International Journal of Pharmaceutics, 75 (1991) 97-115
`<ti 1991 Elsevier Science Publishers B.V. All rights reserved 0378-.H73/91/S03.50
`ADONIS 037851739100307L
`
`97
`
`IJP 02512
`
`Invited Reviews
`Peptide and protein drugs:
`I. Therapeutic applications, absorption
`and parenteral administration
`
`X.H. Zhou and A. Li Wan Po
`Drug Delivery Research Group, The School of Pharmacy, The Queen's University of Belfast, 97 Lisburn Road, Belfast 81'9 7BL (U.K)
`(Received 20 February 1991)
`(Modified version received 4 May 1991)
`(Accepted 10 May 1991)
`
`Key words: Peptide delivery; Protein delivery; Stability; Bioavailability; Absorption barrier;
`Proteolytic activity; Absorption enhancer; Proteinase inhibitor; Liposome
`
`Summary
`
`In th is first part of a two-part review of peptide and protein drugs, the pertinem terminology is introduced and the therapeutic
`applications of those drugs summarised. Their abSOfPtion and the methodology commonly used for study on it are discussed.
`Approaches to optimising delivery of the peptide and protein drugs are highlighted.
`
`Introduction
`
`With the recent advances in recombinant DNA
`technology, the commercial production of pro(cid:173)
`teins and peptides for pharmaceutical purpose is
`now routine. The list of available. therapeutic
`agents produced by this technology is expanding
`rapidly to include interferon, macrophage activa(cid:173)
`tion factors, tissue plasminogen activator, ncu(cid:173)
`ropeptides and experimental agents that may have
`potential in cardiovascular disease, inflammation,
`contraception and so on. Unfortunately, protein
`and peptide drugs possess some chemical and
`
`Correspondence: A. Li Wan Po, Drug Delivery Research
`Group, The School of Pharmacy, The Queen's University of
`Belfast, 97 Lisburn Road. Belfast BT9 7BL U.K.
`
`physical properties, including molecular size, sus(cid:173)
`ceptibility to proteolytic breakdown, rapid plasma
`clearance,
`immunogenicity and denaturation,
`which make them unsuitable for delivery using
`the normal absorption routes and in particular,
`the oral route. In part one of this review protein
`and peptide drugs are considered with particular
`emphasis on their pharmacological profiles, po(cid:173)
`tential routes of delivery and their associated
`problems.
`Recent major reviews on the subject include
`the general article by Gardner (1984) on the
`intestinal absorption of intact peptides and pro(cid:173)
`teins and that by Humphrey and Ringrose (1986)
`on the absorption, metabolism and excretion of
`peptide and related drugs. In a further review,
`Lee (1988) discussed enzymic barriers to peptide
`and protein absorption. Banga and Chien (1988)
`
`FRESENIUS EXHIBIT 1053
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`

`

`98
`
`broadened the scope and considered systemic
`delivery of those agents in general.
`
`Enzymes
`
`Terminology
`
`Peptide or protein drugs are derived from
`amino acids by peptide bond linkages. Proteins
`are large peptides. Peptides containing less than
`eight amino acid residues are called small pep(cid:173)
`tides. Peptide drugs
`in
`this group
`include
`enalapril, lisinopril and thyroid releasing hor(cid:173)
`mone analogues. The term polypeptide drugs
`refers to peptide drugs with eight or more amino
`acid residues and includes cyclosporin, leuproline
`and luliberin. Polypeptide drugs containing from
`about 50 to as many as 2500 amino acid residues
`are named protein drugs. These include insulin,
`growth hormone and interferons. Some protein
`drugs, such as insulin or lgO containing two or
`more polypeptide chains, are called oligomeric
`proteins and their component chains are termed
`subunits or protomers.
`
`Some exogenous enzymes have been used as
`enzyme replacement therapy in the treatment of
`enzyme deficiency diseases such as lysosomal
`storage and mannosidosis (Table 1). Because en(cid:173)
`zyme deficiency in humans is usually genetic in
`origin, enzyme replacement is often the only
`available therapy. ~ome exogenous enzymes have
`also been utilized in the treatment of diseases
`other than inborn enzyme deficiency. Good ex(cid:173)
`amples include t-PA (tissue plasminogen activa(cid:173)
`tors). urokinase and streptokinase. These en(cid:173)
`zymes activate circulating plasminogen and fibrin
`clot-associated plasminogen equally well and, be(cid:173)
`cause of this, they have been marketed in the
`U.K. and U.S.A. (Robinson and Sobel, 1986;
`British National Formulary, 1989). Thrombin-like
`enzymes of snake venoms have also been devel(cid:173)
`oped for dissolving blood clots through enhanced
`release of fibrinopeptides from
`fibrinogen
`(Komalik, 1985).
`
`Honnones
`
`Therapeutic Uses of Peptide and Protein Drugs
`
`Peptide and protein drugs can be conveniently
`classified according to their activity profiles as
`follows:
`
`Hormones represent the largest class of pro(cid:173)
`tein or peptide drugs used in medical therapy. All
`hormones have ' target cells' on which they act
`and these may be located in a specific organ or be
`more widely distributed in the body. Some hor-
`
`TABLE 1
`
`Therapeutic application of some enzymes
`
`Enzymes
`
`Adenosine deaminase
`Dextranase
`{3-Fructofuranosidase
`a-Mannosidase
`
`L-Asparaginase
`/3-Glucosidase
`Tissue plasminogen activators
`Urolcinase
`Streptolcinase
`Thrombin-like enzymes of snake venoms
`
`Therapeutic application
`
`Reference
`
`Enzyme deficiency
`Lysosomal storage
`Stora&e disease
`Mannosidosis
`
`Cancer
`Adult Gaucher's disease
`Thrombosis
`Thrombosis
`Thrombosis
`Thrombosis
`
`Hershfield et al. (1987)
`Colley and Ryman (1974)
`Gregoriadis and Ryman (1972b)
`Patel and Ryman (1974)
`Fishman and Citri (1975)
`Abuchowski et al. (1984)
`Braidman and Gregoriadis (1976)
`Robinson and Sobel (1986)
`Robinson and Sobel (1986)
`Robinson and Sobel (1986)
`Kornalik (1985)
`
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`
`

