biotechnology, pharmaceuticals, use of biotechnology in pharmaceuticals

1. Pharmaceutical
Biotechnology
Advanced drug delivery
Dr Athmar Dhahir Habeeb
PhD in Industrial Pharmacy and pharmaceutical formulations

2.  Biotechnology is the use of microorganisms, plants,
animals or parts of them for the production of useful
compounds and Pharmaceutical biotechnology is
concerned as the biotechnological manufacturing of
pharmaceutical products.
 An insight into the nature of the traditional processes was
achieved in about 1870 when Pasteur illustrated that
chemical conversions in these processes were performed
by living cells, and thus the traditional processes should be
consider biochemical conversions.
Pharmaceutical Biotechnology

3. Decades following Pasteur's discovery, biotechnological
knowledge increased when the catalytic role of enzymes for
most biochemical conversions became apparent, based on
that knowledge tools became available for the control and
optimization of the traditional processes
A further and very important breakthrough took place after the
development of (Molecular Biology). The notion or concept,
brought forward by the pioneers in the molecular biology in
around 1950, that DNA encodes proteins and in this way
controls all cellular processes was the impetus for a new
period in biotechnology.

4. The fast evolving DNA technologies, after the development of
the recombinant DNA technology in 70th, allowed
biotechnologists to control gene expression in the organisms
used for biotechnological manufacturing.
These developed technologies opened new ways for the
introduction of foreign DNA into all kinds of organisms.
Thus genetically modified organisms constructed in this way
to open up completely new possibilities for biotechnology.

5. Complex biological molecules, commonly known as proteins that
usually aim at eliminating the underlying mechanisms for treating
diseases.
Essentially used to make (Complex Larger Molecules) with the
help of living cells (like those found in the human body such as
bacteria cells, yeast cells, animals or plant cells).
Unlike the smaller molecules that are given to a patient
through tablets, the large molecules are typically injected into
the patient’s body.
Biopharmaceuticals

7.  Antibodies- are proteins produced by white blood cells and
are used by immune system to identify bacteria, viruses and
other foreign substances and to fight them off.
 Monoclonal antibodies- are one of the most exciting
developments in pharmaceutical biotechnology at these
recent years. (produced as a result of perpetuating the expression of a
single beta lymphocyte. Consequently, all of the antibody molecules
secreted by a series of daughter cells derived from a single dividing
parent beta lymphocyte are genetically identical).
Pharmaceutical Biotechnology Products
Antibodies, Proteins and Recombinant DNA
products

9. Proteins- made of amino acids or large, complex
molecules that do most of the work in the cells and
are required for the structure, function, and
regulation of the body’s tissues and organs.
Protein biotechnology- is emerging as one of the key
technologies of the future for understanding the development of
many diseases like cancer or amyloid formation for better
therapeutic intervention.

10. Recombinant DNA Technologies
Genetic modification of organisms is done by Fusion of any
DNA fragment to DNA molecules able to maintain
themselves by autonomous replication. Such molecules
called replicons

11. Recombinant DNA
Plasmid or Vector

13. Recombinant DNA technology or DNA cloning technology:
(Application of plasmids in biotechnology)
 Fusing foreign DNA fragment to the isolated plasmid in order to
create a recombinant DNA molecule called replicons.
 Replicons used as carriers for foreign DNA fragments are
termed vectors (include plasmids from bacteria or yeast, or
DNA from bactriovirus, animal virus or Plant virus).
 Foreign DNA- isolated either from microbial, plant or
animal cell
 Restriction enzyme used to cut DNA at a specific site.
 Ligase enzyme used to close circular recombinant DNA.
- Introduction of recombinant DNA into host cell leads to
form (Transformant).
- Vector replicate in the host, thus all daughter cells will
inherit precise copy (a clone) of the recombinant DNA
molecule.

14. Monoclonal Antibodies
Typically made by fusing myeloma cells with the spleen
from a mouse that has been immunized with the desired
antigen.

