Safety and Tolerability of Single Intravenous Doses of T Cell Modulatory Peptide (TCMP-80) in Healthy Volunteers Michael Anne
A. Eldon, E. Daigle,
PhD, PhD,
T Cell
Modulatory
turally
related
Peptide
FCP,
Richard
Darlene
V. Katz,
A. Smith,
PhD,
MD,
and
T. Leese,
Philip
Steven
P. Richieri,
MD, RPh
L-lysine-L-serine, is a synthetic dipeptide strucacid sequence in human immunoglobulin G. Based on in vitro and preclinical in vivo testing, TCMP-80 has immunomodulatory properties. This report describes the first administration of TCMP-80 to man in a randomized, double-blind, placebo-controlled, single rising-dose tolerability trial. Healthy male volunteers received TCMP-80 or placebo as a 10-minute intravenous infusion. At weekly intervals, two of four subjects were given TCMP-80; the remaining two received placebo. Each subject could receive only one dose during the study. Dosing started at 0.01 mg/kg and was increased to 0.03, 0.1, 0.3, 1, 3, 6.5, and 10 mg/kg. CBCs, blood chemistries, urinalyses, and lymphocyte subset populations were monitored predose and postdose on Days 1, 5, and 14. Three placebo and three TCMP-80 subjects reported adverse events. Adverse events reported after TCMP-80 administration were mild in nature (headache, dizziness, hematoma at injection site), appeared to be independent of dose, and resolved without medical intervention. No clinically significant alterations in vital signs, physical examination parameters, or clinical laboratory values were observed. Based on the results of this study, TCMP-80 is safe and well-tolerated within the dose range studied when administered as single intravenous infusions. Additionally, this study design represents an approach to assess the safety of an investigational immunomodulatory drug. to a selected
(TCMP-80),
amino
T
Cell Modulatory Peptide (TCMP-80), L-lysineL-serine, is a synthetic dipeptide composed of naturally occurring amino acids structurally related to a selected amino acid sequence in human immu-
incidence but not the severity of Experimental Allergic Encephalomyelitis (EAE) in SJL/J mice, a model of multiple sclerosis (MS).2 During preclinical evaluation, TCMP-80 adminis-
noglobulin
tered
C (IgG).
T Cell
Modulatory
Peptide
was
selected from a series of synthetic peptides based on its ability to suppress mixed lymphocyte response (MLR) in vitro. During in vitro evaluation, TCMP-80 was shown to modulate immunological responses in a manner which suggests that it influences Helper and/or Suppressor T cell activity.’ In vivo, TCMP-80 at subcutaneous doses of I to 10 mg/kg reduced the
From
Immunetech
Pharmaceuticals,
Inc.,
(Drs.
Eldon,
Daigle,
Katz,
and Richieri), San Diego, California, the Center for Neurologic Study (Dr. Smith), San Diego, California,and the Quincy Research Center Leese), Kansas City, Missouri. Address for reprints: Michael A. Eldon, PhD, Parke-Davis Pharmaceutical Research, Clinical Pharmacology Department, 2800 Plymouth Road, Ann Arbor, Ml 48105. An abstract of this work was presented at the American College of Clinical Pharmacology 1989 Annual Meeting, Baltimore, MD, October 12, 1989. (Dr.
352
#{149} J Clin Pharmacoi
1990;30:352-357
to rats
in single
intravenous
doses
of 100
and
500 mg/kg did not cause acute toxicity but did result in increased T and B lymphocyte proliferation. In a subacute toxicity study, TCMP-80 was administered once daily for 14 days at intravenous doses of 1, 2, 10, or 100 mg/kg. After 14 days of dosing, proliferation of both T and doses of 10 mg/kg
B lymphocytes but was
was stimulated
suppressed at the
at 100
mg/kg dose level. No toxic effects attributable to TCMP-80 were observed at any dose. Other intravenous toxicity studies of TCMP-80 at doses up to 1.6 gm/kg have been performed in mice, rats, and dogs, with no drug-related toxicity observed. These results
suggest
that
TCMP-80
can
modulate
cellular
immune response in a dose dependent fashion but lacks acute toxicity at active doses.3 Based on its ability to suppress T cell activity, TCMP-80 is being developed as a putative therapy
IV TCMP-80
for autoimmune abnormal T cell lupus erythematosus first administration gle rising-dose IND 31,472.
