`
`.43
`ELSVIER
`
`immunology Letters 50 (1996) [-15
`
`
`
`
`Mini-review
`
`The development of Cop 1 (Copaxone®), an innovative drug for
`the treatment of multiple sclerosis: personal reflections
`
`Department of Immunulcgy. The Weizmann Immune of St-iem-e. Relwrul. Israel
`
`Ruth Arnon
`
`Received l5 December l995; accepted 25 January [996
`. ..
`
`Ker-wardr: Copaxone’”: Multiple sclerosis; lmmunoépecific drug
`1. Introduction
`
`.—
`
`.m-———-
`
`
`
`The 14th of June 1995 was a milestone for me -— as
`on this day the file on Ccpolymer 1
`(Cop l) was
`submitted. under
`the name COPAXONE°, by the
`TEVA Pharmaceutical Company to the FDA' for ap-
`proval as a New Drug Application for the treatment of '
`Multiple Sclerosis. For Prof. Michael Sela and myself.
`together with our colleague Dr. Dvora Teitelbaum, this
`was a high point after over 27 years of persistent
`research effort, perseverance and tenacity of purpose.
`The purpose of this paper is to describe the history of
`the development of this drug, interspersed with a review
`of the scientific findings along the way. all from a
`personal perspective. when I am “a quarter of a century
`wiser". Since multiple sclerosis is an immunological
`disease and Cop l
`is an immunospecific drug, my
`perspective is that of an immunologist. and it delineates
`the course of events in a more or less chronological
`order. Beyond the story as such. the paper shows that
`studies with Cop 1 advanced side by side with the
`progress of immunology as a discipline and hence, that
`the increasing sophistication of the research tools and
`methods that emerged from time to time permitted a
`more in-depth study of the immune processes involved
`in activity of this innovative drug. However, the thera—
`peutic potential of this material was evident
`to us
`already 25 years ago.
`It all started in the early 19605. 1 was then a budding
`immunologist. or, to put it more precisely, a young
`chemist ‘corrupted’ into immunology. Immunology in
`those days was a completely different science from what
`it
`is today. much simpler. with more ‘yes’ or ‘no‘
`answers to issues that even now are not yet completely
`understood. Antibody structure and function Were just
`
`GIGS-247859651200 0L7 I996 Elsevier Science 5N. All rights reserved
`PII SOI65-2478l96)02505-0
`
`being revealed and elaborate studies on the structural
`aspects of antigens were conducted. under the coined
`term ‘immunochemistry', to arrive at better understand-
`ing of their interaction with the antibodies. In parallel.
`information on the cellular compartment of the im-
`mune system was just starting to accumulate. with the
`fundamental discoveries of Burnel and Jame.
`In our laboratory we were deeply involved in studies
`on the structural basis of the antigenicity of proteins.
`utilizing synthetic antigens comprising polymers and
`copclymers of amino acids [l]. Research with these
`polymers had been pioneered by Prof. Ephraim
`Katchalski. in whose laboratory both Michael Sela and
`myself received our training. Employing these synthetic
`protein-like molecules we (Michael Sela and i) could
`induce immune responses of almost any desired specifi-
`city, including that of non-protein moieties. 0! specific
`interest was the immune response to lipid components
`which. due to solubility problems. was not easy to
`either elicit or investigate. Hon/ever. conjugates in
`which synthetic lipid compounds were attached onto
`synthetic copolymers of amino acids elicited specific
`response to lipids such as cytolipin H. which is a
`tumor-associated glycolipid [2]. or sphingcmyelin [3].
`Furthermore, we demonstrated that both the sugar and
`lipid components of such molecules contributed to their
`immunological specificity. The resultant anti-lipid anti-
`bodies were capable of detecting the corresponding
`lipids both in water-soluble systems and in their physio-
`logical milieu. This was fhscinating. since it gave us a
`glimpse into some disorders involving lipid-containing
`tissue and consequently led to our interest in demyeli-
`nating diseases. namely disorders in which the myelin
`sheath. which constitutes the lipidvrich coating of all
`axons.
