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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5472_Библиотеки_им_академика_М_И_Перельмана.pdf
Nanobiotechnology
285
polar to give high aqueous solubility and prevent nanoparticle aggregation. In serum or on the cell
surface, highly charged coatings promote nonspecific binding, while polyethylene glycol linked to
terminal hydroxyl or methoxy groups repel nonspecific interactions. Nanoparticles when linked
to biological molecules can act as address tags to direct the nanoparticles to specific organelles
within the cell and specific sites in the body. The common address tags are monoclonal antibodies,
aptamers, streptavidin or peptides. These targeting agents should ideally be covalently linked
to the nanoparticle and should be present in a controlled number per nanoparticle. Monovalent
nanoparticles, bearing a single binding site, avoid clustering and so are preferable for tracking the
behaviour of individual proteins. Multivalent nanoparticles, bearing multiple targeting groups, can
cluster receptors that can activate cellular signalling pathways and give stronger anchoring.
Safety
Most of the nanoparticles are due to the high surface-to-volume ratio that can make the particles
very reactive or catalytic. They are also able to pass through cell membranes in organisms, and
their interactions with biological systems are relatively unknown. A recent study revealed the
effects of ZnO nanoparticles on human immune cells that were at varying levels of susceptibility
to cytotoxicity. Smaller nanoparticles evinced increased cytotoxicity. The lymphocytes (especially
naive T cells) were found to be more resistant to nanoparticle cytotoxicity than monocytes. It may
be due to the capacity of the latter to produce higher levels of reactive oxygen species in response to
internalized nanoparticles. Previously activated memory T cells were more susceptible than naive
T cells, implying a relationship between cell cycle and nanoparticle susceptibility. In addition,
nanoparticle concentrations below those causing appreciable cell death nonetheless induced the
production of proinflammatory cytokines, such as IFN-a and TNF.
Synthetic Biology
Synthetic biology is a wide area of biological research that combines science and engineering.
It encompasses a variety of different approaches, methodologies and discipline, and it describes
pinpoints at the design and construction of new biological functions and systems not found in
nature.
In 1974, the Polish geneticist Waclaw Szybalski introduced the term synthetic biology.
The Nobel Prize in physiology or medicine was awarded to Arber, Nathans and Smith in 1978
for their discovery of restriction enzymes, Waclaw Szybalski then wrote in an editorial in the journal
Gene, “The work on restriction nucleases not only permits us easily to construct recombinant DNA
molecules and to analyse individual genes, but also has led us into the new era of synthetic biology
where not only existing genes are described and analysed, but also new gene arrangements can be
constructed and evaluated.”
Synthetic biology includes the broad redefinition and expansion of biotechnology, with
the ultimate goals of being able to design and build engineered biological systems that process
information, manipulate chemicals, fabricate materials and structures, produce energy, provide
food, and maintain and enhance human health and our environment.
Textbook of Pharmaceutical Biotechnology
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Good examples of engineering in synthetic biology include the pioneering work of Tim Gardner
and Jim Collins on an engineered genetic toggle switch, a riboregulator, the Registry of Standard
Biological Parts and the International Genetically Engineered Machine (iGEM) competition.
The synthetic biology raises questions for ethics, biosecurity, biosafety, involvement of
stakeholders and intellectual property. To date, key stakeholders (especially in the US) have focused
primarily on the biosecurity issues, especially the so-called dual-use challenge. For example, while
the study of synthetic biology may lead to more efficient ways to produce medical treatments (e.g.
against malaria), it may also lead to synthesis or redesign of harmful pathogens (e.g. smallpox) by
malicious actors.
There are several key enabling technologies that are critical to the growth of synthetic biology.
The key concepts include standardization of biological parts and hierarchical abstraction to permit
using those parts in increasingly complex synthetic systems. Achieving this is greatly aided by
basic technologies of reading and writing of DNA (sequencing and fabrication) that are improving
in price/performance exponentially. The measurements under a variety of conditions are needed for
accurate modelling and computer-aided design (CAD).
QUESTIONS
1. Define nanobiotechnology.
2. Define nanolithography and write a note on branches of nanolithography.
3. Write notes on nanomedicine and nanodevices.
4. Write medical uses of nanomaterials.
5. Give an account of challenges in development of nanobiotechnology.
INTRODUCTION
Biopharmaceutical is a pharmaceutical substance used for therapeutic purpose, which is produced
by means of direct extraction from native biological sources. The term biopharmaceutical
is most commonly used to refer to all therapeutic, prophylactics and in vivo diagnostic agents
produced using live organisms or their functional components. In general, the biopharmaceuticals
are complex macromolecules that are over hundred times larger than small molecule drugs with
complex structure activity and maintenance requirements.
