimidazole (CDI) in aprotic solvents such as DMSO, acetone, or XHF. CDI forms an imidazolyl carbamate complex with the hydroxyl group which may be displaced by binding the free amino group of a ligand such as a protein. The reaction is an N- nucleophilic substitution and results in a stable N-alkylcarbamate linkage of the ligand to the polymer. The "coupling" of the ligand to the "activated" polymer matrix is maximal in the pH range of 9-10 and normally requires at least 24 hrs. The resulting ligand-polymer complex is stable and resists hydrolysis for extended periods of time.

Another coupling method involves the use of l-ethyl~3-(3- dimethylaminopropyl) carbodiimide (EDAC) or "water-soluble CDI" in conjunction with N-hydroxylsulfosuccinimide (sulfo NHS) to couple the exposed carboxylic groups of polymers to the free amino groups of ligands in a totally aqueous environment at the physiological pH of 7.0. Briefly, EDAC and sulfo-NHS form an activated ester with the carboxylic acid groups of the polymer which react with the amine end of a ligand to form a peptide bond. The resulting peptide bond is resistant to hydrolysis. The use of sulfo-NHS in the reaction increases the efficiency of the EDAC coupling by a factor of ten-fold and provides for exceptionally gentle conditions that ensure the viability of the ligand-polymer complex.

By using either of these protocols it is possible to "activate" almost all polymers containing either hydroxyl or carboxyl groups in a suitable solvent system that will not dissolve the polymer matrix.

A useful coupling procedure for attaching ligands with free hydroxyl and carboxyl groups to polymers involves the use of the cross-linking agent, divinylsulfone. This method would be useful for attaching sugars or other hydroxylic compounds with bioadhesive properties to hydroxylic matrices. Briefly, the activation involves the reaction of divinylsulfone to the hydroxyl groups of the polymer, forming the vinylsulfonyl ethyl ether of the polymer. The vinyl groups will couple to alcohols, phenols and even amines.

Activation and coupling take place at pH 11. The linkage is stable in the pH range from 1-8 and is suitable for transit through the intestine.

Any suitable coupling method known to those skilled in the art for the coupling of Hgands and polymers with double bonds, including the use of UV crosslinking, may be used for attachment of bioadhesive ligands to the polymeric microspheres described herein. Any polymer that can be modified through the attachment of lectins can be used as a bioadhesive polymer for purposes of drug delivery or imaging.

Lectins that can be covalently attached to microspheres to render them target specific to a bacterial niche could be used as bioadhesives. Useful lectin ligands include lectins isolated from: Abrus precatroius, Agaricus bisporus, Anguilla anguilla, Arachis hypogaea, Pandeiraea simplicifoHa, Bauhinia purpurea, Caragan arobrescens, Cicer arietinum, Codium fragile, Datura stramonium, Doϊichos biflorus, Erγthrina corallodendron, Erythrina cristagalli, Euonymus europaeus, Glycine max, Helix aspersa, Helix pomatia, Lathyrus odoratus, Lens culinaris, Limulus polyphemus, Lysopersicon esculentum, Madura pomifera, Momordica charantia, Mycoplasma gallisepticum, NaJa mocambique, as well as the lectins Concanavalin A, Succinyl-Concanavalin A, Triticum vulgaris, Ulex europaeus I, II and III, Sambucus nigra, Maackia amurensis, Limaxfluvus, Homarus ameήcanus, Cancer antennarius, and Lotus tetragonolobus.

The attachment of any positively charged ligand, such as

polyethyleneimϊne or polylysine, to any microsphere may improve bioadhesion due to the electrostatic attraction of the cationic groups coating the beads to the net negative charge of the mucus. The mucopolysaccharides and mucoproteins of the mucin layer, especially the sialic acid residues, are responsible for the negative charge coating. Any ligand with a high binding affinity for mucin could also be covalently linked to most microspheres with the appropriate chemistry, such as CDI, and be expected to influence the binding of microspheres to the gut. For example, polyclonal antibodies raised against components of mucin or else intact mucin, when covalently coupled to microspheres, would provide for increased bioadhesion.