`is damaged. resulting in various neurological
`dysfunctions.
`
`M_YLAN INC. EXHIBIT NO. 1020 Page-1
`
`MYLAN INC. EXHIBIT NO. 1020 Page 1
`
`

`

`:V:..r.-.'.»_:.x-.~.-;x.'a"..1w..-.:'..r;.v.\'.r.r::s:;.:rt:.r.r:ALVA—Jr.y.:'.-.1='.-.‘.t;1:'.rn.~. 21 rrEIAezsclmurnitr ' V ‘
`
`
`2
`
`R. Amen I Immunology Letter: 50 (1996) 1—”
`
`Multiple sclerosis (MS) is the most frequent demyeli‘
`nating disease. Not much was known at the time, or
`even to date, about
`its aetiology or mechanism of
`triggering. It is a chronic inflammatory disease of the
`central nervous system (CNS),
`in which infiltrating
`lymphocytes lead to damage of the myelin sheath by
`means of immune processes. Hence, it is considered an
`autoimmune disease. In studies performed at a later
`stage, while I was spending my sabbatical leave with
`Dr. John Fahey at 'UCLA, in collaboration with Drs.
`George Ellison, Lawrence Myers and W. Tourtellotte.
`we showed that anti—ganglioside serological activity
`might also be involved in this disease, since antibodies
`against several brain gangliosidcs were detected in sera
`of MS patients, but not in normal individuals. (This
`activity was demonstrated by the capacity of the sera to
`cause complement-dependent lysis of liposomes con-
`taining the respective ganglioside in their lipid bilayer.)
`Moreover, an apparent correlation was indicated be-
`tween the severity of disease and the extent of liposome
`lysis [4].
`Since MS occurs only in the human species, it was
`necessary to develop animal models of the disease for
`the purpose of research. Already in 1937 Rivers [5] had
`observed that a single inoculation of laboratory animals
`with brain or spinal cord tissue in Complete Freund‘s
`adjuvant (CPA) led to an acute neurological autoim-
`mune disease resembling MS, which was designated
`Experimental Allergic Encephalomyelitis (EAE). Cell-
`mediated immune responses were shown to be involved
`in the pathogenicity of EAE. since the disease could be
`transferred by a angle inoculation of
`sensitized
`lymphoid cells [6.7]. In the early 19605 one of the
`myelin components, myelin basic protein (MBP). was
`identified as an enoephalitogenic agent, since when in-
`jected in its purified form it induced EAE in guinea pigs
`[8]. Moreover. the disease proved to be the result of
`cell-mediated response to the MBP, and its specificity
`was emphasized by the ability to prevent or suppress
`EAB by MBP or its modified derivatives [9—11].
`Our previous successes in the development of syn-
`thetic antigens and their valuable contribution to the
`understanding of several
`immunological phenomena,
`prompted us to take a similar approach with regard to
`EAE. We actually intended to synthesize an encephali-
`togen, and anticipated that if the encephalitogenic ac-
`tivity of MBP could indeed be mimicked by a synthetic
`molecule - it might provide us with a useful tool for
`investigating the mechanism of EAE. In parentheses I
`would like to acknowledge the support that we received
`from two people already at this stage of the research.
`The first was Prof. Otto Westphal who was excited by
`our original approach and even helped us obtain the
`first grant for these studies, from a small private foun-
`dation -— the Freudenberg Foundation. The second
`person was the late Prof. Elisabeth Roboz-Einstein. She
`
`was deeply involved in research on EAE from the
`viewpoint of a neurochemist. She was so taken with our
`research approach that years later she bequeathed to us
`her entire scientific and reprint collection.
