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had much slower encainide elimination (T 1/2 = 7.6 hr.), higher plasma encainide
concentrations and, yet, no antiarrhythmic or electrocardiogr^hic effects at
dosages up to 125 mg every six hours. Chraratographic analysis of plasma frcm
the responding patients revealed that the 0-demethyl metabolite (ODE) of encainide
vas present in greater quantities than parent drug; yet in the non-responder it
ViQS hardly detectable except at very high dosages of encainide. Subsequent
studies in a larger nuiriber of patients found that about ten percent of patients
appear to lack the ability to rapidly O-demethylate encainide, and in this group
encainide accumulates to high plasma concentrations. Dr. Woosley and his co-
Vvorkers have accumulated a great deal of indirect evidence indicating that the
metabolites of encainide contribute to the antiarrhythmic efficacy seen diaring
encainide ther^y. To test their liypothesis that one or more of the metabolites
of encainide was pharmacologically active, these investigators conpared the
antiarrhythmic and electrocardiographic effects of ODE and encainide in an animal
model of ventricular arrhythmias, the aconitine-intoxicated rat. ODE was 50-fiDld
more active than encainide against aconi tine-induced ventricular tachycardia.
Plasma analysis indicated that the netabolite was present shortly after encainide
administration in concentrations that appeared to be active in this model.
However, inhibition of metabolism showed a protective effect of encainide alone.
Therefore, both parent cctipound and the 0-demethyl metabolite were active, the
metabolite being the more active of the tv\o.

The correlation of plasma concentration and antiarrhythmic activity of OCE
indicates the potential for a safer, more predictable use of this metabolite as
corpared to encainide. Final proof of the relative safety and efficacy of all
the metabolites will be determined by their administration to patients with
ventricular arrhythmias.

" Regulation of B-Adrenergic Receptor Function "
ROl (3^ 29522-01 (Perkins, J.), University of North Carolina at Chapel Hill

Dr. Perkins ' group has been studying the agonist-induced down-regulation of the
response of the adenylate cyclase syston to catecholamines. Short-term exposure
of human astrocytcma cells to catecholamines results in a reduction of the capa-
city of the catecholamine-occupied 3-adrenergic receptor (BAR) to increase adenyl-
ate cyclase activity. While no change in the number of BAR or in any other param-
eters of adenylate cyclase activity vas ^parent, the steady-state level of adenyl-
ate cyclase activity following incnjbation of cells with isoproterenol dropped to
a new level of 40-50 percent of control. Recovery was r^id and carplete upon
removal of the catecholamine from the medium. The investigators propose that
an uncoupling occurs between the receptor and the adenylate cyclase such that
occupation of the BAR is no longer capable of stimulating enzyme activity.
Receptor binding studies also indicated that the early phase of desensitization
involved uncoupling of BAR and adenylate cyclase. Following short incubations


(5-30 minutes) with isoproterenol (ISO), the apparent affinity of the agonist
for inhibition of iodohydroxypindolol (IHYP) binding was reduced ten-fold with
no charge in the nuittoer of receptors. The time course of this change in affinity
of agonist binding was siinilar to the tine course of loss of adenylate cyclase
activity. Both binding affinity and isoproterenol-stiraulated adenylate cyclase
activity returned to control levels with similar time courses following removal
of isoproterenol.

Dr. Perkins suggests the following kinetic model for the agonist-specific desen-

BA% ^i:^ BARu ^ BARl

vAiere BARj^j is the native receptor, BARy is the altered form of the receptor v^hich
binds agonist but does not activate adenylate cyclase, and BARl is the form of
receptor vvhich occurs after long exposure to isoproterenol and v\hidi is not detect-
able even by antagonist binding techniques. The initial process can be envisioned
as involving the reversible formation of the altered receptor. During short-term
incubation with catecholamine, the receptor exists in both the native and altered
states and the steady state levels of these two forms (and subsequent response of
adenylate cyclase) is determined by the balance of the forward and reverse reac-
tions. Long term incubation with catecholamine results in a loss of receptors
and conversion of the BAR to the IHYP undetectable form (BARl) from \^ich recovery
is very slew.