MATERIALS Study This dose
tolerability
trial
conducted
AND
groups intravenous
mg/kg.
METHODS
of four to infusion
Subjects
receive doses
successively of TCMP-80
asin
saline (Immunetech PharmaCA) or placebo (matching vehieach dose group, two subjects and two subjects received 0.01, 0.03, 0.1, 0.3, 1.0, 3.0, 6.5, were
assigned
challenge
test
consisting
of 40 mcg
TCMP-80
or 0.1 mL placebo was administo detect potential hypersensibefore administration of the test challenges with histamine and also performed to serve as posicontrols, respectively. Test doses on the morning of each dosing
day as fusions
pump-controlled of 5 mL of
at the
of Study
rate
dosing
intravenous solution
inper
minute.
ScreenIng
lntradermal
Thirty-four healthy male subjects participated in this study conducted at Quincy Research Center, Kansas City, Missouri. The protocol was approved by the Quincy Research Center Institutional Review Committee and all subjects gave written informed consent before participating in the study. Before enrollment in the study, each subject was determined to be healthy and have normal immune status based on medical history and clinically normal hematology, blood chemistry, urinalysis, elec-
MATORY/IMMU
0
Day 14
x Xa
X
X x
X x
x X x
x X x
X
X
X
X
X
X
X X
challenge
x X
0.5. and 1 hour after hematology, chemistry,
X
dosing. and urinalysis.
See text for cell types analyzed. TCMP-80 or placebo administered randomization
Day 5
X X x
Study medicationd Symptom evaluation #{149} Performed Including
Day 1
X
Medical history Urine drug screen Physical examination Electrocardiogram Clinical laboratory’ Phenotype analysisc lgG. 1gM, and IgA analysis
as IV in fusion
according
to a blinded
code.
trocardiogram, lymphocyte analysis, and physical examination results. A urine drug screen was used to exclude subjects who might be substance abusers. Subjects were housed in the Quincy Research Center clinic overnight before and for approximately 36 hours after dosing. All medications were prohibited for 4 weeks before and 2 weeks after treatment administration. Safety Subjects
NOSUPPRESSIVES
and
Tolerability were
Criteria
monitored
for
adverse
events
and
changes in clinical status for the first 24 hours after dosing and at follow-up examinations 5 and 14 days after dosing as shown in Table I. Heart rate, respiration, 5-minute supine blood pressure, 2-minute erect blood pressure, and temperature were monitored every
Subjects
ANALGESIC/ANTI-INFLAM
Procedures
ascending
in 0.1 mL vehicle tered to each subject tivity to TCMP-80 dose. Intradermal normal saline were tive and negative were administered 10-minute
I
Day
identification numbers corresponding to their enrollment order, and were randomly assigned to receive TCMP-80 or placebo based on identification number according to a computer-generated randomization code which was blinded to the medical investigators and subjects. The next higher dose was administered to a new group of subjects provided that the previous dose was well tolerated. An intradermal
Schedule Procedure
phosphate-buffered ceuticals, San Diego, cle solution). Within received placebo TCMP-80 at doses of 10
TABLE
under
was a double-blind, placebo-controlled, singlestudy using healthy subjects randomly asin
VOLUNTEERS
diseases mediated by or displaying function such as MS and systemic (SLE). This report describes the of TCMP-80 to humans in a sin-
Design
signed cending
or
HEALTHY
IN
15 minutes
for
the
first
hour,
every
hour
for
the next 7 hours, and then every 4 hours for the next 16 hours. Physical examinations were performed 0.5, I, and 24 hours (day 1) after dosing, as well as on days 5 and 14. Electrocardiograms and samples for clinical laboratory tests and lymphocyte phenotype analysis
were
obtained
predose
and
on days
1, 5, and
14. The following clinical laboratory tests were performed on each occasion: hemoglobin, hematocrit, CBC with differential and reticulocyte count, platelet count, chemistry SMA-20 (fasting), and urinalysis. Lymphocyte phenotypes analyzed on each occasion were:
