Toxicologic Pathology, 40: 40-54, 2012
`Copyright # 2012 by The Author(s)
`ISSN: 0192-6233 print / 1533-1601 online
`DOI: 10.1177/0192623311424169
`
`Comparative Long-Term Preclinical Safety Evaluation of
`Two Glatiramoid Compounds (Glatiramer Acetate,
`1
`, and TV-5010, Protiramer) in Rats and Monkeys
`Copaxone
`
`YUVAL RAMOT
`
`1, MOTI ROSENSTOCK
`
`2, ETY KLINGER
`
`2, DIZZA BURSZTYN
`
`2, ABRAHAM NYSKA
`
`3, AND DORON M. SHINAR
`
`2
`
`1Hadassah—Hebrew University Medical Center, Jerusalem, Israel
`2Teva Pharmaceutical Industries, Ltd, Petach Tikva, Israel
`3Consultant in Toxicological Pathology, Timrat, and Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel
`
`ABSTRACT
`
`1
`Glatiramer acetate (GA), the active ingredient in Copaxone
`, is a complex mixture of polypeptides used for the treatment of relapsing
`remitting multiple sclerosis. Glatiramoids are related mixtures that may differ in some characteristics of the prototype molecule. Our aim is to describe
`the long-term toxicity studies with protiramer (TV-5010), a new glatiramoid, in comparison with similar studies conducted with GA. The toxicity of
`twice-weekly subcutaneous injections of protiramer to Sprague-Dawley rats (twenty-six weeks) and cynomolgus monkeys (fifty-two weeks) was com-
`pared with similar studies done with daily subcutaneous injections of GA. Daily treatment with GA was safe and well tolerated, without systemic effects
`or death. Protiramer administration was not as well tolerated as GA and led to dose- and time-related mortalities, probably mediated through severe
`injection-site lesions both in rats and in monkeys. Bridging fibrosis in the liver and severe progressive nephropathy were seen in rats. A dose-related
`increase in eosinophils was observed in monkeys. The protiramer toxicity studies show that minor variations in the manufacturing of glatiramoids
`may lead to significant toxic effects. It is therefore essential that the safety of any new glatiramoid be studied in long-term preclinical studies before
`exposing humans.
`
`Keywords:
`
`glatiramoids; toxicity; injection site; glatiramer acetate; protiramer.
`
`INTRODUCTION
`
`The glatiramoid class of compounds is a family of synthetic
`heterogenous polypeptide mixtures that include four natural
`amino acids, L-glutamic acid, L-alanine, L-lysine, and L-
`tyrosine,
`in a distinct molar ratio of 0.14:0.43:0.09:0.34,
`respectively, with an average molecular weight (MW) in the
`range of 5,000–9,000 Daltons (Varkony et al. 2009). The first
`glatiramoid, and the best studied one, is glatiramer acetate
`1
`, which is approved
`(GA), the active ingredient in Copaxone
`for the treatment of relapsing remitting multiple sclerosis
`(MS) in over fifty countries. It has been shown that GA has a
`unique immunomodulatory mechanism, leading to disruption
`of the pathogenic process in MS by reinforcing immunoregula-
`tory networks (Arnon and Aharoni 2009; Blanchette and Neu-
`haus 2008; Liblau 2009; Schrempf and Ziemssen 2007; Van
`Kaer 2011; Weber et al. 2007). The exact mode of action of
`GA has not been completely elucidated, but it has been sug-
`gested that the heterogeneous nature of the mixture plays an
`important role in securing activity in such a heterogeneous dis-
`ease (Johnson 2010; Varkony et al. 2009).
`
`This work was performed at Teva Pharmaceutical Industries, Ltd, Petach
`Tikva,
`Israel. Funds for
`this project were provided in part by Teva
`Pharmaceutical Industries, Ltd, Petach Tikva, Israel. Some of the authors are
`employees of Teva Pharmaceutical Industries, Ltd, Petach Tikva, Israel.
`Address correspondence to: Abraham Nyska, DVM, Haharuv 18, P.O. Box
`184, Timrat, Israel 36576; e-mail: anyska@bezeqint.net.
`Abbreviations: CPN, chronic progressive nephropathy; GA, glatiramer
`acetate; MS, multiple sclerosis; MW, molecular weight.