`

`mones like luliberin Outeinizing hormone releas(cid:173)
`ing hormone, LHRH) function solely to bring
`about the release of other hormones from differ(cid:173)
`ent endocrine glands. It is also well known that
`many hormones act by means of a second mes(cid:173)
`senger and quite often this is cyclic AMP (cAMP)
`which is formed from ATP. On reaching its re(cid:173)
`ceptor in the cell membrane, the hormone causes
`the release of cAMP, which is the actual regula·
`tor of the metabolic process. In this way, the
`physioloi?ical effect of one molecule of the hor(cid:173)
`mone is amplified many times (Wills, 1985). Be(cid:173)
`cause hormones are very specific and a tiny
`amount can produce large pharmacological ef •
`fects, they. are ideal for biotechnological develop(cid:173)
`ment which is more suitable for relatively small
`outputs. Perhaps the best known hormone drug is
`insulin which has been used as an endocrinother(cid:173)
`apeutic agent since the 1920's (Banting and Best,
`1922).
`
`Enzyme inhibitors
`
`Enzyme inhibitors have been used as drugs for
`a long time. These include proteins such as apro(cid:173)
`tinin, and peptide drugs such as enalapril and
`lisinopril. Captopril is an inhibitor of angiotensin
`converting enzyme (ACE), which catalyses in vivo
`generation of angiotensin II from the decapep(cid:173)
`tide, angiotensin I, to constrict arterioles and
`increase cardiac output, leading to hypertension
`in man. Captopril is now a widely used antihyper(cid:173)
`tensive agent (Romankiewicz et al., 1983).
`Enalapril and lisinopril are subsequent develop(cid:173)
`ments which are also becoming widely adopted
`for the treatment of hypertension and congestive
`heart failure (Todd and Heel, 1986; Lancaster
`and Todd, 1988).
`
`Antimicrobial agents
`
`A number of antimicrobial agents are peptide
`drugs, for example,
`the penicillins, cephalo•
`sporins, polymyxin B sulphate, actinomycin and
`bleomycin. Structurally, these drugs are small
`peptides, mostly containing a non-peptide moiety.
`All of these antimicrobial drugs are microbial
`metabolites.
`
`99
`
`lmmunomodulating peptides and proteins
`Endogenous immunomodulating agents
`These agents are now produced by molecular
`genetk approad1es. Well-known examples are the
`interferons (IFNs) which are families of inducible
`secretory proteins produced by eukaryotic cells in
`response to viral and other stimuli. Interferons
`are not directly antiviral but they act prophylacti(cid:173)
`cally by inducing antiviral proteins. These protect
`cells from viral infection by inhibiting virus-di(cid:173)
`rected translation and transcription (Moore and
`Dawson, 1989). Another example is interleukin-2
`(IL-2) which exerts its biological effect through
`cell surface receptors on activated T and B cells
`and on NK cells (natural killer cell). Interleukin-2
`has been administered clinically in attempts to
`restore immunocompetence in patients suffering
`from the acquired immunodeficiency syndrome
`(AIDS), and to improve the immunocompetence
`of cancer patients (Dawson and Moore, 1989).
`
`Exogenous immunomodulating agents
`Some exogenous · immunomodulating agents
`are also used to promote immunocompetence in
`man. For example, cyclosporin (CS-4), a cyclic
`undecapeptide which is isolated from Tolypocla(cid:173)
`dium inf/atum Garns, is widely used as an im(cid:173)
`munosuppressive (Caine et al., 1978; Cantarovich
`et al., 1987; Mehta et al., 1988; Borel, 1989),
`whereas muramyl dipeptide has been used as an
`immunological adjuvant (Kreuger et al., 1984;
`Bomford, 1989).
`
`Vaccines
`Va<.:<.:ines derived from the infective microor•
`ganisms are introduced into the mammalian body
`to induce antibody formation against the path(cid:173)
`ogens. Well-known exa_mples include measles vac•
`cine and polio vaccine. It is anticipated that an
`increasing number of such vaccines will be
`biotechnologically produced, to give more specific
`and pronounced antigenic responses.
`
`Absorption of Peptide and Protein Drugs
`Analytical problems
`Several methods have been employed for
`studying the absorption of peptide and protein
`
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`