16.  Laboratory animals (mammal, e.g. mice) are first exposed to the antigen
that an antibody is to be generated against. Usually this is done by a series
of injections of the antigen in question, over the course of several weeks.
Once spleen cells are isolated from the spleen the B cells are fused with
immortalised myeloma cells. The myeloma cells are selected beforehand
to ensure they are not secreting antibody themselves and that they lack the
(HGPRT) gene, making them sensitive to the incubation in HAT medium .
Fused cells are incubated in HAT medium for roughly 10 to 14
days. Hence, unfused myeloma cells die, because they lack HGPRT.
Removal of the unfused myeloma cells is necessary because they have
the potential to outgrow other cells, especially weakly established
hybridomas. Unfused B cells die as they have a short life span. In this way,
only the B cell-myeloma hybrids survival. These cells produce antibodies
(a property of B cells) and are immortal (a property of myeloma cells). The
next stage is a rapid primary screening process, which identifies and
selects only those hybridomas that produce antibodies of appropriate
specificity. The B cell that produces the desired antibodies can be cloned to
produce many identical daughter clones. Once a hybridoma colony is
established, it will continually grow in culture medium and produce
antibodies.

17. 1. Sterility
Most proteins are administered parenterally and it should be
sterile (But are sensitive to heat and other sterilization
treatments) so cannot withstand [autoclaving, gas sterilization,
or sterilization by ionizing radiation].
Protein pharmaceuticals assembled under aseptic conditions,
following established and evolving rules in the pharmaceutical
industry for aseptic manufacture.
Formulation of Biotech Products
Biopharmaceutical Considerations

18. 2.Viral Decontamination
 As recombinant DNA products are grown in microorganisms, these
organisms should be tested for viral contaminants and appropriate
measures should be taken if viral contamination occurs.
 Excipients with a certain risk factor, such as blood derived human
serum albumin, should be carefully tested before use and their
presence in the formulation process should be minimized.
3.Pyrogen removal
 Pyrogens are compounds that induce fever.
 Exogenous pyrogens (pyrogens introduced into the body, not
generated by the body itself) can be derived from bacterial, viral or
fungal sources. Bacterial pyrogens are mainly endotoxins shed from
gram negative bacteria. They are lipopolysaccharides.
 Pyrogen removal of recombinant products derived from bacterial
sources should be an integral part of preparation process:
 Excipients used in the protein formulation should be essentially
endotoxins-free.

19. Continuous with Pyrogen removal
 Pyrogen removal of recombinant products derived from bacterial
sources should be an integral part of preparation process:
Ion exchange chromatographic procedures (utilizing its negative
charge) can effectively reduce endotoxins levels in solution.
 For solutions, water for injection (compendial standards) is (freshly)
distilled or produced by reverse osmosis.

20. Ion exchange chromatography

21.  In a protein formulation (active substance), a number
of excipients selected to serve different purposes.
 The nature of the protein (e.g. lability-rapid change or
destroyed-) and its therapeutic use (e.g. multiple
injection systems) can make these formulations quite
complex in term of excipients profile and technology
(freeze-drying, aseptic preparation).
Excipients Used in Parenteral Formulations
of Biotech Product

22. components found in parenteral formulations
of biotech products
1. Active ingredient
2. Solubility enhancers
3. Anti-adsorption and anti-aggregation agents
4. Buffer components
5. Preservatives and anti-oxidants
6. Lyoprotectants/ cake formers
7. Osmotic agents
8. Carrier system
Note: All of the above are not necessarily present in
one particular protein formulation

23. 2. Solubility Enhancers
 Proteins, in particular those that are non-glycosylated, may
have a tendency to aggregate and precipitate.
 Approaches that can be used to enhance solubility include:
1. Selection of the proper pH and ionic strength conditions
2. Addition of amino acids, such as lysine or arginine (used to solubilize
tissue plasminogen activator, t-PA)
3. Addition of surfactants such as sodium dodecylsulfate, to solubilize non-
glycosylate, IL-2 (interleukin-2) can also help to increase the solubility.

24.  Tissue plasminogen activator (abbreviated tPA or PLAT) is a
protein involved in the breakdown of blood clots.
 As an enzyme, it catalyzes the conversion of plasminogen to
plasmin, the major enzyme responsible for clot breakdown.
 Because it works on the clotting system, tPA is used in clinical
medicine to treat embolic or thrombotic stroke.
 tPA may be manufactured using recombinant biotechnology
techniques. tPA created by this way may be referred to as
recombinant tissue plasminogen activator (rtPA).
Notes
 Interleukin 2 (IL-2) is an interleukin, a type of cytokine
signalling molecule in the immune system.
 It is a protein that regulates the activities of white blood cells
(leukocytes, often lymphocytes) that are responsible for
immunity.