`
`Recently, there has been growing interest in glatiramoids in
`the pharmaceutical industry (Johnson 2010), and many manu-
`facturers are seeking to develop new glatiramoids (Kovalcin,
`Krieger, Collins et al. 2010; Kovalchin, Krieger, Genova et
`al. 2010), and others aim to prepare follow-on (‘‘generic’’)
`versions of GA itself. An additional new glatiramoid product,
`protiramer (also denoted as TV-5010), was recently devel-
`oped by Teva Pharmaceutical Industries, Ltd (Petach Tikva,
`Israel) as a potential follow-on product to GA with a slightly
`higher MW distribution. The rationale for developing a high-
`MW glatiramoid was based on results of fractionation studies
`of GA that showed that the higher the MW of the fraction,
`the higher potency and immune reactivity the fraction has
`in pharmacological models. This new glatiramoid was devel-
`oped to provide better efficacy or less dosing frequency for
`MS patients.
`Protiramer was made by making minor modifications to the
`GA synthetic process, mainly a slight change in the tempera-
`ture in which the precursor molecule is cleaved to yield the
`final mixture. This minor change results in a product with a dif-
`ferent MW distribution and different biological properties than
`GA (De Stefano et al. 2009; Varkony et al. 2009). Based on
`encouraging preclinical safety results from short-term toxicity
`studies, protiramer’s safety, tolerability, and efficacy were
`evaluated in two small phase II clinical trials in MS patients;
`it demonstrated good general safety and tolerability (De Ste-
`fano et al. 2009). Later, long-term preclinical toxicity studies
`in monkeys and rats revealed marked progression in the
`severity of
`injection site
`reactions, which was
`also
`
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`TOXICITY OF GLATIRAMOIDS COMPOUNDS
`
`41
`
`accompanied by kidney and liver damage, and death of some
`high-dose animals. The findings in animals led to the termina-
`tion of the drug development program of protiramer (Varkony
`et al. 2009) and brought about the understanding that even
`slight changes in the manufacturing process of glatiramoids
`may have profound safety implications.
`The aim of the present report is to describe the clinical and
`anatomical pathology findings in the long-term preclinical
`toxicity studies of protiramer and compare these results with
`the results of similar preclinical chronic toxicity studies in
`rats and monkeys that were carried out previously with
`GA. By comparing these two toxicity studies, we show that
`minor variations in the manufacturing of glatiramoids may
`lead to significant toxic effects. In addition, we present and
`discuss the potential mechanistic association between the
`severe injection site lesions and systemic toxicities seen in
`animals.
`
`MATERIALS AND METHODS
`
`Chemicals
`
`Both GA and protiramer were produced by Teva Pharma-
`ceutical Industries, Ltd, Petach Tikva, Israel, at a fully accre-
`dited GMP manufacturing site.
`
`Animals and Housing
`
`The toxicology studies were carried out as fully compliant
`GLP studies in accredited contract research laboratories. All
`animals used in this research were treated humanely, with due
`consideration to alleviation of distress and discomfort. For the
`protiramer studies, male and female Sprague-Dawley rats
`(approximately eight weeks old) were obtained from Charles
`River Laboratories (Saint Germain sur l’Arbresle, France), and
`male and female cynomolgus monkeys (Macaca fascicularis,
`twenty-six to thirty-nine months old) were obtained from
`Siconbrec, Simian Conservation Breeding and Research Cen-
`ter, Inc. (Tanay Rizal, Philippines). For the GA studies, male
`and female Sprague-Dawley rats (approximately four weeks
`old) were obtained from Charles River (Margate, UK), and
`male and female cynomolgus monkeys (Macaca fascicularis,
`approximately twenty-four months old) were obtained from
`Mediprime (Billinghurst, West Sussex, UK). For the rat stud-
`ies, water and feed were available ad libitum (diet reference
`A04C-10 for the protiramer study, and SQC rat and mouse
`maintenance diet No. 1 from Special Diet Services Ltd,
`Witham, UK for the GA study). For the GA monkey study,
`animals were offered daily SQC Mazuri Primate Diet (Special
`Diet Services Ltd, Witham, UK) supplemented by fruit and a
`Bonio biscuit (Spillers Foods Ltd, New Malden, UK), and for
`the protiramer study, animals were offered daily expanded
`complete commercial primate diet (Special Diet Services:
`OWM (E) short SQC) supplemented by fruit. The room tem-
`perature was 18C–25C and the humidity was 40–80%
`throughout all studies. The fluorescent light cycle was twelve
`hours per day for all studies, and there were at least ten room
`
`air changes per hour for the monkey studies, and at least fifteen
`changes for the rat studies. All animals were randomly assigned
`to the treatment groups. For the rat studies, animals were
`housed in groups of three to five (protiramer) or groups of five
`(GA) in stainless steel mesh cages. For the monkey studies,
`animals were housed in groups of two or three (protiramer)
`or individually (GA).
