Pharmacology and Physiology,

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Pharmacology and Physiology, Pharmacology
Lectures BIOL243 / BMSC 213
Dr Paul Teesdale-Spittle School of Biological
Sciences KK713 Phone 6094
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• Bioassays
• Bioassays are the experiments by which the
  pharmacological activity of a compound is
  determined.
• Determination of type and level of response.
• Measurement of concentrations.
• Determination of other responses, including
  toxicity.
• Assays of this sort are generally undertaken on
  whole animals or isolated animal tissue samples.
• Because of the variability it is essential in
  assays of this sort to include a reference
  compound of established activity.
• Relative activities can be compared.

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Experiments are done with many replicates on
large test groups to be statistically valid. This
raises issues of ethics, particularly on toxicity
tests such as LD50 determination. Given the
ethical questions (and expense) of traditional
animal experiments, these are now usually only
performed in the later stages of drug
development. Where possible replacement assays
based on in vitro isolated target, cell culture
or tissue samples are preferred. It is beyond the
scope of this course to evaluate these. Elements
of serendipity step in at the bioassay
stage. Sometimes the effect of metabolism or the
presence of additional targets within a whole
animal leads to unexpected outcomes.
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• The differences between animal and human are
  large e.g. in the routes of drug metabolism and
  target responses.
• There has to be controlled experimental exposure
  of new agents to human test subjects.
• These experiments are broken down into three
  distinct phases, based on the type of information
  and test subject.
• Phase I Undertaken in healthy individual
  volunteers.
• Drug distribution, side effects and potency.
• Phase II As with Phase I, but undertaken with
  small groups of patients.
• Phase III Undertaken using patients where the
  drug is used in their therapeutic treatment.
• These are the true clinical trials.

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• In a phase III trial, the new drug or treatment
  is compared to an existing drug or to a placebo
  or similar control.
• Two randomised groups of patients are generated
  and placed under a controlled therapeutic regime
  of either new drug or control.
• The groups might not be completely random, but
  stratified to ensure equal mixes of functions
  such as gender, age and ethnicity.
• Often trials will be based on a crossover
  procedure.
• The two groups are swapped at some stage in the
  trial.
• Neither patient nor investigator should be aware
  of which group a patient falls into.
• Such trials are referred to as double blind.
• This is done to avoid biasing of the results.

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• Questions
• Why might such knowledge bias results?
• What are the ethical considerations involved in
  trials as described?

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• Some clinical trials are designed in such a way
  as to allow them to be stopped as soon as a
  desired level of proof has been achieved.
• In the sequential trial design, patients from
  each of the two randomised groups are paired with
  each other.
• The outcomes from their treatments with the new
  drug, an existing drug and/or a placebo, are
  continuously monitored noted in terms of which,
  if either, patient is experiencing the better
  therapeutic outcome.
• As soon as a predetermined level of proof is
  obtained to categorise the new drug as better,
  worse or no different than the control
  treatments then the trial is stopped.

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• A thorough statistical analysis of the data from
  clinical trials is required.
• Ideally need large sample sizes.
• The first stage of trials will only answer the
  question Is the new drug, administered under a
  chosen regime better than the existing practice?
• Improvements in regime require further trials.
• Clinical trials are also the first point at which
  human toxicity can be fully investigated.
• Whilst animal experiments are useful they do not
  necessarily
• Measure toxicities (side effects) that are not
  lethal.
• Detect infrequent severe reactions (such as if a
  drug kills 1 in 1000 individuals at low
  concentrations).
• Consider low level, long-term dosage effects.
• Reflect differences between human and animal
  metabolism.

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• Monitoring should continue, even after a drug has
  been released for general use.
• A Phase IV clinical trial on a very large and
  diverse population.
• It is not unknown for a drug to be pulled out of
  clinical usage on the basis of a gradual build up
  of knowledge of adverse effects.
• Some countries have a yellow card system where
  clinicians are obliged to contact a
  government-based medical panel if an adverse
  effect of a drug is noted.
• Allows national, and even international data to
  be collated and compared.

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• Drug administration
• Once administered, a drug has to reach its site
  of action.
• e.g. Crossing barriers, such as membranes,
  before it enters the bloodstream and permeates to
  the lymphatic system.
• Once available, the drug can be distributed
  systemically, or in a targeted fashion to one or
  more sites within the body.
• Features that affect the ability of a drug to
  reach its target
• Route by which the drug is administered
• Ability to cross membranes
• Tendency to become localised within
  compartments within the body
• Ability to be excreted, possibly as a result of
  metabolic modifications.