The term biopharmaceutical was first used in 1980 for the product of pharmaceutical
biotechnology or biotechnological medicines. The biopharmaceutical industry can trace its
beginning to Edward Jenner who has been credited with the development of small pox vaccine
in 1796. The discovery of penicillin by Alexander Fleming in 1928 changed the course of medicine
and heralded the start of the age of biopharmaceuticals. Wilhelm Roux cultivated first cells from
chick embryos in salt solution in 1885. Ross Harrison successfully undertook the animal cell culture
in a frog lymph medium in 1907. It was in 1950 that cell cultures were widely available. During
1960s and 1970s, commercialization of these technologies had further impact on cell culture that
continues to this day. The companies started developing and selling disposable plastic and glass cell
culture products, improved filtration products and materials, liquid and powdered tissue culture
media and laminar flow hoods.
Recent Advances in
Biopharmaceuticals
19
CHAPTER
Introduction
Advantages of DNA Technology in the
Production of Biopharmaceuticals
Biomolecules Produced through
Recombinant DNA Technology
Biopharmaceuticals of Animal Origin
Biopharmaceuticals of Plant Origin
Pharmaceutical Substances of
Microbial Origin
Recent Advances in Biopharmaceuticals
Questions
Textbook of Pharmaceutical Biotechnology
288
Today, alternatives to large-scale submerged microbial and animal cell cultures are increasing.
In addition to vaccines that are grown in animals, embryos and eggs, products grown in transgenic
animals and plants, as well as small volume products of developing cell and gene therapies are
collectively poised to displace the predominated use of large volume bioreactor.
Biotechnology is profoundly changing the way drugs are produced with the use of chemical
synthesis to biomanufacturing. Drugs are now grown and not made. There are also fundamental
differences in the average size of these two types of drugs. The chemically synthesized products
are known as small molecule drugs. Because of this transformative phenomenon in biotechnology,
the pharma industry for biotech products is growing more than twice as fast as traditional
pharmaceutical companies. The proteomics, genomics, bioinformation and metabolism have led
to the identification of hundreds of biological targets and potential biologicals. The potential for
improved disease targeting and greater efficacy in many fields is instrumental in biopharmaceuticals
accounting for an increasing share of the new drug application as submitted to Health Canada and
the US FDA, the latter passing the 50% mark for the first time in 2002. A significant portion
of biopharmaceutical drug development is now performed by some experienced big pharma and
a small number of established biopharma companies. In addition, there are hundreds of small
companies and institutions without sufficient competence to bring novel biopharmaceuticals all
the way to market. Today, even these organizations have full access to the required expertise from
contract manufacturing and clinical research organizations.
ADVANTAGES OF DNA TECHNOLOGY IN THE
PRODUCTION OF BIOPHARMACEUTICALS
The advantages of DNA technological applications to production of biopharmaceuticals are listed below.
1. It provides an alternative to direct extraction from source materials, such as Escherichia coli,
snake venom, human urine, etc.
2. It overcomes the problem of source availability.
3. Large-scale production of therapeutic proteins, such as insulin, hormones, vaccine and
interleukins, using recombinant microorganisms is possible.
4. Production of humanized monoclonal antibodies for therapeutic application is a reality.
5. Its feasibility of generating therapeutic proteins shows advantage over native protein product.
6. Bioremediation by the use of recombinant organisms is possible.
7. Genetic engineering techniques are used in forensic medicine.
8. Production of insect resistant cotton plant by incorporation of insecticidal toxin of Bacillus
thuringiensis is possible.
9. The golden rice (rice in vitamin A) by incorporating three genes required for its synthesis in
rice plant can be produced.
Recent Advances in Biopharmaceuticals
289
BIOMOLECULES PRODUCED THROUGH
RECOMBINANT DNA TECHNOLOGY
The list of important products obtained by recombinant DNA technology is given below.
1. Recombinant hormones: Insulin (and its analogues), follicle-stimulating hormone, growth
hormone, salmon calcitonin, etc.
2. Cytokines and growth factors: Interferons, interleukins and colony-stimulating factors
( interferons: _, ` and a; erythropoietin; interleukin-2; GM-CSF; G-CSF), etc.