`
`Experimental Design
`
`The route of administration in all studies was subcutaneous
`injections. Several injection sites were used in each animal and
`were alternated on each session to minimize local damage.
`Doses for the chronic toxicity studies were selected based on
`tolerability observed in shorter range-finding studies (not
`shown). The GA rat chronic toxicity study included one control
`and three treatment groups of twenty animals/sex/group that
`were treated daily at doses of 0, 3, 10, and 30 mg/kg for
`twenty-six weeks. Vehicle was sterile physiological saline, and
`a constant dose volume of 2 mL/kg was used. The protiramer
`rat chronic toxicity study included four treatment groups and
`one control group; each group consisted of twenty males and
`twenty females per dose treated twice a week at doses of 0,
`2.5, 40, and 300 mg/kg or once every two weeks at 2.5 mg/
`kg dose (low-dose, low frequency) for twenty-six weeks. Addi-
`tional animals (five/sex/group) of the control and high-dose
`groups were assigned to a four-week treatment-free period. The
`vehicle was mannitol solution in water for injection, and the
`dose volume was 5 mL/kg. The GA monkey study included
`three treatment groups and one control group of four ani-
`mals/sex/group, treated at doses of 0, 3, 10, and 30 mg/kg for
`fifty-two weeks. The vehicle was sterile physiological saline,
`and a dose volume of 1.5 mL/kg was used. The protiramer
`monkey study included four treatment groups and one control
`group; each group consisted of seven males and seven females.
`Protiramer treatment of the monkeys was done twice weekly at
`doses of 0, 2, 10, and 60 mg/kg for either thirteen weeks
`(interim group of three animals/sex/dose) or fifty-two weeks
`(main group of four animals/sex/dose). The fifth group was a
`low-dose, low-frequency group and was administered protira-
`mer once every four weeks at a dose level of 2 mg/kg (the same
`number of monkeys for this group). The high dose was lowered
`from 60 to 30 mg/kg on day 280 of the study because of poor
`tolerability. Additional animals of the control, high-dose, and
`low-frequency, low-dose groups (two males and two females
`per group) were assigned to a four-week treatment-free period
`after fifty-two weeks of dosing. The vehicle was mannitol solu-
`tion in water for injection, and the dose volume was 1 mL/kg.
`All animals were observed at least twice daily to detect any
`mortality or morbidity, and once daily for ill health or reaction
`to treatment. Individual body weights were recorded weekly.
`Blood chemistry, coagulation, and hematology parameters
`were examined at the end of each rat study and at predose and
`at several predetermined time points during the monkey stud-
`ies. Urine samples were also collected on several occasions
`in both the rat and monkey studies. At
`the scheduled
`
`
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`42
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`RAMOT ET AL.
`
`TOXICOLOGIC PATHOLOGY
`
`termination, animals were subjected to a full gross pathology
`evaluation including weighing of selected organs and histo-
`pathological analysis. Moribund animals were also examined
`for any pathological abnormality.
`In the GA studies, the weights of the adrenals, pituitary,
`brain (including brain stem), prostate, heart, spleen, kidneys,
`testes with epididymides, liver, ovaries, and thyroids with para-
`thyroids were determined at terminal sacrifice. In the protira-
`mer studies, the weights of the thymus and uterus were also
`determined. For histological examination, tissues were fixed
`in 10% neutral buffered formalin, except for the eyes, which
`were fixed in Davidson’s fixative, and the bone marrow
`smears, which were fixed in methanol (in the GA studies) or air
`dried (in the protiramer studies). In the protiramer studies, the
`testes and epididymides were also fixed in Davidson’s fixative.