Similarly, antibodies directed against specific cell surface receptors exposed on the lumenal surface of the intestinal tract would increase the residence time of beads, when coupled to microspheres using the appropriate chemistry. The ligand affinity need not be based only on electrostatic charge, but other useful physical parameters such as solubility in mucin or else specific affinity to carbohydrate groups.

The covalent attachment of any of the natural components of mucin in either pure or partially purified form to the microspheres would decrease the surface tension of the bead-gut interface and increase the solubility of the bead in the mucin layer. The list of useful ligands would include but not be limited to the following: sialic acid, neuraminic acid, n-acetyl-neuraminic acid, n-glycolylneuraminic acid, 4-acetyl-n-acetylneuraminic acid, diacetyl- n-acetylneuraminic acid, glucuronic acid, iduronic acid, galactose, glucose, mannose, fucose, any of the partially purified fractions prepared by chemical treatment of naturally occurring mucin, e.g., mucoproteins,

mucopolysaccharides and mucopolysaccharide-protein complexes, and antibodies immunoreactive against proteins or sugar structure on the mucosal surface.

The attachment of polyamino acids containing extra pendant carboxylic acid side groups, e.g., polyaspartic acid and polyglutamic acid, should also provide a useful means of increasing bioadhesiveness. Using polyamino acids in the 15,000 to 50,000 kDa molecular weight range would yield chains of 120 to 425 amino acid residues attached to the surface of the microspheres. The polyamino chains increase bioadhesion by means of chain entanglement in mucin strands as well as by increased carboxylic charge.

Formation of Microspheres.

As used herein, the term "microspheres" includes microparticles and microcapsules (having a core of a different material than the outer wall), having a diameter in the nanometer range up to 5 mm. The microsphere may consist entirely of bioadhesive polymer or have only an outer coating of bioadhesive polymer.

Microspheres can be fabricated from different polymers using different methods. Polylactic acid blank microspheres can be fabricated using three methods: solvent evaporation, as described by E. Mathiowitz, et al., J. Scanning Microscopy, 4, 329 (1990); L.R. Beck, et al., Fertil. SteriL, 31, 545 (1979); and S. Benita, et al., J. Pharm. Sd., 73, 1721 (1984); hot- melt microencapsulation, as described by E. Mathiowitz, et al., Reactive Polymers, 6, 275 (1987); and spray drying. Polyanhydrides made of bis- carboxyphenoxypropane and sebacic acid with molar ratio of 20:80 P(CPP- SA) (20:80) (Mw 20,000) can be prepared by hot-melt microencapsulation. Poly(furaaric-co-sebacic) (20:80) (Mw 15,000) blank microspheres can be prepared by hot-melt microencapsulation. Polystyrene microspheres can be prepared by solvent evaporation.

Hydrogel microspheres can be prepared by dripping a polymer solution from a reservoir though microdroplet forming device into a stirred ionic bath.

a. Solvent Evaporation. In this method the polymer is dissolved in a volatile organic solvent, such as methylene chloride. The drug (either soluble or dispersed as fine particles) is added to the solution, and the mixture is suspended in an aqueous solution that contains a surface active agent such as polyvinyl alcohol). The resulting emulsion is stirred until most of the organic solvent evaporated, leaving solid microspheres. Several different polymer concentrations were used: 0.05-0.20 g/ml. The solution is loaded with a drug and suspended in 200 ml of vigorously stirred distilled water containing 1% (w/v) polyvinyl alcohol) (Sigma). After 4 hours of stirring, the organic solvent evaporates from the polymer, and the resulting microspheres are washed with water and dried overnight in a lyopMHzer. Microspheres with different sizes (1-1000 microns) and morphologies can be obtained by this method. This method is useful for relatively stable polymers like polyesters and polystyrene. However, labile polymers, such as polyanhydrides, may degrade during the fabrication process due to the presence of water. For these polymers, the following two methods, which are performed in completely anhydrous organic solvents, are more useful.

