WO2006124047A9 - Pharmaceutical formulations containing microparticles or nanoparticles of a delivery agent - Google Patents

Abstract

This invention relates to microparticles and/or nanoparticles containing a delivery agent and /or an active agent. This invention also relates to pharmaceutical formulations and solid dosage forms, including controlled release solid dosage forms of active agent and a delivery agent

Description

PHARMACEUTICAL FORMULATIONS CONTAINING MICROPARTICLES OR

NANOPARTICLES OF A DELIVERY AGENT

[1] This application claims the benefit of U.S. Provisional Application No.

60/612,810, filed September 23, 2004, and U.S. Provisional Application No. 60/601,258,

filed August 13, 2004, both of which are hereby incorporated by reference.

FIELD OF THE INVENTION

[2] This invention relates to pharmaceutical formulations and methods for

preparing the same.

BACKGROUND OF THE INVENTION

[3] There is a continuing need for improved oral delivery systems for drugs,

such as insulin.

SUMMARY OF THE INVENTION

[4] The present invention relates to microparticles and/or nanoparticles for oral

administration containing a delivery agent compound alone or a combination of a delivery

agent compound and an active agent. Formulations containing these particles (and, for

particles containing only a delivery agent compound, and an active agent) provide

significantly greater bioavailability of the active agent with less variability than oral

administration of a simple mixture of the delivery agent compound and active agent as a powder, tablet, or capsule. Without being bound by any particular theory, it is believed

that in at least some embodiments, this improvement may be due to (1) the small size of the

micro- or nano-particles which permits them to pass from the stomach, through the pylorus

(which typically has a diameter of 1000-2000 μm), to the small intestine, where particle

dissolution and delivery agent-mediated drug absorption is believed to best occur, and (2)

the intimate contact between the delivery agent compound and active agent in the particles

which ensures that the delivery agent compound is present with the active agent at the site

of absorption. Because the micro- and nano-particles freely pass through the pylorus into

the small intestine, unlike a conventional tablet or capsule which must first become

dissolved into particles sufficiently small to do so, variations caused by tablet disintegration

and gastric transit modulated by gastric motility are minimized.

[5] According to one embodiment, the particles comprising a delivery agent

compound and an active agent have a median particle size less than about 900 or 1000 μm.

For example, the median particle size can range from about 45 to about 850 μm, from

about 45 to about 150 μm, from about 150 to about 250 μm, from about 250 to about 425

μm, from about 425 to about 850 μm, from about 100 to about 1000 nm, or from about 500

to about 1000 nm. According to another embodiment, the particles have a median particle

size less than about 1 μm. In some embodiments, particles may be as small as about 1

nanometer and as large as about 999 micrometers. For example, the particles may have a

median particle size of less than about 999 micrometers, from about 1 nanometer to about

999 micrometers, about 1 to about 999 micrometers, about 1 to about 999 nanometers,

about 45 to about 850 micrometers, about 45 to about 150 micrometers, about 150 to about 250 micrometers, about 250 to about 425 micrometers, about 425 to about 850

micrometers, about 100 to about 1000 nanometers, or about 500 to about 1000 nanometers.

[6] Another embodiment is a pharmaceutical formulation comprising a delivery

agent compound and an active agent in which the delivery agent compound is in the form of

particles. The particles can have a median particle size of less than about 999 micrometers,

about 1 nanometer to about 999 micrometers, about 1 to about 999 nanometers, or about 7

to about 16 micrometers. Optionally, the active agent may also be in the form of particles.

For example, the median particle size of the active agent particles may be less than about

999 micrometers, about 1 nanometer to about 999 micrometers, about 1 to about 999

micrometers, or about 1 to about 999 nanometers. According to one embodiment, the

delivery agent particles and the active agent particles both have a median particle size of

about 1 to about 999 micrometers. According to another embodiment, the delivery agent

particles and the active agent particles both have a median particle size of about 1 to about

999 nanometers.

- . [7] Yet another embodiment is a pharmaceutical formulation comprising a

delivery agent and an active agent in which the active agent is in the form of particles

having a median particle size of less than about 999 micrometers. According to one

embodiment, the median particle size of the active agent particles is about 1 nanometer to

about 999 micrometers, about 1 to about 999 micrometers, or about 1 to about 999

nanometers.

[8] The particles can be in the form of fine granules or micro-beads (e.g., beads

having a round/ball shape and a diameter of about 0.2 mm to about 2.0 mm). The micro- beads may be formed by compression. In one embodiment, the pharmaceutical formulation

includes micro-beads containing a delivery agent compound, which are coated with an

active agent, such as insulin or heparin. The micro-beads may have a diameter ranging

from about 0.2 mm to 2.0 mm.

[9] The particles may also include a mucoadhesive, such as a cellulose derivative

(e.g., CMC sodium (available from Aqualon of Wilmington, DE)) or a polyacrylic acid

(e.g., Carbopol™ available from B. F. Goodrich of Cleveland, OH). The mucoadhesive

can (1) facilitate adhesion to mucosa (including in the gastrointestinal tract) thereby

prolonging delivery agent-active agent contact with the mucosa, (2) stabilize and protect the

active agent (e.g. , in the case of insulin), and (3) increase the permeability of

biomembranes (including mucosa) thereby improving delivery and increasing bioavailability

of the active agent.

[10] It has also been discovered that oral administration of insulin in conjunction

with a delivery agent compound by solid oral dosage forms that do not degrade in the

stomach, but do degrade in the intestine, provides significantly greater bioavailability of the

insulin. Such solid oral dosage forms containing insulin or a different active agent provide

greater bioavailability than forms that degrade in the stomach and forms that do not contain

the delivery agent compound. Without being bound by any particular theory, it is believed

that this improvement is due to the sensitivity of insulin and other active agents to

degradation by enzymes or acid found in gastric fluid. Because the solid oral dosage forms

do not degrade in the stomach, the insulin and other active agents are protected from

degradation until they reach the intestine. [11] Another embodimentof the invention is a pharmaceutical formulation (such

as a solid oral dosage form) comprising a therapeutically effective amount of an active

agent and a delivery agent, where the pharmaceutical formulation has a disintegration time

of about 250 seconds to about 650 seconds when orally administered. In another

embodiment, the disintegration time is about 350 to about 550 seconds when orally

administered. In yet another embodiment, the disintegration time is greater than 60 seconds

when orally administered. In yet another embodiment, the disintegration time is greater

than 400 seconds when orally administered. Disintegration time can be determined in water

at 37 ± 2⁰C using the method described in USP <701>. Disintegration times may range from about 1 second to as much as about 24 hours, or more, depending on many factors including, but not limited to, the particular active agent(s), delivery agent compound(s), and excipients included in the pharmaceutical formulation.

[12] Another embodiment is a pharmaceutical formulation (such as a solid oral

dosage form) comprising a therapeutically effective amount of an active agent and a

delivery agent, where the solid oral dosage form does not substantially disintegrate or

dissolve in the stomach, but does substantially disintegrate or dissolve in the intestine. In a

preferred embodiment, the active agent is insulin. In another preferred embodiment, the

active agent is an insulin derivative.

[13] In another embodiment, the pharmaceutical formulation is a solid oral dosage

form which is covered with an enteric coating to retard disintegration in the stomach.

Enteric coatings include, but are not limited to, hydroxypropyl methylcellulose phthalate,

hydroxypropyl methylcellulose acetate succinate, polyvinyl acetate phthalate, cellulose

Ό— acetate trimellitate, cellulose acetate phthalate, poly(methacrylic acid-ethylacrylate), and

poly(methacrylic acid-methyl methacrylate).

[14] In yet another embodiment, the pharmaceutical formulations may be

formulated to erode from the surface of the dosage form, rather than disintegrate.

[15] The pharmaceutical formulations may include enzyme-inhibiting agents to

prevent enzymatic degradation of active agents in the pharmaceutical formulation.

[16] In one embodiment, the delivery agent is a compound having the following

structure or a salt thereof:

O R² O

2OH Ar c N R¹ c OH Formula A

wherein

Ar is phenyl or naphthyl;

Ar is optionally substituted with one or more of -OH, halogen, C1-C4 alkyl, Ci-C₄

alkenyl, Ci-C₄ alkoxy. or C1-C4 haloalkoxy;

R¹ is C3-C20 alkyl, C4-C20 alkenyl, phenyl, naphthyl, (C1-C10 alkyl) phenyl, (C1-C10

alkenyl)phenyl, (C1-C10 alkyl) naphthyl, (C1-C10 alkenyl) naphthyl, phenyl(Ci-Cio alkyl),

phenyl(Ci-Cio alkenyl), naphthyl(Ci-Cio alkyl), or naphthyl(Ci-Cio alkenyl);

R¹ is optionally substituted with Ci to C4 alkyl, Gz to C₄ alkenyl, Ci to C₄ alkoxy,

Ci-C₄ haloalkoxy, -OH, -SH, -CO2R⁸, or any combination thereof;

R² is hydrogen, Ci to ^,C₄ alkyl, or Q to C₄ alkenyl; and

R¹ is optionally interrupted by oxygen, nitrogen, sulfur or any combination thereof. The term "2-OH-Ar" in formula A refers to a phenyl or naphthyl group having a hydroxyl

group at the 2-position.

[17] According to one embodiment, the compounds are not substituted with an

amino group in the position alpha to the acid group.

[18] Preferably, Ar is substituted with a halogen.

[19] Preferably, R² is hydrogen.

[20] Preferably, R¹ is unsubstituted.

[21] Preferably, R¹ is not interrupted.

[22] Preferably, R¹ is CMO, C3-9, C3-7, C3, C7, or C9 alkyl. According to one

embodiment, R¹ is not branched.

[23] Preferred delivery agent compounds include, but are not limited to, N-(8-[2-

hydroxybenzoyl]amino)caprylic acid (the free acid of SNAC), N-(10-[2-

hydroxybenzoyl]amino)decanoic acid (the free acid of SNAC), 4-[(2-hydroxy-4-chloro-

benzoyl)-amino]butanoic acid (the free acid of 4-CNAB), and salts thereof, and solvates

and hydrates thereof. The salt can be, for example, a sodium salt, such as a monosodium

(i.e., SNAC, SNAD, or 4-CNAB) or disodium salt.

[24] In another embodiment, the delivery agent is a compound having the

following structure or a salt thereof:

wherein

R¹, R², R³, and R⁴ are independently H, -OH, halogen, C₁-C₄ alkyl, C₂-C₄ alkenyl, C₁- C₄ alkoxy, -C(O)R⁸, -NO₂, -NR⁹R¹⁰, or -N⁺R⁹R¹⁰R¹¹ (R¹²)^";

R⁵ is H, -OH, -NO₂, halogen, -CF₃, -NR¹⁴R¹⁵, -N⁺R¹⁴R¹⁵R¹⁶ (R¹³)^", amide, C₁-C₁₂ alkoxy, C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, carbamate, carbonate, urea, or -C(O)R¹⁸;

R⁵ is optionally substituted with halogen, -OH, -SH, or -COOH;

R^s is optionally interrupted by O, N, S, or -C(O)-;

R⁶ is a C₁-C₁₂ alkylene, C₂-C₁₂ alkenylene, or arylene;

R⁶ is optionally substituted with a C₁-C₄ alkyl, C₂-C₄ alkenyl, C₁-C₄ alkoxy, -OH, - SH, halogen, -NH₂, or -CO₂R⁸;

R⁶ is optionally interrupted by O or N;

R⁷ is a bond or arylene;

R⁷ is optionally substituted with -OH, halogen, -C(O)CH₃, -NR¹⁰R¹¹, or -N⁺R¹⁰R¹¹R¹²

(R¹³)-;

R⁸ is H, C₁-C₄ alkyl, C₂-C₄ alkenyl, or -NH₂;

R⁹, R¹⁰, R¹¹, and R¹² are independently H or C₁-C₁₀ alkyl;

R¹³ is a halide, hydroxide, sulfate, tetrafluoroborate, or phosphate;

R¹⁴, R¹⁵, and R¹⁶ are independently H, C₁-C₁₀ alkyl, C₁-C₁₀ alkyl substituted with - COOH, C₂-C₁₂ alkenyl, C₂-C₁₂ alkenyl substituted with -COOH, or -C(O)R . 1¹7'.; R¹⁷ is -OH, C₁-C₁₀ alkyl, or C₂-C₁₂ alkenyl; and R¹⁸ is H, C₁-C₆ alkyl, -OH, -NR¹⁴R¹⁵, OrN⁺R¹⁴R¹⁵R¹⁶ (R¹³)^".

[25] In yet another embodiment, the delivery agent is a compound having the

following structure or a salt thereof:

Formula C wherein

R¹, R², R³, R⁴ and R⁵ are independently H, -CN, -OH, -OCH₃, or halogen, at least one of R¹, R², R³, R⁴ and R⁵ being -CN; and R⁶ is a C₁-C₁₂ linear or branched alkylene, alkenylene, arylene, alkyl(arylene) or aryl(alkylene).

[26] In yet another embodiment, the delivery agent is a compound having the

following structure or a salt thereof:

Formula D wherein each occurrence of X is hydrogen, halogen, hydroxyl, or C₁-C₃ alkoxy,

R is substituted or unsubstituted Cj-C₃ alkylene or substituted or unsubstituted C₂-C₃ alkenylene, and n is an integer from 1 to 4.

[27] In yet another embodiment, the delivery agent is a compound having the

following structure or a salt thereof:

Formula E

wherein

X is halogen, and R is substituted or unsubstituted C₁-C₃ alkylene or substituted or unsubstituted C₂-C₃ alkenylene.

[28] Preferred delivery agent compounds include but are not limited to, N-(8-[2- hydroxybenzoyl]-amino)caprylic acid, N-(10-[2-hydroxybenzoyl]-amino)decanoic acid, 8-(2- hydroxy-4-methoxybenzoylamino)octanoic acid, 8-(2-hydroxy-5-chlorobenzoylamino)- octanoic acid, 4-[(2-hydroxy-4-chlorobenzoyl)amino]butanoic acid, and pharmaceutically acceptable salts thereof. The pharmaceutical formulations of the present invention may include any of the aforementioned delivery agent compounds, or any other delivery agent compounds, alone or in combination with one or more additional delivery agent compounds.

[29] Suitable active agents include but are not limited to, proteins, polypeptides,

peptides, hormones, polysaccharides, as well as synthetic, natural or recombinant sources

thereof: growth hormones; growth hormone releasing hormones; growth hormone releasing factor, interferons; interleukin-1; interleukin-2; insulin, optionally having counter

ions including zinc, sodium, calcium and ammonium; insulin-like growth factor; heparin;

calcitonin; erythropoietin; atrial naturetic factor; antigens; monoclonal antibodies;

somatostatin; protease inhibitors; adrenocorticotropin, gonadotropin releasing hormone;

oxytocin; leutinizing-hormone-releasing-hormone; follicle stimulating hormone;

glucocerebrosidase; thrombopoietin; filgrastim; prostaglandins; cyclosporin; vasopressin;

cromolyn sodium; vancomycin; desferrioxamine; bisphosphonates; parathyroid hormone;

anti-migraine agents; glucagon-like peptide 1 (GLP-I); antimicrobials; vitamins; and

analogs, fragments, mimetics or polyethylene glycol (PEG)-modified derivatives of these

compounds; or any combination thereof. Preferred active agents include, but are not

limited to, insulin and heparin (including, but not limited to, unfractionated heparin and low

molecular weight heparin).