`Fixed tissues were embedded in paraffin, sectioned, and stained
`with hematoxylin and eosin. The following tissues from
`all high-dose and control groups animals were sampled: adre-
`nals, brain (including brain stem), esophagus, eyes, femur,
`Harderian gland, heart and aorta, large intestine (cecum, colon,
`and rectum), small intestine (duodenum, jejunum, and ileum),
`injection site, kidney, liver, lung (and main stem bronchi),
`lymph nodes (mandibular and mesenteric), mammary gland,
`nose, optic nerves, ovary, pancreas, parathyroid gland, pituitary
`gland, prostate gland, salivary gland, skin, spinal cord and scia-
`tic nerve, spleen, stomach (including forestomach and glandu-
`lar stomach), testis (with epididymis and seminal vesicle),
`thigh muscle, thymus, thyroid gland, tongue, trachea, urinary
`bladder, uterus, vagina, and Zymbal’s gland. In the GA rat
`study, the following tissues were examined microscopically: all
`tissues from all animals in control and high-dose groups, all tis-
`sues from animals that died or were killed in extremis, injection
`sites, and abnormalities from the low- and intermediate dose
`groups. In the protiramer group, histopathological examina-
`tion was performed for all organs from the following animals:
`for all animals found dead or sacrificed moribund during the
`study, and all the control group animals, all intermediate dose
`group males, and all high-dose group females and all high-
`dose group males sacrificed after twenty weeks of treatment.
`Histopathological examination was also performed for kid-
`neys, liver, and injection sites from all other dose groups as
`well as for the animals that were assigned to the treatment-
`free ‘‘recovery’’ groups. The severity of microscopic lesions
`observed was graded based on the following numerical
`scale: 0 (no lesion), 1 (minimal), 2 (mild), 3 (moderate), and
`4 (severe), based on the criteria explained by Shackelford
`et al. (2002).
`
`Statistical Analysis
`
`In all studies, statistical analysis was performed separately
`for males and females.
`For the analysis of the clinical chemistry and hematology
`parameters, the Levene test for homogeneity of variances and
`the Shapiro-Wilk test to assess for normality were performed
`in protiramer studies, and the Levene test was performed in
`
`GA studies. Parametric methods were performed in protiramer
`studies in the case of homogeneous variances and normal dis-
`tribution in all groups and in GA studies in the case of homo-
`geneous variances between groups. Such analysis consisted of
`the one-way analysis of variance (ANOVA) followed, if the
`ANOVA test was significant, by the Dunnett test for pairwise
`comparisons. Nonparametric methods were performed in pro-
`tiramer studies in the case of evidence of heterogeneous var-
`iances or a non-normal distribution in at least one group and
`in GA studies in the case that there was a significant group
`effect or a significant sex–group interaction in the Levene
`test. Such analysis consisted of the Kruskal-Wallis ANOVA
`followed, if the Kruskal-Wallis test was significant, by the
`Wilcoxon rank sum test
`for pairwise comparisons. The
`Jonckheere-Tersptra trend test was used to test for an increas-
`ing or decreasing trend in response (excluding the low-dose,
`low-frequency group). In the GA studies,
`in the case of
`homogeneous variances between groups, a regression test for
`post-dose variables was used instead. The ANOVA results
`are not reported.
`For histopathology data, nonparametric methods were used.
`Such analysis consisted of the Kruskal-Wallis test followed, if
`the Kruskal-Wallis test was significant, by the Wilcoxon rank
`sum test for pairwise comparisons. The Jonckheere-Tersptra
`trend test was used to test for an increasing or decreasing trend
`in response (excluding the low-dose, low-frequency group).
`The Kruskal-Wallis results are not reported. Statistically signif-
`icant results for pairwise comparisons are indicated on the
`tables next to the appropriate mean value, using the following
`abbreviations: * p  .05; ** p .01; *** p .001. Statistically
`significant results for trend test are indicated on the tables next
`to the appropriate mean value, using the following abbrevia-
`tions: a p .05; b p .01; c p  .001.
`
`RESULTS
`
`Survival, Clinical Observations, Body Weight Gain, and
`Food Consumption
`
`Glatiramer Acetate Studies
`
`In the rat chronic toxicity study, treatment was well toler-
`ated at the tested dose levels. Six animals died during the six
`months of the study, but none of these was considered to be
`related to treatment, as the deaths were evenly distributed
`among the dose groups including control animals, and since the
`pathological examination did not reveal any association to
`treatment. Two of these animals died accidentally as result of
`the bleeding procedure. There were no effects on body weight
`gain or food consumption at any dose level, except for a slight,
`probably incidental, increase in weight for high-dose females
`during the interval of weeks 1–4. Clinical observations were
`restricted to occasional rough hair coat, stained fur, and appear-
`ance of sores at the injection sites that were noted occasionally
`in rats in the 10 and 30 mg/kg/day GA groups. Few dosing
`sites were abandoned or left to recover because of the above
`mentioned findings.