3. Blood products: Albumin, fibrinolytics, thrombolytics and clotting factors (factor VII, factor
IX, tissue plasminogen activator, recombinant hirudin), etc.
4. Recombinant enzymes: Dornase alfa (Pulmozyme), _-L-iduronidase (Aldurazyme), acid
glucosidase (Myozyme), urate oxidase, etc.
5. Recombinant vaccines: Recombinant protein or peptides, DNA plasmid and anti-idiotype
(HBsAg vaccine, HPV vaccine), etc.
6. Monoclonal antibodies and related products: Mouse, chimeric or humanized, whole molecule
or fragment, single chain or bispecific and conjugated (rituximab, trastuzmab, infliximab,
bevacizumab), etc.
7. Miscellaneous products: Bone morphogenic protein, conjugate antibody, pegylated
recombinant proteins, antagonists, etc.
The production of biopharmaceuticals is possible from three different natural origins (Fig. 19.1).
Figure 19.1 Production of biopharmaceuticals.
Biopharmaceuticals
Plant originAnimal origin
Microbial origin
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BIOPHARMACEUTICALS OF ANIMAL ORIGIN
Sex Hormones
Male and female gonads produce a range of steroid hormones, which regulate the development and
maintenance of reproductive and related functions. Two main classes of sex steroids are androgens
and oestrogens, of which the most important human derivatives are testosterone and estradiol,
respectively. Progestogen is a third class of sex steroids, distinct from androgens and oestrogens.
It is the most important and naturally occurring. In general, androgens are considered male sex
hormones, since they exert masculinizing effects, whereas oestrogens and progestogen are known
as female sex hormones, although all types are present in each gender at different levels.
Androgens
Androgens were first discovered in 1936. The major androgen produced by testes is testosterone
that is secreted (4–10 mg) daily into blood stream. Androgen, also known as androgenic hormone
or testoid, is the generic term for any natural or synthetic compound (usually a steroid hormone)
that stimulates or controls the development and maintenance of male characteristics in vertebrates
by binding to androgen receptors. The spectrum of activity covers activities related to accessory
male sex organs and development of male secondary sex characteristics. Androgens are also the
original anabolic steroids and the precursor of all oestrogens, the female sex hormones. Androgen
ablation can be used as an effective therapy in prostate cancer (Table 19.1).
Other examples of medicinally important synthetic steroid hormones are as follows:
1. Glucocorticoids: Prednisone, dexamethasone, triamcinolone
2. Mineralocorticoid: Fludrocortisone
3. Vitamin D: Dihydrotachysterol
4. Androgens: Oxandrolone, nandrolone (also known as anabolic steroids)
5. Oestrogens: Diethylstilbestrol (DES)
6. Progestins: Norethindrone, medroxyprogesterone acetate
As their name suggests, these steroids have both anabolic and androgenic effects. Through a
combination of these effects, anabolic steroids stimulate muscle formation leading to increased
muscle mass and strength. Some of the most commonly used steroids, both medicinally as well as
otherwise, include the following drugs:
1. Prednisone: It is a dehydrogenated analogue of cortisol that is used as an anti-inflammatory
drug in the treatment of arthritis and as an immunosuppressant. It is used in a wide range
of autoimmune diseases, such as severe asthma, allergies and rheumatoid arthritis, and to
prevent and also treat rejection in organ transplantation.
Table 19.1 Synthetic medicinal steroids
Hormone Description Therapeutic indication
Testosterone Produced by testes, longer half-life. Hypogonadism, postmenopausal breast
Methyl testosterone Synthetic androgen, half-life more
than testosterone
Male hypogonadal disorder and
female breast cancer
Recent Advances in Biopharmaceuticals
291
2. Nandrolone: It is an androgen (trade names: Durabolin or Kabolin) used to treat testosterone
deficiency or breast cancer or osteoporosis. The positive effects of the drug include muscle
growth, increased red blood cell production, appetite stimulation and increased bone density.
3. Anavar: Being extremely mild and anabolic, it is used for the treatment of alcoholic hepatitis,
Turner’s syndrome and weight loss caused by HIV. It is used for promotion of strength and
duality of muscle mass gains; although its mild nature makes it less than ideal for bulking
purposes.
4. Winstrol: The anabolic properties of winstrol are mild in comparison to many other steroids.
It increases strength without excess weight gain, promotes vasculature and therefore famous
amongst bodybuilders.