[30] In one embodiment of the present invention, the active agent is insulin. The insulin-containing pharmacuetical formulations of the present invention may also include a second hypoglycemic agent, an inhibitor of renal glucose reabsorption, or any combination of

the foregoing (such as those described in U.S. Patent Publication No. 2005/0143424, which

is hereby incorporated by reference). Suitable second hypoglycemic agents include, but are

not limited to, insulin secretion-promoting agents, insulin resistance-ameliorating agents, insulin mimetics, α-glucosidase inhibitors, glucogenesis inhibitors, and any combination of any of the foregoing. According to one embodiment, the solid dosage form includes a sulfonyl urea, nieglitinide analogue, biguanide (preferably metformin), or any combination of any of the foregoing. According to a preferred embodiment, the solid dosage form includes ^• metformin. [31] Also provided is a pharmaceutical formulation, such as a solid dosage unit

form, comprising the microparticles or nanoparticles of the present invention and/or having

the disintegration times discussed above. The dosage unit form may be in the form of a

tablet, capsule, powder, or sachet. The dosage unit form may have, alone or in

combination, one or more enteric coatings, disintegrants, super disintegrants (such as

sodium starch glycolate or croscarmellose sodium), and extra particle super disintegrants.

[32] In one embodiment, the solid oral dosage unit form is a fast disintegrating

tablet. In another embodiment, the solid dosage unit form has a controlled or delayed

release.

[33] According to one embodiment, the present invention provides a tablet

comprising the aforementioned particles and a disintegrant. In one embodiment, the

disintegrant is a super disintegrant, such as sodium starch glycolate (Primojel^® available

from Azebe UK Ltd. of South Humberside, UK), croscarmellose sodium (Primellose^®

available from Azebe UK Ltd. of South Humberside, UK), or an extra particle super

disintegrant.

[34] Another embodiment is a solid dosage form comprising a therapeutically

effective amount of insulin and a delivery agent compound, where the solid dosage form

has a disintegration time of at least 60 seconds when administered orally. The solid dosage

form may have an enteric coating or be a surface eroding formulation. The solid dosage

form may further comprise one or more enzyme inhibiting agents.

[35] Yet another embodiment is a solid dosage form comprising a therapeutically

effective amount of insulin and a delivery agent compound, where the solid dosage form does not substantially disintegrate or dissolve in the stomach but does disintegrate or

dissolve in the small intestine. The solid dosage form may have an enteric coating or be a

surface eroding formulation. The solid dosage form may further comprise one or more

enzyme inhibiting agents.

[36] Another embodiment is a method for administering an active agent to an

animal, particularly an animal in need of the active agent, by administering a

pharmaceutical formulation comprising the rnicroparticles or nanoparticles of the present

invention and/or those having the disintegration times discussed above (i.e. those having a

controlled or sustained release). Oral administration is a preferred route of administration.

[37] Yet another embodiment is a method of treating a disease or for achieving a

desired physiological effect in an animal by administering a pharmaceutical formulation of

the present invention, including solid unit dosage forms comprising the microparticles or

nanoparticles of the present invention and/or those having the disintegration times discussed

above (i.e. those having a controlled or sustained release). Yet another embodiment is a

method of increasing the oral bioavailability of active agents by orally administering a

pharmaceutical formulation of the present invention.

[38] Yet another embodiment is a method of treating diabetes and/or reducing the

incidence of systemic hyperinsulinemia associated with chronic dosing of insulin in a

mammal (such as in a human, particularly a human in need thereof) by administering to the

mammal a therapeutic effective amount of an insulin-containing pharmaceutical formulation

of the present invention, e.g., those comprising the microparticles or nanoparticles of the

present invention and/or those having the disintegration times discussed above. In one embodiment, the delivery agent compound is the free acid of 4-CNAB or a

pharmaceutically acceptable salt thereof. The pharmaceutical formulation may be

administered on a chronic basis.

[39] Yet another embodiment is a method of treating impaired glucose tolerance,

early stage diabetes, or late stage diabetes or achieving glucose homeostasis in a mammal

(such as in a human, particularly in need thereof) by administering to the mammal a

therapeutic effective amount of an insulin-containing pharmaceutical formulation of the

present invention, such as a pharmaceutical formulation comprising the microparticles or

nanoparticles of the present invention and/or having the disintegration times discussed

above. In one embodiment, the delivery agent compound is the free acid of 4-CNAB or a

pharmaceutically acceptable salt thereof. The pharmaceutical formulation may be

administered on a chronic basis.

[40] Yet another embodiment is a method of treating a human diabetic patient by

orally administering to the human diabetic patient on a chronic basis a therapeutic effective

amount of an insulin-containing pharmaceutical formulation described herein.

[41] Yet another embodiment is a method of preparing the micro- and nano¬

particles of the present invention by drying a solution of a delivery agent compound and an

active agent, for example, until a solid is formed, and optionally, isolating the particles.

Preferably, the mixture is homogenous (e.g., the delivery agent compound and the active

agent are uniformly distributed throughout the mixture). The method includes co-drying a

mixture of the delivery agent compound, the active agent, and a solvent. Suitable solvents

include, but are not limited to, hydroxylic solvents, water, and mixtures thereof. According to one embodiment, the mixture is dried at from about 10 to about 40° C (e.g.,

at room temperature). Preferably, the drying is performed at a controlled temperature.

According to one embodiment, the drying is performed over an inert gas (preferably

nitrogen gas). The dried material may optionally be milled and/or sieved to obtain the

desired particle size. This method results in particles containing a homogeneous mixture of

the delivery agent compound and the active agent.

[42] Another method of preparing the micro- and nano-particles of the present

invention is by lyophilizing a mixture of the delivery agent compound, the active agent, and

a solvent. Suitable solvents include, but are not limited to, hydroxylic solvents, water, and

mixtures thereof.

[43] Yet another method of preparing the micro- and nano-particles of the present

invention is by (1) dissolving a delivery agent compound and an active agent in a

supercritical fluid, and (2) decreasing the system pressure to deposit the delivery agent

compound and active agent as extremely fine particles. The deposition is a result of the

rapid expansion of the supercritical solution.

[44] The following embodiments are collectively referred to herein as the "solid

pharmaceutical composition embodiments".

[45] The above features and many other attendant advantages of the invention will

become better understood by reference to the following detailed description when taken in

conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS [46] Figure 1 depicts a schematic of direct dosing to the stomach and the jejunum.

[47] Figure 2 is a graph of the concentration of insulin level (±SEM) following

direct dosing of coprocessed microparticles to the stomach and the jejunum over time.

[48] Figure 3 is a graph of the change in glucose level (±SEM) following direct

dosing of coprocessed microparticles to the stomach and the jejunum over time.

[49] Figure 4 is a graph of the change in glucose (±SEM) following oral gavage

from 3 different dosage forms: 1) a tablet made by compressing insulin and carrier, 2) a

capsule containing microparticles of coprocessed insulin and carrier, and 3) a capsule

containing a simple mixture of insulin and carrier, over time.

[50] Figure 5 is a graph of the insulin level (±SEM) following oral gavage from

3 different dosage forms: 1) a tablet made by compressing insulin and carrier, 2) a capsule

containing microparticles of coprocessed insulin and carrier, and 3) a capsule containing a

simple mixture of insulin and carrier, over time.

[51] Figure 6 is a graph of the insulin level (±SEM) following oral gavage of a

capsule containing microparticles of coprocessed insulin and carrier over time. Two of the

ten rats exhibited significantly high insulin absorption. The average values with (N=IO)

and without (N =8) inclusion of these two high responders are depicted in the graph.

[52] Figure 7 is a graph of the change in glucose (±SEM) following oral gavage

of a capsule containing microparticles of coprocessed insulin and carrier over time. Two of

the ten rats exhibited significantly high insulin absorption. The average values with (N= 10)

and without (N =8) inclusion of these two high responders are depicted in the graph. [53] Figure 8 is a chart of the estimated absolute bioavailability (±SEM) from in

situ dosing of coprocessed insulin and carrier to the stomach and the jejunum. Two

compositions were evaluated: 1) insulin (0.25mg/kg) + delivery agent (37.5mg/kg), and 2)

insulin (0.5mg/kg) + delivery agent (75mg/kg).

[54] Figure 9 is a chart of the estimated absolute bioavailability of insulin level

(±SEM) from 1) subcutaneous administration, 2) direct dosing to the stomach, 3) direct

dosing to the jejunum, 4) a tablet made by compressing insulin and carrier, 5) a capsule

containing microparticles of coprocessed insulin and carrier with and without inclusion of

the two high responders, and 6) a capsule containing a simple mixture of insulin and

carrier.

[55] Figure 10 is a chart of the estimated bioavailability of insulin in the portal

vein (±SEM) from 1) direct dosing to the stomach, 2) direct dosing to the jejunum, 3) a

tablet made by compressing insulin and carrier, 4) a capsule containing microparticles of

coprocessed insulin and carrier with and without inclusion of the two high responders, and

5) a capsule containing a simple mixture of insulin and carrier.

[56] Figure 11 is a chart of the estimated bioavailability (±SEM) of insulin

relative to subcutaneous administration from 1) direct dosing to the stomach, 2) direct

dosing to the jejunum, 3) a tablet made by compressing insulin and carrier, 4) a capsule

containing microparticles of coprocessed insulin and carrier with and without inclusion of

the two high responders, and 5) a capsule containing a simple mixture of insulin and

carrier. [57] Figure 12 is a chart of the estimated bioavailability of insulin in the portal

vein relative to subcutaneous administration (±SEM) from 1) direct dosing to the stomach,

2) direct dosing to the jejunum, 3) a tablet made by compressing insulin and carrier, 4) a

capsule containing microparticles of coprocessed insulin and carrier with and without

inclusion of the two high responders, and 5) a capsule containing a simple mixture of

insulin and carrier.

[58] Figure 13 is a graph of the individual insulin levels following oral gavage of

a capsule containing microparticles of coprocessed insulin and carrier over time. Rat 14 and

rat 17 exhibited significantly high insulin absorption. The average values with (N=IO) and

without (N =8) inclusion of these two high responders are depicted in the graph.

[59] Figure 14 is a graph of the individual glucose change following oral gavage

of a capsule containing microparticles of coprocessed insulin and carrier over time. Rat 14

and rat 17 exhibited significantly high insulin absorption. The average values with (N= IO)

and without (N =8) inclusion of these two high responders are depicted in the graph.

- . [60] Figure 15 is a graph of the individual insulin level following oral gavage of a

capsule containing microparticles of coprocessed insulin and carrier over time. Rats 14 and

17 were omitted. The average value from N=8 is depicted in the graph.

[61] Figure 16 is a graph of the individual glucose change following oral gavage

of a capsule containing microparticles of coprocessed insulin and carrier over time. Rats 14

and 17 were omitted. The average value from N =8 is depicted in the graph. [62] Figure 17 is a graph depicting the changes over time in serum glucose levels

in rhesus monkeys that have been fed formulations 1-6, described below, containing insulin

and a delivery agent. These formulations have varying disintegration times.

[63] Figure 18 is a graph depicting the changes over time in serum insulin

concentration rhesus monkeys that have been fed formulations 1-6, described below,

containing insulin and a delivery agent. These formulations have varying disintegration

times.

[64] Figure 19 is a graph of anti-factor Xa activity (U/ml) versus time in monkeys

after administration of the SNAD/heparin formulation described in Example 10.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

[65] The "particles," "micro-beads," and "granules" described herein may be

any shape and can include one or more ingredients in addition to the delivery agent

compound and/or active agent. The specific ingredients of any given particle, micro-bead,

or granule, may also depend on the processes used and will not necessarily be the same in

each individual particle, micro-bead, or granule from a batch.

[66] For example, where particles, micro-beads, or granules of an active agent

are prepared separately from particles, micro-beads, or granules of a delivery agent

compound, the active agent particles, micro-beads, or granules will, generally, not

comprise delivery agent compound, and the delivery agent particles, micro-beads, or granules will, generally, not comprise active agent, though each particle, micro-bead, or

granule may comprise other ingredients, as disclosed herein.

[67] In other embodiments, particles, micro-beads, or granules may be formed

from a solution, suspension or mixture, in liquid or dry form, without limitation, which

comprises at least an active agent and a delivery agent compound. Thus, for example, any

given particle, micro-bead, or granule comprises both active agent and delivery agent

compound, and may further comprise one or more other ingredients.

[68] The terms "diameter" and "median particle size" are generally used to refer

to the dimensions of particles, micro-beads, and granules. The "median particle size" or

"diameter" was determined as follows for Examples 8, 9, 10.

[69] Instrument: Mastersizer 2000 (EQ 202, model MS2K, serial number

34315-67)

[70] Manufacturer: MALVERN instruments, England

[71] Software: Mastersizer 2000

- . [72] Accessory: Scirocco 2000 (A) (model ADA 2000, serial number

34270/73)

[73] Dispersant: Dry dispersion

[74] Analysis model: General purpose

[75] Particle RI: 1.520

[76] Obscuration: 1 - 6%

[77] Standards: Malvern Quality Audit Standard for Sample Dispersion Units [78] The Malvern Mastersizer 2000 determines particle size by laser diffraction

and model fitting. A well-dispersed sample in any two-phase system (e.g., powders,

suspensions, or emulsions) is introduced into the path of a He-Ne laser focused with a lens

of a length suitable for particle sizes present in the sample. The scattering pattern of

particles in the laser path is measured by an array of detectors, with each detector

measuring data from a particular range of angles.

[79] The Malvern apparatus assumes that the particles being measured are perfect

spheres. For non-spherical particles the resulting particle size distribution may be different

from those obtained by methods based on others principles. The electronic measurements

will often have to be accompanied by microscopic investigation to determine the type of

particles being investigated. For irregularly shaped particles, the particle size data obtained

from Mastersizer 2000 will be interpreted as the diameter of an imaginary sphere that is

equivalent in volume to the measured particle. (Note: d(0.1) is the size of particle for

which 10% of the sample is below this size, d(0.5) is the size of particle for which 50% of

the sample is below this size, and d(0.9) is the size of particle for which 90% of the sample

is below this size.

[80] Generally, this apparatus measures one dimension of a, e.g., particle as it

travels past a laser; i.e., it measures the length of a straight line through the particle. For

irregular particles, this results in a variation of results since the orientation of a particle

relative to the laser may result in the single measurement being taken of that individual

particle's longest, shortest, or any other dimension. However, a measurement is taken of a

number of particles and a median diameter or size is calculated. Thus, "size" or "diameter" figures are estimates of the median "size" or "diameter" of particles.

Alternatively, "diameter" or "size" was measured by a sieve method described in Example

1. "Diameter" should not be read to necessarily imply a spherical shape or a circular

dimension, though in certain embodiments, e.g., particles may have rounded edges or

generally spherical shapes.

[81] It should be understood, also, that the invention is not limited to particles,

micro-beads, or granules which fall within a narrow range of "sizes" or "diameters".

Thus, for example, some embodiments may comprise, depending at least on the ingredients

and processes used, some particles which fall within, for example, both the nanometer and

micrometer scale, in the same batch. The actual "sizes" or "diameters" of the individual

particles may fall within a relatively narrow or relatively large range.