353
ELDON
TABLE Subject
Demographics
Characteristic
by Treatment Placebo
32 19 to 48 71.0
range (kg)
Mean height (cm) Height range (cm)
Cell
Presented TCMP.80
Mean age (yr) Age range (yr) Mean weight (kg) Weight
II
30 20 to 45 71.8
56.2 to 84.6
54.4 to 89.5
175 159 to 190
175 163 to 182
Type
Antibody
Human leukocytes Total T cells (E receptor) Mature T cells Helper T cells Suppressor T cells Activated T cells Mature B cells Natural killer cells Total lymphocytes (absolute count)
14. Lymphocyte
AL
lished for each method as values within ±2 standard deviations about the mean values obtained from healthy subjects included in the database for the clinical laboratory performing the analyses. The duration, severity, and clinical outcome of adverse events occurring during the study were recorded on case report forms. An assessment of the relationship of an adverse event to treatment was made by the medical investigators. The clinical status of each subject from the time of dosing through follow-up on day 5 was evaluated as the basis for deciding to advance to the next higher dose level. RESULTS
HLe1 TI 1
CD2
T3 T4 T8 Act BI
CD3 CD4 CD8 CD2O
populations
-
performed using concentrations of predose and on and
lin concentration values from each were compared to upper and lower each assay method. Normal limits
subject receiving TCMP-80 and one subject receiving placebo in the 1 mg/kg dose group did not return for follow-up on days 5 or 14. Both subjects were replaced to allow evaluation of the 1 mg/kg dose before administering the next higher dose. Thus, adequate data were available only from the 32 subjects completing the study. These subjects were well-matched between treatment groups for age, body weight, and height (Table II). Dose escalation proceeded as planned since each dose was found to be well tolerated. Three subjects receiving placebo and three subjects receiving TCMP-80 reported adverse events. In general, adverse events reported after TCMP-80 administration were mild in nature (headache, dizziness, hematoma at injection site), appeared to be independent of dose, and resolved without medical intervention. One
T
NKH1
All phenotype analyses were flow cytometry methods.45 Plasma IgG, 1gM, and IgA were determined day
Marker
ET
immunoglobu-
observation time normal limits for had been estab-
TABLE Adverse
Events
III
Presented
by Subject
and Dose
Dose Subject
Related
mg/kg
3 18
0.01 1.0
25
6.5
2
Placebo
16
Placebo
Hematoma at injection site Lightheaded, dizziness, slight fever, headache, myalgia Tightness in jaw and temple, dull
pain in temple Itching at injection site, loss of appetite, fatigue, runny nose, sneezing, tightness in abdomen, increased respiration rate Stiffness and soreness of head and neck,
28
354
Placebo
#{149} .1 Clin Pharmacol
Outcome
Symptoms
Tightness
1990;30:352-357
diplopia,
nystagmus
and ache in jaw and temple
To TCMP-80
Resolved Resolved
No Possibly
Resolved
Unlikely
Resolved
-
Discontinued, ultimately
preexisting Resolved
hospitalized, resolved as
-
conditions -
IV TCMP-80
IN
HEALTHY
After receiving placebo treatment, subject 16 was discontinued due to neurologic findings that led to hospitalization. The subject reported stiffness and soreness of the head and neck, diplopia, and nystagmus which were subsequently attributed to preexisting neurologic and psychiatric conditions. Adverse events and their outcomes are reported in Table III. No clinically significant changes in vital signs, physical examination results, electrocardiograms, clinical laboratory test results, or lymphocyte phenotype analyses were observed at any observation point after administration of any dose of TCMP-80, with the exception of one subject. After receiving 6.5 mg/kg of TCMP-80, subject 25 displayed mild elevation of serum transaminases without contributory clinical symptoms on days 5 and 14 as well as at an additional follow-up approximately 6 weeks after dosing. This finding was considered to be related to TCMP-80 administration since most other causes of transaminase elevation were ruled out. Other ab-