`

`100
`
`drugs. However, high molecular weight proteins
`and polypeptides present some unique difficul(cid:173)
`ties. Techniques such as gel filtration and ion-ex(cid:173)
`change HPLC usually have to be used. Even so, it
`is still very difficult to assay them in the presence
`of body fluids such as blood and urine. In such
`cases, radioassays or radioimmunoassays are of(cid:173)
`ten the most appropriate and hence, these tech(cid:173)
`niques have been widely used in the measure(cid:173)
`ment of the bioavailability of peptide or protein
`drugs. However, radioassays may be non-specific,
`and many chemical assay procedures may by
`themselves influence the conformation of protein
`
`drugs, thereby causing the loss of their biological
`activities. The entity being chemically assayed
`may not be the biologically active moiety and in
`such cases, in vitro or in vivo bioassays are often
`used during absorption studies. For protein/
`peptide hormones, the measurement of pharma(cid:173)
`cological responses may be the assay method of
`choice. For enzymes or enzyme inhibitors, spe(cid:173)
`cific enzyme reactions may be the best analytical
`method. The bioavailability of immunomodulat(cid:173)
`ing and antimicrobial agents may be evaluated
`using some specific animal models and indicator
`microorganisms. For example, the prophylactic
`
`TABLE 2
`
`Instability of protein and peptide drugs
`
`Effect factor
`
`Physical instability
`Aggregation
`
`Precipitation
`
`Insulin
`
`Chemical instability
`f3 Elimination
`
`Deamidation
`
`Disulphide exchange
`
`Racemization
`
`Oxidation
`
`Lysozyme
`Phosvitin
`Bovine growth hormone
`Human growth hormone
`
`Insulin
`
`r-Immunoglobulin
`Epidermal growth factor
`Prolactin
`Gastrin releasing peptide
`ACTH
`
`Lysozyme
`Ribonuclease A
`
`ACTH
`
`Corticotropin
`a-, /3-Melanotropins
`Parathyroid hormone
`G~strin
`Calcitonin
`Corticotropin releasing factor
`
`Protein or peptide drugs
`
`Reference
`
`Interferon-y
`
`Bovine growth hormone
`
`Hsu and Arakawa (1985)
`Arakawa et al. (1987)
`Brems et al. (1986)
`Brems et al. (1988)
`Brennan et al. (1985)
`Lougheed et al. (1980)
`
`Nashef et al. (1977)
`Sen et al. (1977)
`Lewis and Cheever ( 1965)
`Lewis et al. (1970)
`Becke r et al. (1988)
`Berson and Yalow (1966)
`Fisher and Porter (I 981)
`Minta and Painter (1972)
`Diaugustine et al. (1987)
`Graf et al. (1970)
`McDonald et al. (1983)
`Graf et al. (1971)
`Bhatt et al. (1990)
`
`Volkin and Klibanov (1987)
`Zale and Klibanov (1986)
`
`Geiger and Clarke (1987)
`Meinwald et al. (1986)
`
`Dedman ct al. (1961)
`Dixon (I 956)
`Tashjian ct al. (1964)
`Morley et al. (1965)
`Riniker et al. (1968)
`Vale et al. (1981)
`
`FRESENIUS EXHIBIT 1053
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`
`