[82] As used herein and in the appended claims, the singular forms "a," "an,"

and "the," include plural referents unless the context clearly indicates otherwise. Thus, for

example, reference to "a particle" includes one or more of such particles, reference to "an"

active agent includes one or more of such active agents, and "a" delivery agent includes

one or more delivery agents,

[83] The term "about" generally means within 10%, preferably within 5%, and

more preferably within 1 % of a given value or range.

[84] The term "hydrate" as used herein includes, but is not limited to, (i) a

substance containing water combined in the molecular form and (ii) a crystalline substance

containing one or more molecules of water of crystallization or a crystalline material

containing free water.

[85] The term "solvate" as used herein includes, but is not limited to, a molecular

or ionic complex of molecules or ions of a solvent with molecules or ions of the delivery

agent compound or salt thereof, or hydrate or solvate thereof.

[86] The term "delivery agent" refers to any of the delivery agent compounds

disclosed herein.

[87] The term "SNAC" refers to the monosodium salt of N-(8-[2-

hydroxybenzoyl]-amino)caprylic acid, including the various polymorphic forms of the

monosodium salt described in U.S. Provisional Application No. 60/569,476, filed May 6,

2004 (which is hereby incorporated by reference) unless otherwise indicated.

[88] The term "SNAD" refers to the monosodium salt of N-(10-[2-

hydroxybenzoyl]-amino)decanoic acid, unless otherwise indicated. The term "disodium salt

of SNAD" refers to the disodium salt of N-(10-[2-hydroxybenzoyl]-amino)decanoic acid.

[89] The term "5-CNAC" refers to the monosodium salt of N-(8-[2-hydroxy-5-

chlorobenzoyl]-amino)octanoic acid, unless otherwise indicated.

[90] The term "4-CNAB" refers to the monosodium salt of sodium N-4-[(2-

hydroxy-4-chlorobenzoyl)amino]butanoate, including anhydrous, monohydrate, and

isopropanol solvates thereof and various polymorphic forms of the monosodium salt described in International Publication No. WO 03/057650 (which is hereby incorporated by

reference), unless otherwise indicated.

[91] An "effective amount of active agent" is an amount of active agent which is

effective to treat or prevent a condition in a living organism to whom it is administered

over some period of time, e.g., provides a therapeutic effect during a desired dosing

interval.

[92] The term "insulin" refers to all forms of insulin, including, but not limited

to, naturally derived insulin and synthetic forms of insulin, such as those described in U.S.

Patent Nos. 4,421,685, 5,474,978, and 5,534,488, each of which is hereby incorporated by

reference in its entirety.

[93] The term "insulin derivatives" refers to insulin-derived proteins and peptides

with insulin actions, and include, for example, lispro, BlOAsp and HOE-901.

[94] An "effective amount of delivery agent" is an amount of the delivery agent

which enables and/or facilitates the absorption of a desired amount of active agent via any

route of administration (such as those discussed in this application including, but not limited

to, the oral (e.g., across a biological membrane in the gastrointestinal tract), nasal,

pulmonary, dermal, buccal, vaginal, and/or ocular route).

[95] The terms "alkyl" and "alkenyl" as used herein include linear and branched

alkyl and alkenyl substituents, respectively.

[96] The phrase "pharmaceutically acceptable" refers to additives or compositions

that are physiologically tolerable when administered to a mammal. [97] The phrase "substantially disintegrate" means that about 75% to about 95%

of the total volume of the tablet will break apart and dissolve into its component parts (e.g.

insoluble coated particles, insoluble disintegrant, etc.), and the tablet is no longer intact

except for small aggregates.

[98] "Surface eroding formulation" refers to formulations that do not disintegrate

but instead erode, e.g., the formulation dissolves from the surface over a pre-determined

period of time and the tablet generally remains intact and retains its overall shape. The

surface eroding formulations allow for sustained release of an active agent over the pre¬

determined time period.

[99] The terms "micronize" and "micronized" generally refer to a process, or

particles which have been processed, such that their diameters/sizes are within the general

range of microparticles and/or nanoparticles.

[100] The term "microparticle" generally includes particles having a diameter

ranging from about 1 to about 999 micrometers (microns, μm).

- [101] The term "nanoparticle" generally includes particles having a diameter

ranging from about 1 to about 999 nanometers (nm)..

[102] The term "insulin derivatives" includes insulin-derived proteins and peptides

with insulin actions, and include, for example, lispro, BlOAsp and HOE-901.

[103] "Insulin secretion-promoting agents" exert their hypoglycemic action, by

mainly influencing pancreatic β-cells to promote insulin secretion into blood, and include,

for example, sulfonylureas (for example, tolbutamide, chlorpropamide, glibenclamide

(glyburide), glipizide, glimeperide, and gliclazide); and meglitinide analogues (for example, repaglinide, nateglinide, meglitinide and mitiglinide (KAD- 1229))). Other insulin

secretion-promoting agents are, for example, K⁺-ATP channel inhibitors (for example,

BTS-67-582), glucagon-like peptide-1 receptor agonists (for example, glucagon-like

peptide-1, exendin-4 and NN-2211) and dipeptidyl peptidase-IV inhibitors with an effect of

enhancing the action of glucagon-like peptide-1. According one embodiment, the insulin

secretion-promoting agent is a sulfonylurea or meglitinide analogue.

[104] The term "insulin resistance-ameliorating agents" includes agents exerting

hypoglycemic action by enhancing the action of insulin in target tissues, and include for

example peroxisome proliferator activator receptor (PPAR)-γ agonists (for example,

thiazolidine-based compounds such as pioglitazone, rosiglitazone, and ciglitazone; or non-

thiazolidine-based compounds such as GI-262570, JTT-501, YM-440, NN-622 and KRP-

297), PPAR-γ antagonists and protein tyrosine phosphatase inhibitors. The insulin

resistance-ameliorating agents include, for example, pharmaceutical agents with a function

ameliorating insulin resistance, for example biguanides (for example, metformin,

phenformin and buformin, preferably metformin), PPAR-α agonists (fibrate-series

compounds such as simfibrate, clofibrate, bezafibrate and clinofibrate and non-fibrate-series

compounds), anti-obesity agents (for example, 5-hydroxytryptamine (5-HT) reuptake

inhibitors such as sibutramine, lipase inhibitors such as orlistat and adrenalin β-receptor

agonists such as AJ-9677). Preferred insulin resistance-ameliorating agents include, but are

not limited to, biguanides, such as metformin.

[105] The term "insulin mimetics" refers to agents expressing the hypoglycemic

action through physiological insulin action, namely the action promoting glucose uptake into cells, in a manner more or less independent to insulin, except for insulin derivatives,

and include for example insulin receptor-activating agents (for example, CLX-0901 and L-

783281) and vanadium.

[106] The term "α-glucosidase inhibitors" refers to agents expressing the

hypoglycemic action through suppression of glucose absorption into bodies, mainly via the

inhibition of α-glucosidase in the intestinal tube and include, for example, acarbose,

voglibose and miglitol.

[107] The term "glucogenesis inhibitors" refers to agents expressing hypoglycemic

action mainly through the inhibition of glucogenesis, and include for example glucagon

secretion suppressors (for example, M&B-39890A and octreotide), fatty acid decomposition

inhibitors (for example, nicotinic acid derivatives and carnitine palmitoyltransferase- 1

inhibitor) and glucose-6-phosphatase inhibitors.

[108] The term "inhibitor of renal glucose reabsorption" refers to agents which

inhibit glucose reabsorption in uriniferous tubules. The primary action of the inhibitor of

renal glucose reabsorption is not involved in the promotion of the uptake into target tissue

cells, the suppression of the absorption from intestinal tube, or the hypoglycemic action via

the suppression of the synthesis in tissues. Suitable inhibitors of renal glucose reabsorption

include, but are not limited to, those described in U.S. Patent Publication No.

2005/0143424, which is hereby incorporated by reference.

Delivery Agent Compounds [109] The delivery agent compound may be any of those described in U.S. Patent

Nos. 5,650,386 and 5,866,536 and International Publication Nos. WO94/23767,

WO95/11690, W095/28920, WO95/28838, W096/10396, W096/09813, WO96/12473,

WO96/12475, WO96/30036, WO96/33699, WO97/31938, WO97/36480, WO98/21951,

WO98/25589, W098/34632, W098/49135, WO99/16427, WO00/06534, WOOO/07979,

WOOO/40203, WO00/46182, WO00/47188, WO00/48589, WOOO/50386, WO00/59863,

WOOO/59480, WO01/32130, WO01/32596, WO01/34114, WO01/44199, WO01/51454,

WO01/70219, WO01/92206, WO02/02509, WO02/15959, WO02/16309, WO02/20466,

WO02/19969, WO02/070438, WO03/026582, WO02/100338, WO03/045306,

WO03/26582, and WO 03/057170, all of which are hereby incorporated by reference.

[110] Non-limiting examples of delivery agent compounds include N-(8-[2- hydroxybenzoyl]amino)caprylic acid, N-(10-[2-hydroxybenzoyl]amino)decanoic acid, 8-(2- hydroxy-4-methoxybenzoylamino)octanoic acid, 8-(2-hydroxy-5-chlorobenzoyl- amino)octanoic acid, 4-[(2-hydroxy-4-chlorobenzoyl)amino]butanoic acid, and salts thereof.

Preferred salts include, but are not limited to, monosodium and disodium salts.

[Ill] According to one embodiment, the delivery agent compound is N-(8-[2-

hydroxybenzoyl]amino)caprylic acid or a pharmaceutically acceptable salt thereof.

. [112] According to another embodiment, the delivery agent compound is N-(10-[2-

hydroxybenzoyl]amino)decanoic acid or a pharmaceutically acceptable salt thereof.

[113] According to another embodiment, the delivery agent compound is 4-[(2-

hydroxy-4-chlorobenzoyl)amino]butanoic acid or a pharmaceutically acceptable salt thereof.

[114] According to another embodiment, the delivery agent compound is 8-(2-

hydroxy-5-chlorobenzoylamino)octanoic acid or a pharmaceutically acceptable salt thereof. [115] The delivery agent compounds may be in the form of the carboxylic acid or

pharmaceutically acceptable salts thereof, such as sodium salts, and hydrates and solvates

thereof. The salts may be mono- or multi-valent salts, such as monosodium salts and

disodium salts (e.g., the disodium salt of 8-(2-hydroxy-5-chlorobenzoylamino)-octanoic

acid, the disodium salt of N-(8-[2-hydroxybenzoyl]amino)caprylic acid, the disodium salt of

N-(10-[2-hydroxybenzoyl]amino)decanoic acid). See, for example, International

Publication No. WO 00/59863, which is hereby incorporated by reference The delivery

agent compounds may contain different counter ions chosen for example due to their effect

on modifying the dissolution profile of the carrier.

[116] The delivery agent compounds may be prepared by methods known in the

art, such as those discussed in the aforementioned publications (e.g., International

Publication Nos. WO 98/34632, WO 00/07979, WO 01/44199, WO 01/32596, WO

02/02509, WO 02/20466, and WO 03/045306). SNAC, SNAD, 4-CNAB, and the free

acid and other salts thereof may be prepared by methods known in the art, such as those

described in U.S. Patent Nos. 5,650,386 and 5,866,536 and International Publication No.

WO 02/02509, each of which are hereby incorporated by reference.

[117] Salts of the delivery agent compounds of the present invention may be

prepared by methods known in the art. For example, sodium salts may be prepared by

dissolving the delivery agent compound in ethanol and adding aqueous sodium hydroxide.

[118] The delivery agent compound may be purified by recrystallization or by

fractionation on one or more solid chromatographic supports, alone or linked in tandem.

Suitable recrystallization solvent systems include, but are not limited to, acetonitrile, methanol, and tetrahydrofiiran. Fractionation may be performed on a suitable

chromatographic support such as alumina, using methanol/n-propanol mixtures as the

mobile phase; reverse phase chromatography using trifluoroacetic acid/acetonitrile mixtures

as the mobile phase; and ion exchange chromatography using water or an appropriate buffer

as the mobile phase. When anion exchange chromatography is performed, preferably a 0-

500 mM sodium chloride gradient is employed.

[119] The delivery agent may contain a polymer conjugated to it by a linkage

group selected from the group consisting Of -NHC(O)NH-, -C(O)NH-,-NHC(O), -OOC-, -

COO-, -NHC(O)O-, -OC(O)NH-, -CH₂NH -NHCH₂-, -CH₂NHC(O)O-, -OC(O)NHCH₂-,-

CH₂NHCOCH₂O-, -OCH₂C(O)NHCH₂-, - NHC(O)CH₂O-, -OCH₂C(O)NH-, -NH-, -0-,

and carbon-carbon bond, with the proviso that the polymeric delivery agent is not a

polypeptide or polyamino acid. The polymer may be any polymer including, but not

limited to, alternating copolymers, block copolymers and random copolymers, which are

safe for use in mammals. Preferred polymers include, but are not limited to, polyethylene;

polyacrylates; polymethacrylates; poly (oxy ethylene); poly (propylene); polypropylene

glycol; polyethylene glycol (PEG); and derivatives thereof and combinations thereof. The

molecular weight of the polymer typically ranges from about 100 to about 200,000 daltons.

The molecular weight of the polymer preferably ranges from about 200 to about 10,000

daltons. In one embodiment, the molecular weight of the polymer ranges from about 200

to about 600 daltons and more preferably ranges from about 300 to about 550 daltons.

Active Agents [120] Active agents suitable for use in the present invention include biologically

active agents and chemically active agents, including, but not limited to, pesticides,

pharmacological agents, and therapeutic agents. Suitable active agents include those that

are rendered less effective, ineffective or are destroyed in the gastro-intestinal tract by acid

hydrolysis, enzymes and the like. Also included as suitable active agents are those

macromolecular agents whose physiochemical characteristics, such as, size, structure or

charge, prohibit or impede absorption when dosed orally.

[121] For example, biologically or chemically active agents suitable for use in the

present invention include, but are not limited to, proteins; polypeptides; peptides;

hormones; polysaccharides, and particularly mixtures of muco-polysaccharides;

carbohydrates; lipids; small polar organic molecules (i.e. polar organic molecules having a

molecular weight of 500 daltons or less); other organic compounds; and particularly

compounds which by themselves do not pass (or which pass only a fraction of the

administered dose) through the gastro-intestinal mucosa and/or are susceptible to chemical

cleavage by acids and enzymes in the gastro-intestinal tract; or any combination thereof.