`In the late [9605 methods for the synthesis of se-
`quenced polypeptides were not yet available. However,
`as graduates of the laboratory of Prof. Ephraim
`Katchalski. we were experts in the synthesis of random
`copolymers of amino acids. and hence we prepared a
`series of such copolyrners. with compositions approach-
`ing that of MBP, all with a highly basic nature due to
`their high lysine contents. However. efi'orts over the
`course of more than a year led to negative results —
`none of these synthetic copolyrners possessed any en-
`cephalitogenie activity [l2]. Furthermore, even the con-
`jugation of
`sphingolipid moiety — which could
`potentially enhance the anti-sphingomyelin response
`- and consequently the demyelination process — did not
`endow these polymers with any encephalitoge'nic activ-
`ity whatsoever, Disappointment. Was our hypothesis
`wrong? Did the synthetic approach fail us in this case?
`Should we give up?
`Concomitantly. we were expanding our studies on
`EAE and its characterization: in collaboration with the
`late Dr. H. l-lirshfeld. we developed a simplified proce-
`dure for the purification of MB? from myelin [13].
`based on the use of a new ion-exchange resin.
`SulphoethyLSephadex, and it enabled us to prepare
`large quantities of the purified protein for more exten-
`sive investigation of EAE. The purified MBP was
`highly potent in the induction of EAE, as contrasted
`with the complete lack of such activity in any of the
`synthetic copolymers. Did these polymers possess any
`other form of biological activity?
`_
`It is a common practice in immunology, once an
`interaction between antigen and antibody is established,
`to elucidate its specificity by competition. or inhibition
`studies. Only substances of a similar specificity will
`evince inhibitory properties. This had been the proce-
`dure by which we had identified the specificity of the
`synthetic antigens. and the contribution of various
`parts of the macromolecules as ‘antigenic determi-
`nants‘, or epitopes, to their overall antigenic activity.
`This technique had been employed to characterize the
`specificity of blood group antigens and many other
`systems. In most'cases such inhibition studies are per~
`formed with relatively short molecules — sugars. or
`peptides. Would it apply to the macromolecular copoly-
`mers? Furthermore. the issue we were addressing was
`not a relatively simple antigen-antibody interaction. but
`rather a more complex biological process. namely the in
`vivo induction of encephalitogenic activity. Neverthe-
`less,
`the earlier findings of Elisabeth Roboz-Einstein
`and Marian Keen that MBP. as well as some other
`brain basic proteins. can inhibit EAE [9,10] indicated
`that this could be a plausible approach.
`
`“my.4-
`
`MYLAN INC. EXHIBIT NO. 1020 Page.2
`
`MYLAN INC. EXHIBIT NO. 1020 Page 2
`
`

`

`
`
`R. Anton [Immunology letters 50 (1996) l—lS
`
`3
`
`2. Suppression of EAE, the animal model for multiple
`sclerofis
`
`The results of the inhibition experiments were over-
`whelming -— not one. but several of the synthetic co-
`polymers showed high efficacy in suppressing EAE.’ The
`most active among the series was Copolymer 1 (Cop I).
`composed of L-alanine, L-lysine, L-glutamic‘acid and
`L-tyrosine in a residue molar ratio of 6.0:4.7:l.9:l.0.
`and hence most of our subsequent research was con-
`ducted only with this substance. It had a marked
`suppressive effect on EAE when injected to guinea pigs
`in incomplete Freund’s adjuvant or even in aqueous
`saline solution, after an initial challenge with a disease-
`induciug dose of MBP [12]. It reduced the incidence of
`EAE from about 75% in the control group to only 20%
`min the treated group. Was the efl'ect real? Could it be an
`artefact? A second batch of Cop l was immediately
`synthesized, very similar in its composition and molecu-
`lar size to the original one. and was found to be
`identical in its suppressive cheer on EAE. indicating
`that the observed suppressive effect is a real one. This
`seemed interesting indeed. Not only did we have in
`hand a tool for studying the mechanism of RAE and
`the immune processes involved in it. but already at that
`early stage we realized that this might lead eventually to
`a therapeutic agent. We submitted patent applications
`in Israel and abroad and were granted them in various
`countries during 1972 to 1974.
`It was now necessary to learn more about the efl'ect
`of Cop l and how it exerts it. Cynthia Webb. a PhD.
`student who-joined our team. showed that the. suppres-
`sive activity of Cap 1 could be explained by its
`immunological cross~reactivity with the MBP. Cross-re-
`action was clearly manifested on the cellular level.