`
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`Vol. 40, No. 1, 2012
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`TOXICITY OF GLATIRAMOIDS COMPOUNDS
`
`43
`
`In the monkey GA study, one female of the intermediate
`dose group was removed from the study because of poor food
`consumption and loss of body weight. Histopathology revealed
`lymphoid and bone marrow atrophy and adrenal cortical hyper-
`trophy, signs that are consistent with stress. Because of the iso-
`lated nature of the mid-dose finding, the condition of this
`animal was not considered to be treatment related. Clinical
`signs in the remaining animals were restricted to the sites of
`injection. Thickening of the skin was noted in all treated
`groups, with high-dose animals experiencing greater frequency
`and earlier occurrence, starting generally by week 11. Signs in
`the low-dose group of 3 mg/kg were milder and noted from
`week 20. Sores were occasionally accompanied by swelling
`and abscess. By week 52, thickening was consistently noted
`in all high-dose animals.
`
`Protiramer Studies
`
`In the rat chronic study, nine out of seventy-two high-dose
`and one out of fifty-two mid-dose animals died during the
`study, and these deaths were attributed to treatment with protir-
`amer. Seven other deaths were considered unrelated. Clinical
`signs consisting of pallor, subdued behavior, thin appearance,
`stained fur, lacrimation, hair loss, scabs, and piloerection were
`noted in all treated groups. Local reactions to treatment were
`dose dependent and included induration associated with
`erythema and/or hematoma in both the high- and mid-dose
`groups. There was no obvious local reaction to treatment in ani-
`mals given the low dose of 2.5 mg/kg (twice weekly or once
`every four weeks). Most of the local reactions to treatment had
`disappeared by the end of the four-week treatment-free period.
`There was a dose-related reduction in mean body weight and
`mean body weight gain in males given protiramer, with signif-
`icant differences and even weight loss in males at the dose lev-
`els of 40 and 300 mg/kg, but not at 2.5 mg/kg or in the females
`of any group. The poor tolerance of animals in the high-dose
`group (300 mg/kg) necessitated the early termination of this
`group in week 20 of the study.
`In the monkey chronic study of protiramer, four unsched-
`uled deaths were noted, two of which were clearly treatment
`related. The two treatment-related cases were animals in the
`high-dose group that were diagnosed at necropsy with multiple
`subcutaneous areas of hemorrhage and vascular necrosis, and
`lymphoid and eosinophilic infiltrates, which were considered
`to be factors contributing to death. It should be noted that the
`first treatment-related death occurred only after more than six
`months of treatment. The two other deaths that were not treat-
`ment related were low-dose animals, one animal that died prob-
`ably from an accident and one animal that was removed from
`the study after being diagnosed as a carrier of hepatitis B. Body
`and organ weights were unaffected by treatment. Occasional
`animals in the high-dose group were salivating or subdued,
`or had loss of balance, were retching or circling, had stiff limbs,
`or were motionless. In the severe cases, there was also a
`decrease in blood pressure, decrease in body temperature, and
`increase in respiratory rate. These signs started appearing after
`
`seven weeks and were more pronounced and frequent after thir-
`teen weeks. Accordingly, a decision was taken in week 40 to
`reduce the high-dose level from 60 to 30 mg/kg.
`Injection site reactions were observed in all treated monkeys
`in a dose-dependent manner. In the control animals, the lesions
`were infrequent and consisted mostly of scabs, and slight
`erythema and induration at a very low incidence (usually single
`animals on a very small number of occasions). Skin lesions at
`the injection site in animals treated at 2 mg/kg once every four
`weeks were similar to those in controls. In animals treated at
`2 mg/kg twice weekly, there was a slight increase in the
`incidence and severity of the lesions of the type seen in control
`animals, and in addition, tumefaction was noted. Injection site
`lesions in animals treated at 10 mg/kg were more frequent and
`pronounced. The frequency and severity of the injection site
`lesions were greatest in the animals treated with 60/30 mg/kg.
`Because of the severity of these lesions, the original injection
`sites could not be used starting from week 40, and new
`injection sites were introduced. Lesions at the new sites of
`injection were similar to those seen in this dose group at the
`beginning of the study, indicating that these reactions do not
`involve immunological sensitization.