b. Hot Melt Microencapsulation. In this method, the polymer is first melted and then mixed with the solid particles of dye or drug that have been sieved to less than 50 microns. The mixture is suspended in a non-miscible solvent (like silicon oil), and, with continuous stirring, heated to 5⁰C above the melting point of the polymer. Once the emulsion is stabilized, it is cooled until the polymer particles solidify. The resulting microspheres are washed by decantation with petroleum ether to give a free-flowing powder. Microspheres with sizes between one to 1000 microns are obtained with this method. The external surfaces of spheres prepared with this technique are usually smooth and dense. This procedure is used to prepare microspheres made of polyesters and polyanhydrides. However, this method is limited to polymers with molecular weights between 1000-50,000.

c. Solvent Removal. This technique is primarily designed for polyanhydrides. In this method, the drug is dispersed or dissolved in a solution of the selected polymer in a volatile organic solvent like methylene chloride. This mixture is suspended by stirring in an organic oil (such as silicon oil) to form an emulsion. Unlike solvent evaporation, this method can be used to make microspheres from polymers with high melting points and different molecular weights. Microspheres that range between 1-300 microns can be obtained by this procedure. The external morphology of spheres produced with this technique is highly dependent on the type of polymer used.

d. Spray-Drying In this method, the polymer is dissolved in methylene chloride (0.04 g/mL). A known amount of the active drug is suspended (insoluble drugs) or co-dissolved (soluble drugs) in the polymer solution. The solution or the dispersion is then spray-dried. Typical process parameters for a mini-spray drier (Buchi) are as follows: polymer concentration = 0.04 g/mL, inlet temperature = -24°C, outlet temperature = 13-15 ⁰C, aspirator setting = 15, pump setting = 10 mL/minute, spray flow = 600 Nl/hr, and nozzle diameter = 0.5 mm. Microspheres ranging between 1- 10 microns are obtained with a morphology which depends on the type of polymer used. This method is primarily used for preparing microspheres designed to improve imaging of the intestinal tract, since for this application, particle size should not exceed 10 μ.

e. Hydrogel Microspheres. Microspheres made of gel-type polymers, such as alginate, are produced through traditional ionic gelation techniques. The polymers are first dissolved in an aqueous solution, mixed with barium sulfate or some bioactive agent, and then extruded through a microdroplet forming device, which in some instances employs a flow of nitrogen gas to break off the droplet. A slowly stirred (approximately 100- 170 RPM) ionic hardening bath is positioned below the extruding device to catch the forming microdroplets. The microspheres are left to incubate in the bath for twenty to thirty minutes in order to allow sufficient time for gelation to occur. Microsphere particle size is controlled by using various size extruders or varying either the nitrogen gas or polymer solution flow rates.

Claims

We claim:

1. A method of delivering a pharmaceutical formulation to a bacterial niche of an animal host comprising:

selecting at least one feature that differentiates the bacterial constituents of the niche from the rest of bacterial residents that coexist in the same anatomical location, and

providing a delivery system that exploits the feature to release a pharmaceutical formulation preferentially in the vicinity of the constituents of a bacterial niche

2. The method of claim 1, wherein release of the pharmaceutical formulation is triggered by at least one differentiating feature of the bacterial niche.

3. The method of claim 1 , wherein the host is a human.

4. The method of claim 1, wherein the feature that differentiates the bacterial niche is selected using a method selected from the group consisting of 16srRNA sequencing, polymerase chain reaction, a metagenomϊc method, mass spectrometry, nuclear magnetic resonance, phage display, and screening a subject's serum IgG for antibodies against microbiota antigens

5. The method of claim 1 , wherein the active ingredient of the pharmaceutical formulation is selected from the group consisting of a small molecule, a nucleic acid, a protein, a polypeptide, a carbohydrate, a lipid, a full organism, a mixture of organisms, and combinations thereof.

6. The method of claim 5, wherein the small molecule has low bioavailability.

7. The method of claim 5, wherein the carbohydrate has a prebiotic effect on the selected bacterial niche.

8. The method of claim 7, wherein the carbohydrate is selected from the group consisting of a fructooligosaccharide, a galactooligosaccharide, inulin, a xylooligosaccharide, polydextrose, a manooligosaccharide, and an arabinoxylan

9. The method of claim 5, wherein the organism or mixture of organisms delivered includes at least one commensal organism of the human flora.

10. The method of claim 5, wherein the organism or mixture of organisms delivered includes at least one probiotic organism.