VOLUNTEERS
HELPER
-
UPPER
LIMIT
S
1500 1000
500
LOWER
LIMIT
0 -1
0
1
2
3
4
5
6
7
8
9
101112131415
DAY
HELPER
ALL DOSE
2500
T CELLS GROUPS
-
PLACEBO UPPER
LIMIT
LOWER
LIMIT
2000 1500
500
UPPER
LIMIT
0 -10
6000
B
1
2
3
4
5
6
7
8
9101112131415
DAY
4000 2000 --
LOWER
-
LIMIT
0 1
0
1
2
3
4
5
6
7
8
9
10
11 12131415
DAY
TOTAL LYMPHOCYTE ALL DOSE GROUPS
8000
-
COUNT PLACEBO UPPER
-
UMIT
6000
x
TCMP-80
1000
COUNT TCMP-80
-
X
-
E 2000
-
TOTAL LYMPHOCYTE ALL DOSE GROUPS
T CELLS GROUPS
-
S
-
ALL DOSE
2500
2000 LOWER
-1
0
1
2
3
4
5
6
7
8
LIMIT
9101112131415
DAY
Figure 1. Total lymphocyte count as a function ing. Data from all subjects receiving TCMP-80 from all subjects receiving placebo have been upper and lower plots, respectively, tipper and cate range of normal values for healthy subjects.
ANALGESIC/ANTI-INFLAM
MATORY/IMM
of time after dosat any dose and combined on the lower limits indi-
UNOSU
PPRESSIVES
Figure 2. Helper T cells count as a function of time after dosing. Data from all subjects receiving TCMP-8O at any dose and from all subjects receiving placebo have been combined on the upper and lower plots, respectively. Upper and lower limits indicate range of normal values for healthy subjects.
normal clinical laboratory values were sporadic and felt to be unrelated to treatment when viewed in relation to associated laboratory values collected over the course of the trial. Values for all clinical parameters were similar between TCMP-80 and placebo treatments when compared within or among dose groups. No clinically significant changes in lymphocyte populations or in plasma immunoglobulin concentrations were observed in any subject at any dose level. Populations of lymphocyte subtypes were similar within and among subjects before and after administration of both treatments. Since phenotype analysis results were similar for all TCMP-80 treated subjects, the data were pooled for graphic comparison to pooled data from placebo treated subjects. Figure 1 shows pooled total lymphocyte data for both treatments. Phenotype values for both treatments were generally within normal limits at all
355
ELDON
SUPPRESSOR 1600
UPPER
LIMIT
LOWER
LIMIT
1200 -1000 800 600
400 200 -1
0
1
2
3
4
5
6
7
8
9101112131415
DAY
SUPPRESSOR ALL DOSE
I CELLS
GROUPS
and longer status, with
PLACEBO
-
1600 UPPER
AL
components of immune response by quantitating selected T and B cell subsets in peripheral blood and serum immunoglobulin concentrations, respectively. The T and B cell markers chosen are suitable for monitoring the type of immunomodulation observed during preclinical development of TCMP-80, plus they have clinical applications in monitoring diseases that could be treated using immunosuppression therapy.6 The immune system tests used in this study monitor late indicators of immunomodulation and often take days after exposure before changes are observable. Since the effect of TCMP-80 on the immune system of healthy subjects was unknown, the tests were expected to indicate gross changes in immune status such as generalized stimulation or suppression. Quantitation of lymphocyte phenotypes was scheduled to monitor potential acute (days I and 5)
T CELLS
1400
ET
LIMIT
term the
(up to day 14) intent to monitor
changes in for longer
immune periods
1400 1200 -
1000
800
MATURE B CELLS ALL DOSE GROUPS - TCMP-80
600
400
1000
200
LOWER
LIMIT 800
-1
0
1
2
3
4
5
6
7
8
S
9101112131415
600
DAY
UPPER
-.-_
LIMIT
51
400 U
Figure 3. Suppressor T cells count as a function ing. Data from all subjects receiving TCMP-80 from all subjects receiving placebo have been upper and lower plots, respectively. Upper and cate range of normal values for healthy subjects.
