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Pharm304 > Delivery of proteins and peptides > Flashcards

Delivery of proteins and peptides Flashcards

1
Q

What is the rationale behind studying protein/peptide drug delivery?

A
  • protein and pptides account for around $138.3 billion USD in profit, but their method of administration is limited to the parenteral route which can often be inconvenient for the patient.
  • Safer, painless and more convenient method of drug delivery is desired.
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2
Q

What are polypeptides made out of?

A

covalently linked amino acids

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3
Q

What is the difference between proteins and peptides?

A

Polypeptides with 40 amino acids are referred to as proteins

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4
Q

What is a function of a protein determined by?

A

its non covalent structure

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5
Q

What is an amino acid?

A

in chemistry terms, it is a molecule which contains both amino and carboxyl functional groups

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6
Q

What are the main classes of amino acids?

A 
aliphatic
aromatic
charged
polar
sulfonated
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7
Q

What are aliphatic amino acids?

A
  • protein side chain contains only C or H atoms
  • methioine is also part of this category as although its side chain contains a sulfur atom it is largely non-reactive
  • e.g. valine, leucine, alanine, proline, methionine
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8
Q

What are aromatic amino acids?

A

amino acids which contain an aromatic ring as part of their side chain

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9
Q

What are charged amino acids?

A

Amino acids which contain a side chain which is charged.

  • amino acids that are usually negative (deprotonated) at physiological pH include glutamate and aspartate
  • amino acids which are usually positive (protonated) at physiological pH include arginine and lysine
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10
Q

What are polar amino acids?

A

Amino acids with a side chain that is also polar. This includes charged amino acids, but they do not always have to be charged

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11
Q

What is the difference between essential and non essential amino acids?

A

Essential amino acids are made by the body, (isoleucine, leucine, lysine etc.)

non essential are obtained from the diet
(alanine, asparagine, aspartate)

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12
Q

What are examples of protein pharmacueticals?

A

insulin
interferon b
interferon g

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13
Q

What are examples of marketed protein pharmaceuticals?

A

actimmune (interferon G)
Betaseron (interferon b)
humulin,
novolin

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14
Q

What are protein pharmaceuticas?

A

There are more than 100 FDA approved protein drugs of which, 80% are recombinant proteins.
-sales are currently around $50billion/year- increased to over $70billion now

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15
Q

What are the classes of protein pharmaceuticals?

A
  • vaccines (peptides, parts of proteins, killed bacteria)
  • peptides (oxytocin, pitocin)
  • blood products (factor X, Factor VIII, gamma globulin, serum albumin
  • Recombinant therapeutic proteins (herceptin, humulin, alferon)
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16
Q

What features of proteins make it challenging for drug delivery?

A
  • proteins are large & unstable molecules
  • protein structure is held together by weak non-covalent forces
  • easily destroyed by relatively mild storage conditions
  • easily destroyed and eliminated by the body
  • hard to produce in large quantities
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17
Q

What are the in vivo probelms with proteins?

A
  • elimination by B cells and T cells
  • proteolysis by end/exo peptidases
  • small proteins (<30kD) filtered out by kidneys very quickly
  • unwanted allergic reactions or even toxicity may develop
  • losses due to insolubility/ adsorption
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18
Q

What are the in vitro problems with proteins?

A
  • denaturation
  • aggregation
  • precipitation
  • adsorption
  • deamidation
  • oxidation
  • disulfide exchange
  • proteolysis
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19
Q

What is denaturation?

A

-3D structure disrupted into linear form due to change in pH or temperature resulting in loss of function

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20
Q

What is aggregation?

A

-two amino acids combine

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21
Q

What is precipitation?

A

the protein failures to dissolve and forms a solid

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22
Q

What is adsorption?

A

the protein sticks onto the surface of a container

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23
Q

What is deamidation?

A

the amine functional group of the protein is cleaved

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24
Q

What is oxidation?

A

the protein side group is oxidised

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25
Q

What is disulfide exchange?

A

the cys groups from a bridge and distort the protein structure resulting in loss of function

26
Q

What is proteolysis?

A

The break down of proteins into polypeptides or amino acids.

27
Q

What is Fragmentation?

A

amino acids are broken up into fragments

28
Q

What is Isomerisation?

A

An L form amino acid becomes a D form amino acid

29
Q

What is Oligomerisation?

A

Where two or more polypeptide chains combine

30
Q

What is cross linking?

A

When an amine from one amino acid links with the carboxyl group of another amino acid

31
Q

What are the main ways to deal in vitro problems with proteins?

A

storage
formulation
delivery

32
Q

How can storage be implemented to reduce in vitro protein problems

A
  • refrigerate protein
  • Change the way protein is packed
  • incorporate additives
  • use freeze drying process
33
Q

How does refrigeration reduce protein problems?

A

low temperature reduces :

  • microbial growth and metabolism
  • thermal/spontaneous denaturation
  • adsorption

-freezing is best for long term storage, but the freeze thaw process can denature process

34
Q

How much does the freeze-thaw cycle dentaure proteins?

A

generally:

after 1 cycle, there is about 103% recovery
after 2 cycles, only 94 is recovered
after 3 cycles, 92 % is recovered

35
Q

How does packaging help to reduce protein problems?

A

-choice of packaging materials is important to avoid chemical reaction between protein and packaging and adsorption etc.

  • smooth glass walls best to reduce adsorption/precipitation
  • Dark, opaque walls reduce oxidation
  • air-tight containers or argon atmosphere reduces air oxidation

-polystyrene or containers with silanyl/plasticizer coatings should be avoided as proteins can adsorb and react with the surface

36
Q

How can storage additives help to reduce protein problems?