`in
`both in vitro (lymphocyte transformation) and in vivo
`(delayed hypersensitivity) assays [14]. In studies involv-
`ing the series of copolymers. there was a good correla-
`tion between the level of such cross-reactivity and the
`capacity to suppress EAE. As for the humoral antibody
`response, we could not detect cross-reactivity between
`MBP and Cop 1 using the methods available at the
`time. namely the precipitin test or the Farr test (which
`is the ‘ancestor’ of the radioimmunoassay). However.
`the more sensitive passive cutaneous anaphylaxis test
`showed that guinea pig anti—Cop 1 sets did cross-react
`to a certain extent with MBP. but not vice versa [14].
`All these tests were performed, of course, with poly-
`clonal antibodies. the only methodology available at
`the time. Later studies. using monoclonal antibodies.
`showed a highly significant cross-reactivity between
`MBP and Cop l
`-- about a third of the hybridomas
`raised against rat MB? cross-reacted with Cap 1 to the
`same level of reactivity as with the homologous antigen,
`and a proportion of the anti-Cop 1 antibodies reacted
`with MBP [15]. Moreover. some of the monoclonal
`
`antibodies raised against either MBP or Cop I reacted
`in a heteroclitic manner and favoured the cross-reacting
`antigen over the immunogen. It is of interest that the
`cross-reactivity was observed only with the monoclonal
`antibodies — antisera ol' the immunized mice from
`which these antibodies originated showed no cross—rear,
`tivity. Thus. the use of monoclonal antibodies uncov-
`ered specificities that were not evident in lhe polyclonal
`response and revealed the pronounced cross-reactivity
`between Cop l and MBP. on the B-oell level as well as
`the previously observed T-cell level. This provided a
`plausible basis for the suppressive effect of Cap 1 on
`MBP-induced EAE.
`
`3. Specificity of EAE suppresslon by Cup I
`
`The results described above. for the suppressive effect
`of Cop l on EAE, were demonstrated for the disease
`induced in guinea pigs by the inoculation of bovine
`MBP. It was known, however. that the induction of
`EAE is species dependent. in respect to both the species
`from which the MB? is derived and the species in
`which the disease is induced. The specificity is reflected
`not so much in the actual susceptibility to the disease.
`which is relevant to most species. but in the particular
`region in the MBP molecule which is responsible for the
`encephalitogenic activity. The encephalitogcnie determi-
`nants l'or guinea pigs, mice. rats and primates are all
`dili'erent [16].
`It was therefore interesting to observe that Cap 1 was
`effective in suppression of EAE in guinea pigs also
`when it had been induced by MB? of human origin
`[17]. These results are of particular interest. since when
`EAE was induced in guinea pigs by the human en-
`cephalitogen.
`the histological changes observed in-
`cluded demyelinations and fibrosis in the guinea pig
`brain. thus resembling the symptoms of MS more than
`when the disease is induced by either bovine or rodent
`MBP. In later experiments we have induced EAE in
`guinea pigs with MBP of other species and demon-
`strated that the suppressing effect of Cop l was firm
`and abiding.
`Is the chest of Cop 1 then a specific one. or is it due
`to some non—specific immunosupprcssive properties?
`This is an important question. since it reflects on the
`mechanism of its activity in the suppression of EAE.
`Evidence far its specificity were provided by two exper-
`iments: The first demonstrated that Cup I
`lacked any
`suppressive effect on the immune response in several
`systems —- a particulate antigen such as bacteriophage
`T4. soluble proteins such as BSA and RNase. and
`carrier-hapten
`systems
`such
`as DNP-BGG or
`polyalanyl HSA. In all cases the level of the immune
`response was not affected by the presence of Cop l.
`Neither was skin graft rejection in rats affected by the
`
`MYLAN- INC. EXHIBIT NO, 1020 Page 3
`
`
`
`
`
`......___.‘_4--‘4-.-.'