The physical properties of the BARy differ fran those of BAR^. While native
rec^tors sediment as a single peak in sucrose gradients with adenylate cyclase
from isoproterenol-stimulated control or desensitized cells, the receptors fron
desensitized cells migrate as two peaks. Receptors from the light peak exhibit
properties similar to those of uncoupled receptors. The appearance of this
"light" form of receptor coincides on a terrporal basis with the agonist-induced
uncoupling reaction. The cellular location of these receptors is speculative at
this point since they sediment at densities lighter than markers for the plasma
menbrane. This "light" receptor peak does sediment at equilibrium densities sim-
ilar to thDse for Golgi apparatus, lysoscmes, and endoplasmic reticulum, suggest-
ing that the uncoupled receptors might exist in cytoplasmic vesicles resulting
frcm agonist-induced selective endocytosis of reenter protein.

" Pharmacokinetics - Pharmacodynamics of Iimiunoregulating Agents "
P50 GM 26691-03 (Benet, L. ) , University of California, San Franci sco

Among the challenges of pharmacokinetics has been to relate the kinetic measures
of a drug in the body to the pharmacological activity of that drug, and hence to
specific measures of clinical efficacy and toxicity. In the case of iimiunoregu-
lating drugs, these challenges have been aitplified by the difficulties in quanti-
tating these drugs in biological fluids at the levels present after administration
of therapeutic doses, and, perhaps more iirportant, defining precise measures of
pharmacological activity \Ahich can be related to clinical efficacy.

Dr. Leslie Benet and his colleagues have been pioneering in the field of "immuno-
pharmacokinetics " to find better ways of applying pharmacokinetics to this expand-
ing area of therapeutically iirportant drugs. These investigators have developed
sensitive and specific assays for azathioprine, 6-mercaptopurine , and for prednisone


and prednisolone as well as for the endogenous glucocorticoids. In addition,
because of the difficulty of defining any quantitative rneasure of efficacy ard
toxicity in the vvhole patient, intermediate treasures of immunological effect
vhidh can be applied to inmunoregulatory agents have been developed. These
workeirs have selected the inhibition of the mixed lyitphocyte reaction (MLR) as
a rational model for quantifying iitrnunosuppressive activity in plasma.

These investigators have recently studied the effects of prednisone, predniso-
lone and hydrocortisone on human peripheral blood lyitphocyte blastogenesis in
a two-way MU^. As measured by the inhibition of the MLR, prednisolone is
about twice as potent as hydrocortisone. Despite their differences in potency,
both prednisolone and hydrocortisone yield the same maximal inhibition. Predni-
sone, on the other hand, shows no significant itimunosuppressive activity despite
the significant slow conversion of prednisone to prednisolone in the MLR by
11-hydroxylation. The presence of prednisone had no effect on the irtiihition
due to prednisolone and hydrocortisone.

Ettplqying itiodifications of the MLR assay at 50 percent piastre concentration in
the mixed lyitphocyte cultures, a quantitative tteasure of iimunosuppression,
apparently related to pharmacokinetics, was developed. This measure, the area
under the irihibition-time curve, is calculated in a ttanner similar to the
ccrtimonly used phamacckinetic parameter, the area under the drug-plasma concen-
tration time curve. Dr. Benet and his group have used this new irethodology
to study the pharmacokinetics of prednisolone and prednisone in stable renal
transplant patients and in patients with oral mucocutaneous vesiculoerosive
disease. In addition, the fharmacckinetics of prednisolone was cotpared
between cushingoid and non-cushingoid patients. While, in contrast to previous
literature r^xDrts, a significant decrease in prednisolone clearance in cushing-
oid patients was not found, they did find a significantly higher level of
hydrocortisone in cushingoid patients follcwing exogenous glucocorticoid dosing.

Additional studies have been aimed at extending and validating the correlation
between the intermediate index of iiTiraunosi:5)pressive activity and pharmacckin-
etics for this important class of drugs and their rtetaibolites .

" The Role of Prostaglandins in Hemcpoiesis "
ROl GtA 30410-01 (Zibch, V.), University of California at Davis

The granulocyte-iTBcrcphage progenitor cells are catimitted hematopoietic cells
vAiich are capable of proliferating and differentiating in the presence of a
glycoprotein termed "colony stimulating factor" (CSF). The name derives from
the fact that cloning of these progenitor cells in vitro in the presence of
CSF produces colonies of hematopoietic cells. Various sitall molecules, includ-
ing the prostaglandins, are known to alter CSF-induced proliferation.
Dr. Vincent Zibch has been investigating the role vAiich prostaglandins play
in the stimulation of granulopoiesis.