5. Steranobol: Chemically, steranobol has a chloro-group added at the 4-position of testosterone.
It is an artificial, synthetic androgenic steroid with anabolic effects similar to testosterone. It is
frequently misused to improve physical performance in sports and athletics. It is prohibited by
many sports authorities, including the International Olympic Committee.
6. Clenbuterol: It is widely used as a bronchodilator in many parts of the world. It is known to
directly stimulate the adipose tissue and accelerate the breakdown of triglycerides to form free
fatty acids.
Oestrogen
`-Oestradiol is the principal oestrogen produced by the ovary. Oestrogens are synthesized in all
vertebrates, as well as some insects. Oestrogenic sex hormones have an ancient evolutionary history.
Oestrogens are used in oral contraceptives, oestrogen-replacement therapy for postmenopausal
women and in hormone replacement therapy for transwomen. It is 10 times more potent than
oestron and 25 times more potent as compared to oestriol. Like all steroid hormones, oestrogens
readily diffuse across the cell membrane. Once inside the cell, they bind to and activate oestrogen
receptors, which in turn, modulate the expression of many genes.
The medicinally important oestrogens are as follows:
1. Ethinylestradiol is used for oestrogen replacement therapy in deficient states, both pre- and
postmenopausal, and breast cancer.
2. Oestradiol is natural oestrogen used in oestrogen-replacement therapy in menopausal and
menstrual disorders.
3. Mestranol is a synthetic oestrogen and used in treatment of menopausal conditions.
Progesterones and Progestogens
Progesterone is a hormone produced by corpus luteum. Progestogens or gestagens are a group of
hormones, including progesterone. The progestogens are one of the five major classes of steroid
hormones, in addition to the oestrogens, androgens, glucocorticoids and mineralocorticoids. The
progestogens are characterized by their basic 21-carbon skeleton called a pregnane skeleton
(C21), and they are both naturally occurring and synthetic. They are named for their function
in maintaining pregnancy (progestational), although they are also present at other phases of the
oestrous and menstrual cycles. The oestrogens possess an estrane skeleton (C18) and androgens, an
andrane skeleton (C19).
Textbook of Pharmaceutical Biotechnology
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Progesterone is mainly used in the treatment of menstrual disorder and breast and endometrical
cancer. They are used in hormone replacement in addition to being used as contraceptive agents.
The uses of synthetic progesterones are summarized in Table 19.2.
The other steroidal hormones used therapeutically are corticosteroids, catecholamines and
prostaglandins. The therapeutic uses of synthetic prostaglandins are in gynaecology. They are used
in inhibition of platelet and for induction of vasodilation secretion of gastric acid.
BIOPHARMACEUTICALS OF PLANT ORIGIN
Recombinant Proteins
The plant produces wide varieties of bioactive molecules by secondary metabolic pathways. They
are now gaining widespread acceptances as a general platform for the large-scale production of
recombinant proteins. The proteins obtained from plants may be used as diagnostic reagents and as
vaccine drugs for the production of recombinant proteins on industrial scale. Important advantage
with plants in the production of recombinant protein is that vaccine candidates can be expressed
in edible plant organs (Table 19.3). The deliveries of recombinant protein in edible plant organs
are exceptional because they would be used locally for vaccination campaigns. Potato was the first
major system that was used for vaccine and transgenic production.
Plant-derived proteins can be used for the production of insulin, human growth hormones,
interferons, monoclonal antibodies, etc.
Plant-Derived Therapeutics
The plants are the sources of many important secondary metabolites, which are used as therapeutic
agents in the treatment of wide spectrum of human ailments. Alkaloids, glycosides, flavonoids, steroids,
etc., are important groups of secondary metabolites obtained from plants and used systematically in
treatment of diseases. Some important drugs of plant origin have been described in Table 19.3.
Alternative Plant-Based Expression System
Most of the plant-derived recombinant pharmaceutical proteins have been produced by
biotransformation and from transgenic plant lines. There are some advantages and limitations in
use of plant-based production system, which are described in Table 19.4.
Table 19.2 Synthetic progesterones
Hormone Description Use
Megestrol acetate Synthetic progesterone Treatment of endometrial carcinoma and
some forms of breast cancer
Medroxyprogesterone acetate
Synthetic progesterone Treatment of menstrual disorder,
endometriosis and contraceptive
Recent Advances in Biopharmaceuticals
293
PHARMACEUTICAL SUBSTANCES OF MICROBIAL ORIGIN
The microorganisms produce wide spectrum of secondary metabolites of therapeutic significance.