`
`MYLAN INC. EXHIBIT NO. 1020 Page 3
`
`

`

`
`
`4
`
`It. Anton / Immlogy Letters 50 (1996) I ~15
`
`[17,18]. The second evidence is
`injection of Cop 1
`derived from an experiment using a copolymer identical
`to Cop l, but composed of D-amino acids instead of
`the natural Moan, and denoted D-Cop 1. Both com-
`position aad size of Cop l and DvCop l were identical.
`D-Cop l was devoid of any suppressive eflect on EAE,
`nor was it cross-reactive with MBP at either the hu-
`
`moral or the cellular level (unpublished results).
`Equally, or even more important is the question
`whether Cop 1 would be effective in suppressing EMS
`in species other than guinea pigs. Indeed, in a detailed
`study We showed that Cop 1 demonstrated efi‘ective
`suppression'of EMS in rabbits [17], in mice [19] and in
`two species of primates — rhesus monkeys [20] and
`baboons [21] (Fig. 1). It is thus apparent that Cop I
`does not manifest species specificity, either for the
`source of encephalitogen or for the animal tested.
`The results in primates were very significant. since. as
`shown in Fig. 1, these animals are highly susceptible to
`EAE and all those sensitized with MBP succumbed to
`the disease. The experiment with Rhesus monkeys in—
`cluded 10 animals, of which five served-as controls and
`the other five were treated with Cop l. The treatment
`was given daily starting immediately after the onset of
`the first stages of paralysis. All five. monkeys in the
`control group deteriorated very rapidly and died within
`4—11 days after the onset of symptoms. In contrast,
`four out of the five Cop l-treated monkeys showed
`improvement after 4—5 days of treatment and finally
`recovered completely from the paralysis. The fifth Cop
`Hreated monkey continued to deteriorate after an
`initial 'iniprovcment. sult'ered a relapse 35 days later,
`and finally'died. of. BAE:
`A similar level of eflicacy of Cop l was observed in
`the experiments with‘ the baboons which included a
`total of 15 anir'nals. The six baboons in the control
`group developed EAE'with progressive paralysis and
`died within 4_—-ll days after initial symptoms were
`
`-
`
`100
`
`I] control Group
`Treated Group
`
`%disease 888 92 Gulnee Rabbits Mine Rhesus Baboons
`
`pigs
`
`monkeys
`
`Fig. l. Suppression of EAE by Cop 1 in various species. Incidence of
`disease in Cop l-treeted animals as compared to untreated controls
`
`noted. In nine baboons daily treatments were started
`immediately after the first paralysis symptoms were
`observed. Although initially they continued to deterio-
`rate and most of them reached the state of full paraly-
`sis. they eventually began to recuperate, and seven out
`of the nine showod full recovery. I remember these
`experiments very vividly since for us they constituted a
`considerable effort: baboons are large animals, weight-
`ing about 30 kg each and require special cages equipped
`with appropriate fixtures for experimental manipulation
`of the animals. Since we had only five such cages, the
`above experiment was actually performed in three
`stages - each one including live baboons. of which two
`served as controls and three were treated with Cop 1.
`After the second stage we knew What to expect and
`hence during the third one we filmed one of the Cop
`l-treated baboons through all the phases of the trial -
`before the EAR-inducing challenge with the MBP. dur—
`ing the paralysis period (2—3 days) and up to its
`complete recovery when he jumped vivaciously in his
`cage: The film was quite effective and helped me later in
`demonstrating the effect of Cop l and its therapeutic
`potential. Furthermore. it helped me in raising interest
`among neurologists and motivating them to conduct a
`clinical trial with Cop l.
`The results achieved in the sub-human primates are
`highly significant for two reasons: (1) since they are the
`species closest
`to humans,
`the efl'ect of Cop l on
`primates is more relevant to multiple sclerosis; (2) in all
`primates the treatment with Cop 1 was begun only after
`symptoms of disease were evident and hence its efl'ectiv-
`ity was a positive indication, since any treatment sug-
`gested for MS would be feasible only after disease had
`been diagnosed Further-facts, one of the Cop-1 treated
`rhesus monkeys which had fully recuperated after being
`paralyzed was examined and showed no histological
`damage in its brain. A baboon similarly tested showed
`very minimal histological lesions. in contrast. drastic
`damage and multiple lesions were noticed in the brains
`of all
`rhesus monkeys and baboons of the control
`groups, as well as those treated with Cop 1 who, died of
`EAR. This indicates that brain lesions which arc‘irot
`long-lasting could be amenable to remyelination.