`

`TABLE3
`Liposomcs as peptide and protein carrier
`
`JOI
`
`Liposome
`composition
`
`Phosphatidyl-
`choline : cholesterol
`7:2
`
`Phosphatidyl-
`choline : cholesterol
`7:7
`
`Phosp hatidyl-
`choline : cholesterol :
`phosphatidic acid
`7:2: 1
`
`Dimyristoyl
`phosphatidyl-
`choline : choles-
`terol: dicetyl
`phosphate
`1 : 0.75 : 0.1 1
`
`Phosphatidyli•
`nositol
`
`Phosphatidyl·
`choline : choles-
`terol: dicetyl
`phospha te
`10: 2 : 1
`
`Phosphatidyl-
`choline : choles-
`terol: dicetyl
`phosp hate
`3: 9:1
`
`Phosphatidyl-
`choline : pbos-
`phatidylserine
`7:3
`
`Phosphatidyl-
`choline : choles-
`terol : dicetyl
`phosphate
`7: I :2
`
`Phosphatidyl-
`choline : choles-
`terol : phospha-
`tidic acid
`20: 1.5 :0.2
`
`Phosphatidyl-
`choline : choles-
`terol : phospha-
`tidic acid
`7 : I :2
`
`Peptide or
`protein
`
`semipurified
`glucocerebroside
`t3-glucosidase
`
`highly purified
`glucocerebroside
`t3-glucosidase
`
`bacterial
`amyloglucosidase
`
`cholera toxin
`human malaria
`sporozoite antigen
`
`insulin
`
`insulin
`
`Route
`
`i.v.
`
`i.v.
`
`i.v.
`
`i.v.
`
`i.v.
`
`o ral
`
`Animal
`model
`
`man
`
`Reference
`
`Belchetz et al. Om)
`
`man
`
`Gregoriadis e t al. (1982)
`
`man
`
`TyrelJ e l al. (1976)
`
`rabbit
`
`Alving et al. (1986)
`
`mouse
`ra t
`
`rat
`
`Dapergolas and Gregoriadis
`(1976)
`
`Patel and Ryman (1976)
`
`insulin
`
`oral
`
`rat
`
`Tanaka et al. (1975)
`
`muramyl peptide
`
`i.v.
`
`mouse
`guinea-pig
`
`Fidler et al. (I 985)
`
`lysozyme
`
`adenovirus type
`5 hexon protein
`
`lysozyme
`
`Sessa and Weissmann
`(1970)
`
`i.v.
`
`mouse
`
`Six et al. (1988)
`
`Sessa and Weissmann
`(1970)
`
`f,.""'inued)
`
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`
`

`

`102
`
`TABLE 3 (continued)
`
`Llposome
`com Position
`Microcap,sule,;
`
`Phosphatidyl-
`choline : choles-
`terol: dicetyl
`phosphate
`7: 2 :1
`
`Phosphatidyl-
`choline : choles-
`terol: phospba-
`tic1ic acid
`7:2:1
`
`Phosphatidyl-
`choline : choles-
`terol : phospha•
`tidic acid
`7:2:1
`
`Phosphatidyl-
`choline : choles-
`terol: phospha-
`tidic acid
`7 : 2 : 1
`
`Phosp.hatidyl-
`cbuline ; chutes-
`terol ; phospha·
`tidic acid
`7 :2:t
`
`DipalmitoYI·
`phosphotidyl•
`choline
`
`Phosphatidyl-
`choline : choles-
`lerol: phospha•
`tidic aci.d
`7:2:1
`
`Phosphatidyl-
`choline : choles-
`terol: Phospha-
`tidicacid
`7 :2 :1
`
`Phosphalidyli•
`nositol
`
`Phosphatidyl-
`choline : choles-
`terol
`7:2
`
`Phosphatidyl-
`choline: choles-
`terol : phospha-
`tidic acid
`7:2:1
`
`Peptide or
`protein
`
`cab.lase
`
`amyloglucosidase
`
`Route
`
`i.s.
`
`i .v.
`
`· Animal
`model
`
`R.\0\1:SC.
`
`rat
`
`Reference
`
`-·~--•· ···-
`
`Cha~ and Poman,;ky
`(1968)
`
`Gregoriadis and Ryman
`(1972a)
`
`yeast invertase
`
`i.v.
`
`rat
`
`Gregoriadis and Rvman
`(1972b)
`
`neuraminidase
`
`i.v.
`
`rat
`
`G regoriadis et al. (1974a)
`
`dextran.ase
`
`i.v.
`
`rat
`
`Colley and Ryman (1974)
`
`a-mannosidase
`
`i.v.
`
`rat
`
`Patel and Ryman (1974)
`
`a -amylase
`
`horseradish
`peroxidase
`
`amoeba
`
`Batzri and Korn (1975)
`
`rat
`
`Wisse and Gregoriadis
`(1975)
`
`asparaginase
`
`i.v.
`
`mouse
`
`Neerunjun and Gregoriadis
`(1976)
`
`glucose oxidase
`
`albumin
`
`albumi.n
`
`i.v.
`
`i.v.
`
`i.v.
`
`mouse
`
`rat
`
`Dapergolas ct al.
`(1976)
`
`Gregoriadis and Neerunjun
`(1974)
`
`man
`
`Gregoriadis et al. (1974b)
`
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`
`