[122] Further examples include, but are not limited to, the following, including

synthetic, natural or recombinant sources thereof: growth hormones, including human

growth hormones (hGH), recombinant human growth hormones (rhGH), bovine growth

hormones, and porcine growth hormones; growth hormone releasing hormones; growth

hormone releasing factor, interferons, including α (e.g., interferon alfacon-1 (available as

Infergen^® from InterMune, Inc. of Brisbane, CA)), β and D; interleukin-1; interleukin-2;

insulin, including porcine, bovine, human, and human recombinant, optionally having counter ions including zinc, sodium, calcium and ammonium; insulin-like growth factor,

including IGF-I; heparin, including unfractionated heparin, heparinoids, dermatans,

chondroitins, low molecular weight heparin, very low molecular weight heparin and ultra

low molecular weight heparin; calcitonin, including salmon, eel, porcine and human;

erythropoietin; atrial naturetic factor; antigens; monoclonal antibodies; somatostatin;

protease inhibitors; adrenocorticotropin, gonadotropin releasing hormone; oxytocin;

leutinizing-hormone-releasing-hormone; follicle stimulating hormone; glucocerebrosidase;

thrombopoietin; filgrastim; prostaglandins; cyclosporin; vasopressin; cromolyn sodium

(sodium or disodium chromoglycate); vancomycin; desferrioxamine (DFO);

bisphosphonates, including alendronate, tiludronate, etidronate, clodronate, pamidronate,

olpadronate, and incadronate; parathyroid hormone (PTH), including its fragments; anti¬

migraine agents such as BIBN-4096BS and other calcitonin gene-related proteins

antagonists; glucagon-like peptide 1 (GLP-I); antimicrobials, including antibiotics, anti-

bacterials and anti-fungal agents; vitamins; analogs, fragments, mimetics or polyethylene

glycol (PEG)-modified derivatives of these compounds; or any combination thereof. Non-

limiting examples of antibiotics include gram-positive acting, bacteriocidal, lipopeptidal and

cyclic peptidal antibiotics, such as daptomycin and analogs thereof.

[123] According to one embodiment, the active agent is insulin.

[124] According to another embodiment, the active agent is heparin, such as

unfractionated heparin or low molecular weight heparin.

[125] The amount of active agent used in a pharmaceutical composition or dosage

unit form of the present invention is an amount effective to treat the target indication. However, the amount can be less than that amount when the composition is used in a

dosage unit form because the dosage unit form may contain a plurality of delivery agent

compound/ active agent, such compositions may contain a divided effective amount. The

total effective amount can then be administered in cumulative units containing, in total, an

effective amount of active agent. Moreover, those skilled in the field will recognize that an

effective amount of active agent will vary with many factors including the age and weight

of the animal, the animal's physical condition, as well as other factors.

[126] The total amount of active agent to be used of can be determined by methods

known to those skilled in the art. However, because the compositions of the invention may

deliver active agent more efficiently than compositions containing the active agent without

the delivery agent, lower amounts of active agent than those used in prior dosage unit forms

or delivery systems can be administered to the subject, while still achieving the same blood

levels and/or therapeutic effects.

[127] According to one embodiment, insulin is administered at a dose of about

0.025 to about 1.0 mg per kilogram of body weight of the recipient per day (mg/kg/day),

about 0.06 to about 0.25 mg/kg/day, or about 0.09 to about 0.19 mg/kg/day (based on the

weight of active agent). The desired dose may be administered either as a single or divided

dose.

[128] Generally an effective amount of delivery agent to facilitate the delivery of

the active agent is administered with the active agent. According to one embodiment, the

amount of delivery agent to active agent on a molar basis ranges from about 100: 1 to about

1:1, from about 80:1 to about 2:1, or from about 20:1 to about 10:1. Delivery agent to active agent molar basis ranges may be higher than 100:1 for particular combinations of

delivery agents and active agents. Alternatively, delivery agent to active agent ranges may

be about 1:1 or lower, such as, e.g., 0.1:1 or lower, with particular combinations of

delivery agents and active agents.

[129] Dosage unit forms can also include any one or combination of excipients,

disintegrants, lubricants, plasticizers, colorants, flavorants, taste-masking agents, sugars,

sweeteners, and salts.

[130] The compositions of the subject invention are useful for administering

biologically or chemically active agents to any animals, including but not limited to birds

such as chickens, insects, fish, reptiles, mammals (including, but not limited to, rodents,

aquatic mammals, domestic animals such as dogs and cats, farm animals such as sheep,

pigs, cows and horses, and preferably humans).

[131] Another embodiment of the present invention is a method for the treatment

or prevention of a disease or for achieving a desired physiological effect, such as those

listed in the table 1 below, in an animal by administering the particles of the present

invention. Preferably, an effective amount of the particles for the treatment or prevention

of the desired disease or for achieving the desired physiological effect is administered.

Specific indications for active agents can be found in the Physicians' Desk Reference (58^th

Ed., 2004, Medical Economics Company, Inc., Montvale, NJ), which is herein

incorporated by reference. The active agents in the table below include their analogs,

fragments, mimetics, and polyethylene gly col-modified derivatives.

Table 1

Controlled or Sustained Release Formulations

[132] The solid dosage forms of the present invention may be formulated so as to

prevent or retard break down in the stomach. Controlled release formulations suitable for

use in the present invention may, for example, include an enteric coating or may be

formulated to erode from the surface. [133] According to one embodiment, the solid oral dosage forms comprises a

therapeutically effective amount of an active agent and a delivery agent, wherein the solid

oral dosage form has a disintegration time of about 250 seconds to about 650 seconds when

orally administered. In another embodiment, the disintegration time is about 350 to about

550 seconds when orally administered. In one embodiment the disintegration time is

greater than 60 seconds when orally administered. In another embodiment, the

disintegration time is greater than 400 seconds when orally administered. Disintegration

time can be determined in water at 37 ± 2°C using the method described in USP <701>.

[134] The solid dosage forms of the present invention may be covered by an

enteric coating. The enteric coating may serve as the primary control for delaying the

release of the drug composition or compositions in the solid dosage form. The enteric

coating stays intact in the stomach and prevents or retards release into the stomach in the

solid dosage form. Release of the active agent is delayed until the solid dosage form

reaches the intestine. Once in the intestine, the higher pH causes release of the active

agent. Enteric coatings include, but are not limited to, hydroxypropyl methylcellulose

phthalate, hydroxypropyl methylcellulose acetate succinate, polyvinyl acetate phthalate,

cellulose acetate trimellitate, cellulose acetate phthalate, poly(methacrylic acid-

ethylacrylate), and poly(metihιacrylic acid-methyl methacrylate). Other enteric coatings

which may be used in accordance with the present invention are described in U.S. Patent

No. 5,851,579, which is hereby incorporated by reference.

[135] In one embodiment of the present invention, the enteric coating is applied to

the entire tablet, or other dosage form. In one embodiment the enteric coating is applied to a multiparticulate system, such as a system comprising microparticles and/or nanoparticles

discussed above.

[136] The solid dosage forms of the present invention may be formulated to erode

from the surface of the tablet (or other dosage uniform), or at the surface of the multi-

particulate system (e.g. a system comprising microparticles discussed above). These

surface erosion formulations slowly dissolve from the surface rather than disintegrate. By

controlling the rate of surface erosion, release of the active agent and drug composition of

the solid dosage form can be delayed. The surface erosion formulations can be formulated

such that substantial release of the active agents or drug compositions do not occur until the

solid oral dosage form reaches the intestines.

Enzyme Inhibiting Agents

[137] The solid dosage forms of the present invention (comprising the

microparticles or nanoparticles of the present invention and/or having the disintegration

times discussed above) may also include enzyme inhibiting agents. Enzyme inhibiting

agents incorporated into the solid dosage unit forms may prevent the breakdown of insulin

or other active agents that may be sensitive to enzymatic degradation. Enzyme inhibiting

agents are described in U.S. Patent No. 6,458,383 which is hereby incorporated by

reference.

[138] Generally, inhibitory agents can be divided into the following classes:

inhibitors that are not based on amino acids, including P-aminobenzamidine, FK-448,

camostat mesylate and sodium glycocholate; amino acids and modified amino acids, including aminoboronic acid derivatives and n-acetylcysteine; peptides and modified

peptides, including bacitracin, phosphinic acid dipeptide derivatives, pepstatin, antipain,

leupeptin, chymostatin, elastatin, bestatin, hosphoramindon, puromycin, cytochalasin

potatocarboxy peptidase inhibitor, and amastatin; polypeptide protease inhibitors, including

aprotinin (bovine pancreatic trypsin inhibitor), Bowman-Birk inhibitor and soybean trypsin

inhibitor, chicken egg white trypsin inhibitor, chicken ovoinhibitor, and human pancreatic

trypsin inhibitor; complexing agents, including EDTA, EGTA, 1,10-phenanthroline and

hydroxychinoline; and mucoadhesive polymers and polymer-inhibitor conjugates, including

polyacrylate derivatives, chitosan, cellulosics, chitosan-EDTA, chitosan-EDTA-antipain,

polyacrylic acid-bacitracin, carboxymethyl cellulose-pepstatin, polyacrylic acid-Bowman-

Birk inhibitor.

[139] The choice and levels of the enzyme inhibitor are based on toxicity,

specificity of the proteases and the potency of inhibition, and will be apparent to those

skilled in the art.

. [140] Without wishing to be bound by theory, it is believed that an inhibitor can

function solely or in combination as: a competitive inhibitor, by binding at the substrate

binding site of the enzyme, thereby preventing the access to the substrate (examples of

inhibitors believed to operate by this mechanism are antipain, elastatinal and the Bowman

Birk inhibitor); a non-competitive inhibitor that can be simultaneously bound to the enzyme

site along with the substrate, as their binding sites are not identical; and/or a complexing

agent due to loss in enzymatic activity caused by deprivation of essential metal ions out of

the enzyme structure. Examples

[141] The following examples illustrate the invention without limitation. All parts

are given by weight unless otherwise indicated.

Example 1

1. Test Articles

a. Co-processed insulin/delivery agent microparticles used for site specific, in

situ experiment and oral gavage experiments

[142] Recombinant human zinc insulin (50 mg) and sodium 4-CNAB (7.5 g) were

dissolved in 50 ml of deionized water. The clear solution was dried with nitrogen flow at

room temperature for 24 hours. The obtained coprocessed cake was milled into fine

particles, which were then sieved through a 40/60 mesh screen to obtain microparticles of a

specific size range. The size of the microparticles used in the current study ranged from

250 to 420 μm. These microparticles contained by weight 0.55% of insulin, 9.5% of water

and 89.5 % of delivery agent. A total of approximately 90% (w/w) of insulin was recovered

from this process.

[143] Particles were measured by passing them through seives with different size

openings (850 μm, 425 μm, 250 μm, 150 μm, 45 μm). With this method, it can be

determined that the median particle size ranges from about 45 to about 850 μm, from about

45 to about 150 μm, from about 150 to about 250 μm, from about 250 to about 425 μm, or

from about 425 to about 850 μm. [144] Insulin content in the microparticles was measured with reversed phase

HPLC (Phenomenex column: Luna 3u C18 (2) lOOA, 150 x 4.6 mm, 3 micro; mobile

phases: A, 0.1% TFA in water; B, 0.1% TFA in acetonitrile; Detector: UV280 nm). Water

contents of the particles were measured with a 737 KF coulometer.

b. Capsules loaded with the microparticles for oral gavage

[145] Gelatin capsules (size #9) were used in the rat studies. The necessary amount

of microparticles loaded manually into the gelatin capsules were determined based on an

average rat body weight of 350 mg. Each loaded capsule contained approximately 16 mg of

the microparticles (equivalent to 0.0875 mg of insulin).

c. Insulin/Delivery Agent mini-tablets for oral gavage experiment

[146] Insulin was well mixed with delivery agent at a ratio of 1:150 (w/w), which

corresponded to 0.67% (w/w) of insulin. Based on an average rat body weight of 350 mg, a

total amount of 26.43 mg of the mixed powder, which contained 0.175 mg of insulin and

26.26 mg of delivery agent, was directly compressed into tablets under a pressure of 1000

psi in a Carver press. The cylindrical mini-tablets were 2 mm in diameter and 6 mm in

height.

d. Capsules loaded with insulin/delivery agent physical blend for oral gavage

experiment

[147] Insulin was well mixed with delivery agent at a ratio of 1: 150 (w/w). The

amount of insulin and delivery agent mixture loaded manually into the gelatin capsules (size

#9) were determined based on an average rat body weight of 350 mg. Each capsule

contained 26.43 mg of the mixture (equivalent to 0.175 mg insulin). 2. Direct Dosing Procedures for In Situ Experiments

[148] A schematic of the direct dosing procedure is shown in Figure 1. Surgery

was carried out in a clean environment using a clean lab coat, mask, safety goggle, gloves

and surgical cap. Anesthesia was induced to the Sprague Dawley rats with 5% isoflurane

as an induction concentration, and maintained at 2% isoflurane in pure oxygen to the

completion of the study.

a. Stomach direct dosing

[149] After the right jugular vein was catheterized for sampling blood, the skin

over the esophagus and trachea was dissected, and the musculus digastricul venter rostralis

(protective muscular bundles) was identified and dissected to make an access toward the

esophagus. The esophagus was partially severed, and inserted with a 12 cm PE204 tubing

for a segment of the esophagus measuring- 6-9 cm. The dosing formulation was introduced

through this tubing using a blunt wire to push in the microparticles. After dosing, the

esophagus was ligated with a 3-0 silk suture for preventing any leakage from the stomach.

. b. Jejunum direct dosing

[150] After the right jugular catheterization for the blood sampling, the abdominal

cavity was opened by dissecting the linea alba toward the sternum, thus exposing the

xiphoid cartilage. The most proximal segment of jejunum was first identified. A less

vascularized section of the proximal jejunum was partially nipped, and a dosing tube was

introduced toward the distal end. After dosing, the dosing tube was removed, and a 2 cm

PE206 tubing was pushed in, and placed so that the nipped wound was located in the

middle of both ends of the 2 cm tubing. A suture was tied around the tubing with jejunum at both ends, and the wound was closed with a drop of a vetbond™ tissue adhesive

(available from 3M of St. Paul, Minnesota).

3. Oral Gavage Procedures

[151] Studies were carried out in Sprague Dawley rats (body weight was

approximately 350 grams) by oral gavage administration. The mini tablets or capsules were

administrated orally in rats using a modified gavage tubing with a trocar. Rats were fasted

for about 24 hours and anesthetized by intramuscular administration of ketamine (44

mg/kg) and thorazine (1.5 mg/kg). At pre-determined time intervals, blood samples were

drawn from the tail artery and were appropriately prepared as either plasma or serum for

glucose and insulin bioassays. The animal was sacrificed at the end of the experiment and

rat gastrointestinal mucosa was observed for any sign of local toxicity.

4. Bioassay Procedures

[152] Rat serum concentrations of insulin were determined using Insulin ELISA

Test Kit (DSL Inc.). The limit of quantitation (LOQ) has been established at 12.5 DU/mL,

with the calibrated linear range of the assay up to 250 DU/mL. Changes in blood glucose

levels were measured using a glucometer.

5. Results

a. Site Specific Study (In Situ) Results

[153] The concentration of insulin and the change in glucose level following direct

dosing of the coprocessed microparticles to the stomach and the jejunum are shown in

Figures 2 and 3, respectively. The individual data are listed in Tables 2 to 5. [154] Insulin concentration from dosing to the jejunum reached a maximum value

at the first sampling point (W < 15 min) from each formulation. The corresponding tmin of

glucose occurred approximately 30 min. later.