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• The routes of drug administration are broken down
  under two categories
• Enteral, where the drug is administered through
  an interface that leads to absorption via the
  gastrointestinal (GI) tract.
• The term enteral comes from the Greek for an
  intestine - enteron.
• Examples include oral, sublingual and rectal
  routes.
• Parenteral, where the drug is delivered in a
  manner that avoids the GI tract.
• Examples include intravenous, intramuscular,
  subcutaneous, epidural, ocular, inhalation and
  intra articular.

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• Enteral administration
• Generally, the oral route is preferred by
  patients.
• Best compliance to therapy.
• When a drug is administered orally, it has to
  cross a barrier of epithelial tissue.
•  
• The oral cavity
• The epithelium is smooth, thin and multi-layered.
• The salivary environment is generally slightly
  acidic.
• The blood flow from the mouth is not passed
  directly to the liver where metabolic degradation
  of the drug would be likely.
• Once a drug has entered solution, it will have
  only a very limited lifetime within the mouth.
• Small tablets can be held under the tongue this
  is sublingual administration.

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• The stomach
• The alimentary canal is essentially a hollow tube
  walled by a series of 4 layers.
• These are the mucosa (inner surface), submucosa,
  muscularis and serosa.
• Multiply folded and a single layer of cells
  thick.
• The stomach acidity (pH 2) is sufficient to
  suppress ionisation of organic acids and promote
  ionisation of bases.
• BUT only uncharged species are able to cross
  lipid membranes.
• e.g. Strychnine is poorly absorbed from the
  acidic stomach.

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The small intestine The purpose of the small
intestine is to adsorb exogenous material from
food. Has macroscopic (the folds of Kerckring),
milliscopic (projecting villi) and microscopic
folds (microvilli) and projections lead to an
enormous surface area. A small intestine is
typically 280 cm long but has a surface area of
about 200 m2. The pH gradates from 4-5 near the
stomach to weakly alkaline. This allows for
absorption of both weakly acidic and weakly basic
drugs. It usually takes several hours for
material to pass through the small intestine.
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• The large intestine
• Not primarily an absorption site, and so its
  epithelial layer lacks the features of the small
  intestine, such as the microvilli.
• Adsorption of residual drug that has passed
  through the small intestine can continue in the
  large intestine.
• It can be desirable to administer some drugs via
  the rectum.
• A patient is unable to take or retain orally
  administered compounds.
• The drug would be broken down by proteolytic
  enzymes.
• The drug is too unpalatable orally
• To avoid metabolic degradation in the liver (the
  blood flow from the rectum does not pass through
  the liver on the way to the heart).

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Parenteral administration Parenteral
administration presents a different set of
problems to the use of enteral routes. Generally,
there are fewer issues regarding pH, adsorption
and metabolism. There is usually more immediacy
of action. These advantages are offset against
discomfort and reduced ability to retract
administration.
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Ocular administration The eye has a combination
of lipophilic and hydrophobic layers, and
contains systems designed to clear the eye of
exogenous material e.g. solution drainage and
tear production. Less than 10 of most drugs
delivered to the eye are actually absorbed, and
about 90 of absorbed drug enters systemic
circulation. Thus typically only about 1 of a
drug administered through the eye actually enters
the eye itself.
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Dermal administration The skin is a very good
barrier. Its outer layer consists of a tightly
packed, partially desiccated array of dead cells
with a high keratin content. This is the stratum
corneum. Whilst the sweat glands do puncture the
stratum corneum, they do not provide a pathway
for absorption of drugs. It is also not uncommon
to deliver drugs via the skin. The only
qualitative difference between absorption through
the skin and the epithelial layers of the GIT is
that the rate of passive diffusion is low.
Exercise List as many dermally delivered
drugs or classes of drugs as you can. Hint
Think in terms of patches, creams, lotions, gels
and similar. Try and categorise whether these
drugs are expected to cause their effect locally
or distant to the site of administration.
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• The respiratory tract
• The site absorption of drugs within the
  respiratory tract is governed by where they are
  deposited.
• Gasses, volatile liquids and small particles
  reach the alveoli.
• Smaller than 10 ?m, typically around 2 ?m.
• Includes bacteria, viruses, fumes, pollen, smoke
  and aerosols (e.g. asthma inhalers).
• In an alveolus the epithelail layer is 0.5-1.0
  ?m thick, about 100 times thinner than the
  equivalent spacing in the skin or intestine.
• Larger particulates are generally deposited and
  absorbed higher up the respiratory tract.
• Undissolved solids are removed by the action of
  cilia, which push them back to the nose or mouth.