`

`103
`
`Peptide or
`protein
`albumin
`
`Route
`
`i.v.
`
`Animal
`model
`mouse
`
`Reference
`
`Heath et al. (1976)
`
`diphtheria
`toxoid
`
`i.v.
`
`mouse
`
`Oreguriadis and Alisun
`(1974)
`
`fetuin
`
`J.v.
`
`rat
`
`Oregorladls a nd
`Neerunjun (1975)
`
`anti-a-
`glucosidase
`
`monoclonal
`anti-Thy 1 IgG 1
`
`i.v.
`
`rat
`
`De Barsy et al. (1975)
`
`i.v.
`
`mouse
`
`Debs et al. ( 1987)
`
`monoclonal
`an ti-Thyl lgG I
`
`i.v.
`
`mouse
`
`Wolff and Gregoriadis
`(1984)
`
`TRH
`
`superoxide
`dismutase
`
`i.v.
`
`intratracheal
`injection
`
`cat
`
`rat
`
`Kumashiro e t a l. (1986)
`
`Padmanabhan e t al.
`(1985)
`
`factor VIII
`
`oral
`
`man
`
`subunits of
`monoclona l IgM
`
`i.p.
`
`mouse
`
`Sakuragawa et al.
`(1985)
`
`Hashimoto et al.
`(1986)
`
`TABLE 3 (conrin.ued)
`
`Liposome
`composition
`Ph05phatidyl-
`choline : choles-
`te rol: dicetyl
`phosphate
`6: 6:2
`
`Pho,;phatidyl-
`choline : choles-
`terol : dicetyl
`phosphate
`7: 2: 1
`
`Phosphatldyl-
`choline: choles-
`terol : p hospha-
`tidic acid
`7:2: 1
`
`Phosphatidyl-
`choline : choles-
`terol : phospha-
`tidic acid
`4: 2: 1
`
`Phosphatidyl-
`choline : choles-
`terol : phosphati-
`dylethanolamine
`10 : 10:1
`
`Distearoylphos-
`phatidylcholine:
`(2-puridyldithio)-
`propionol-dipal-
`mitoylphospha-
`tidylcholine:
`cholesterol
`0.99: 0.01 : I
`
`• Liposomes
`
`Phosphatidyl-
`choline : phos-
`phatidylserine
`7: 3
`
`Phosphatidylcholine:
`phosphatidic acid
`15.3 : 0.1
`
`Dipalmitoyl-
`phosphatidyl-
`choline: cholesterol: m-ma-
`leimidobenzoyl-
`(dipalmitoyl-
`phosphatidyl)-
`ethanolamine
`25: 17.5: 2.5
`
`• The composition of liposomes was not mentioned in the n~ner.
`
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`

`104
`
`usefulness of intranasal IFN-/3 against rhinovirus
`infection was determined using healthy volun(cid:173)
`teers or animals (Higgins et al., 1986).
`Assay methods available for monitoring the
`absorption of small peptide drugs are freely avail(cid:173)
`able and routine methods include reverse-phase
`HPLC, TLC, and fluorescence techniques.
`
`Stability
`
`Irrespective of which dosage form is used, pep(cid:173)
`tide or protein decomposition may be a problem.
`Drug breakdown can take place both in the for(cid:173)
`mulation and when present in tissue fluids. First
`pass metabolism and enzymic breakdown are dis(cid:173)
`cussed in greater detail further on. Non-enzymic
`breakdown may be of two types: chemical and
`physical changes. Physical changes include aggre(cid:173)
`gation and precipitation and are usually induced
`by high concentrations of co-solvents which may
`be used in some formulations or by injudicious
`choice of ionic strengths. Loss of conformation
`not only leads to poor absorption but also to loss
`of activity. Chemical changes include ,8-elimina(cid:173)
`tion, deamidation, disulphide exchange, racem(cid:173)
`ization and oxidation. Examples of peptides and
`proteins which have been reported to be unstable
`are shown in Table 2 along with some of the
`reported mechanisms of breakdown.
`
`Parenteral routes of delivery
`
`For systemic delivery of peptide and protein
`drugs, parenteral administration is currently al(cid:173)
`most universally required in order to achieve
`consistent therapeutic activities. This is because
`of the drugs' susceptibility to breakdown by gas(cid:173)
`tric acid and the proteolytic enzymes in the gas(cid:173)
`trointestinal tract. In addition, peptides and pro(cid:173)
`teins are high-molecular-weight substances and
`thus do not easily cross the intestinal mucosa.
`Therefore, the oral bioavailabilities of most intact
`peptides and proteins are very low.
`Of the parenteral routes, only intravenous (i.v.)
`administration is usually efficient in delivering
`protein and peptide drugs to the syste.mic circula(cid:173)
`tion. For example, optimal blood levels of protein
`
`or peptide drugs, such as ')'-globulin (Buckley,
`1982), can be achieved by the intravenous route.
`Generally, intramuscular or subcutaneous injec(cid:173)
`tions are less efficient due to the absorption and
`diffusion barriers presented by the muscle mass
`and connective tissues under the skin. However,
`insulin can be efficiently administered by subcu(cid:173)
`taneous injections (Nora et al., 1964; Koivisto and
`Felig, 1978) although hydrolysis is still significant
`(Berger et al., 1979).
`While most peptide/ protein drugs can be effi(cid:173)
`ciently delivered to the systemic circulation by
`parenteral injections, poor disposition profiles
`lead to sub-optimal therapeutic benefits without
`high dosing frequencies. Such frequent injections,
`besides being unpleasant to the patient:s, alsu
`throm(cid:173)
`to usual complications such as
`lead
`bophlebitis and tissue necrosis.
`In attempts to improve the disposition profile
`and the efficiency of delivery of parenterally ad(cid:173)
`ministered peptides and protein drugs, many in(cid:173)
`vestigators have reported on liposomal systems.
`Examples of enzymes and monoclonal antibodies
`which have been formulated as liposomal systems
`for intravenous administration are shown in Table
`3. Also included are some liposomal systems in(cid:173)
`tended for oral administration. Despite the ex(cid:173)
`tensive evaluation of such systems and experi(cid:173)
`mental results (Gregoriadis, 1976; Goosen, 1987)
`indicating that insulin absorption is greatly en(cid:173)
`hanced in animals by liposomal encapsulation of
`the hormone, no insulin liposomal system is cur(cid:173)
`rently in commercial use.
`One biodegradable implant in current use in
`humans in goserelin acetate formulated in a
`biodegradable matrix of lactide-glycolide co-poly(cid:173)
`mer. Systems which are designed with an enzymi(cid:173)
`cally controlled feed-back mechanism have also
`been described. Fischcl-Ghodsian et al. (1988),
`for example, reported on an insulin system con(cid:173)
`sisting of insulin and glucose oxidase dispersed in
`an ethylene /vinyl acetate polymer matrix. In the
`presence of glucose oxidase, glucose is converted
`into gluconic acid. This acid lowers the pH and
`increases the solubility of entrapped insulin which
`is then released faster. Consequently, some feed (cid:173)
`back control between glucose and insulin is
`thereby established.
`
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`
`