Table 2

Direct dosing of coprocessed microparticles to the stomach *Insulin (0.5mg/kg), Delivery Agent (75mg/kg))

1) Insulin

Insulin Stomach

Time Rat#

(min) 1 #2 #3 #4 #5 #6 #7 #8 mean SD SEM CV

0 12.5 12.5 12.5 12.5 12.5 12.5 12.5 12.9 0.0 0.0 0.0

15 114.5 72.8 80.9 12.6 210.0 12.5 118.7 158.5 97.6 68.1 24.1 69.8%

30 95.3 19.3 35.5 12.5 211.0 12.5 15.1 66.0 58.3 68.4 24.2 117.2%

45 62.8 12.5 894.0 12.5 213.0 12.5 15.0 12.5 154.3 306.7 108.5 198.8%

60 18.2 12.5 157.0 12.5 174.0 140.3 12.5 12.5 67.4 74.7 26.4 110.9%

90 12.5 12.5 12.5 12.5 61.3 12.5 12.5 74.2 26.3 25.8 9.1 98.1%

AUC_0→90 4780 2132 18970 1127 14438 4001 2795 5046 6661 6451 2281 96.8% (two rat data were removed, rat #1-2 stomach)

2) Glucose

Change from base line Stomach

Time

(min) Rat# l #2 #3 #4 #5 #6 #7 #8 Mean SD SEM CV

0 0 0 0 0 0 0 0 0 0 0 0

15 -9.9 -21.0 -12.6 -38.1 -6.6 -24.8 3.0 -13.9 -15.5 12.5 4.4 -80.6%

30 -44.3 -43.8 -25.8 -56.1 -9.8 -54.4 -3.4 -47.6 -35.7 20.2 7.1 -56.6%

45 -75.0 -50.9 -30.2 -73.6 -17.1 -73.6 -14.8 -64.2 -49.9 25.8 9.1 -51.6%

60 -80.7 -51.3 -31.1 -66.1 -18.7 -72.8 -21.9 -55.1 -49.7 23.5 8.3 -47.3%

90 -62.3 -32.1 -35.2 -59.7 -20.3 -63.6 -29.5 -28.3 -41.4 17.5 6.1 -42.3% (two rat data were removed, rat #1-2 stomach)

Table 3

Direct dosing of coprocessed microparticles to the jejunum data (Insulin (0.5mg/kg), Delivery Agent (75mg/kg))

D Insulin

Insulin Jejunum

Time

(min) #9 #10 #11 #12 #13 #14 #15 #16 mean SD SEM CV

0 12.5 12.5 12.5 12.5 12.5 12.5 12.5 12.5 0.0 0.0 0.0 15 413.2 1193.3 669.4 1177.5 2270.6 228.9 954.4 374.9 910.3 661.1 233.8 72.6% 30 354.3 148.4 70.5 64.7 481.0 168.4 782.9 57.4 265.9 258.2 91.3 97.1% 45 79.5 28.0 20.5 26.5 170.8 148.6 531.6 12.5 127.3 174.3 61.6 137.0% 60 23.1 14.7 12.5 16.8 71.6 117.0 200.1 12.5 58.5 68.4 24.2 116.9% 90 12.5 12.5 12.5 12.5 30.8 12.5 37.5 12.5 17.9 10.2 3.6 56.8%

AUCQ_→9O 13506 21158 11969 19690 46003 11102 39192 7235 21232 14054 4969 66.2%

(two rat data were removed, rat# 2 jejunum and rat# 4 jejunum)

2) Glucose

Change from base line Jejunum

Time

(min) #9 #10 #11 #12 #13 #14 #15 #16 Mean SD SEM CV

0 0 0 0 0 0 0 0 0 0 0 0

15 -50.8 -35.6 -16.7 -16.0 -61.1 -38.2 -62.8 -36.3 -39.7 17.9 6.3 -45.0%

30 -67.3 -65.5 -59.5 -35.8 -81.8 -62.5 -78.1 -52.9 -62.9 14.4 5.1 -22.9%

45 -64.4 -68.5 -76.3 -56.4 -74.4 -71.6 -78.1 -53.3 -67.9 9.2 3.2 -13.5%

60 -62.7 -62.5 -71.3 -62.7 -62.1 -65.6 -74.0 -42.2 -62.9 9.5 3.4 -15.1%

90 -62.4 -49.8 -69.7 -55.6 -41.8 -44.2 -69.4 -26.7 -52.5 14.9 5.3 -28.3%

(two rat data were removed, rat# 2 jejunum and rat# 4 jejunum)

Table 4

Direct dosing of coprocessed microparticles to the stomach (Insulin (0.25mg/kg), Delivery Agent (37.5mg/kg))

1) Insulin stomach

Time

(min) sto-1 sto-2 sto-3 sto-4 sto-5 sto-6 sto-7 sto-8 mean SD SEM CV

0 12.5 12.5 41.2 21.5 12.5 12.5 12.5 12.5 17.2 9.5 3.4 55.4%

15 12.5 20.8 14.7 12.5 12.5 12.5 26.4 12.5 16.0 4.9 1.7 30.7%

30 12.5 12.5 12.5 12.5 12.5 12.5 12.5 12.5 12.5 0.0 0.0 0.0%

45 48.8 12.5 12.5 12.5 12.5 12.5 12.5 12.5 17.0 12.0 4.3 70.4%

60 12.5 12.5 12.5 12.5 12.5 12.5 12.5 12.5 12.5 0.0 0.0 0.0%

90 12.5 12.5 12.5 12.5 12.5 12.5 12.5 12.5 12.5 0.0 0.0 0.0%

AUC_0→90 1670 1250 1373 1193 1125 1125 1334 1125 1274 187 66.0 14.6&

2) Glucose

Change from base line Stomach

Time Rat#

(min) 1 #2 #3 #4 #5 #6 #7 #8 mean SD SEM CV

0 0 0 0 0 0 0 0 0 0 0 0.0

15 -2.2 -12.3 2.2 0.8 -2.2 -12.3 2.2 0.8 -2.9 5.7 2.0 196.5%

30 -14.4 -10.1 -6.5 0.8 -14.4 -10.1 -6.5 0.8 -7.6 5.6 1.9 -73.7%

45 -15.8 -8.8 -12.7 0.0 -15.8 -8.8 -12.7 0.0 -9.3 5.9 2.1 -63.4%

60 -15.8 -11.4 -17.9 -5.8 -15.8 -11.4 -17.9 -5.8 -12.7 4.6 1.6 -36.2%

90 -19.1 -16.3 -8.6 -6.6 -19.1 -16.3 -8.6 -6.6 -12.7 5.2 1.8 -40.9%

Table 5

Direct dosing of coprocessed microparticles to the jejunum data (Insulin (0.25mg/kg), Delivery Agent (37.5mg/kg)) 1) Insulin jejunum

Time

(min) jej-l Jej-2 Jej-3 Jej-4 Jej-5 Jej-6 Jej-7 Jej-8 mean SD SEM CV

0 12.5 12.5 12.5 12.5 12.5 12.5 12.5 12.5 12.5 0.0 0.0 0.0%

15 428.5 532.4 232.6 12.5 62.0 186.6 79.6 160.5 219.2 170.7 60.4 77.8%

30 67.9 100.8 44.7 12.5 15.5 12.5 14.2 49.3 39.7 30.4 10.7 76.4%

45 16.8 40.8 26.6 12.5 12.5 12.5 12.5 12.5 18.4 9.7 3.4 52.6%

60 12.5 24.7 17.3 12.5 12.5 12.5 12.5 12.5 14.6 4.1 1.5 28.1%

90 12.5 12.5 12.5 12.5 12.5 12.5 12.5 12.5 12.5 0.0 0.0 0.0%

AUCn→on 8261 10947 5229 1125 1913 3737 2157 3897 4658 3392 1199 72.8%

2) Glucose

Change from base line Jejunum

Tune

(min) #9 #10 #11 #12 #13 #14 #15 #16 Mean SD SEM CV

0 0 0 0 0 0 0 0 0 0 0 0.0

15 -29.7 -20.5 -35 .8 1.3 -28.9 -18.2 -25. 9 -25.6 -22.9 11.2 3.9 -48.9%

30 -52.2 -54.5 -41 .9 -1.3 -50.0 -60.3 -36. 8 -52.8 -43.7 18.7 6.5 -42.8%

45 -63.6 -65.3 -43 .8 -0.7 -56.8 -69.2 -40. 8 -59.7 -59.0 22.3 7.8 -37.8%

60 -56.9 -69.4 -33 .8 11.6 -56.8 -62.1 -27. 2 -55.7 -41.9 28.1 10.6 -67.1%

90 -63.9 -59.7 -26 .9 8.3 -50.0 -31.8 -11. 8 -28.4 -28.6 22.7 8.5 -79.4%

b. Results from Oral Gavage Experiments Using Tablet and Capsules

[155] The glucose and insulin data from the three formulations tested are shown in

Figures 4 and 5, respectively. The individual data are listed in Tables 6 to 7. The results

from the direct dosing studies to the stomach and jejunum are included for comparison. The

individual glucose and insulin data for the simple mix of insulin and delivery agent is

shown in Table 8.

[156] In the group of 10 rats that was dosed with capsules containing

microparticles of coprocessed insulin and carrier, the average minimum glucose lowering was 70% from baseline at 30 minutes. One rat died at 15-30 minutes, likely due to

hypoglycemia, six rats were rescued at 30 minutes with dextrose, an additional rat was

rescued at 60 minutes, and two of the six that were rescued at 30 minutes died after 60

minutes. There were no signs of GI irritation or GI damage from the oral gavage procedure

from necropsies of the rats after the experiment. The average minimum glucose lowering

from tablets that contained the same amounts of insulin and carrier was 50% .

[157] The corresponding insulin concentrations are shown in Figure 5. Insulin

concentration is highest from the coprocessed microparticles in a capsule, followed by the

tablet and the capsule of the simple mix.

[158] In the oral gavage studies using capsules containing coprocessed

microparticles, two (of 10) rats were found to exhibit high insulin absorption. Retainer

samples were reassayed and insulin levels were approximately the same as those from the

original samples, as shown in Table 6(3), shown above. Insulin levels with and without

two high responders are shown in Figures 6 and 7, respectively. The individual and average

insulin and glucose profiles from N= 10 and N=8 are shown in Figures 13 to 16.

Table 6 Oral gavage of tablets: Insulin (0.5mg/kg), Delivery Agent (75mg/kg)

1) Insulin

Time Rat#

(min) 11 #12 #13 #14 #15 #16 #17 #18 #19 #20 mean SD SEM CV

0 12.5 12.5 12.5 12.5 12.5 12.5 12.5 12.5 12.8 12.5 12.5 0.1 0.0 0.8%

15 468.8 162.1 700.1 12.5 1363.4 197.4 565.4 57.0 114.4 12.5 365.4 426.3 134.8 116.7%

30 90.5 14.5 108.6 12.5 174.0 14.4 62.7 117.5 20.0 16.1 63.1 57.2 18.1 90.7%

45 15.2 32.0 22.9 12.5 44.5 12.5 16.3 43.3 12.5 12.5 22.4 12.9 4.1 57.6%

60 12.5 12.5 13.9 12.5 23.2 16.7 12.5 12.5 12.5 12.5 14.1 3.5 1.1 24.6%

90 12.5 12.5 12.5 12.5 12.5 12.5 12.5 12.5 12.5 12.5 12.5 0.0 0.0 0.0%

JUC_o→9o 9180 3692 13068 1125 24532 4022 10229 3830 2768 1179 7363 7259 2295 98.6%

2) Glucose

Change from baseline

Time Rat#

(min) 1 #2 #3 #4 #5 #6 #7 #8 #9 #10 mean SD SEM CV

0 0 0 0 0 0 0 0 0 0 0 0 0 0.0 0.00%

15 -51.4 -38.9 -36.5 -37.8 -23.8 -26.7 12.1 16.5 25.0 54.3 -10.7 35.0 11.1 -326.7%

30 -70.8 -69.4 -66.2 -68.3 -67.9 -68.9 -57.1 -41.8 -20.7 47.8 -48.3 37.4 11.8 -77.4%

45 -66.7 -63.9 -64.9 -57.3 -57.1 -52.2 -44.0 -27.5 -27.2 52.2 -40.9 35.7 11.3 -87.4%

60 -54.2 -47.2 -41.9 -42.7 -34.5 -33.3 -9.9 -4.4 -1.1 51.1 -21.8 31.7 10.0 -145.1%

90 -50.0 -26.4 -17.6 -19.5 -13.1 5.6 9.9 11.0 32.6 106.5 3.9 42.9 13.6 1099.9%

10

Table 7

Oral gavage of capsules containing coprocessed insulin and delivery agent data (Insulin (0.5mg/kg), Delivery Agent (75mg/kg))

5 1) Insulin

Time Rat#

^min) 11 #12 #13 #14 #15 #16 #17 #18 #19 #20 mean SD SEM C

0 12.5 12.5 12.5 12.5 12.5 12.5 12.5 12.5 12.5 12.5 0.0 0.0 0.0 0.0⁽

15 461.1 35.2 400.0 1080.6 143.1 36.7 7970.5 1611.3 369.8 922.5 1303.1 2396.7 757.9 183.9^«

30 244.0 120.0 3268.4 35.3 32.7 5915.1 201.0 150.9 240.3 1134.2 2070.4 654.7 182.5'

45 26.9 13.7 3132.7 18.9 201.2 3309.5 58.7 12.5 38.3 756.9 1399.0 442.4 184.8'

60 13.8 12.5 2129.7 12.5 28.7 3693.9 16.0 12.5 12.5 659.1 1335.7 422.4 202.6'

90 12.5 12.5 984.9 12.5 12.5 12.5 12.5 151.4 367.5 116.2 242.7'

^0→90

Cn=9) 11752 3096 175011 3522 4986 285725 28279 8561 18579 59946 100827 33609 168.2'

-0→90 [IF=T) 11752 3096 3522 4986 28279 8561 18579 11254 9279 3507 82.4'

2) Glucose

Time

(min) #11 #12 #13 #14 #15 #16 #17 #18 #19 #20 mean SD SEM CV

0 0 0 0 0 0 0 0 0 0 0 0 0 0.0 0.0%

15 -27.9 41.6 -32.5 -30.4 -23.3 19.8 -53.9 -5.5 -19.6 -25.3 -15.7 27.7 9.8 -176.3%

30 -79.0 -17.5 -76.6 -64.0 -40.1 -78.0 -77.0 -63.8 -77.0 -63.7 21. .4 7.5 -33.5%

45 -42.1 -46.4 -62.6 -36.7 -66.7 -53.9 -4.9 10.9 -50.0 -39.2 25 .9 9.1 -66.2%

60 3.2 -34.9 -61.4 -13.3 -75.3 -70.3 -7.2 64.5 -48.3 -30.8 45, .9 16.2 -149.0%

90 68.4 -7.2 -62.6 20.7 92.0 105.1 -8.6 29.3 70. .0 24.7 239.0%

3) Reassay insulin levels of Rats 14 and 17

10 Rat #14

Rat #17

Table 8

Oral gavage of capsules containing containing a simple mix of insulin and delivery agent data

(Insulin (0.5mg/kg), Delivery Agent (75mg/kg))

1) Insulin rat time #1 #2 #3 #4 #5 #6 #7 #8 #9 #10 mean SD SE CV

0 12.5 12.5 12.5 12.5 12.5 12.5 12.5 12.5 12.5 12.5 12.5 0 0 0.0%

15 41.4 230.9 58.3 110.7 82.2 12.5 12.5 12.5 14.9 25.2 60.1 68.8 21.8 114.4%

30 12.5 49.6 12.5 12.5 12.5 12.5 12.5 12.5 12.5 12.5 16.2 11.7 3.7 72.3%

45 12.5 12.5 12.5 12.5 12.5 12.5 12.5 12.5 12.5 12.5 12.5 0 0 0.0%

60 12.5 _. 12.5 12.5 12.5 12.5 12.5 12.5 12.5 12.5 12.5 12.5 0 0 0.0%

90 12.5 12.5 12.5 12.5 12.5 12.5 12.5 12.5 12.5 12.5 12.5 0 0 0.0%

AUC_n +90 1559 4958 1812 2598 2171 1125 1125 1125 1161 1316 1895 1189 376 62.7%

2) Glucose

Change from base line

Time Rat#

(min) 1 #2 #3 #4 #5 #6 #7 #8 #9 #10 mean SD SEM CV

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0%

15 7.7 -32.3 3.4 9.3 14.5 58.7 34 1.7 51.7 1.7 15.0 26.7 8.4 177.7%

30 -38.2 -78.5 -46.1 -42.9 -47.2 28.7 45.7 54.6 2.7 -17.9 -13.9 44.9 14.1 -322.4%