11. The method of claim 1 , wherein the bacterial niche is located in an anatomical region of the host selected from the oral cavity, oesophagus, stomach, duodenum, jejunum, ileum, cecum, colon, nasal cavity, eye, ear, lungs, vagina, and skin.

12. The method of claim 11 , wherein the bacterial niche is located in the skin, and wherein the delivery system targets at least one bacteria selected from the group consisting of Staphylococcus, Micrococcus,

Corynebacterium, Brevibacteria, Propionibacteria, and Acinetobacter spp.

13. The method of claim 12 wherein the bacterial niche is located in a structure of the human dermis selected from the stratum corneum, the hair follicle, the sebaceous gland, an eccrine sweat gland, and an apocrine sweat gland.

14. The method of claim 11 , wherein the bacterial niche is located in the vagina, wherein the delivery system targets at least one bacteria selected from the group consisting of Lactobacillus, Gardnerella, Mobiluncus, Bacteroides, and Mycoplasma spp., and wherein the pharmaceutical composition causes at least one change selected from the group consisting of increased Lactobacillus, decreased Gardnerella, decreased Mobiluncus, decreased Bacteroides, and decreased Mycoplasma spp.

15. The method of claim 14, wherein the delivery of the pharmaceutical composition is triggered by a hydrolysis reaction performed by a bacterium selected from the group consisting of a Lactobacillus spp.md a

Bifidobacterium spp.

16. The method of claim 11, wherein the bacterial niche is located in the lungs, wherein the pharmaceutical formulation is contained in a porous particle suitable for aerosolization in a dry powder inhaler, wherein the particle has a density less than about 0.4g/cm3 and wherein at least 50% of the particles have an aerodynamic diameter of less than 4 microns.

17. The method of claim 16, wherein the particle comprises at least one component selected from the group consisting of a prebiotic compound, a probiotic organism, and a host commensal organism.

18. The method of claim 17, wherein the particle comprises more than 1% in weight of a prebiotic mucoadhesive carbohydrate.

19. The method of claim 11. wherein the bacterial niche is located in the nasal cavity, wherein the pharmaceutical formulation is contained in a particle comprising at least one component selected from the group consisting of a prebiotic compound, a probiotic organism, and a host commensal organism.

20. The method of claim 1 , wherein the bacterial niche performs at least one function selected from the group consisting of vitamin synthesis, isoprenoid synthesis, metabolism of xenobiotics, metabolism of glycans, metabolism of ammoacids, regulation of fat storage by the host, energy harvest, development of the host immune system, creation of an epithelial barrier, and regeneration of an epithelial barrier.

21. The method of claim 1 , wherein the differentiating feature of the bacterial niche that triggers release of the pharmaceutical formulation is selected from the group consisting of a change in pH, a lag time, a and change in pressure.

22. The method of claira 1 , wherein the differentiating feature of the bacterial niche that triggers release of the pharmaceutical formulation is either the ability to perform a reduction reaction or the ability to perform a hydrolysis reaction.

23. The method of claim 22, wherein the reduction reaction is selected from a nitro group reduction, and azo group reduction, an azo bond cleavage, a reduction of a sulfoxide to a sulfide, a reductive deamination, and a dehydroxylation.

24. The method of claim 23, wherein the reduction reaction is performed at least in part by an eri2yme selected from the group consisting of a nitroreductase, an azoreductase, and a sulfoxide reductase.

25. The method of claim 24, wherein the delivery carrier contains an azo- polymer that is selectively degraded by a bacterial azoreductase.

26. The method of claim 22, wherein the hydrolysis reaction is performed at least in part by an enzyme selected from the group consisting of a B- glucoronidase, a B-galactosidase, a B-glucosidase, a fucosidase, and a sialidase.

27. The method of claim 26, wherein the delivery carrier contains a GRAS polysaccharide that is selectively degraded by a bacterial hydrolase.

28. The method of claim 27, wherein the delivery carrier is selected from the group consisting of amylase,, xanthan gum, dextran, pectin, and galactomannan.

29. The method of claim 26, wherein the hydrolysis reaction is performed by bacteria selected from a member of the lactic acid bacteria and a

Bacteroides sp.