`

`General Approaches to Optimizing Absorption
`ond Disposition
`
`To optimize the absorption of high-molecular(cid:173)
`weight protein and peptide drugs across absorp(cid:173)
`tion barriers, several approaches are available: (i)
`inhibition of their enzymic degradation; (ii) in(cid:173)
`creasing their permeability across the relevant
`membrane; and (iii) improving their resistance to
`breakdown by structural modification.
`
`Inhibitors of proteolytic enzymes
`
`Protease inhibitors have been known for sev(cid:173)
`eral years to increase the absorption of protein
`drugs (Laskowski ct al., 1958). Table 4 lists the
`different protease inhibitors which have been used
`in investigations of the delivery of peptide and
`protein drugs.
`Aprotinin, a bovine pancreatic kallikrein in(cid:173)
`hibitor, consists of a single-chain polypeptide
`containing 58 amino acid residues with a molecu(cid:173)
`lar weight of 6500 (Kassell ec al., 1965). le has
`been used
`to
`inhibit plasmin,
`trypsin, chy(cid:173)
`motrypsin and various
`intracellular proteases
`(Trautschold et al., 1967). It was demonstrated, in
`an early study, that when insulin and aprotinin
`
`105
`
`were injected together into a loop of the jejunum,
`a significant drop in blood glucose was observed.
`In contrast, no significant drop in blood glucose
`was found when the insulin was injected alone
`(Laskowski et al., 1958). Similar results have also
`been reported by several other workers (Berger
`et al., 1980; Fredenberg et al., 1981; William et
`al., 1983; Dandona et al., 1985; Linde and Gun(cid:173)
`narson, 1985; Owens e t al., 1988). However, some
`recent studies provided conflicting results, at least
`with respect to insulin and calcitonin absorption
`by nasal administration (Hanson et al., 1986;
`Aungst and Rogers, 1988). When the effects of
`laureth-9, sodium salicylate, Na 2 EDTA and
`aprotinin on insulin absorption via the rectal,
`nasal and buccal tissues were examined by Aungst
`and Rogers (1988), aprotinin was found to be
`ineffective, either alone or in combination with
`laureth-9. Ha nson and his co-workers (1986) ex(cid:173)
`amined the effects of several protease inhibitors,
`including bile salt, fatty acid derivative, aprotinin,
`kallikrein inhibitor, RG- 1, bestatin, fusidic acid,
`chemostatin, benzamidine, chymotrypsin
`in(cid:173)
`hibitor, trypsin inhibitor III-0 and leupeptin on
`intranasal delivery of calcitonin, and found that
`aprotinin in vitro did not inhibit proteolytic activ(cid:173)
`ity of nasal extracts. In vivo the inhibitor did not
`
`TABLE 4
`
`Inhibitors of pro1eoly1ic enzymes used in investigation of the delivery of peptide and protein drugs
`
`Compound
`
`Rome
`
`Aprotinin
`
`intesti nal
`
`Peptide
`studied
`insu lin
`
`RNase
`insu lin
`
`insulin
`RNase
`
`insulin
`
`s.c. a
`
`intestinal
`
`intesti nal
`
`Animal
`model
`rat
`
`rat
`man
`
`rat
`rat
`
`rat
`
`rat
`rat
`
`Reference
`
`Ziv and Kidron (1987),
`Laskowski et al. (1958)
`Ziv and Kidron (1987)
`Owens et al. (1~88),
`L1ndc and Gunnarsson (1985),
`Berger et al. (1 980)
`Ziv and Kictron (I 987)
`Ziv and Kidron (1987)
`
`Yokoo et al. (1988)
`
`Hussain et al. (1989)
`Hussain et a l. (1989)
`
`SOybean
`trypsin
`inhibitor
`FK-448 b
`(chymotrypsin
`inhibitor)
`Borole ucine <
`Borovaline c
`
`nasal
`nasal
`
`Leu-enkephal in
`Leu-enkephalin
`
`• Subcutaneous delivery.
`b 4-(4-lsopropylpiperazinocarbonyl)phenyl-1,2,3,4-tetrahydro-1-naphthoate methanesulpho nate.
`c a-Aminoboronic acid derivatives.
`
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`
`