`Another aspect of the experimental evidence which
`lends support to the potential of Cop l in relation to
`MS, is its beneficial efi'ect in the chronic—relapsing form
`of EAE (CR-EAE). This type of EAE [22] is induced in
`guinea pigs by sensitizing juvenile animals with the
`eneephalitogenic challenge. and is characterized by ini-
`tial onset followed by a reversal stage and subsequent
`relapses. Due to its relapsing nature CRvEAE is consid-
`ered a more faithful experimental model for MS. In
`collaboration with Dr. Wisniewski and his colleagues
`We studied the effect of Cop l on this type of disease.
`We showed that pretreatment had a marked effect both
`in delaying the initial onset and in preventing the
`
`MYLAN INC. EXHIBIT NO. 1020 Page4
`
`MYLAN INC. EXHIBIT NO. 1020 Page 4
`
`

`

`. .4‘.‘...\.r“‘—l-I-\.'.‘
`
`
`
`
`
`R. Arno» [Inmmnology letters 50 {I996} I —I5
`
`5
`
`appearance of relapses. Therapeutic treatment. which
`was given after the onset of initial symptoms. reduced
`both the occurrence and the severity of relapses [23].
`The next logical step was to investigate whether Cop
`l was of any benefit
`to MS patients. We therefore
`conducted some basic toxicological studies which are
`prerequisite for any clinical trial. A full ‘toxicological
`package‘ is an extremely costly afi‘air (millions of dol-
`lars), which we couldn't afford. We did. hawever. carry
`out LDN determinations and experiments of acute and
`subacute toxicity in mice and rats as well as in seven
`beagle dogs. which were performed at the Weizmann
`Institute, with the expertise of Dr. Asher Meshorer
`(unpublished results).
`The LDso tests actually failed, since no death oc-
`curred at doses up to 2000 mglkg (the highest dose that
`could be administered). The acute and subacute toxicity
`tests showed that Cop i can be administered by either
`single or several successive administrations in doses up
`to BOOO-I‘old higher than the expected recommended
`dose for
`treatment. producing neither pathological
`eli'ects nor any other macroscopic or microscopic
`changes. Furthermore. in the Ames test, Cop I showed
`no mutagenic eifect. The conclusion was therefore that
`Cop l is a non-toxic material. and the results fulfilled
`the requirements for a Phase I clinical trial.
`
`4. Initial antral trials
`
`Our first clinical trial was conducted in Israel, at the
`Hadassah Medical School,
`in collaboration with Dr.
`Oded Abramsky {24]. Dr. Abramsky, an enthusiastic
`neurologist at Hadassah who is now the Chairman of
`the Department of Neurology there and currently
`serves as the Dean of the Medical School. was at the
`time at the Weizmann Institute, working towards his
`PhD. thesis under my supervision. In this capacity he
`took part
`in some of the experiments with Cop l.
`Impressed by the experimental data he was interested in
`testing the effect of Cop 1 in patients. This Preliminary
`Trial, according to the approval conditions of the Is-
`raeli Helsinki Committee. included only four MS pa-
`tientsin the terminal stages of the disease. They were
`treated with 2—3 mg of- Cop 1. 2—3 times a week. for
`4—6 months (the initial 3 Weeks were under hospitaliza-
`tion). Under these conditions no beneficial efl'eCt was
`expected. Indeed. the patients did not show any signifi-
`cant change in their motor function. Two of them
`exhibited some improvement in vision and speech ca~
`pacity. but in the absence of a control group. it was
`impossible to relate this improvement to the treatment.