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Intravenous administration In considerations of
drug bioavailablity, the amount of drug entering
the system is always compared to that when the
drug is administered intravenously. Intravenous
administration makes the maximum possible amount
of drug bioavailable, since all of the
administered drug enters the body. Exercise
List at least 3 advantages and 2 disadvantages of
intravenous administration.
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Intramuscular and subcutaneous administration
Intramuscular administration usually involves
injection into the muscles of the buttocks, side
of the thigh or upper arm. Subcutaneous
administration delivers the drug directly under
the skin. Drug adsorption is governed by the rate
of transport across the walls of the vasculature,
in particular the capillaries. Rate of absorption
increased through increases in blood flow and
contact between drug and capillaries. e.g.
Exercise and massaging the area around the
injection Co-injection with a compound that
constricts blood flow through the capillaries
will slow release from the site of
administration, which can be beneficial for
application of local anaesthetics.
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The walls of capillaries are composed of
endothelial cells. More easily crossed than
epithelial cells (exception the blood brain
barrier). Capillaries allow the passage of both
polar and non-polar species. The mechanisms of
transport are different for each. Non-polar
species are generally absorbed by passive
diffusion. Polar species probably pass between
the endothelial cells, and their rate of
transport depends on their size. Even
macromolecules, such as proteins can enter and
leave from capillaries, but their rate of
transport is low. Large molecules may also be
transported away from the site of injection in
the lymph.
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• Absorption, distribution elimination
• Bioavailability and crossing membranes
• The activity of a drug depends on two unrelated
  factors
• Its concentration at the site of action
• Its ability to interact with its target site.
• To reach its site of action, a drug will usually
  cross lipid membrane(s)
• The rate of transport of a drug across a membrane
  is given by

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Rm and Raq are approximately the same for related
compounds. So the rate of transport can be
considered to depend only on the partition
coefficient and the concentration differential.
Maximum rate of transport requires a high
concentration gradient, and thus high water
solubility (hydrophilicity), and a large
partition coefficient (i.e. high lipophilicity).
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pH effects Ionised species do not readily cross
membranes passively, although there are some
mechanisms by which they can be actively
transported. The partition coefficient at a given
pH (PpH) depends on the value of P of the neutral
compound (Pneutral) and the fraction of the
compound that is unionised at the pH under
consideration (f). PpH Pneutral.f
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A weak acid typically a carboxylic acid.The
ionisation is given by the equation HA ? H
A-
PpH PHA.fHA log PpH log PHA log fHA
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pKa -log10 Ka and pH -log10 Hso Ka
10-pKa and H 10-pH. Also xa/xb
x(a-b)
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• Drug distribution
• Once a drug become bioavailable, it is likely to
  be distributed amongst the various components of
  the body.
• The percentages of body weight of body
  components
• Water 55-60
• Fat 15-20
• Protein 12
• Carbohydrate 0.5