45 -13.9 -22.6 -20.8 -34.8 -38.9 20.4 42.1 44.8 -0.7 11.0 -1.3 30.1 9.5 -2247.0%

60 -3.1 41.4 -18.5 -14.3 -26.9 20.9 65.5 47.7 26.2 32.9 17.2 31.3 9.9 182.1%

90 6.2 73.1 32.6 53.4 40.1 43.1 51.7 39.6 30.6 41.2 18.4 5.8 44.6%

Example 2

1. Summary of Intravenous, Portal Vein and Subcutaneous Experiments

a. Experiment

[159] Intravenous, intraportal and subcutaneous dosing in rodents were conducted

to estimate the absolute bioavailability, the absorption of insulin in the portal vein, and the

bioavailability to relative subcutaneous administration. The data are summarized in Tables

9 to 11. The average insulin AUCo->∞/Dose was 0.0093 min.kg/ml from intravenous

dosing. This value was assumed to be constant in the estimates of absolute bioavailability. Table 9 Insulin pharmacokinetics results from intravenous (IV) administration

AUCW AUC_0->∞/

Dose C₀ kd AUC_0->co Dose v_d CW,/ Dose Dose (min. ιg/kg) (ng/ml) (min ¹) ng/ml) (min. kg/ml) (ml/kg) (kg/ml) (min. kg/ml)

2.5 15.79 0.89 17.74 0.0071 158 0.0063 0.0071

2.5 7.48 0.71 10.56 0.0042 334 0.0030 0.0042

2.5 13.36 0.84 15.87 0.0063 187 0.0053 0.0063

2.5 12.78 0.72 17.73 0.0071 196 0.0051 0.0071

2.5 11.26 0.72 15.75 0.0063 222 0.0045 0.0063 X±SD 12.1 ± 0.78 ± 0.0062 ± 219 ± (n-5) 2.8 0.07 15.5 ±2.6 0.0011 68

8 50.67 0.51 99.35 0.0124 157.88 0.0063 0.0124

8 52.64 0.53 99.32 0.0124 151.98 0.0066 0.0124

8 49.63 0.48 103.4 0.0129 161.19 0.0062 0.0129 X±SD 51.0 ± 0.51 ± 100.7 ± 0.0126 ± (n=3) 1.5 0.03 2.4 0.0003 157 ± 5

9 46.79 0.47 99.55 0.0111 192.35 0.0052 0.0111

9 36.32 0.48 75.67 0.0084 247.80 0.0040 0.0084

9 55.30 0.45 122.89 0.0137 162.75 0.0061 0.0137 X±SD 46.1 ± 0.47 ± 99.4 ± 0.0110 ± 201 ±

(n=3) 9.5 0.02 23.6 0.0026 43

Average 0.0053 0.0093

SD 0.001133858 0.0033

SEM 0.000341871 0.00099

Table 10 Insulin results from portal and systemic administrations

Table 11 Insulin results from subcutaneous (SC) administration dy Insulin AUC_0- AUCo_T

# dose Time Insulin SD SE Glucose SD SE N >τ T ^ max AUC_0->τ Dos

(min. (min. (mil

(mg/kg) (uU/ml) (mg/dl) uU/ml) (min) ng/ml) kg/ni

1 0.025 0 0.49 0.77 0.31 121 12.6 5.1 6 3640.8 15 76.1 140.0 0.00560

15 76.14 17.52 7.15 110 26.4 10.7 6

30 54.34 21.92 8.95 90 31.4 12.8 6

45 32.26 4.59 1.87 101 36.6 14.9 6

60 21.61 4.29 1.75 102 30.4 12.4 6

120 5.61 2.93 1.19 130 37.0 15.1 6

180 1.64 1.82 0.74 194 54.7 22.3 6

2 0.025 0 2.15 2.37 0.96 116 12.9 5.2 6 4234.4 15 79.5 162.9 0.00651

15 79.54 24.22 9.88 103 8.3 3.3 6

30 53.03 20.27 8.27 90 17.4 7.1 6

45 45.53 23.98 9.79 100 26.2 10.7 6

60 23.48 21.47 8.76 120 23.6 9.6 6

120 9.36 9.60 3.92 196 45.6 18.6 6

180 3.48 5.23 2.13 188 45.1 18.4 6

3 0.05 0 1.51 1.59 0.71 83 6.9 3.1 5 7230.0 15 98.8 278.1 0.00556

15 98.85 14.16 6.33 66 17.1 7.6 5

30 73.59 25.49 11.40 60 21.4 9.5 5

45 61.37 21.56 9.64 61 27.2 12.1 5

60 58.20 32.09 14.35 73 24.4 10.9 5

120 21.34 17.43 7.79 98 21.6 9.7 5

180 8.30 7.49 3.35 105 30.7 13.7 5

4 0.05 0 0.37 0.90 0.36 73 4.8 1.9 6 6407.7 15 103.4 246.5 0.00492

15 103.42 25.17 10.27 66 6.9 2.8 6

30 81.64 24.94 10.18 59 10.5 4.3 6

45 68.91 20.41 8.33 64 9.0 3.6 6

60 58.58 21.87 8.93 72 7.0 2.8 6

90 32.01 9.92 4.05 88 14.0 5.7 6

120 21.15 9.42 3.84 110 17.0 6.9 6 0.05 00 00 00 00 6600 55..11 22..22 55 1317.0 15 34.9 50.7 0.00101

15 34.90 46.01 20.57 42 6.1 2.7 5

30 9.71 18.15 8.12 38 6.9 3.1 5

45 16.57 37.04 16.56 42 9.8 4.4 5

60 10.46 20.05 8.96 48 17.6 7.8 5

90 2.94 4.25 1.90 52 25.2 11.3 5

120 5.05 6.91 3.09 66 41.8 18.7 5

0.05 0 0 1 122..8800 1 188..0011 7 7..3355 6 688 5 5..00 2 2..00 6 6 9937.5 15 191.7 382.2 0.00764

15 191.67 100.40 40.98 40 6.2 2.5 6

30 119.71 79.53 35.56 39 2.5 1.1 5

45 121.33 118.89 48.53 36 7.1 2.9 6

60 74.65 57.97 23.66 32 4.3 1.7 6

90 42.88 37.77 15.42 35 5.5 2.2 6

120 25.65 17.59 7.18 39 9.2 3.7 6

0.05 00 55..8855 33..1111 11..2277 6655 88..77 33..55 66 7711.1 30 126.0 296.6 0.00593

15 90.29 73.70 30.08 45 7.2 2.9 6

30 125.96 107.54 43.90 39 8.9 3.6 6

45 67.83 95.55 39.01 40 10.9 4.4 6

60 78.19 105.69 43.14 42 13.2 5.4 6

90 45.77 49.52 20.22 42 18.7 7.6 6

120 18.24 13.53 5.52 50 26.2 10.7 6

0.05 00 22..4400 55..8888 22..4400 7700 44..88 11..99 66 5303.3 15 131.7 204.0 0.00407

15 131.71 131.15 5334 59 6.8 3.0 6

30 70.90 45.95 18.76 64 12.1 4.9 6

45 66.28 42.41 17.31 69 16.5 6.7 6

60 0.59 1.27 0.52 73 13.1 5.3 6

90 33.96 14.99 6.12 89 17.8 7.2 6

120 14.64 9.35 3.82 106 18.6 7.6 6

Avg AUC_0->T/

Dose 0.00515 (min. kg/ml)

b. Results

[160] The ratio of systemic to portal insulin was found to be approximately 0.62

(calculated from data in Table 10). Hence, the bioavailability in the portal vein can be

calculated by dividing the absolute bioavailability by 0.62. The portal bioavailability

provides an estimate of drug absorption from oral delivery. The average insulin AUCo-

>t/Dose was 0.00516 min. kg/ml from subcutaneous dosing. This value is used to estimate

bioavailability relative to subcutaneous. With the exception of the intravenous data, all

AUC were calculated from t=0 to the last sampling point (i.e. AUCo->t). [161] In the rat model, these results from intraportal administration suggest that the maximum absolute bioavailability of insulin is approximately 60% from oral delivery or by any other means of 100% GI absorption of insulin into the portal vein. Secondly, the absolute bioavailability from SC is approximately 56%.

[162] The estimates of bioavailability (absolute bioavailability, portal

bioavailability, relative bioavailability to subcutaneous, and relative portal bioavailability to

subcutaneous) are summarized in Figures 8 to 12, and in Table 12. The estimated absolute

bioavailability from in situ dosing to the stomach and the jejunum are shown hi Figure 8.

The values of bioavailability were 5% when dosed in the stomach and 18% when dosed in

the jejunum from microparticles containing coprocessed insulin (0.5 mg/kg) and delivery

agent (75 mg/kg).

[163] The estimated absolute bioavailability from the tablet and capsule

formulations dosed by oral gavage in rats are shown in Figure 9 using formulations

containing 0.5 mg/kg insulin and 75 mg/kg delivery agent. The values of bioavailability

were 6% when dosed from tablets, and 1.6% when dosed from capsules containing a

simple mix of insulin and carrier.

Table 12

Estimates of bioavailability

AUCo->τ/

AUCo->τ T_roaχ C_maχ AUC₀->τ Dose BA BAportal ReI BA ReI BAportai

(min. (min. (min. uU/ml) (min) (uU/ml) ng/ml) kg/ml) (%) (%) (%) (%)

[AUCo->τ/ [AUCo^r /

(IP/JV=0.62) Dose]sc=0.0052 Dose]sc=0.0052

SC 0.0052 55.67

Insulin (0.5mg/kg) + Delivery Agent (75mg/kg) Stomach

4778.70 15 114.50 183.80 3.68E-04 3.97 6.40 7.12 11.49 2130.75 15 72.75 81.95 1.64E-04 1.77 2.85 3.18 5.12

18958.58 45 893.86 729.18 1.46E-03 15.74 25.38 28.27 45.59

1126.65 15 12.61 43.33 8.67E-05 0.94 1.51 1.68 2.71

14425.95 45 212.73 554.84 1.11E-03 11.97 19.31 21.51 34.69

4000.73 60 140.31 153.87 3.08E-04 3.32 5.36 5.96 9.62

2793.45 15 118.66 107.44 2.15E-04 2.32 3.74 4.16 6.72

5046.23 15 158.50 194.09 3.88E-04 4.19 6.76 7.52 12.13

Mean 6657.63 28.13 215.49 256.06 5.12E-04 5.53 8.91 9.93 16.01

SD 6446.07 18.70 280.36 247.93 4.96E-04 5.35 8.63 9.61 15.50

SE 2279.03 6.61 99.12 87.65 1.75E-04 1.89 3.05 3.40 5.48 CV

(%) 96.82 66.48 130.10 96.82 9.68E+01 96.82 96.82 96.82 96.82

Jejunum

13504.88 15 413.23 519.42 1.04E-03 11.21 18.08 20.14 32.48

21156.68 15 1193.25 813.72 1.63E-03 17.56 28.32 31.54 50.88

11967.90 15 669.38 460.30 9.21 E-04 9.93 16.02 17.84 28.78

19689.45 15 1177.47 757.29 1.51E-03 16.34 26.36 29.36 47.35

46001.85 15 2270.55 1769.30 3.54E-03 38.18 61.59 68.59 110.62

11101.35 15 228.90 426.98 8.54E-04 9.21 14.86 16.55 26.70

39190.05 15 954.35 1507.31 3.01 E-03 32.53 52.47 58.43 94.24

7234.05 15 374.88 278.23 5.56E-04 6.00 9.68 10.79 17.40

Mean 21230.78 15 910.25 816.57 1.63E-03 17.62 28.42 31.65 51.05

SD 14053.61 0 661.14 540.52 1.08E-03 11.66 18.81 20.95 33.80

SE 4968.70 0 233.75 191.10 3.82E-04 4.12 6.65 7.41 11.95 CV

(%) 66.19 0 72.63 66.19 6.62E+01 66.19 66.19 66.19 66.19

Tablet

9181.20 15 468.81 353.12 7.06E-04 7.62 12.29 13.69 22.08

3692.64 15 162.11 142.02 2.84E-04 3.07 4.94 5.51 8.88

13068.89 15 700.10 502.65 1.01 E-03 10.85 17.50 19.48 31.43

1125.00 0 12.50 43.27 8.65E-05 0.93 1.51 1.68 2.71

24533.57 15 1363.43 943.60 1.89E-03 20.36 32.84 36.58 59.00

4022.33 15 197.39 154.70 3.09E-04 3.34 5.38 6.00 9.67

10228.04 15 565.39 393.39 7.87E-04 8.49 13.69 15.25 24.60

3830.06 30 117.47 147.31 2.95E-04 3.18 5.13 5.71 9.21

2767.70 15 114.35 106.45 2.13E-04 2.30 3.71 4.13 6.66

1178.43 30 16.06 45.32 9.06E-05 0.98 1.58 1.76 2.83

Mean 7362.78 16.50 371.76 283.18 5.66E-04 6.11 9.86 10.98 17.71

SD 7669.89 9.01 445.61 295.00 5.90E-04 6.37 10.27 11.44 18.44

SE 2425.43 2.85 140.91 93.29 1.87E-04 2.01 3.25 3.62 5.83 CV

(%) 104.17 54.63 119.86 104.17 1.04E+02 104.17 104.17 104.17 104.17

Capsule (co-dried)

(N=9) 11570.64 15 461.06 445.02 8.90E-04 9.60 15.49 17.25 27.82

3096.24 30 120.02 119.09 2.38E-04 2.57 4.15 4.62 7.45

175011.18 30 3268.40 6731.20 1.35E-02 145.27 234.30 260.93 420.86 3523.46 15 143.14 135.52 2.71 E-04 2.92 4.72 5.25 8.47

4988.01 45 201.24 191.85 3.84E-04 4.14 6.68 7.44 11.99

285725.26 15 7970.50 10989.43 2.20E-02 237.16 382.52 426.00 687.10

28278.49 15 1611.31 1087.63 2.18E-03 23.47 37.86 42.16 68.00

8559.93 15 369.78 329.23 6.58E-04 7.11 11.46 12.76 20.58

18578.54 15 922.50 714.56 1.43E-03 15.42 24.87 27.70 44.68

Mean 59925.75 21.67 1674.22 2304.84 0.00 49.74 80.23 89.35 144.11

SD 100838.27 10.90 2570.42 3878.39 0.01 83.70 135.00 150.34 242.49

SE 33612.76 3.63 856.81 1292.80 0.00 27.90 45.00 50.11 80.83

CV

(%) 168.27 50.29 153.53 168.27 168.27 168.27 168.27 168.27 168.27

Capsule (co-dried)