25. The mechanism of action of these solubility enhancers
Type of enhancer and protein involved and is not always
fully understood.
depends on

26. 0
10
20
30
40
50
60
70
80
90
100
0 0.05 0.1 0.15 0.2 0.25
Apparent
solubility
(mg/ml)
Figure 1: Shows the effect of arginine concentration on the
solubility of t-PA (alteplase) at pH 7.2 and 25oC.
Arginine-phosphate (M)
A : type I alteplase
B : type II alteplase
C : 50:50 mixture of
type I and type II alteplase

27. In the above examples aggregation is physical in nature, i.e.
based on hydrophobic and/ or electrostatic interactions
between molecules by formation of covalent bridges
between molecules through disulfide bonds, and ester or
amide linkages.
 In these cases proper conditions should be found to avoid
these chemical reactions (the figure above clearly
indicates the dramatic effect of this basic amino acid on
the apparent solubility of t-PA).

28.  Anti-adsorption agents (added to reduce adsorption of the
active protein to interfaces).
 Some proteins normally have hydrophobic sites in the core
structure. They tend to expose hydrophobic sites when an
interface is present.
These interfaces can be water/air, water/container wall or
interfaces formed between the aqueous phase and utensils
used to administer the drug (e.g. catheter, needle).
3. Anti-adsorption and anti-aggregation agents

30.  These adsorbed, partially
unfolded protein molecules
form aggregates, leave the
surface, return to the
aqueous phase, form larger
aggregates and precipitate.
 Example:
The proposed mechanism for
aggregation of insulin in
aqueous media through contact
with a hydrophobic surface (or
water-air interface) is presented
in Figure 2.

31. Figure 2 Reversible self-association of insulin, its adsorption to the
hydrophobic interface and irreversible aggregation in the adsorbed
protein film
crystal
Hydrophobic surface
Aqueous solution
monomer Dimer Hexamer
Tetramer

32.  Native insulin in solution is in an equilibrium state between
monomeric, dimeric, tetrameric and hexameric form.
 The relative abundance of the different aggregation states
depends on the pH, insulin concentration, ionic strength
and specific excipients (Zn2+ and phenol).
 Suggestion: dimeric form of insulin adsorbs to hydrophobic
interfaces and subsequently forms larger aggregates at the
interface.
This adsorption explains why anti-adhesion agents can also
act as anti-aggregation agents.

33.  Ex: Albumin (strong tendency to adsorb to surfaces)
and is therefore added in relatively high concentration (e.g.
1%) as an anti-adhesion agent to protein formulations.

Mechanism: albumin competes with the therapeutic protein
for binding sites and prevents adhesion of the therapeutically
active agent by combination of its binding tendency and
abundant presence.

34.  Insulin is one of the many proteins that can form
fibrillar precipitates (long rod-shaped structures with
diameters in the 0.1 µm range).
1. Low concentrations of phospholipids and
surfactants (as a fibrillation-inhibitory effect).
2. The selection of the proper pH to prevent this
unwanted phenomenon.
This can be
prevented by:
 Apart from albumin, surfactants can also prevent adhesion
to interfaces and precipitation.
Readily adsorb to hydrophobic interfaces with their own
hydrophobic groups and render this interface hydrophilic by
exposing their hydrophilic groups phase.

35. 4. Buffer components
Buffer selection is an important part of the formulation
process, because of the pH dependence of protein solubility ,
physical and chemical stability.
Buffer systems regularly encountered in biotech formulations
are:
1. phosphate
2. citrate
3. acetate

36. The isoelectric point (pI)
pH of a solution at which the net primary charge of a protein
becomes zero.
At a solution pH that is above the pI the surface of the protein is
predominantly negatively charged and like-charged molecules will
exhibit repulsive forces.
At a solution pH that is below the pI, the surface of the protein is
predominantly positively charged and repulsion between proteins
occurs.
At the pI the negative and positive charges cancel, repulsive
electrostatic forces are reduced and the attraction forces
predominate. The attraction forces will cause aggregation and
precipitation.
The pI of most proteins is in the pH range of 4-6.