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Fat Partition of drugs into fat is not generally
significant. This is even true for many quite
lipophilic drugs, although there are
exceptions. Very little contact surface area
between the vasculature and the body fat
deposits. Lipophilic drugs tend not to be found
as free solutes but rather are associated with
plasma proteins, so they are not partitioning out
of water.
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Plasma proteins Plasma consists of 90 water,
8 plasma proteins and 2 other organic or
inorganic species. Many drugs bind to the plasma
proteins as they have low water
solubility. Albumin provides most of the
available sites for absorption, particularly of
acidic drugs. ?-globulin and an acid glycoprotein
can become important can become important in
binding basic drugs. The characteristics of this
type of binding would be expected to follow that
for the binding of an agonist to a receptor.
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BUT there are multiple potential binding sites
and competition for binding sites between a drug
and other ligands. A saturating hyperbolic
binding curve generally holds true. At clinically
relevant drug concentrations, the ability of
albumin to bind a drug is not saturated. Albumin
is found at around 0.6 ?M and can typically bind
two drug molecules, the binding is not fully
saturated until the plasma drug concentration is
above 1.0 ?M.
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Body fluid compartments About 2/3rds of the total
body water is intracellular The remaining 1/3rd
is extracellular. The compartments for the
extracellular fluid are transcellular fluid,
plasma and interstitial fluid. These are found at
ratios of approximately 1310. One measure of
distribution of a drug around the various body
fluid compartments is the distribution volume,
Vd. Vd D/Cp Where D the dose that has become
bioavailable and Cp is the concentration of the
drug in the plasma. If the drug is administered
intravenously, all of it becomes bioavailable. If
this is retained within the plasma, then Vd is
the plasma volume.
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Where the drug is more widely distributed, more
drug was administered than is available in the
plasma, so the value of Vd is higher than the
total plasma volume. If the drug is strongly
sequestered, then the amount residual in the
plasma (Cp ) will be low. A low value of Cp will
lead to high values of Vd. It is possible to
obtain values of the volume of distribution that
are in excess of the total body volume! Values
of Vd are usually quoted as a fraction of total
body weight, as this compensates for differing
sizes of individuals to some extent. Exercise
Explain why this assumption is oversimplistic Wher
e the value of Vd is less than 0.1 Lkg-1, it
would indicate that the drug is not widely
distributed outside the plasma.
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Elimination The concentration of drug found
within the body is partly dependent on its
elimination. A combination of metabolic
transformation and excretion of both the original
drug and its metabolic by-products.   Metabolism T
he ideal end-point of metabolic transformation is
to enable a xenobiotic to be excreted, rather
than accumulate within the body.
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• The processes of drug metabolism are usually
  considered in three phases
• Phase I the introduction or revelation of a
  reactive functional group. These are almost
  inevitably nucleophilic -OH and NH groups.
• Phase II conjugation of functional groups,
  such as those revealed in phase I, in order to
  enhance water solubility and so aid excretion.
• Phase III further transformation of a phase II
  product.
•  
• There are no simple rules to govern which routes
  of metabolic transformation will be followed by a
  given drug.
• Here are a few pointers to common themes in drug
  metabolism.

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• Phase I
• The principal phase I metabolic transformations
  are oxidation, reduction and hydrolysis.
• Oxidation - The mixed function oxygenase (MFO)
  system.
• Found throughout the body, activity is
  particularly noticeable in the smooth endoplasmic
  reticulum of the liver.
• The most commonly invoked enzymes of the MFO
  system belong to the cytochrome P-450 (more
  colloquially CYP-450, or simply P-450) family.
• Especially valuable in oxidation of hydrophobic
  substrates.
• Convert an unactivated C-H bond into a C-OH
  group.
• Their iron atom chelated to a haem group
  complexes molecular oxygen, O2.
• The overall transformation is given by the
  reaction
• R-H O2 2H 2e- ? R-OH H2O

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f) Displacement of the substrate from the active
site.
a) Binding of the C-H containing substrate.
e) Attachment of the iron-bound OH group to the
radical centre of the substrate.
d) Abstraction of the H atom from the C-H bond.
b) Binding of molecular oxygen.
c) Loss of one oxygen atom in water.
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• Alkenes and aryl groups often become activated
  to epoxides
• Alkyl groups become converted into alcohols
• Sulphides and tertiary amines can be converted
  to their oxides
• Alkyl halides and, in some cases, primary amines
  can become carbonyl compounds (aldehydes or
  ketones)
• Alkyl groups can be cleaved from amines and
  ethers.Other enzymes
• Flavin monoxygenases
• Monoamine oxidase
• Xanthine oxidase and
• Alcohol dehydrogenase.

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• Reduction There are a number of enzymes
  responsible.
• Aldehydes and ketones give alcohols.
• Azo compounds (RNNR) and nitro groups give
  amines.
• Hydrolysis - Hydrolysis by esterases,
  carboxypeptidases and aminopeptidases.
• Esterases have broad substrate specificities
• Found within the liver, kidney, other tissues and
  in the plasma.
• Esters into more polar carboxylic acids and
  alcohols.
• Amides into more polar carboxylic acids and
  amines.
• Ester hydrolysis is often rapid.
• Hydrolysis of amides generally slower.