`
`Clinical Chemistry and Hematology
`
`Glatiramer Acetate Studies
`
`In the rat study, there was no effect of the GA treatment on
`hematology or clinical chemistry results. In the monkey study,
`there were occasional minor changes in AST and ALT values at
`the high dose level (Table 1). However, these changes were spora-
`dic, minimal, and without a clear dose- or time-dependent rela-
`tionship. There were no corroborating histological findings.
`
`Protiramer Studies
`
`In the rat study, hematology analyses revealed a minor
`decrease in activated partial thromboplastin time (APTT) and
`prothrombin time (PT) and a small increase in platelet counts
`in the males and females given 40 and 300 mg/kg (Table 2).
`A small decrease in red blood cell count and hemoglobin con-
`centration was evident in females given 40 and 300 mg/kg.
`This effect was accompanied by a slightly higher percentage
`of reticulocytes in these groups of females. Red blood cell para-
`meters were also slightly affected in males at both dose levels,
`but to a lesser extent than in the females, with a slightly
`increased reticulocyte percentage at 300 mg/kg. The absolute
`and relative polymorphonuclear neutrophil counts were
`increased in females given 40 mg/kg and in males and females
`given 300 mg/kg, associated with reduced relative lymphocyte
`counts. There was also an increase in the absolute and relative
`monocyte counts at the end of the treatment-free period in
`females given 300 mg/kg (Table 2). The above treatment-
`related changes in hematology and coagulation parameters
`were no longer evident at the end of the four-week treatment-
`free period, except for the decreased PT time and increased
`monocyte and platelet counts in females given 300 mg/kg.
`
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`RAMOT ET AL.
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`TOXICOLOGIC PATHOLOGY
`
`TABLE 1.—Mean clinical chemistry in male and female glatiramer acetate–treated monkeys at week 52.
`
`Dose level (mg/kg/d)
`
`AST (IU/L)
`ALT (IU/L)
`Alk Phos (IU/L)
`GGT (IU/L)
`Na (mmol/L)
`K (mmol/L)
`Cl (mmol/L)
`Ca (mmol/L)
`P (mmol/L)
`Glucose (mmol/L)
`Urea (mmol/L)
`Total bilirubin (micromole/L)
`Creatinine (micromole/L)
`Total protein (G/L)
`Albumin (G/L)
`
`Males
`
`Females
`
`0
`
`52
`48
`1492
`154
`147
`4.3
`109
`2.34
`1.8
`3.3
`7.2
`1.2
`85
`83
`46
`
`3
`
`52
`46
`1820
`175
`146
`4.2
`110
`2.34
`1.8
`3.3
`6.4
`2.7
`82
`81
`44
`
`10
`
`50
`49
`1378
`148
`147
`4.2
`111
`2.3
`1.7
`4.1
`6.5
`2.1
`80
`83
`49
`
`30
`
`83***
`75*
`1372
`118
`143
`4.4
`113
`2.35
`1.7
`3.8a
`6.8
`2.7
`88
`87
`46
`
`0
`
`50
`41
`1068
`95
`146
`3.8
`111
`2.3
`1.5
`2.5
`6.5
`2.5
`74
`79
`40
`
`3
`
`58
`42
`1038
`93
`146
`4.2
`108
`2.33
`1.8
`3.7
`6.7
`3
`83
`82
`43
`
`10
`
`52
`49
`936
`93
`146
`4.3
`110
`2.27
`1.7
`3.5
`5.8
`1.5
`79
`86**
`43
`
`30
`
`63*
`53
`1112
`71*
`146
`4.3
`111
`2.3
`1.8
`3.3
`6.4
`1.9
`80
`84*
`45
`
`a Significant at p < .05 using the dose-response test.
`* p < .05
`** p < .01
`*** p < .001
`
`TABLE 2.—Mean hematology parameters in male and female protiramer-treated rats at week 20 for group 4 males and week 26 for other groups.