37. Figure 1. A plot of the solubility of various
forms
of hGH as a function of pH. The closed
symbols mean that precipitate was
present in the dialysis tube after
equilibration, whereas open symbols
mean that no solid material was present,
and thus the solubility is at least this
[hGH]
mg/ml
pH
3 4 6
5 7
1
5
10
20
Circles = recombinant hGH
Triangles = Met-hGH
Squares = pituitary hGH
A good example of importance of
the isoelectric point (its
negative logarithm [pH] is
equal to pI) is the solubility
profile of human growth
hormone (hGH, pI around
5) as presented in Figure 1:
pI: is the pH at a particular
molecule carries no net
electrical charges (overall
charge).
Thus molecule is affected by
pH of its surrounding
environment and can
become more positively or
negatively charged due to
the gain or loss, respectively,
of (H+).
Such molecules have
minimum solubility in water
+ve
charg
e -ve
charg
e

38. Even short, temporary pH changes can cause
aggregation. Explain why?
 These conditions can occur, for example, during the
freeze-drying process, when one of the buffer
components is crystallizing and the other is not.
 In a phosphate buffer, Na2HPO4 crystallizes faster than
NaH2PO4.
drop in pH during the freezing step.
While other buffer components do not crystallize, but
form amorphous systems and then pH changes are
minimized.

39. 5. Preservatives and Anti-oxidants
 Methionine, cysteine, tryptophane, tyrosine and
histidine are amino acids that are readily oxidized.
 Proteins rich in these amino acids are susceptible to
oxidative degradation.
1. Replacement of oxygen by inert gases in the vials
helps to reduce oxidative stress.
2. Addition of anti-oxidant such as ascorbic acid or
sodium formaldehyde sulfoxylate can be considered.
The solution
!!!
Antioxidants

40. Certain proteins are formulated in the container
designed for multiple injection schemes.
 After administering the first dose, contamination with
microorganism may occur and the preservatives are
needed to minimize growth.
 Usually, these preservatives are present in
concentrations that are bacteriostatic rather than
bactericide in nature.
 Antimicrobial agents mentioned in the USP XXIV are the
mercury-containing pheylmercuric nitrate, thimerosal,
p-hydroxybenzoic acids, phenol, benzyl alcohol and
chlorobutanol.
Preservatives

41. Shelf Life of Protein Based Pharmaceuticals
Protein can be stored:
(1) as an aqueous solution
(2) in freeze-dried form
(3) in dried form in a compacted state (tablet).
The stability of protein solutions strongly depends on
factors such as pH, ionic strength, temperature, and the
presence of stabilizers.
E.g.: Figure 2 shows the pH dependence of α1-antitrypsin
and clearly demonstrates the critical importance of pH
on the shelf-life of proteins.

42. Freeze-Drying of Proteins
 Proteins in solution often do not meet the preferred
stability requirements for industrially pharmaceutical
products (>2 years), even when kept permanently under
refrigerator conditions (cold chain).
 The abundant presence of water promotes chemical and
physical degradation processes.

43. Importance of Freeze Drying
 Freeze-drying may provide the desired stability by extending
shelf life. During freeze-drying water is removed via
sublimation and not by evaporation.
it works by freezing the material, then reducing the pressure
and adding heat to allow the frozen water in the material to
sublimate.
 Three stages can be discerned in the freeze-drying process:
(1) freezing step
(2) primary drying step
(3) secondary drying step.

44. Table 1. Three stages in the freeze drying process of protein
formulations.
1. Freezing
The temperature of the product is reduced from ambient
temperature to a temperature below the eutectic temperature
(Te), or below the glass transition temperature (Tg) of the
system. A Tg is encountered if amorphous phases are present.
2. Primary drying
Crystallized and water not bound to protein/excipients is
removed by sublimation. The temperature is below the Te or Tg;
the temperature is for example -40oC and reduced pressures are
used.
3. Secondary drying
Removal of water interacting with the protein and excipients.
The temperature in the chamber is kept below Tg and rises
gradually, e.g., from -40oC to 20oC.