`

`106
`
`enhance the serum calcium drop observed. These
`results are supported by the study -of Deurloo et
`al. (1989). The co-administration of sodium tau(cid:173)
`rodihydrofusidate with aprotinin also failed to
`increase significantly insulin bioavailability in rab(cid:173)
`bits via the nasal route. Clearly, further studies
`are required to define better the effects of apro(cid:173)
`tinin on the absorption of peptide and protein
`drugs.
`More recently, a-aminoboronic acid deriva(cid:173)
`tives, such as boroleucine, which are potent and
`reversible inhibitors of aminopeptidase, have been
`used to stabilize peptide drugs during their in(cid:173)
`tranasal absorption (Hussain et al., 1989). When
`these inhibitors were compared with other known
`peptidase inhibitors, bestatin [an inhibitor of
`leucine aminopeptidase, aminopeptidase B, and
`aminopeptidase N (Suda et al., 1976)) and
`puromycin [an inhibitor of aminopeptidase B and
`N but not leucine aminopeptidase (McDonald et
`al., 1964)], using leucine enkephalin as substrate
`in rat nasal perfusate, it was found that bestatin
`and puromycin were less effective than boro(cid:173)
`leucine, even at concentrations 100- and 1000-
`times higher, respectively.
`
`Absorption enhancers
`
`The use of absorption enhancers has been
`studied extensively, panicularly with respect to
`insulin absorption. These enhancers can be di(cid:173)
`vided into several groups as listed in Table 5.
`Despite extensive use, it is very difficult to
`make a judgement about the relative efficacy of
`these bioenhancers in promoting peptide or pro(cid:173)
`tein absorption because the results were obtained
`in different laboratories using different assay
`methods and different experimental conditions.
`However, it is clear that the bioavailability of
`most peptide and protein drugs administered by
`any non-parenteral route may be significantly en(cid:173)
`hanced by some of these compounds (see Tables
`in part II of this review).
`The value of a particular enhancer depends on
`the route of administration used. For example,
`the bioavailability of ocular insulin was found to
`be significantly enhanced by saponin, whereas
`enhancement by glycocholate, which was a poten-
`
`tially good enhancer for nasal and rectal peptide
`and protein drug absorption, was found to be
`only slight (Chiou and Chuang, 1989).
`The mechanisms of action of the peptide ab(cid:173)
`sorption enhancers are not clearly known, but
`several possibilities have been postulated. The
`first is increased solubility of the drugs brought
`about by the enhancers because proteins and
`peptides usually form aggregates in aqueous solu(cid:173)
`tions. In the presence of enhancers, dissociation
`takes place to form monomers which are better
`absorbed. A second mechanism is the protection
`of the peptide and protein drugs from potential
`proteolytic hydrolysis. Both bile salts (Hirai et al.,
`1981b; Hanson et al., 1986; Zhou and Li Wan Po,
`1991) and derivatives of fusidic acid (Deurloo et
`al., 1989) are known to inhibit proteolytic degra(cid:173)
`dation of the drugs by nasal homogenates. Thirdly,
`binding between peptide or protein and enhancer
`to produce a better-absorbed entity may be a
`possibility. Although the effects of absorption en(cid:173)
`hancers such as glycocholate (Hirai et al., 1981b)
`and sodium cholate (Zhou and Li Wan Po, 1991)
`on the absorption of insulin by nasal delivery are
`thought to be partly due to inhibition of protease ,
`recent work suggests that compared to aminopep(cid:173)
`tidase inhibitors such as bestatin and amastatin,
`cholate and its analogues are not very efficient
`(Hanson et al., 1986). Cholate and its analogues
`may also enhance the absorption of peptide and
`protein drugs by binding to insulin (Zhou and Li
`Wan Po, 1991). This would pre.vent the formation
`of enzyme-substrate complex to undergo the nec(cid:173)
`essary conformational change which aligns the
`catalytic site on the protease with the susceptible
`bond of the substrate. Cholate and its analogues
`may possibly also promote the absorption of pro(cid:173)
`teins by selectively denaturing the enzymes, al(cid:173)
`though this is unlikely as it is difficult to identify
`the basis for the necessary selectivity.
`
`Chemical modification
`
`Chemical modification is an important ap(cid:173)
`proach for enhancing the absorption of peptides
`and protein drugs, especially for peptides with
`fewer than ten amino acid residues. Chemical
`modification usually results in denaturation of
`
`FRESENIUS EXHIBIT 1053
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`
`