30. The method of claim 26, wherein the lactic acid bacteria is selected from the group consisting of a Bifidobacterium and a Lactobacillus spp.

31. The method of claim 1 , wherein the delivery system targets the pharmaceutical formulation to a niche containing Bacteroides, wherein the pharmaceutical formulation is released by the action an enzyme from Bacteroides performing a thiazole ring-opening reaction.

32. The method of claim 31 , wherein the Bacteroides is B.

thetaiotaomicron.

33. The method of claim 1 , wherein the delivery system targets the pharmaceutical formulation to a niche containing Firmicutes, wherein the pharmaceutical formulation is released by the action of a molecule selected from the group consisting of an amino acid transporter, a sugar transporter, and ABC transporter, and a PTS sytem.

34. The method of claim 33, wherein the Firmicute is selected from the group consisting E. rectale, F. prausnit∑ii , and E. eligens.

35^ The method of claim 1, wherein the delivery system targets the pharmaceutical formulation to a niche containing Clostridia, wherein the pharmaceutical formulation is released by the action of a molecule selected from the group consisting of a ferredoxin-reducing hydrogenase, a membrane-bound oxidoreductase, a Clostridial enzyme performing an imidazole ring opening reaction, and a Clostridial enzyme performing a thiazole ring-opening reaction.

36. The method of claim 1 , wherein the delivery system targets the pharmaceutical formulation to a niche containing sulfate-reducing bacteria, wherein the pharmaceutical formulation is released by a disulfide exchange reaction.

37. The method of claim 1 , wherein the delivery system targets the pharmaceutical formulation to a bacterial niche involved in vitamin or isoprenoid synthesis, wherein the pharmaceutical formulation is released by the action of an enzyme of the 2-methyl-d-erythritol 4-phosphate pathway.

38. The method of claim 1, wherein the delivery system targets the pharmaceutical formulation to a bacterial niche involved in fiavonoid transformation, wherein the pharmaceutical formulation is released by a deglycosylation reaction.

39. The method of claim 1, wherein the delivery system targets a bacteria selected from Eubacterium ramulus and Enterococcus casseliflavus.

40. The method of claim 1 , wherein the pharmaceutical formulation is used to treat or manage a condition associated with dysbiosis of a bacterial niche.

41. The method of claim 40, wherein the pharmaceutical formulation is used to treat or manage a condition selected from the group consisting of obesity, inflammatory bowel disease, Crohn's disease, ulcerative colitis, asthma, cystic fibrosis, allergies, diabetes, psoriasis, eczema, rosacea, atopic dermatitis, gastrointestinal reflux disease, a cancer of the gastrointestinal tract, an infection, an autism spectrum disorder condition, bacterial vaginosis, rheumatoid arthritis, multiple sclerosis, Parkinson's disease, Alzheimer's disease, necrotizing enterocolitis, enterohemorrhagic colitis, pseudomembranous colitis, a bacterial infection, a viral infection, and a parasitic infection.

42. The method according to claim 1, wherein the administration route is selected from the group consisting of oral, topical, rectal, vaginal, topical, pulmonary, ocular and parenteral.

43. The method of claim 1 , wherein the pharmaceutical composition is used to treat Crohn's disease, and wherein the delivery system targets at least one bacterial population selected from Firmicutes, Bacteroides, Eubacteria, Petostreptococcus, and Bifidobacteria.

44. The method of 43, wherein the pharmaceutical composition causes at least one change selected from the group consisting of decreased Bacteroides levels, decreased Eubacteria levels, decreased Petostreptococcus levels, increased Bacteroides levels, and increased F.prausnitzii levels.

45. The method of claim 1 , wherein the pharmaceutical composition is used to treat or prevent a condition selected from an autoimmune disease and an infection, and wherein the delivery system targets a Segmented

Filamentous Bacteria.

46. The method of claim 1, wherein the pharmaceutical composition is used to treat obesity, and wherein the pharmaceutical composition causes at least one change selected from increased Bacteroidetes levels and decreased Firmicutes levels.

47. The method of claim 1, wherein the pharmaceutical composition is used to treat an autism spectrum disorder condition, and wherein the pharmaceutical composition causes a decrease in Clostridia levels.