45.  The freeze-drying of a protein solution without the
proper excipients causes, as a rule, irreversible
damage to the protein.
 Table 4.3 lists excipients typically encountered in
successfully freeze-drying protein products:

46. Table 4.3. typical excipients in a
freeze-dried protein formulation
1. Bulking agents: mannitol/ glycine
 Reason: elegance/ blowout prevention
 Blowout is the loss of material taken away by the water vapor that
leaves the vial. It occurs when little solid material is present in the
vial.
2. Collapse temperature modifier: dextran, albumin/ gelatine
 Reason: prevent increase collapse temperature.
3. Lyoprotectant: sugars, albumin
 Reason: protection of the physical structure of the protein.
 Mechanism of action of lyoprotectants is not fully understood.
Factors that might play a role are:

47. Mechanisms of action of lyoprotectants
1. Lyoprotectants replace water as stabilizing agent
(water replacement theory),
2. Lyoprotectants increase the Tg of the cake/ frozen
system
3. Lyoprotectants will absorb moisture from the stoppers
4. Lyoprotectants slow down the secondary drying process
and minimize the chances for overdrying of the protein.
Overdrying might occur when residual water levels after
secondary drying become too low.

48.  The parenteral Route of Administration
 Parenteral administration is defined as administration via
those routes where a needle is used, including intravenous (IV),
intramuscular (IM), subcutaneous (SC) and intraperitoneal (IP)
injections.
 The blood half-life of biotech products can vary over a
wide range. For example, the circulation half-life of t-PA is a
few minutes, while monoclonal antibodies (MAB) have half-
lives of a few days
Delivery of Proteins

49.  One reason to develop modified proteins through
site directed mutagenesis
To enhance circulation half-life.
By expanding the mean residence time for short half-
life proteins (switch from IV to IM or SC
administration).
1- changes in disposition which
Have a significant impact on the therapeutic
performance of the drug.

50. These changes are related to:
i. The prolonged residence time at the IM or SC
site of injection compared to IV administration
and enhanced exposure to degradation
reactions (peptidases).
ii. Differences in disposition.

51. Regarding point 1 (Prolonged residence
time at IM or SC site of injection and the enhanced
exposure to degradation reactions.)
A- For instance, diabetics can become “insulin
resistant” through high tissue dipeptidyl peptidase {DPP-IV}
activity .
B- Other factors that can contribute to absorption variation
are related to differences in exercise level of the muscle
at the injection site.
C- The state of the tissue, for instance the occurrence of
pathological conditions, may be important as well.

52. Regarding point 2 (Differences in disposition).
Upon administration, the protein may be
transported to the blood circulation
or
through the
lymphatics
through the capillary wall
at the site of injection.
Note: The fraction of the administered dose
taking this lymphatic route is molecular
weight dependent.

53. Routes of uptake of SC or IM injected
drugs
Blood Capillary wall
lymph
Low Mwt drugs
Site of injection
High Mwt drugs

54. Molecular weight of different
proteins
 rIFN alpha-2a (Mw 19 kDa)
 Cytochrome C (Mw 12.3 kDa)
 Inulin (Mw 5.2 kDa)
 FUdR (Mw 256.2 Da)
The following Figure shows:
Cumulative recovery in the efferent lymph from the
right popliteal lymph node following SC administration
into the lower part of the right hind leg of sheep

55. 0
10
20
30
40
50
60
70
0 2 4 6 8 10 12 14 16 18 20
lymph
recovery
[%
of
dose]
molecular weight [kDa]
FUdR
Inulin
IFN-α-2a
Cytochrome C
Correlation between the molecular weight and cumulative recovery

56.  Lymphatic transport takes time (hours) and
uptake in the blood circulation is highly dependent
on the injection site.
 On its way to the blood, the lymph passes
through draining lymph nodes and contact is
possible between lymph contents and cells of the
immune system such as macrophages, B- and T-
lymphocytes residing in the lymph nodes.

57. The Oral Route of Administration
 Oral delivery of protein drugs would be preferable because:
1. It is patient friendly
2. No intervention by a healthcare professional is necessary to
administer the drug.
 Not Preferable:
Oral bioavailability is usually very low.
 The two main reasons for failure of uptake after oral
administration
1. Protein degradation in the gastrointestinal (GI) tract.
2. Poor permeability of the wall of the GI tract in case of a
passive transport process.