Using both turpentine- induced rat n^eloid hyperplastic bone marrow cells and
sodium caseinate-induced peritoneal itDnocyte/tttacrophages, labelled with -'-'^-
arac±iidonic acid. Dr. Zibch and his colleagues were able to show that CSF chal-
lenge resulted in the release of arachidonic acid from as yet unidentified
insnforane phospholipid(s) . The investigators speculate that the release of
arachidonic acid is due to activation of a cellular phospholipase A by CSF.


The aradhidonic acid v^iiicih is released by CSF is subsequently transformed into
products of both the cyclooxygenase (prostaglandins) and lipoxygenase (leuko-
trienes) pathways. The h^^perplastic granulocytic rnarrcw cells generated a sig-
nificantly greater proportion of lipoxygenase products than did the nonocyte/
macrcphage population, iirplying that the two cell populations may have differing
functional roles in hemopoiesis.

Hydrolysis of free aradhidonic acid frcm cellular phospholipid is the rate-
limiting st^ in the phospholipid^aradiidonic acid-prostaglandin/leukotriene cas-
cade. The investigators also tested the hypothesis that it is the products
of this cascade vAiich produce growth stimulation. Mouse bone marrow cells were
incubated in the presence and absence of various inhibitors of the lipoxygenase
and cyclooxygenase pathways. Drugs v^hidi inhibited both pathways also inhibited
CSF -induced proliferation of the marrcw cells. A selective inhibitor of the
lipoxygenase pathway (15-HETE) inhibited CSF-induced colony fiDrmation vAiile
indonethacin, v\hich at low concentrations selectively inhibits the cyclooxygenase
pathway, had no inhibitory effect on CSF-induced colony formation. This inplies
that it is the products of the lipoxygenase pathway, the leukotrienes , rather
than the prostaglandins which mediate granulopoiesis in response to CSF. Further
support for this was generated by adding a mixture of the lipoxygenase products
isolated fran rat peritoneal neutrophils to marrow cells incubated with a lipoxy-
genase-cyclooxygenase inhibitor. The inhibition of CSF-induced proliferation
was reversed by the addition of these lipoxygenase products and colony fortration
once again occurred.

" Use of Macrophage-like Cell Lines "
ROl GM 29042-02 (Rosen, O.), Albert Einstein College of Medicine

Taxol, \(\*iic±i possesses antileukemic and tumar inhibitory properties, is a novel
diterpene isolated fron the plant Taxus brevifolia . Dr. Susan Horwitz at Albert
Einstein College of Medicine has been investigating the actions of this drug both
in vitro and in the macrcphage-like cell line, J774.1, in an attenpt to identify
the mechanism of cytotoxicity of this drug. Unlike other antimitotic drugs, sudi
as colcliicine or vinblastine, vvhich inhibit microtubule formation, taxol enhances
both the rate and yield of microtubule assertbly. The critical concentration of
microtubule protein required for asseitibly is reduced and the microtubules formed
are stable to depolymerization by calcium or cold. Taxol also asseirbles tubulin
under conditions in v^ich polymerization would not nonnally occur, such as in the
absence of microtubule-associated proteins, exogenously added GTP, organic buffer,
or at low tenperatures . Taxol appears to act by stabilizing the interactions
between tubulin diners that lead to microtubule polymer formation. In Hela cells,
taxol produces unusual microtubule cytoskeletons characterized ty the presence of
discrete microtubular burriles, and cells treated with the drug accumulate in G2
and M phases. Under conditions vshich prevent the characteristic taxol-induced
bundle formation in J774. 1 cells, binding of taxol to intact cells is retained.
Thus, binding of taxol to microtubules is not in itself sufficient to prcmote
bundle formation.

Using radiolabeled taxol and either intact J774. 1 cells or purified tubulin frcm
brain. Dr. Fforwitz has found that taxol binds in a specific and saturable manner
to a sirgle, high affinity binding site on the microtubule. The stoichionetry of
this binding with the tubulin dimer concentration approaches one. Maximal binding
occurs at drug concentrations vvhich produce maximal grcwth inhibition. Structure-
activity relationship studies with a series of natural and semisynthetic taxanes


have shown that both cytotoxicity and microtubule stabilization in vitro require
an intact taxane ring and ester side chain. Small changes in the side chain and
in ring substituents can, however, be tolerated without loss of biological activ-
ity. Dr. Horwitz hopes that these studies will permit the incorporation of a
fluorescent moiety into the drug so that it can be used as a probe for micro-
tubule assentoly processes. In addition, genetic variants of the J774.1 line
that are highly resistant to the grcMth. inhibitory properties of taxol have
been isolated and cloned. These are being used to study the interaction of
taxol with tubulin and its associated proteins in an effort to further understand
the mechanism of action of this unique cytotoxic agent.