`
`Males
`
`Females
`
`Dose level (mg/kg)
`
`0
`
`2.5
`
`40
`
`300
`
`2.5 (low)
`
`0
`
`2.5
`
`40
`
`300
`
`2.5 (low)
`
`Red blood cell count (T/L)
`Hemoglobin (g/L)
`Reticulocytes (%)
`Packed cell volume (%)
`Platelet count (G/L)
`Activated partial thromboplastin time (s)
`Prothrombin time (s)
`Polymorphonuclear neutrophils (G/L)
`Polymorphonuclear neutrophils (%)
`Lymphocytes (%)
`Monocytes (G/L)
`Monocytes (%)
`
`8.99
`155
`1.7
`47.1
`1331
`29.4
`21.7
`2.04
`18.8
`76.7
`0.23
`2.1
`
`9.11
`161
`1.3
`48.3
`1314
`32.3
`25.1
`1.48
`17.1
`77.6
`0.22
`2.5
`
`8.31
`145
`1.9
`43.6*
`1688**
`21.6*
`15.6*
`2.79
`26.6
`67.1
`0.33a
`3.2**a
`
`8.11
`143
`1.9
`41.8
`1623a
`21.2a
`16a
`2.79
`23.3
`70.8
`0.32
`2.5
`
`9.16
`158
`1.4
`47.2
`1235
`29.9
`21.2
`2.33
`22.2
`71.5
`0.36
`3.4**
`
`8.12
`153
`1.4
`45.1
`1170
`17.4
`14.1
`0.88
`12.2
`82.8
`0.15
`2.1
`
`8.01
`151
`1.5
`44.7
`1228
`16.7
`14.2
`1.08
`16.8**
`77.4**
`0.17
`2.6
`
`7.76
`146
`1.7
`42.9*
`1328
`16.1
`14.2
`0.99
`14.6
`79.7
`0.16
`2.4
`
`7.21b
`133*b
`2.7b
`39.7*b
`1951**c
`13.8a
`13a
`1.44**b
`17.8*
`76.1*b
`0.29**b
`3.4a
`
`7.73
`151
`2.2
`44.1
`1201
`15.7
`14.5
`0.89
`12
`82.6
`0.19
`2.5
`
`a Significant at p < .05 using the Jonckheere-Terpstra trend test.
`bSignificant at p < .01 using the Jonckheere-Terpstra trend test.
`cSignificant at p < .001 using the Jonckheere-Terpstra trend test.
`* p < .05
`** p < .01
`
`There were several treatment-related changes in serum clinical
`chemistry parameters in males and females given 40 and 300
`mg/kg (Table 3). These parameters included decreased total
`protein and albumin concentrations, increased globulin con-
`centrations,
`increased total cholesterol
`levels, and slightly
`decreased serum sodium. Most of the above treatment-related
`changes in serum clinical chemistry were still evident after the
`treatment-free period (Table 4).
`In the monkey study, hematology evaluation revealed a
`marked dose-related increase in absolute and relative eosino-
`phil counts in the groups treated twice weekly at 2, 10, and
`60 mg/kg throughout the treatment period (Table 5). Animals
`treated with 2 mg/kg once every four weeks were unaffected.
`
`Throughout the treatment period, animals treated at 60 mg/kg
`tended to have lower mean phosphorus, cholesterol, and albu-
`min concentrations, and from week 13, lower globulin concen-
`trations (and lower A/G ratios). After four weeks without
`treatment, these differences had essentially returned to normal,
`although a slight difference in serum cholesterol concentrations
`remained.
`
`Pathology
`
`Glatiramer Acetate Studies
`
`Gross pathology examination at the end of dosing of both
`rats and monkeys did not reveal treatment-related changes in
`
`
`Page 5 of 15
`
`YEDA EXHIBIT NO. 2019
`MYLAN PHARM. v YEDA
`IPR2014-00644
`
`

`

`Vol. 40, No. 1, 2012
`
`TOXICITY OF GLATIRAMOIDS COMPOUNDS
`
`45
`
`TABLE 3.—Mean serum clinical chemistry parameters in male and female protiramer-treated rats at week 20 for group 4 males and
`week 26 for other groups.
`
`Dose level (mg/kg)
`
`Sodium (mmol/L)
`Potassium (mmol/L)
`Total cholesterol (mmol/L)
`Total protein (g/L)
`Albumin (g/L)
`Globulin (g/L)
`Albumin/globulin ratio
`Creatinine (mmol/L)
`
`0
`
`144
`4.4
`1.92
`71
`33
`39
`0.9
`45
`
`2.5
`
`144
`4.5
`1.91
`70
`32
`38
`0.9
`47
`
`Males
`
`40
`
`143
`4.8a
`11.88*a
`69
`24**c
`44*a
`0.6*b
`58a
`
`300
`
`143
`4.8
`7.01
`64
`22
`42*
`0.5
`42
`
`2.5 (low)
`
`0
`
`144
`4.4
`1.84
`72
`33
`39
`0.9
`46
`
`143
`4.2
`2.89
`83
`42
`40
`1
`51
`
`2.5
`
`143
`4.4
`2.7
`80
`41
`39
`1
`52
`
`Females
`
`40
`
`142
`4.5
`4.49
`78
`37*
`40
`0.9
`49
`
`300
`
`2.5 (low)
`
`139*b
`4.6a
`8.46a
`76*b
`34**c
`43
`0.8**b
`51
`
`143
`4.5
`2.61
`81
`41
`40
`1
`57*
`
`aSignificant at p < .05 using the Jonckheere-Terpstra trend test.