Antibiotics are by far the most important group of substances produced by microbial strains, and
this group of pharmaceuticals has had the greatest single positive impact on human health care in
history. Antibiotics are defined as low molecular mass microbial secondary metabolites that at low
concentration inhibit the growth of other microorganisms. Around 12,000 antibiotics have been
isolated and characterized so far. Several fungal strains are known to produce antibiotics. The first
antibiotic to be used medically, penicillin, was extracted from Penicillium notatum. Sir Alexander
Fleming in 1928 noted the ability of the mould P. notatum to produce an antibiotic substance.
Approximately, 55% of antibiotics are produced by the genus Streptomyces (filamentous bacterial
group), 15% from other actinomycetes bacteria and rest from filamentous fungi.
Table 19.3 Some important therapeutic agents obtained from plants
Drugs Biological source Chemical type Therapeutic indications
Aspirin
Salix alba
Salicylic acid Anti-inflammatory and analgesic
Atropine
Atropa belladonna
Alkaloids Dilation of pupil
Digitoxin
Digitalis purpurea,
Digitalis lanata
Steroids Cardiac stimulants
Morphine
Papaver somniferum
Alkaloids Analgesic
Taxol
Taxus brevifolia,
Taxus baccata
Terpenoids Breast cancer, acute lymphoma in
children and adult
Vincristine, vinblastine
Catharanthus roseus
Alkaloids Blood cancer, Hodgkin’s disease
Sennosides A and B
Cassia angustifolia,
Cassia acutifolia
Glycosides Purgative
Podophyllum
Podophyllum hexandrum
Resin Purgative, cholagogue and bitter tonic
Citronellal
Cymbopogon nardus
Volatile oils Insect repellant
Turmeric
Curcuma longa
Terpenoids Anti-inflammatory, condiment and spice
Aloe
Aloe barbadensis,
Aloe vera
Glycoside Purgative
Rauwolfia
Rauwolfi a serpentine
Alkaloids Antihypertensive and tranquillizer
Scopolamine
Datura metel
Alkaloids CNS depressants and asthma
Xanthine
Theobroma cacao
Alkaloids CNS stimulant and diuretic
Table 19.4 Advantages and limitations of plant-based production system
System Advantages Limitations
Transgenic plant, accumulation
within plant
Good yield, economy scalability;
establishment of permanent lines
Restricted for production, timescale,
regulatory compliance
Virus-infected plants Good yield, manageable timescale Biosafety, construct size limitation
Cell or tissue culture Defined timescale, containment,
secretion into medium, possibility of
bioconversion
High cost of production
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Major Families of Antibiotics
1. `-Lactams 5. Ansamycins
2. Tetracyclines 6. Peptide and glycopeptides
3. Aminoglycosides 7. Miscellaneous antibiotic
4. Macrolides
The major families of antibiotics are described in Figure 19.2.
There are around 124 antibiotics available in the market till August 2010.
1. ȕ-Lactams: These are `-lactams that include penicillin and cephalosporins. Penicillin refers to
a family of both natural and semisynthetic antibiotics. Naturally produced penicillin includes
penicillin G and V. The examples of penicillin are phenethicillin, procillin, oxacillin, etc. The
cephalosporins have an antibiotic mechanism of action identical to that of the penicillin. It is
the prototypic natural cephalosporins.
2. Tetracyclines: They are the family of antibiotics that display a characteristic 4-fused core
ring structure. They exert their antimicrobial effect by inhibiting protein synthesis in sensitive
microorganisms. Chlortetracycline was the first member of this family to be discovered in
1948. Tetracyclines gained widespread medical use due to their broad spectrum of activity
that includes not only Gram-negative bacteria but also mycoplasm, rickettsias, chlamydias
and spirochaetes.
3. Natural tetracyclines: Oxytetracyclines, chlortetracycline and demeclocyclines.
4. Semisynthetic: Methacyclines, doxycyclines and minocyclines.
5. Aminoglycoside antibiotics: The aminoglycosides are a closely related family of antibiotics
produced almost exclusively by members of the genera Streptomyces and Micromonospora.
They are polycationic compounds of a amino alcohol to which amino sugar is attached.
Therapeutically important aminoglycosides are streptomycin, neomycin, tobramycin,
framycetin, paromomycin, gentamicin, sisomicin and aminoglycoside.
Figure 19.2 Major families of antibiotics.
Ma or amilies o antibiotics
Pepti e an gl copepti es
β actams
etrac clines
Miscellaneous antibitotics
Aminogl cosi es
Macroli es
Ansam cins