" Structure-Function Relationships of Lectins "
ROl GM 21882-08 (Etzler, M.), University of California, Davis

Many plants and animals contain agglutinins called lectins that can catibine
specifically with erythrocytes and other types of cells. The hemagglutinatirg
properties of these lectins are due to their ccmplementarity for specific carbo-
hydrate residues on the cell surface. Lectins are quite abundant in the seeds of
leguminous plants and differ fron one another in their specificities. To date
mDre than 800 plant species have been found to contain lectins, but only a rela-
tively small number of these lectins have been rigorously studied.

The specificities of lectins for carbohydrate residues have made than valuable
analytical tools for studies of ccmplex carbohydrates, both in solution and on
the cell surface. In fact the observations that seme lectins can specifically
agglutinate tumor cells have led to a large number of studies using lectins to
distinguish between normal and transformed cells. In addition to these uses,
some lectins have been found to have mitogenic properties, and several lectins
are known to be very toxic to mammalian cells.

All plant lectins characterized to date have been found to be oligomers of pep-
tide or glycopeptide chains. Although some lectins are canposed of identical
si±>units, other lectins contain more than one type of subunit. Superinposed on
this subunit heterogeneity is the finding that many lectins exist in multiple
molecular forms, called isolectins, vvhich may or may not have different
specificities .

Dr. Marilynn Etzler and her colleagues have been studying the lectins of the
seeds of Dolichos biflorus (Horse gram) for a number of years. These investi-
gations have consisted of structural studies, ccntparative studies of various
lectins and their modified forms, and studies of the development of the Dolichos
biflorus lectin during the life cycle of the plant.

The Dolichos biflorus seed lectin is a glycoprotein. The carbohydrate unit con-
tains mannose and N-acetylglucosamine and is linked to the protein through a
N-acetylglucosaminyl-asparaginyl linkage. Structural analysis was by pronase
digestion, alpha-mannosidase treatment, periodate oxidation and exhaustive
nethylation followed by gas-liquid chromatography and mass spectrometry. The
data indicate that the glycopeptide is a branched structure with mannose at
the branch point and that the branches are terminated by nonreducing alpha-
mannosyl residues.


The NH2-terminal amino acid sequences of the first thirty residues of subunits
I and II were determined and found to be identical. OOOH-Terminal amino acid
analysis of the individual subunits showed a sequential release of leucine,
serine and valine frcm Subunit I and aspartate, leucine and proline fron Subunit
II. A corparison of the peptide maps from isolated Subunits I and II showed
that all peptides were canmon to both subunits with the exception of one. This
unique peptide was shown to be derived from the COOH-terminal of the subunit.
The fbllcwing diagram summarizes the data to date:


SUBUNIT I / Met Ser Val Leu

— Identical Sequence —

Identical Peptide Maps

SUBUNIT II Met Pro Leu Asp

7 '.


Metal content of the lectin was investigated by atomic absorption spectranetry.
The lectin contains Ca^"*", Mg2+, Mn^"*", Zn^"*" and Cu^"*". Demetallization of the
lectin inactivat«a it as tested by affinity electrophoresis. Full activity could
be restored by the addition of Ca2+ alone or by a ccnibination of the metal ions.
None of the other metal ions added individually restored full activity.

The develqpimnt and distribution of the lectin during the life cycle of the plant
was followed using a sensitive radioimmunoassay. The lectin was first detected in
the seed of the plant 27 days after flowering and rapidly attained the high level
of lectin present in the mature seed. The lectin is confined to the cotyledons
and decreases at about the same rate as the other protein reserves during the
absorption of the cotyledons after germination of the seeds. No lectin was
detected in the roots of the plant throughout its life cycle. Althou^ the stems
and leaves were initially thought to contain some lectin, these tissues were found
instead to contain a glycoprotein that cross reacts with antibodies against the
seed lectin.

The cross reactive material (CFM) was isolated frcm the stems and leaves and
found to have an amino acid and carbohydrate ccnposition similar to that of the
seed lectin. The CFM is a dimer with one subunit identical in electrcphoretic
mobility to Subunit I of the seed lectin and a second unit of higher molecular
weight. Both subunits have the same NH2-terminal sequence. The available data
suggest that both the CFM and the seed lectin subunits may represent different
degrees of ccrtpletion or modification of a canmon polypeptide chain.