A

We can add/use:

  • stabilising salts or ions (such as Sn2+ for insulin) can be added
  • polyols (glycerol and/or polyehthylene glycol to solubilise the proteins and reduce precipitation
  • sugars or dextran to displace water or reduce microbe growth
  • surfactants to reduce adsorption and aggregation
37
Q

What is freeze drying and how does it help with storage to reduce protein problems?

A
  • Freeze drying is the only cost effective means to prepare solid, chemically active proteins
  • best for long term storage
  • removes a considerable amount of water from the protein lattice as much as possible, that some proteins are actually deactivated.
38
Q

What is the basic freeze drying process?

A
  • the liquid sample is frozen in the container
  • placed under a strong vacuum
  • solvent is sublimated which leaves only the solid or nonvolatile compounds this results in a change from the frozen to vapour state of the API without needing to first convert to liquid.
39
Q

What is the advantage of freeze drying?

A

it reduces moisture content to <0.1%

40
Q

What are the three phases of the freeze drying process?

A
  1. freezing of solution at -35 to -40 degrees
  2. sublimation phase with temperature around -35 degrees and very low pressure to remove the frozen water
  3. secondary drying stage to remove most residual water. The pressure remains low but temperature can rise up to 20 degrees without collapse of the porous cake.
41
Q

What are the formulation strategies to reduce protein problems?

A
  • PEGylation
  • proteinylation
  • microsphere/nanosphere encapsulation
42
Q

What is PEGylation?

A

the addition of PEGs onto the protein structure

43
Q

What is a PEG?

A

poly ethylene glycols.

  • these are polymers of ethylene oxide
  • non toxic, hydrophillic, FDA approved and uncharged polmer.
  • Those with low MW (<600) appear as clear liquids above 24 degrees
44
Q

What are the advantages of formulating proteins with PEGs?

A
  • increases in vivo half life (4-400x)
  • decreases immunogenecity
  • increases protease resistance
  • increases solubility and stability
45
Q

What is immunogenicity?

A

-the ability of an antigen to elicit the immune response

46
Q

What is lactoferrin?

A

80kDa sized member of transferrin

  • family of iron binding glycoproteins first discovered in 1939 as red protein from bovine milk.
  • later discovered in human milk also (1960)
47
Q

What was the effect of synthesising mono-PEGylated bovine lactoferrin with branced kDA PEG?

A
  • higher resistance to pepsin proteolysis in the mature rat
  • proteolytic half life prolonged by 2-fold due to steric hindrance and increased molecular mass after PEGylation.
  • absorption increased 10 fold
  • PEGylated bLF may be adsorbed by intestinal epithelium via 2 possible pathways
48
Q

What are the two possible pathways PEGylated bLF adsorption by the intestinal epithelium?

A
  1. specific receptor mediated transcytosis

2. non selective transcytosis

49
Q

What is proteinylation?

A

attachment of additional or secondary (non immunogenic) proteins for in vivo protection

50
Q

What is the effect of proteinylation?

A

-increases in vivo half life (10x)
cross links with serum albumin
cross links/connects by protein engineering with antibody fragments

51
Q

What is encapsulation?

A

-involves encapsulating protein or peptide drugs in small porous particles for protection from environmental degradation.

52
Q

What is the effect of encapsulation?

A
  • sustained release and prolonged plasma drug concentration
  • increased half life
  • increased permeability and drug bioavailability
  • enhancement of drug targeting and reduction of drug side effect)
53
Q

What are the two types of micro and nanospheres for encapsulation?

A
  1. non biodegradeable e.g. polyethylene, nylon, rayon, polester, PVC
  2. biodegradeable e.g. PLGA or ply(lactic-co-glycolic acid); PLA, gelatin, chitosan
54
Q

What are some methods of nanoparticle preparation?

A
  • interfacial polymerisation

- solvent evaporation

55
Q

How can different routes of delivery help to reduce protein problems?

A

There are many routes of delivery that can be used to avoid degradation of the proteins by the environment

  • parenteral
  • oral
  • nasal
  • patch/transdermal
  • other routes (pulmonary, rectal/vagina, ocular)
56
Q

What are the different types of parenteral delivery of proteins?

A
  • intravaneous
  • intramuscular
  • subcutaneous
  • intradermal
57
Q

What is the advantage with parenteral delivery of proteins?

A
  • currently the route of delivery for 95% of proteins
  • allows rapid and complete absorption
  • allows smaller dose size
  • avoids FPM
58
Q

What are the disadvantages with parenteral delivery of proteins?

A
  • overdosing, necrosis problems
  • local tissue reactions/hypersensitivity
  • inherent limitations of the route.
59
Q

What are the different excipients added into parenteral drug delivery of proteins?

A
  • solubility enhancers like amino acids and detergents
  • antiadsorbents/aggregation blockers like albumin and detergents
  • buffers like phosphate and citrate
  • preservatives like phenol and benzyl alcohol
  • antioxidants like ascorbic acid and cysteine
  • cytoproctectantss like sugars
  • osmotic agents like sugars and NaCl
60
Q

What is the purpose of cytoproctetants?

A

-stabilisation of the protein in dry form

61
Q

Why is there so much interest in protein peptide drugs?

A

-most ‘cures’ for difficult diseases like alzheimers, cancer, auto immun etc will be found through protein drugs

  • newer methods of delivery appearing
  • oral delivery can be very promising if achieved

Pharm304 (21 decks)

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