`

`107
`
`TABLE5
`
`Absorption enhancers for peptides and proteins
`
`Compound
`
`Fatty acid
`MCFC •
`Caprylate
`Caprate
`Laurate
`
`LCFC b
`Oleate in PAGB 0
`Linoleate in PAGB
`Linolenate in PAGB
`Olcic acid
`
`Bile salts
`Taurocholate
`Cholate
`Deoxycholate
`Glycocholate
`Chenodeoxycholate
`Demcycholate (aerosol)
`Glycocholate
`Cholate
`Dcoxycholatc
`Cholate
`
`Glycocholate
`
`Route
`
`Peptide
`
`Animal
`model
`
`Reference
`
`nasal
`nasal
`nasal
`
`rectal
`rectal
`rectal
`vaginal
`
`nasal
`nasal
`nasal
`nasal
`nasal
`nasal
`vaginal
`rectal
`rectal
`intestinal
`intestinal
`nasal
`rectal
`buccal
`sublingual
`
`insulin
`insulin
`insulin
`
`insulin
`insulin
`insulin
`leuprolide
`
`insulin
`insulin
`insulin
`insulin
`insulin
`insulin
`leuprolide
`insulin
`insulin
`insulin
`RNase
`insulin
`insulin
`insulin
`insulin
`
`rat
`rat
`rat
`
`rat
`rat
`rat
`rat
`
`rat
`rat
`rat
`rat
`rat
`man
`rat
`rat
`rat
`rat
`rat
`rat
`rat
`rat
`rat
`
`Mishima et al. (1987)
`
`Morimoto et al. (1983)
`
`Okada e l al. (1982)
`
`Hirai et al. (1981a)
`Moses et al. (1983)
`
`Mishima et al. (1987)
`
`Moses et al. (1984)
`Okada et al. (1982)
`Ziv et al. (1981)
`
`Ziv et al. ( 1987)
`
`Aungst et al. (1988)
`
`re.:tal
`
`rectal
`
`rectal
`
`rectal
`
`rectal
`
`Enamine derivatives of phenylelycine
`Ethylacetoacetate
`enamine of sodium
`D-glycine
`Ethylacetoacetate
`enamine of sodium
`o-alanine
`Eth.ylacetoacetate
`enamine of sodium
`o-leucine
`Ethylacetoacetate
`en amine of sodium
`o-isoleucine
`Ethylacetoacetate
`enamine of sodium
`o-phenylalanine
`Ethylacetoacetate
`crtamine of ~odium
`o-phcnylalan ine
`in ge latin
`Ethylacetoacetate
`enamine of sodium.
`o-phenylglycinate
`Ethylacetoaceta te
`enamine of sodium
`DL-phenylalanine
`
`rectal
`
`rectal
`
`rectal
`
`insulin
`
`rabbit
`
`Kim ct al. (1983)
`
`insulin
`
`rabbit
`
`insulin
`
`rabbit
`
`insulin
`
`rabbit
`
`insulin
`
`rabbit
`
`insulin
`
`dog
`
`insulin
`
`1,IC""'"'-·
`
`rabbit
`
`-k-"-;•
`
`Kamada et al. (1981)
`
`'IA';.., .. 1~ ....
`
`.... ,
`
`, 1,noA\
`
`wed)
`
`FRESENIUS EXHIBIT 1053
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`
`

`

`108
`
`TABLE 5 (continued)
`
`Compound
`
`Ro ute
`
`Peptide
`
`E1hylacetoacetate
`enamine of sodium
`o-phenylglycine
`
`rectal
`
`lysozyme
`
`Reference
`
`Animal
`model
`
`rabbit
`
`rectal
`
`lysozyme
`
`rabbit
`
`Miyake e1 al. (1964)
`
`rectal
`
`rectal
`
`nasal
`
`rectal
`
`rectal
`
`rectal
`
`lysozyme
`
`lysozyme
`
`insulin
`
`insulin
`
`calcitonin
`
`insulin
`
`rabbit
`
`rabbit
`
`rat
`
`Hirai et al. (1981a)
`
`rabbit
`
`Nishihata et al. (1983)
`
`rat
`
`dog
`
`Morimoto et al. (1985)
`
`Shichiri et al. (1978)
`
`Ester type
`Glycerine-I ,
`3-diacetoacetate
`1,2-lsopropyl idene-
`glyceryl-3-
`acetoacetate
`Ethylaceto-
`acetylglycolate
`Polyoxyethylene
`10-monolaurate
`Olyceryl esters of
`acetoacetic acid
`
`Ether type
`Polyoxyethylene
`9-lauryl ether