58. (protein degradation in the GI tract)
i. The human body has developed a very efficient system to break down
proteins in our food to amino acids, or di- or tri-peptides.
ii. These building stones for body proteins are actively absorbed for use
wherever necessary in the body.
iii. In the stomach pepsins (a family of aspartic proteases) are secreted.
They are particularly active between pH 3 and 5 and lose activity at
higher pH values.
iv. Pepsins are endopeptidases capable of cleaving peptide bonds
distant from the ends of the peptide chain. They preferentially
(cleave peptide bonds between two hydrophobic amino acids).
v. Other endopeptidases are active in the GI tract at neutral pH
values, e.g., trypsin, chymotrypsin, and elastase. They have
different peptide bond cleavage characteristics that more or less
complement each other.
vi. Exopeptidases, proteases degrading peptide chains from their
ends, are present as well. Examples are carboxypeptidase A and B.

60. viii. In the GI lumen the proteins
are cut into fragments that
effectively further break
down to amino acids, di- and
tri-peptides by brush border
(microvillus) and cytoplasmic
proteases of the enterocytes
(intestinal absorptive cells).

61. (permeability)
i. High molecular weight molecules do not readily penetrate the
intact and mature epithelial barrier if diffusion is the sole
driving force for mass transfer.
ii. Their diffusion coefficient decreases with increasing
molecule size.
iii. Protein are no exception to this rule.
iv. Active transport of intact therapeutic recombinant proteins
over the GI-epithelium has not been described yet.

62. Conclusion
The above analysis leads to the conclusion that the oral route of
administration for therapeutic protein is unsuitable if high
(or at least constant) bioavailability is required.

63. O However, for the category of oral vaccines the above-
mentioned hurdles of degradation and permeation are
not necessarily prohibitive.
O Ex: For oral immunization, only a (small) fraction of
the antigen (protein) has to reach its target site to elicit an
immune response.
OThe target cells are B-lymphocyte cells that produce
secretory IgA antibodies.
O and antigen presenting accessory cells located in
Peyer’s patches (macroscopically identifiable follicular
structures located in the wall of the GI tract).

64.  Peyer’s patches are overlaid with microfold (M) cells
(separate the luminal contents from the lymphocytes).
 These M cells have little lysosomal degradation capacity and
allow for antigen sampling by the underlying lymphocytes.
 Moreover, mucus producing goblet cell density is reduced over
Peyer’s patches.
 This reduces mucus production and facilitates access to the M
cell surface for luminal contents.
 Attempts to improve antigene delivery via the Peyer’s patches
and to enhance the immune response are made by using
microspheres, liposomes or modified live vectors, such as
attenuated bacteria and viruses.

65. Parenteral administration has disadvantages
(needles, sterility, injection skill) compared to
other possible routes.
Delivery through nose, lungs, rectum, oral
cavity, and skin have been selected as potential
sites of application.
Alternative Route of Administration

66. I. Nasal
Advantage:
1. Easily accessible
2. Fast uptake
3. Proven track record with a number of “conventional” drugs
4. Probably lower proteolytic activity than in the GI tract
5. Avoidance of first pass effect
6. Spatial containment of absorption enhancers [osmolarity &
pH] is possible (when drugs exhibits poor membrane
permeability, large molecular size, lack of lipophilicity and
enzymatic degradation by amino peptidases).
The potential pros and cons for
different relevant routes

67. Nasal
Disadvantage:
1. Reproducibility (in particular under intranasal
pathologies may affect or capacity for nasal
absorption)
2. Safety (e.g., cilliary movement that propelled
proteins into the throat where it is swallowed and
destroyed by the products of the stomach).
3. Low bioavailability for proteins (Because they are
large molecular weight polar drugs thus they
have low membrane permeability).

68. II. Pulmonary (intratracheal inhalation or
instillation)
Advantage:
1. Relative easy to access (aerosol or syringe).
2. Fast uptake.
3. Proven track record with “conventional” drugs.
4. Substantial fractions of insulin are absorbed.
5. Lower proteolytic activity than in the GI tract.
6. Avoidance of hepatic first pass effect.
7. Spatial containment of absorption enhancer.

69. Pulmonary
Disadvantage:
1. Reproducibility (in particular under pathological
conditions, smoker/non-smoker).
2. Safety (e.g., inhaled human insulin [powder or liquid]
has been shown to be more immunogenic than
comparator insulins given by S.C. routes; however,
adverse effects of antibody formation demonstrated)
3. Presence of macrophages in the lung with affinity for
particulates.