48. A method of delivering a pharmaceutical formulation away from a bacterial niche of an animal host comprising:

selecting at least one feature that is lacking in the bacterial constituents of the niche in a given anatomical location and present in other bacterial residents of that same anatomical location, and

providing a delivery system that releases a pharmaceutical formulation away from the vicinity of the constituents of a bacterial niche; wherein release of the pharmaceutical formulation does not occur significantly in the bacterial niche and is triggered by the differentiating feature away from the bacterial niche.

49. The method of claim 48, wherein the delivery system targets the pharmaceutical formulation away from a niche containing Firmicutes, wherein the pharmaceutical formulation is released by the action of a glycan- degrading enzyme.

50. The method of claim 49, wherein the Firmicute is selected from E, rectale, F. prausnitzii and E. eligens.

51. The method of claim 48, wherein the delivery system targets the pharmaceutical formulation away from a niche containing Bacteroidetes, wherein the pharmaceutical formulation is released by the action of a molecule selected from an amino acid transporter, a sugar transporter, and ABC transporter, and a PTS sytem.

52. The method of claim 51 , wherein the Bacteroidetes is B.

thetaiotaomicron.

53. The method of claim 48, wherein the delivery system targets the pharmaceutical formulation away from a niche containing Clostridia, and preferentially to a niche containing a bacteria selected from E.coli,

Bifidobacteria, Lactobacillus, and Bacteroides wherein the pharmaceutical formulation is released by the hydrolysis of lactulose.

54. The method of claim 1, wherein the delivery system comprises a compound that directs the pharmaceutical formulation preferentially to a mucosa-associated bacterial niche.

55. The method of claim 54, wherein the compound is a mucoadhesive compound selected form a poly vinyl pyrrolidone, methyl cellulose, sodium carboxy methylcellulose, hydroxy propyl cellulose, a cellulose derivative, a polyanhydride , a lectin, and a hydrogel.

56. The method of claim 55, wherein the delivery system has a size between 10 nm and 500nm, and comprises a coating of low molecular weight polyethylene glycol.

57. The method of claim 1 , wherein the delivery system further comprises an enteric coating.

58. The method of claim 1, wherein the delivery system further includes a targeting moiety that binds to a bacterial surface antigen that is present in the bacterial niche at higher concentrations than in other bacterial residents of the same anatomical location.

59. The method of claim 58, wherein the targeting moiety is selected from the group consisting of an antibody, an antibody fragment, a peptide, a minibody, a fibronectin domain, a multimer of an Ankyrin repeat fold, a protein A protein fold, a lipocalin protein fold, and a nucleic acid aptamer.

60. The method of claim 59, wherein the bacterial surface antigen is selected from the group consisting of a receptor, and adhesion molecule, a transporter, a cell surface structure, a secreted enzyme, a toxin, and a virulence factor.

61. The method of claim 1 wherein the delivery system is a

bacteriophage.

62. The method of claim 61, wherein the feature that differentiates the bacterial constituents of the bacterial niche is the expression of at least one compound or cell structure selected from a lipopolysaccharide, a teichoic acid, a protein, and a flagella, which can be bound by a bacteriophage.

63. A method of increasing the bioavailability of an active drug metabolite generated by a bacterial niche comprising:

providing a drug known to have an active bacterial metabolite, and selecting at least one feature that differentiates the bacterial constituents of the niche that generate the active drug metabolite in a given anatomical location from the rest of bacterial residents of that same anatomical location, and

providing a delivery system that releases the drug in the vicinity of the constituents of a bacterial niche; wherein release of the drug is triggered by the differentiating feature of the bacterial niche.

64. A method of decreasing the bioavailability of a drug metabolite generated by a bacterial niche comprising:

providing a drug known to have a toxic bacterial metabolite, and selecting at least one feature lacking in the bacterial constituents that generate the toxic metabolite in a given anatomical location and present in other bacterial residents of that same anatomical location, and

providing a delivery system that releases the drug away from the vicinity of the constituents of a bacterial niche; wherein release of the drug does not occur significantly in the bacterial niche and is triggered by the differentiating feature away from the bacterial niche.

65. The method of claim 1₅ wherein the host is a non-human mammal.