`bSignificant at p < .01 using the Jonckheere-Terpstra trend test.
`cSignificant at p < .001 using the Jonckheere-Terpstra trend test.
`* p < .05.
`** p < .01.
`
`TABLE 4.—Mean serum clinical chemistry parameters in male and
`female protiramer-treated rats at week 30 (end of treatment-free period).
`
`Males
`
`Females
`
`0
`
`145
`1.93
`31
`34
`0.9
`
`300
`
`145
`4.29
`29
`37*
`0.8*
`
`0
`
`143
`2.56
`38
`33
`1.1
`
`300
`
`137**
`15.03**
`28***
`38***
`0.7**
`
`Dose level (mg/kg)
`
`Sodium (mmol/L)
`Total cholesterol (mmol/L)
`Albumin (g/L)
`Globulin (g/L)
`Albumin/globulin ratio
`
`* p < .05.
`** p < .01.
`*** p < .001.
`
`any organ, except for the injection site lesions, and there were
`no changes in either absolute or relative organ weights.
`Histopathology examination of tissue sections from the
`rat study indicated that the pathological changes were con-
`fined to the injection sites (Table 6). Fibrosis was the most
`common lesion, with a dose-related increase in incidence
`and severity. Myositis in the subcutaneous panniculus mus-
`cle was less common and was seen mostly at the high dose
`of 30 mg/kg/day, with a slight increase in males of the 10
`mg/kg GA group. There were sporadic cases of cellulitis,
`with increased severity in the 30 mg/kg dose group. In gen-
`eral, injection site lesions occurred more frequently at the
`posterior injection sites than in the anterior sites. This dif-
`ference might be attributed to the presence of multiple
`layers of
`fascia at
`the anterior sites, which may have
`contributed to increased dispersal of the injected material
`(Figures 1–3).
`Likewise, in the monkey study, treatment-related lesions
`were confined to the injection sites (Table 7) that were charac-
`terized by areas of dermal
`fibrosis,
`in which small-
`mononuclear-cell
`infiltration, occasionally giant cells, and
`rarely, lymphoid germinal follicles were present. In most ani-
`mals, a minor diffuse eosinophilic infiltration was also present.
`
`All groups were affected (including controls), but there was a
`clear treatment-related increase in severity. There were also
`minor and less prevalent edematous, hemorrhagic, and necrotic
`lesions at the injection sites, which showed no clear association
`with administration of GA (Figures 4–7).
`
`Protiramer Studies
`
`the termination of the rat
`Macroscopic examination at
`study revealed enlarged livers and kidneys in the high-dose
`groups. Weighing of the organs confirmed this macroscopic
`observation, and the weights of the liver, kidney, and spleen
`were greater in animals treated at 40 and 300 mg/kg when com-
`pared with concurrent controls at the end of the treatment period.
`In the histopathological examination of the rat
`tissues,
`treatment-related changes were seen in the injection sites, liver,
`and kidneys.
`
`Injection Sites: Fibrosis was seen in all injection sites from
`all groups (Table 8). In control animals, the fibrosis was graded
`as minimal to slight, whereas in other treated groups, the fibro-
`sis was graded as minimal to marked. Associated with fibrosis,
`especially at the high dose level, there were edema, thrombus
`formation, lymphoid cell infiltration, hemorrhage, neovascu-
`larization, and degeneration/regeneration of the panniculus
`skin muscle. In some animals, particularly at the high dose,
`these lesions were associated with subcutaneous necrosis,
`which occasionally formed a central necrotic cavity (Figures
`8–11). As each injection site was used several times, the
`inflammation was a mix of chronic and subacute reactions. The
`changes were dose related, and the increase in severity in
`treated animals indicated poor local tolerance to the test item.
`Following a four-week treatment-free period, previously
`treated animals had subacute to chronic inflammation associ-
`ated with fibrosis, necrosis, and hemo