70. III. Rectal
Advantage:
1. Easily accessible
2. Partial avoidance of hepatic first pass
3. Probably lower proteolytic activity than in the upper
parts of GI tract
4. Spatial containment of absorption enhancers is
possible
5. Proven track record with a number of “conventional”
drugs.
Disadvantage:
Low bioavailability for proteins

71. IV. Buccal
Advantage:
1. Easily accessible
2. Avoidance of hepatic first pass
3. Probably lower proteolytic activity than in the lower
parts of the GI tract
4. Spatial containment of absorption enhancer is
possible
5. Option to remove formulation if necessary
Disadvantage:
1. Low bioavailability of proteins
2. No proven track record yet.

72. V. Transdermal
Advantage:
1. Easily accessible
2. Avoidance of hepatic first pass
3. Removal of formulation if necessary is possible
4. Spatial containment of absorption enhancers
5. Proven track record with “conventional” drugs
6. Sustained/controlled release possible
Disadvantage:
Low bioavailability of proteins

73.  The nasal, buccal, rectal, and transdermal routes all have
been shown to be of little clinical relevance if systemic
action is required, and if simple protein formulations
without an absorption enhancing technology are used.
 In general, bioavailability is too low and varies too much!
The pulmonary route may be the exception to this rule
(because in pulmonary the absorption was strongly
protein dependent, with no clear relationship with it’s
molecular weight).
Conclusion

74.  In human the drug should be inhaled instead of
intratracheally administered.
 The delivery of insulin to Type I (juvenile onset) and
Type II (adult onset) diabetics has been extensively
studied and clinical phase III trials evaluating
efficacy and safety have been performed or are
ongoing.
 The first pulmonary insulin formulation was
approved by FDA in January 2006 (Exubera®).
 It was taken off the market 2008 because of poor
market presentation

75.  Many pharmaceutical companies doing research in
the field to develop an inhalational preparation
announced the termination of product
development following the poor acceptance and
risk of lung cancer of the first US FDA approved
inhaled insulin product, Exubera®.
 This formulation produced cough, dyspnoea
(difficulty in breathing), increased sputum, and
epistaxis (nosebleed), and was contraindicated in
patients with chronic obstructive pulmonary
disease (COPD) and asthma.

76. Technosphere insulin: a new inhaled insulin
 MannKind Corporation has developed a
powdered formulation of insulin with a
higher percentage of absorption from the
lungs. This product, Afrezza® (Technosphere®
insulin), appears to have overcome some of
the barriers that contributed to the
withdrawal of Exubera® and is currently
under review by the FDA.

77.  Technosphere insulin is a new inhaled insulin preparation
which mimics normal prandial insulin release. It decreases
post-prandial blood glucose (PPG) levels and has good
glycaemic control with significantly lesser hypoglycaemia.
 Current data show that this formulation has no impact on
pulmonary function.
 Long-term safety studies with regard to pulmonary function
and risk for development of lung carcinoma need to be
monitored.
 The FDA is currently reviewing Technosphere insulin for use
in both type 1 and type 2 diabetes.

78. Technosphere® insulin

79. Classified according to proposed mechanism of action
1. Increase the permeability of the absorption barrier:
 Addition of fatty acids/phospholipids, bile salts, enamine derivatives of
phyenylglycine, ester and ether type (non)-ionic detergents, saponins, salicylate
derivatives of fusidic acid or glycyrrhizinic acid, or methylated β cyclodextrins
 Through iontophoresis
 By using liposomes.
2. Decrease peptidase activity at the site of absorption and along the
“absorption route”: aportinin, bacitracin, soybean tyrosine inhibitor,
boroleucin, borovaline.
3. Enhance resistance against degradation by modification of the molecular
structure.
4. Prolongation of exposure time (e.g., bio-adhesion technologies).
Approaches to enhance bioavailability of proteins

80. Bioavailability (%)
No. Of AA
Molecule
With glycocholate
Without glycocholate
70-90
< 1
29
Glucagon
15-20
< 1
32
Calcitonin
10-30
< 1
51
Insulin
7-8
< 1
191
Met-hgH
Examples of Absorption Enhancing Effects
Effect of glycocholate (absorption enhancer) on nasal
bioavailability of some proteins and peptides.

81.  Major issues now being addressed are
reproducibility, effect of pathological conditions
(e.g., rhinitis) on absorption and safety aspects of
chronic use.
 Absorption enhancing effects were shown to be
species dependent.
 Pronounced differences in effect were observed
between rats, rabbits, and humans.