(N=7) 11570.64 15 461.06 445.02 8.90E-04 9.60 15.49 17.25 27.82

3096.24 30 120.02 119.09 2.38E-04 2.57 4.15 4.62 7.45

3523.46 15 143.14 135.52 2.71 E-04 2.92 4.72 5.25 8.47

4988.01 45 201.24 191.85 3.84E-04 4.14 6.68 7.44 11.99

28278.49 15 1611.31 1087.63 2.18E-03 23.47 37.86 42.16 68.00

8559.93 15 369.78 329.23 6.58E-04 7.11 11.46 12.76 20.58

18578.54 15 922.50 714.56 1.43E-03 15.42 24.87 .27.70 44.68

Mean 11227.90 21.43 547.01 431.84 0.00 9.32 15.03 16.74 27.00

SD 9277.29 11.80 544.29 356.82 0.00 7.70 12.42 13.83 22.31

SE 3506.49 4.46 205.72 134.86 0.00 2.91 4.69 5.23 8.43

CV

(%) 82.63 55.08 99.50 82.63 82.63 82.63 82.63 82.63 82.63

Capsule (simple mix)

1558.08 15 41.37 59.93 1.20E-04 1.29 2.09 2.32 3.75

4956.66 15 230.89 190.64 3.81 E-04 4.11 6.64 7.39 11.92

1812.60 15 58.34 69.72 1.39E-04 1.50 2.43 2.70 4.36

2598.35 15 110.72 99.94 2.00E-04 2.16 3.48 3.87 6.25

2171.06 15 82.24 83.50 1.67E-04 1.80 2.91 3.24 5.22

1125.00 0 12.50 43.27 8.65E-05 0.93 1.51 1.68 2.71

1125.00 0 12.50 43.27 8.65E-05 0.93 1.51 1.68 2.71

1125.00 0 12.50 43.27 8.65E-05 0.93 1.51 1.68 2.71

1161.65 15 14.94 44.68 8.94E-05 0.96 1.56 1.73 2.79

1315.97 15 25.23 50.61 1.01 E-04 1.09 1.76 1.96 3.16

Mean 1894.94 10.50 60.12 72.88 1.46E-04 1.57 2.54 2.83 4.56

SD 1188.69 7.25 68.82 45.72 9.14E-05 0.99 1.59 1.77 2.86

SE 375.90 2.29 21.76 14.46 2.89E-05 0.31 0.50 0.56 0.90

CV

(%) 62.73 69.01 114.47 62.73 6.27E+01 62.73 62.73 62.73 62.73 n (0.25rτ )g/kg) + Delivei ry Agent (37.5mg/kg)

Stomach

1669.50 45 48.80 64.21 2.57E-04 2.77 4.47 4.98 8.03

1249.50 15 20.80 48.06 1.92E-04 2.07 3.35 3.73 6.01

1373.25 0 41.20 52.82 2.11 E-04 2.28 3.68 4.09 6.60

1192.50 0 21.50 45.87 1.83E-04 1.98 3.19 3.56 5.74 1125.00 0 12.50 43.27 1.73E-04 1.87 3.01 3.35 5.41

1125.00 0 12.50 43.27 1.73E-04 1.87 3.01 3.35 5.41

1333.22 15 26.38 51.28 2.05E-04 2.21 3.57 3.98 6.41

1125.00 0 12.50 43.27 1.73E-04 1.87 3.01 3.35 5.41

Mean 1274.12 9.38 24.52 49.00 1.96E-04 2.12 3.41 3.80 6.13

SD 186.56 15.91 13.77 7.18 2.87E-05 0.31 0.50 0.56 0.90

SE 65.96 5.63 4.87 2.54 1.01E-05 0.11 0.18 0.20 0.32 CV

(%) 14.64 169.71 56.16 14.64 1.46E+01 14.64 14.64 14.64 14.64

Jejunum

8260.50 15 428.50 317.71 1.27E-03 13.71 22.12 24.63 39.73

10947.00 15 532.40 421.04 1.68E-03 18.17 29.31 32.64 52.65

5229.00 15 232.60 201.12 8.04E-04 8.68 14.00 15.59 25.15

1125.00 0 12.50 43.27 1.73E-04 1.87 3.01 3.35 5.41

1910.94 15 61.95 73.50 2.94E-04 3.17 5.12 5.70 9.19

3735.93 15 186.56 143.69 5.75E-04 6.20 10.00 11.14 17.97

2157.20 15 79.60 82.97 3.32E-04 3.58 5.78 6.43 10.38

3897.03 15 160.54 149.89 6.00E-04 6.47 10.43 11.62 18.74

Mean 4657.82 13.13 211.83 179.15 7.17E-04 7.73 12.47 13.89 22.40

SD 3392.58 5.30 182.48 130.48 5.22E-04 5.63 9.08 10.12 16.32

SE 1199.46 1.88 64.52 46.13 1.85E-04 1.99 3.21 3.58 5.77 CV

(%) 72.84 40.41 86.14 72.84 7.28E+01 72.84 72.84 72.84 72.84

Example 3

Insulin and 4-CNAB Stability in Simulated Gastric Fluid

[164] The stability of insulin in simulated gastric fluid (SGF) was evaluated in the

presence and absence of 4-CNAB. Solutions were prepared containing insulin (1 mg/ml)

with and without monosodium 4-CNAB (1 mg/ml).

[165] The SGF was prepared with and without pepsin, a gastric enzyme. SGF pH

1.2 was prepared as per the USP NF 26 guidelines. 2 g sodium chloride and 3.2 g of

pepsin were weighed and added to a suitable container, and deionized water was added to

reach one liter in volume. If necessary, the pH was adjusted to 1.2 by addition of

concentrated HCl or NaOH. A second SGF solution omitting the pepsin was also prepared. [166] Four 50 ml samples of SGF (two with pepsin and two witfiout) were placed

into a jacketed vessel connected to a circulating water bath set at 37°C. The solutions were

stirred with magnetic stir bars for ten minutes to allow the solutions to reach 37⁰C and

reach thermal equilibrium. 50 mg of 4-CNAB was added to one of the samples containing

pepsin and one of the samples without pepsin, and the solutions were stirred for a few

minutes to allow the 4-CNAB to dissolve. 50 mg of insulin was added to the each of the

samples. After dissolution of the insulin, samples of the solutions were taken at pre¬

determined time intervals, filtered, and immediately assayed by HPLC for insulin and 4-

CNAB content. The first sample withdrawn after all the insulin was dissolved was

considered to have been drawn at time zero (0). The results are shown in table 13.

Table 13

[167] The term " % of theoretical" as used herein, means the percent of the

concentration (mg/mL) of withdrawn solution at the time-point the sample was taken as

compared to the theoretical concentration (mg/mL) of the measuring component for

experiment. The standard of deviation for the HPLC analysis is +5 % . These results show

that insulin is unstable in SGF containing pepsin, since only 3.0% of the insulin remained at

the first sampling point (97% of the insulin was degraded), while insulin is stable at least up

to 2 hours in SGF without pepsin.

Example 4

Stability of Insulin in Simulated Intestinal Fluid

[168] The stability of insulin in simulated intestinal fluid (SIF) was evaluated in the

presence and absence of 4-CNAB.

[169] The SIF solutions were prepared with and without pancreatic enzyme. SIF

pH 7.5 was prepared as per the USP NF 26 guidelines. SIF was prepared by addition of

6.8 g monobasic potassium phosphate and 10 g of pancreatin into a suitable vessel, and i deionized water was added to reach a total volume of one liter. If necessary, the pH was

adjusted to 7.5 by addition of 0.2 N sodium hydroxide. A second SIF solution omitting the

pancreatin, an intestinal enzyme, was also prepared.

[170] Four 50 ml samples of SIF (two with pancreatin and two without) were

placed into a jacketed vessel connected to a circulating water bath set at 37°C. The

solutions were stirred with magnetic stir bars for ten minutes to allow the solutions to reach

37⁰C and reach thermal equilibrium. 50 mg of 4-CNAB was added to one of the samples containing pepsin and one of the samples without pepsin, and the solutions were stirred for

a few minutes to allow the 4-CNAB to dissolve. 50 mg of insulin was added to the each of

the samples. After dissolution of the insulin, samples of the solutions were taken at pre¬

determined time intervals, and immediately assayed by HPLC for insulin and 4-CNAB

content. The results are shown in table 14.

Table 14

[171] These results show that insulin is stable in SIF without pancreatin and

degrades in presence of the enzyme. Insulin is more stable in SIF with and without enzyme than in SGF with and without enzyme. At the first sampling time point (0 minuts) only

3.0% insulin remained in SGF with enzymes while 58.9% and 66.9% insulin remained in

SIF.

Example 5 Effect of Formulation on Insulin Absorption and Action

[172] Six formulations containing insulin shown in Table 15 were prepared as

follows.

Table 15

[173] Polyplasdone XL, is available from International Specialty Products,

Wilmington DE.; Emcocel HD90, Prosolv HD90, Emcompress and Anhydrous

Emcompress is available from JRS Pharma, Patterson, NY.

[174] The formulations were fed to rhesus monkeys in doses containing 100 mg/kg

of 4-CNAB and 13 U/kg insulin. Groups of four rhesus monkeys, two males and two

females, were fasted for at least 12 hrs prior to dosing and up to 4 hrs after dosing. Water

was withheld approximately 1 hr before dosing and up to 2 hrs after dosing after which it

was permitted ad libitum. The dosing was followed by a 5 ml water flush. Blood samples

(approximately 2 ml each) were collected by venipuncture at 15 minutes before dosing and

at 5, 10, 15, 20, 30, 45 minutes and 1, 1.5, 2, 3, 4 hr after dosing. Each blood sample

was divided into two portions. One portion was allowed to clot at room temperature and

centrifuged at 2-8⁰C for 10 minutes at 3000 rpm. The serum obtained was aliquoted into two portions and stored at -70⁰C until shipment. One sample was shipped to Emisphere on dry

ice for insulin analysis by ELISA while the other was retained by the CRO for serum glucose

analysis. The second portion of the blood was kept on wet ice for up to 30 minutes and

centrifuged at 2-8⁰C for 10 minutes at 3000 rpm. The plasma obtained was shipped to

Emisphere on dry ice for analysis of 4-CNAB content by HPLC. Each formulation was

administered to 4 rhesus monkeys, except formulation 1, which was administered to 8

rhesus monkeys. Blood samples were taken at predetermined intervals as described above

and assayed for insulin and glucose levels. The results are shown in table 16 and in

Figures 17 and 18 . Table 16

[175] Disintegration time was determined in water at 37 + 2°C using the method described in USP <701>. Multiple tubes containing water are placed in a basket-rack

assembly immersed in a water bath maintained at 37 + 2°C. The basket-rack assembly raises

and lowers the tubes at a constant frequency. The tablets are placed in the tubes and are periodically examined to determine if they have disintegrated completely. Each tablet is tested in six different tubes. If 1 or 2 tablets fails to consistently disintegrate, the procedure is repeated on additional tablets. The average maximum concentration of insulin (C_max) was

determined for each group based upon the serum levels of insulin measured as described

above. If the blood glucose levels in the primates falls to very low levels ( < 1 mmol/L)

during the experiment they are administered dextrose in order to bring the blood glucose up to a safe level. The average Cmax for each group, as well as the number of rhesus monkeys

rescued, is shown in table 17.

Table 17

Example 6

Preparation of Enteric Coated Tablets

[176] Capsules were manufactured by encapsulating 300 mg of a formulation

including 150 units insulin, 200 mg 4-CNAB, 0.4% w/w povidone, -29.1% w/w

Emcompress, 1% w/w SLS, and 1% w/w magnesium stearate into size 2 white opaque

capsules. The capsules were first coated with a subcoat consisting of Opadry clear for a

weight gain of 5 % followed by an enteric coat of 20% weight gain for a total weight gain

on the capsules of 25 % .

[177] Tablets were manufactured by pressing 300 mg of the formulation described

above into tablets. An 10% weight gain enteric coat was applied. The formulations for the

subcoats and enteric coats are shown in table 18 below.

Table 18

[178] Opadry™ Clear is available from Colorcon, of West Point, PA.

[179] Milli Q Water is highly purified water and is available from Millipore of

Billerica, Massachusetts.

[180] Eudragit L30D55 is available from Degussa AG, Parsippany, NJ.

[181] To verify the effectiveness of the enteric coat, the coated capsules and tablets

were placed in 0.1 N HCl for two hours or pH 6.8 phosphate buffer for one hour. The

coated capsules and tablets did not dissolve in the 0.1 N HCl, but did dissolve in the pH 6.8

phosphate buffer.

Example 7

SNAC Micro Beads Coated With Heparin

[182] 5 g of SNAC and 0.5 g of magnesium stearate were mixed. 0.02 g of the

mixed powder was fed into a die. Small beads of SNAC and magnesium stearate were

made at 1200 PSI bar pressure The beads had a round/ball shape size of about 0.2 mm to about 2.0 mm. The SNAC beads were then coated with 2.5 g of heparin, in liquid form,

by a rotary method and dried under vacuum oven at 40° C for 10 hours.

Example 8

Micronized SNAP with Heparin

[183] SNAD was screened through a 35 mesh Tyler standard sieve. The SNAD

was milled with a Glen Mills, Model SlOO centrifugal ball mill (Clifton, NJ) equipped with

a 250 mL stainless steel grinding jar and 30 mm (440c) diameter stainless steel balls was

used. The process parameters investigated were (1) number of balls used, (2) duration of

milling, (3) milling speed, and (4) milling jar total charge. A Malvern Mastersizer 2000

equipped with a Scirocco 2000 dry accessory was used for particle size determination. A

Kratos XRD 6000 (version 4.1) X-ray powder diffractometer scanning over the 2Θ range 5-

40° 2Θ was used for monitoring crystallinity changes. The diverging, scattering, and

receiving slits were 1°, 1°, and 0.3 mm respectively. A Brinkmann 737 KF coulometer was

used for moisture content determination while a Quantachrome Nova 3000 Series Surface

Area Analyzer was used for specific surface area determination.

[184] The results indicated that the particle size distribution of pre-screened SNAD

was d(0.1) = 1.6 μm, d(0.5) = 10.5 μm, and d(0.9) = 314.9 μm. The data obtained using different numbers of balls ranging from 1 to 5 indicated that the optimum number of balls for the charge used was 2. The use of 2 balls yielded the particle size d(0.1) = 1.1 μm, d(0.5) = 12.0 μm, and d(0.9) = 154.3 μm. [185] An evaluation of the effect of milling time for a fixed number of balls and charge indicated that a milling time of 120 minutes was optimum resulting in the particle size distribution, d(0.1) = 2.0 μm, d(0.5) = 15.4, and d(0.9) = 62.9 μm.

[186] An evaluation of the milling speeds 100, 300, and 500 rpm indicated that optimum milling was obtained at 300 rpm. This speed yielded the particle size distribution, d(0.9) = 62.9 μm compared to unmilled SNAD d(0.9) = 314.9 μm.

[187] A charge of 37 mL of the 250 mL milling jar provided better milling

compared to 75 and 112 mL. The powder X-ray diffraction analysis indicated that milling

did not result in crystallinity changes for SNAD. The Karl Fischer moisture content

determination indicated no significant changes in moisture content.

[188] The SNAD was then mixed with heparin.