`However. the most important finding was that no side
`effect was observed in any of the patients. There were
`no changes in blood pressure. heart rate and ECG. or
`in liver and kidney functions. Nor were any toxic or
`
`allergic reactions observed. This information paved the
`way for further clinical trials in less severe patients.
`The difficulty was to find a clinician to perform such
`a trial.
`I recall this time as the 'pcddling period'.
`I
`participated in almost any conference. large or small.
`which dealt with MS.
`I presented our experimental
`- data. wherever possible I screened the film on the
`baboon and talked to everyone who was prepared to
`listen. I had success with two neurologists: Dr. Helmut
`J. Bauer from Gdttingen in Germany and Dr. Murray
`13. Bernstein of the Albert Einstein College of Medicine
`in New York.
`I met Dr. Bauer at one of the meetings which I
`attended in Europe. He had a large clinic for the
`treatment of MS and was excited by the opportunity to
`test the effect of Cop l and the glimmer of hope for the
`otherwise desperate patients. The trial he conducted
`was an open-label one. involving in all 21 patients. [0
`of whom (with DSS range 24:) received a daily dose of
`2 mg Cop l and the other II (with 055 range 5—7)
`received a daily dose of 20 mg Cup i. for the duration
`of one month. The results were indicative of some
`improvement, particularly in the group of relapsing-re-
`mitting patients and those with lower DSS. Due to the
`short duration of the trial and to the lack of a control
`group. the significance of the beneficial eli‘cct
`is not
`clear. However. the trial was important for demonstrat-
`ing the safety of Cop l — there were only a few minor
`local reactions and two cases with transient fever with
`out any other adverse effects.
`Dr. Bor'nstein. who passed away recently. was a very
`dynamic personality. whom I also met' at a conference
`in Europe. He was a renowned neurologist. who was
`also in charge of a large MS clinic. He was interested in
`the pathogenic mechanisms leading to MS and their
`association with EAE. His previous studies in tissue
`culture had indeed served to relate MS to EAE [25].
`demonstrating that mammalian CNS tissue cultures
`respond with identical patterns of demyelination when
`exposed to serum from EAE—afl‘ected animals or from
`MS patients. Hence, the rationale for his willingness to
`launch a trial with an agent which suppresses EMS and
`CR-EAE. looking for its effect on MS patients. Alto-
`gether. Dr. Bernstein and his colleagues conducted
`three clinical trials. a preliminary one and two pilot
`double-blind controlled trials. one involving exacerbab
`ing remitting (IS-R) patients and the second involving
`_ chronic/progressive (C-P) patients. as summarized in a
`recent review article [26}.
`The preliminary trial [27] involved 16 patients (four
`ER and 12 C-P) and was conducted as an open study.
`The first patients were hospitalized for the first 3 weeks
`to look for any significant local or systemic effects. but
`since no undesirable side effect was observed. subse-
`
`quent patients were hospitalized for only 24-48 h. and
`continued the Cop 1 treatment as outpatients for the
`
`MYLANINCEXHIBIT 1510-1020 Page 5
`
`MYLAN INC. EXHIBIT NO. 1020 Page 5
`
`

`

`
`
`6
`
`.R. Amen [Immunology Loam 50(1996) 1-15
`
`duration of the trial. The specific aims of this prelimi-
`nary trial were to determine: (1) Did Cop I produce
`any apparent undesirable side reactions? (2) Did Cop I
`produce any apparent desirable elfects? (3) Could a
`dosage schedule be established for further (pilot) trials
`should they appear to be warranted? The results
`showed that: (1) except for occasional local transient
`reactions at the site of injection (pain, itching, redness,
`etc), no systemic reactions of any kind were reported.
`(2) 0f the 16 patients, 11 did not demonstrate any
`apparent favorable efiect, but
`five demonstrated a
`definite improvement. The most noticed etl‘ect was the
`cessation of exacerbations in two of the four ER pa-
`tients. (3) The optimum dosage was determined to be
`20 rug/day. 0n the basis of these preliminary results, it
`was decided to extend the evaluation of Cop l
`to
`rigorous doubleblind, randomized, placebo controlled
`trials.