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Phase II In phase II transformations, reactive,
usually nucleophilic, groups are conjugated (or
linked) to polar moieties to ensure efficient
excretion. Acylation - usually of amines. May
often serve the purpose of deactivation rather
than polarisation. Amino acid conjugation
formation of an amidic linkage to the ?-amino
group of an amino acid. Glucuronidation
conjugation of alcohols, amines, phenols, thiols
and, sometimes, carboxylic acids
Glutathionylation glutathiones nucleophilic
thiol (-SH) group reacts with electrophilic
centres (e.g. epoxides, alkyl and aryl halides)
Methylation occurs with amines, phenols and
thiols. Also a deactivation process. Sulphation
usually the introduction of a sulphate ester to a
phenol or alcohol.
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Phase III Some phase II products are further
transformed.   First pass metabolism A
classification of drug metabolism based upon the
stage during the life-cycle of the drug in the
body at which it is metabolised. Metabolism in
the intestine and in the liver are most commonly
problematical, as these can severely reduce the
amount of drug that becomes bioavailable. Drugs
which are particularly susceptible to first pass
metabolism need to be administred in higher doses
or by routes that avoid the intestine and the
liver before bioavailability is achieved.
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• Metabolic activation
• Drugs that have been designed to be activated by
  phase I or phase II metabolic processes are
  called prodrugs.
•  
• Excretion
• The rate of excretion can vary from rapid to
  slow.
• It is to be expected that the products of drug
  metabolism will generally be cleared more rapidly
  than the parent drug.
• There are three processes that lead to renal
  excretion
• Glomerular filtration
• Tubular secretion/reabsorption and
• Passive diffusion across the renal tubule.

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• Glomerular filtration
• The endothelium of the glomerular capillaries is
  fenestrated.
• Provides a semi-permeable size-exclusion
  membrane
• Mr lt 10,000 free passage
• Mr gt 70,000 no passage, except some neutral
  species of weights up to 100,000 which can pass
  through the fenestrations.
• The glomerular capillaries provide a filtering
  surface area of over 1 m2.
• Equilibrium between the concentration in the
  plasma and in the receiving vessel the
  Bowmans space can be established.
• With small solutes the concentrations in the
  plasma and the fluid of the Bowmans space are
  the same.
• Drugs that are strongly bound to plasma proteins
  will have a much lower glomerular filtration rate
  than might be expected.

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• Active tubular secretion/reabsorption
• Only about 20 of the plasma that enters the
  kidney is subjected to glomerular filtration.
• The remainder enters the peritubular capillaries.
• They surround the tubules that lead from the
  Bowmans space, and therefore carry its filtrate
  from the glomerulus.
• There are transport processes designed to recover
  essential small molecules or ions lost in
  glomerular filtration (e.g. water, Na, K and
  Cl- ions amino acids and some peptides) and also
  to remove further undesirable solutes from the
  plasma.
• In addition to passive diffusion, there are two
  active transport mechanisms for this latter
  secretion process
• One to drive the secretion of acidic species and
  the second for secretion of bases.
• Drugs that are protein-associated can be cleared
  from the plasma, and up to 80 of some drugs can
  be cleared from the plasma through tubular
  secretion.

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Passive tubular secretion/reabsorption Lipophilic
drugs can become reabsorbed via passive
diffusion. It is possible to trap a drug in the
urine through pH changes leading to drug
ionisation.   Clearance 125 mL min-1 of plasma is
subjected to glomerular filtration. The total
plasma flow through the kidney is 700 mL
min-1. The total plasma volume is usually between
2 and 3 L. Urine is produced at around 1 mL
min-1, a rate that is very variable. It is common
to express the overall effect of excretion in
terms of clearance, which is the volume of plasma
that had contained the amount of substance
cleared from the kidney in unit time.
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• Exercise
• Try and describe the clearance profile with time
  for the following cases.
• If possible draw a schematic graph of drug plasma
  concentration against time.
• A drug that is cleared only by glomerular
  filtration, but is completely filtered by this
  process.
• A drug that is completely removed from the
  plasma by active transport mechanisms in the
  peritubular capillaries.
• A drug that enters the urine by passive
  diffusion.
• glomerular filtration 125 mL min-1
• Total plasma flow through the kidney 700 mL
  min-1.
• Total plasma volume 2 - 3 L.
• Urine is production 1 mL min-1