Example 9

Micronized SNAC with Micronized Heparin

. [189] SNAC and heparin were micronized separately by the procedure described in

Example 8 with 2 balls at 200 rpm for 120 minutes and then mixed together. The

micronized SNAC had a d(0.5) of 7.574 μm SNAC/heparin capsules having the

formulations shown in table 19 below were prepared by hand packing them into hard

gelatin capsules.

Table 19

Ingredient Formulation (mg/capsule)

A B

[190] ^l - Propylene glycol monocaprylate is available as Capmul™ PG 8 from

Abitec Corporation of Columbus, OH.

[191] ² - PEG 300 is available as Carbowax™ 300 from Dow Chemical Co. of

Midland, MI.

[192] The heparin, SNAC, and sodium lauryl sulfate were mixed. Separately, the

PEG 300, propylene glycol monocaprylate, and water (for formulation B) were mixed.

50% of the liquid PEG 300/propylene glycol monocaprylate mixture was transferred to a

mortar. The heparin, SNAC, and sodium lauryl sulfate blended powder was added little by

little and triturated with the liquid in the mortar and pestle. The capsules were then packed

with the resulting mixture.

Example 10

Micronized SNAC/Heparin [193] Heparin (118.5 mg/dose (22,500 rpm)) and SNAC (125 mg/dose) were dry

mixed, screened through a 35 mesh screen, and milled for about 4 minutes with a ball mill.

The mixture was packed into capsules (Capsugel Size 1 capsules (Greenwood, SC)).

[194] The capsules were administered to rhesus monkeys (2 capsules per monkey)

by the following procedure. Rhesus monkeys weighing between 3.5 - 5.0 kg were fasted

overnight before the experiments and food was returned about 2 hours after dosing. Water

was withheld from 30 minutes prior to dosing until 30 minutes after dosing, except for

those quantities used for dosing. Each dosage form was delivered to the rear of the mouth

using a pill gun. After release of the dosage form, 5 ml of reverse osmosis water was

administered into the oral cavity to facilitate swallowing. Following delivery, the oral

cavity was inspected to ensure that the capsule was swallowed. Antifactor Xa from blood

samples was measured over 6 hours.

[195] The results are shown in Figure 19.

Example 11

Micronized SNAC/Heparin

[196] Capsules containing micronized SNAC/heparin as shown in table 20 below

were prepared as follows.

[197] A solution of heparin and SNAC was prepared as follows. The required

amounts of heparin and SNAC were weighed out and water, which was previously adjusted

to a pH of about 8 with sodium hydroxide, was added. The pH of the resulting solution

was in the range of about 7.3 - 7.5. The solution pH was adjusted to a pH of about 8 with sodium hydroxide. The solution was then dried in a RotoVap apparatus at 50° C under

vacuum. The evaporating was done using the program outlined below.

[198] 1. Immediate reduction of vacuum from 760 torr to 200 torr

[199] 2. Reduction of vacuum pressure from 200 to 100 torr in 2 minutes

[200] 3. Reduction of vacuum pressure from 100 to 50 torr in 2 minutes

[201] 4. Reduction of vacuum pressure from 50 to 25 torr in 4 minutes

[202] 5. Reduction of vacuum pressure from 25 to 15 torr in 4 minutes

[203] 6. Reduction of vacuum pressure from 15 to 10 torr in 2 minutes [204] 7. Evaporating at 10 ± 2 torr and 70 rpm in 30 minutes

[205] 8. Switch to 50 rpm manually and continue with evaporating for 4 hours

[206] The sample was vacuum dried overnight. The resulting powder was then micronized and filled into capsules to give the desired dose.

Table 20

[207] The present invention is not to be limited in scope by the specific

embodiments described herein. Indeed, various modifications of the invention in addition

to those described herein will become apparent to those skilled in the art from the foregoing

description and the accompanying figures. Such modifications are intended to fall within

the scope of the appended claims.

[208] Patents, patent applications, publications, product descriptions, and protocols

are cited throughout this application, the disclosures of which are incorporated herein by

reference in their entireties for all purposes.

Claims

What is Claimed is:

1. Particles comprising a delivery agent and an active agent, wherein the

particles have a median particle size of less than about 999 micrometers.

2. The particles of claim 1, wherein the particles have a median particle size of

about 1 nanometer to about 999 micrometers.

3. The particles of claim 2, wherein the particles have a median particle size of

about 1 to about 999 micrometers.

4. The particles of claim 2, wherein the particles have a median particle size of

about 1 to about 999 nanometers.

5. The particles of claim 2, wherein the particles have a median particle size of

about 45 to about 850 micrometers.

6. The particles of claim 2, wherein the particles have a median particle size of

about 45 to about 150 micrometers.

7. The particles of claim 2, wherein the particles have a median particle size of

about 150 to about 250 micrometers.

8. The particles of claim 2, wherein the particles have a median particle size of

about 250 to about 425 micrometers.

9. The particles of claim 2, wherein the particles have a median particle size of

about 425 to about 850 micrometers.

10. The particles of claim 2, wherein the particles have a median particle size of

about 100 to about 1000 nanometers.

11. The particles of claim 10, wherein the particles have a median particle size

of about 500 to about 1000 nanometers.

12. A pharmaceutical formulation comprising particles having a median particle

size of less than about 999 micrometers, the particles comprising a delivery agent and an

active agent.

13. The pharmaceutical formulation of claim 12, wherein the particles have a

median particle size of about 1 nanometer to about 999 micrometers.

14. The pharmaceutical formulation of claim 13, wherein the particles have a

median particle size of about 1 to about 999 micrometers.

15. The pharmaceutical formulation of claim 13, wherein the particles have a

median particle size of about 1 to about 999 nanometers.

16. The pharmaceutical formulation of claim 13, wherein the particles have a

median particle size of about 45 to about 850 micrometers.

17. The pharmaceutical formulation of claim 13, wherein the particles have a

median particle size of about 45 to about 150 micrometers.

18. The pharmaceutical formulation of claim 13, wherein the particles have a

median particle size of about 150 to about 250 micrometers.

19. The pharmaceutical formulation of claim 13, wherein the particles have a

median particle size of about 250 to about 425 micrometers.

20. The pharmaceutical formulation of claim 13, wherein the particles have a

median particle size of about 425 to about 850 micrometers.

21. The pharmaceutical formulation of claim 13, wherein the particles have a

median particle size of about 100 to about 1000 nanometers.

22. The pharmaceutical formulation of claim 21, wherein the particles have a

median particle size of about 500 to about 1000 nanometers.

23. A pharmaceutical formulation comprising a delivery agent and an active

agent, wherein the delivery agent is in the form of particles having a median particle size of

less than about 999 micrometers.

24. The pharmaceutical formulation of claim 23, wherein the delivery agent

particles have a median particle size of about 1 nanometer to about 999 micrometers.

25. The pharmaceutical formulation of claim 23, wherein the active agent is in

the form of particles, and the active agent particles have a median particle size of about 1

nanometer to about 999 micrometers.

26. The pharmaceutical formulation of claim 24, wherein the delivery agent

particles have a median particle size of about 1 to about 999 micrometers.

27. The pharmaceutical formulation of claim 24, wherein the delivery agent

particles have a median particle size of about 1 to about 999 nanometers.

28. The pharmaceutical formulation of claim 26, wherein the delivery agent

particles have a median particle size of about 7 to about 16 micrometers.

29. The pharmaceutical formulation of claim 23, wherein the delivery agent

particles are compressed to form micro-beads.

30. The pharmaceutical formulation of claim 29, wherein the micro-beads are

coated with an active agent.

31. The pharmaceutical formulation of claim 30, wherein the active agent is

insulin or heparin.

32. The pharmaceutical formulation of any of claims 29-31, wherein the micro-

beads have a diameter of about 0.2 mm to about 2.0 mm.

33. The pharmaceutical formulation of claim 25, wherein the delivery agent

particles and the active agent particles both have a median particle size of about 1 to about

999 micrometers.

34. The pharmaceutical formulation of claim 25, wherein the delivery agent

particles and the active agent particles both have a median particle size of about 1 to about

999 nanometers.

35. A pharmaceutical formulation comprising a delivery agent and an active

agent, wherein the active agent is in the form of particles having a median particle size of

less than about 999 micrometers.

36. The pharmaceutical formulation of claim 35, wherein the active agent

particles have a median particle size of about 1 nanometer to about 999 micrometers.

37. The pharmaceutical formulation of claim 35, wherein the active agent

particles have a median particle size of about 1 to about 999 micrometers.

38. The pharmaceutical formulation of claim 17, wherein the active agent

particles have a median particle size of about 1 to about 999 nanometers.

39. The pharmaceutical formulation of any of any of claims 12-38, wherein the

delivery agent compound is selected from N-(8-[2-hydroxybenzoyl]-amino)caprylic acid, N-

(10-[2-hydroxybenzoyl]-amino)decanoic acid, 8-(2-hydroxy-4-

methoxybenzoylamino)octanoic acid, 8-(2-hydroxy-5-chlorobenzoylamino)-octanoic acid, 4-

[(2-hydroxy-4-chlorobenzoyl)-amino]butanoic acid, and pharmaceutically acceptable salts

thereof.

40. The pharmaceutical formulation of any of claims 12-38, wherein the delivery

agent compound is N-(8-[2-hydroxybenzoyl]-amino)caprylic acid or a pharmaceutically

acceptable salt thereof.

41. The pharmaceutical formulation of any of claims 12-38, wherein the delivery

agent compound is N-(10-[2-hydroxybenzoyl]-amino)decanoic acid or a pharmaceutically

acceptable salt thereof.

42. The pharmaceutical formulation of any of claims 12-38, wherein the delivery

agent compound is 4-[(2-hydroxy-4-chloro-benzoyl)-amino]butanoic acid or a

pharmaceutically acceptable salt thereof.

43. The pharmaceutical formulation of any of any of claims 12-38, wherein the

active agent is selected from proteins, polypeptides, peptides, hormones, and

polysaccharides.

44. The pharmaceutical formulation of any of any of claims 12-38, wherein the

active agent is selected from the following, including synthetic, natural or recombinant

sources thereof: growth hormones; growth hormone releasing hormones; growth hormone

releasing factor, interferons; interleukin-1; interleukin-2; insulin, optionally having counter

ions including zinc, sodium, calcium and ammonium; insulin-like growth factor; heparin;

calcitonin; erythropoietin; atrial naturetic factor; antigens; monoclonal antibodies;

somatostatin; protease inhibitors; adrenocorticotropin, gonadotropin releasing hormone;

oxytocin; leutinizing-hormone-releasing-hormone; follicle stimulating hormone;

glucocerebrosidase; thrombopoietin; filgrastim; prostaglandins; cyclosporin; vasopressin;

cromolyn sodium; vancomycin; desferoxamine; bisphosphonates; parathyroid hormone;

anti-migraine agents; glucagon-like peptide 1 (GLP-I); antimicrobials; vitamins; analogs, fragments, mimetics or polyethylene glycol (PEG)-modified derivatives of these

compounds; or any combination thereof.

45. The pharmaceutical formulation of any of claims 12-38, wherein the active

agent is insulin.

46. The pharmaceutical formulation of any of claims 12-38, wherein the active

agent is heparin.

47. The pharmaceutical formulation of any of claims 12-38, wherein the active

agent is unfractionated heparin.

48. The pharmaceutical formulation of any of claims 12-38, wherein the active

agent is low molecular weight heparin.

49. The pharmaceutical formulation of any of claims 12-48, wherein the particles

have an enteric coating.

50. A solid dosage unit form comprising the pharmaceutical formulation of any

of claims 12-49.

51. The solid dosage unit form of claim 50, further comprising a disintegrant.

52. The solid dosage unit form of claim 51, wherein the disintegrant is a super

disintegrant.

53. The solid dosage unit form of claim 52, wherein the super disintegrant is^'

sodium starch glycolate or croscarmellose sodium.

54. The solid dosage unit form of claim 52, wherein the super disintegrant is an

extra particle super disintegrant.

55. The solid dosage unit form of any of claims 50-54, wherein the solid dosage

unit form is in the form of a tablet.

56. The solid dosage unit form of any of claims 50-54, wherein the solid dosage

unit form is in the form of a capsule.

57. A method of treating diabetes in a mammal in need thereof, comprising

administering to the animal a therapeutic effective amount of a pharmaceutical formulation

of any of claims 12-49.

58. The method of claim 57, wherein the delivery agent compound is 4-[(2-

hydroxy-4-chloro-benzoyl)-amino]butanoic acid or a pharmaceutically acceptable salt

thereof.

59. A method of treating impaired glucose tolerance, early stage diabetes, or late

stage diabetes or achieving glucose homeostasis in humans, comprising administering a

therapeutic effective amount of a pharmaceutical formulation of claim 12-49.

60. The method of claim 59, wherein the method comprises administering the

pharmaceutical formulation on a chronic basis.

61. A method of treating a human diabetic patient comprising orally

administering to the human diabetic patient on a chronic basis a therapeutic effective

amount of a pharmaceutical formulation of any of claims 12-49.

62. A method of treating diabetes and reducing the incidence of systemic

hyperinsulinemia associated with chronic dosing of insulin in a patient in need thereof

comprising orally administering on a chronic basis to the patient a therapeutic effective

amount of a pharmaceutical formulation of any of claims 12-49.

63., A solid dosage form comprising

(a) a therapeutically effective amount of insulin; and

(b) a delivery agent;

wherein the solid dosage form has a disintegration time of at least 60 seconds when

administered orally.

64. The solid dosage form of claim 63, further comprising an enteric coating.

65. The solid dosage form of claim 63, wherein the solid dosage form is a

surface eroding formulation.

66. The solid dosage form of any of claims 63-65, further comprising enzyme

inhibiting agents.

67. The solid dosage form of any of claims 63-66, wherein the solid dosage form

comprises particles having a median particle size of less than 999 micrometers.

68. A solid dosage form comprising

(a) a therapeutically effective amount of insulin; and

(b) a delivery agent; wherein the solid dosage form does not substantially disintegrate or dissolve in the stomach

but will disintegrate or dissolve in the small intestine.

69. The solid dosage form of claim 68, further comprising an enteric coating.

70. The solid dosage form of claim 68, wherein the solid dosage form is a

surface eroding formulation.

71. The solid dosage form of any of claims 68-70, further comprising enzyme

inhibiting agents.

72. The solid dosage form of any of claims 68-71, wherein the solid dosage form

comprises particles having a median particle size of less than 999 micrometers.

73. A method of treating diabetes in a mammal in need thereof, comprising

administering to the animal a therapeutic effective amount of a solid dosage form

comprising the solid dosage form of any of claims 63-72.

74. A method of treating impaired glucose tolerance, early stage diabetes, or late

stage diabetes or achieving glucose homeostasis in humans, comprising administering a

therapeutic effective amount of the solid dosage form of any of claims 63-72.

75. The method of claim 74, wherein the method comprises administering the

pharmaceutical composition on a chronic basis.

76. A method of treating a human diabetic patient comprising orally

administering to the human diabetic patient on a chronic basis a therapeutic effective

amount of a solid dosage form of any of claims 63-72.

77. A method of treating diabetes and reducing the instance of systemic

hyperinsulinemia associated with chronic dosing of insulin in a patient in need thereof

comprising orally administering on a chronic basis to the patient a therapeutic effective

amount of a solid dosage form of any of claims 63-72.