`A double blind trial. It's easier said than done. The
`first trial involved patients with exacerbating/remitting
`(ER) MS. It took Dr. Bomstein more than a year of
`careful design and planning.
`including consultations
`with statisticians, epidemiologists and social workers, in
`addition to the medical and nursing stafi' who were to
`participate in the trial on a day to day basis. Appropri-
`ate planning was a prerequisite for success and for the
`interpretation of the results and their significance. Dr.
`Bernstein did an excellent job in the entire design and
`conduct of this pilot trial. (I have been told that this
`trial served as a model for the design of subsequent
`clinical trials in MS patients by other investigators.) He
`was also the driving force both in recruiting the neurol-
`ogists. scientists and nurses, as Well as in securing the
`necessary funds for conducting the trial. This required
`many meetings, in some of which i participated and
`presented the experimental data which provided the
`theoretical basis for the trial. Finally, the NIH agreed
`to support the trial. approval was obtained from the
`Committee on Clinical
`investigations of the Albert
`Einstein College of Medicine and the Federal Food and
`Drug Administration, and the ball started rolling.
`The trial
`included 50 patients,
`the recruitment of
`whom was also not a simple or easy process. which
`involved the interviewing of several hundreds of pa-
`tients and lasted almost 3 years. i will not describe this
`trial in detail, since its results have been published in
`the New England Journal of Medicine [28] and subse-
`quently summaIiZed in several review articles [26,29,30].
`I will therefore give only the main points and conclu-
`sions and emphasize only our own personal involve-
`ment.
`
`Our role in this trial was to supply the needed
`amounts of Cop l. The substance, which consists of a
`copolymer of amino acids, is synthesized by polymer-
`ization of the N-carboxyanhydride derivatives of four
`amino acids - alanine,
`lysine. glutamic acid and ty-
`
`rosine. These derivatives, prepared by interaction with
`phosgene, are extremely sensitive to both tetnperature
`and humidity, and hence have to be prepared under
`very stringent conditions. The polymerization stage is
`also a delicate process, if reproducibility of composition
`and molecular size is to be achieved. It involves two
`additional steps of deblocking of the functional car-
`boxylic group of the glutamic acid and amino group of
`the lysine. which have to be protected during the entire
`synthesis. It is a rather complicated prowdure which
`takes at least 2 weeks in addition to the prior synthesis
`of the monomeric N-carboxanhydrides. Using labora-
`tory scale equipment,
`'we could prepare only gram
`amounts at a time. and this meant that our laboratory
`was converted into a mini-factory that was constantly
`busy for those several years with the preparation and
`testing of Cop 1. Into this picture enters our super
`technician Mr. lsrael Jacobson. That we were able to
`keep up at all with demand is due in no small measure
`to his professionalism and unstinting devotion to the
`job.
`Mr. Jacobson is a unique character —- I refer to him
`as ‘The last Mohican'. He received his education in
`Europe as a pharmacist in the early 19305, but had to
`leave his country when Hitler rose to power, and joined
`the Weizmann Institute in 1938, shortly after it was
`established. He is an extraordinary person and a first
`rate technician, both knowledgeable and creative,
`highly meticulous in his performance and devoted to his
`work above and beyond the call of duty. In 1953 he
`started to work with Prof. Ephraim Katchaiski-Katzir,
`and as such became an expert in the preparation of
`polyamino acids and was our partner all along the way.
`He used to come to the lab at 4 a.m. — during‘the
`Winter in pitch dark — so as to be able to work without
`disturbance and thus increase his productivity. With
`Dr. Teitelbaum, who was responsible for the final
`stages of the synthesis as well as the analysis of every
`single batch. for both chemical characteristics (compo-
`sition,
`size) and biological activity (immunological
`properties and suppressive eli'ect on EAE), we prepared
`altogether about 120 batches of Cop 1 (10-80 g each),
`totalling over 3 kg. It was a continuous production line
`over a period of about 10 years, from 1979 to 1989, a
`tremendous effort for a research laboratory in an aca-
`demic