"Norpace® Kinetics and Bioavailability in Heart Failure "
ROl GM 28424-02 (Lima, J. ) , Ohio State University

The degree of drug binding to plasma proteins is an important determinant not only
of the drug's metabolism and distribution, but also of its pharmacodynamic activ-
ity because only free, unbound drug in plasma is available to interact at the
cellular level to produce a response. Moreover, the clearance of most drugs fron
blood is restricted to their free, unbound fractions. The fraction of drug bound


to plasma proteins is inversely related to the total (free + bound) concentration
of the drug in plasma over a wide concentration range. This relationship between
total plasma concentration and the fraction of the drug bound to plasma protein is
not clinically impDrtant for most drugs because the bound fraction is usually
constant over the relatively narrow plasma concentration range achieved following
administration of therapeutic doses to patients. It has been thou^t for many
years that albumin is the major protein v\hich binds drugs in human plasma, ^4ore
recently, however, it has been recognized that there is another carpDnent of
human plasma \«hich is also responsible for a significant amount of drug binding,
particularly in the case of basic drugs. This cotpDnent is a-j^-acid glycoprotein

Disopyr amide phosphate (Norpace®), a relatively nev antiarrhythmic drug, is a
basic drug v^ich is moderately to highly bound to plasma protein. It is an unus-
ual ccnpound in that its bound fraction appears to be concentration-dependent at
ther^jeutic plasma concentrations. Consequently, its free fraction is also con-
centration-dependent in the therapeutic dose range. In addition to its apparent
dependence on concentration, the bound fraction of disopyramide appears to vary
according to the source of protein used in binding studies in vitro . Widely
differing extents of binding of disopyramide to plasma protein have been reported.

Dr. John Lima has been trying to explain seme of these anomalies in disopyramide
distribution by comparing the binding of disopyramide to protein in various sources
commonly used in protein binding studies, and by studying the influence of concen-
tration-dependent protein binding on the pharmacokinetics and pharmacodynamics of
the drug in human subjects. Previous work from Dr. Lima's laboratory had indicated
that as much as 35 percent of disopyramide was bound to two independent sites on
albumin. More recent work indicates that these two sites are on different proteins.
Equilibrium dialysis binding studies of disopyramide to proteins in human plasma
and serum have now been used to treasure binding constants for these sites. The
first binding site has a higher affinity (approximately 800-fold) and lower capacity
for disopyramide as compared to the second site. The second site accounts for
only about 5-10 percent of the binding of the drug at concentrations ranging from
10~' - 10~3m and is identical to the binding of the drug to albumin.

The similarity in the binding constants between AAG and the first class of bind-
ing sites suggests that these are identical as well. Thus most of the binding of
disopyramide to human plasma protein is to AAG. This binding is torperature inde-
pendent in the tonperature range of 25° to 37" and is not affected by freezing,
storage, and thawing of the plasma, indicating an ionic-type binding.

Dr. Lima's group has neasured disopyramide binding to plasma from normal volun-
teers, to donor plasma obtained from a blood bank and to plasma and serum from
patients in the cardiac intensive care unit. The affinity constants character i-
zir^ the high (AAG) affinity binding sites were similar for cardiac patient and
donor plasmas but both were lower than for normal plasma. There was no difference
noted for the low affinity site. The capacity constant, a measure of how much drug
is bound, for cardiac patient serum was nearly twice that for normal volunteer
serum. Despite similar affinity constants for cardiac patient serum and for blood
bank donor serum, the capacity constant for cardiac patient serum was much higher
than for donor serum. In an atteipt to explain this anomalous relationship, the
investigators used dialysis and absorption techniques to restore binding in the
donor serum. This led to the theory that there exists in donor serum a conpetitive
inhibitor for disopyramide binding. The binding of several basic drugs to serum


and plasma proteins has been found to be reduced following contact of blood with
the rubber steppers of blood collection tubes. Work by Lima's group and others
now indicates that plasticizers leak out of the storage bags used by blood banks
and these plasticizers ccmpete with basic drugs like discpyramide for the hi(^
affinity binding site of AAG. In addition, Shard and co-workers have shown that
drug binding to protein can be altered by canpetition with heparin viien indwell-
ing heparinized catheters are used to obtain samples .

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Online LibraryNational Institute of General Medical Sciences (U.Annual report : National Institute of General Medical Sciences (Volume 1982) → online text (page 12 of 16)