observation points as shown Helper T cells, Suppressor cells, respectively. Occasional mal limits were judged to be did not appear to represent group trend.
of time after dosat any dose and combined on the lower limits indi-
200 LOWER
--..-.--------.---------
LIMIT
0 ‘1
in Figures 2, 3, and 4 for T cells, and Mature B values outside norepisodic in nature and a clinically significant
0
1
2
3
4
5
MATURE 1000
ALL DOSE
6
7 DAY
8
9101112131415
B CELLS GROUPS - PLACEBO
-
600
UPPER
LIMIT
DISCUSSION Designing
a safety
and
tolerability
trial
for
the
_________
first
LOWER
administration of a potential immunomodulator to humans proved to be challenging. The study was designed to identify potential side effects of TCMP-80 by combining practical monitoring of immune
system
parameters
with
conventional
monitoring, while keeping subject foremost concern. The panel of immune was assembled to monitor the cellular
356
#{149} J ClIn Pharmacol
1990;30:352-357
safety
safety as the system tests and humoral
LIMIT
-10123456789101112131415 DAY
4. Mature B cells count as a function of time after dosing. Data from all subjects receiving TCMP-8O at any dose and from all subjects receiving placebo have been combined on the upper and lower plots, respectively. Upper and lower limits indicate range of normal values for healthy subjects. Figure
IV
TCMP-80
IN
HEALTHY
if required. Serum immunoglobulins were determined predose and at the last follow-up visit since they have relatively long intrinsic half-lives and thus would change more slowly in response to stimuli. We elected to limit exposure to active drug to two subjects per dose level in case significant adverse events, especially those involving the immune system, occurred. For the same reasons, each subject could receive only one dose rather than administering a series of single doses to one subject. The protocol was designed to allow repetition of a given dose or inclusion of doses intermediate to those originally planned should clinically significant adverse effects be observed. Dose escalation was not to be resumed until the relationship between adverse events and TCMP-80 dose was resolved, and the drug was determined to be well tolerated at the dose level in question. It was not necessary to invoke any of these options during the course of the study since all dose levels were well tolerated. Placebo treatment was included in the study design to provide an unbiased control for evaluation of the safety and tolerability of TCMP-80. In particular, the most serious adverse events were reported by a subject receiving placebo treatment. Without a placebo control treatment, this occurrence as well as other nondrug-related random and systematic changes in subject status could have been attributed to TCMP-80 treatment. Based on the results of this study, TCMP-80 is safe and well tolerated in healthy human subjects over the dose range studied when administered as single
ANALGESIC/ANTI-INFLAMMATORY/IMMUNOSUPPRESSIVES
VOLUNTEERS
intravenous infusions. Since no limiting adverse events were observed, the maximum tolerated dose (MTD) was determined to be 10 mg/kg, the highest dose studied. Further safety evaluation of TCMP-80 in healthy subjects, including administration of multiple doses, is desirable before initiating testing in target patient populations. It is likely that potent immunomodulatory drugs for the treatment of a variety of medical conditions will be discovered. As these drugs are developed for clinical use, protocols must be designed to meet the special requirements of drugs which have the potential to effect the immune system. This study represents an approach for initial safety and tolerability assessment of this class of drugs. REFERENCES 1. Unpublished Inc., San Diego,
research CA 92121,
data, Immunetech submitted under
Pharmaceuticals, IND 31,472.
2. Hahn CS, Moore CX, Plummer JM: Suppression tal allergic encephalomyelitis (EAE) by a synthetic peptide. Fed Proc 1987;46:1376.
of experimenIgG Fc-derived
3. Atkinson JE, McCoy JL, Richieri SP: A two-week intravenous safety study of T Cell Modulatory Peptide in rats. Toxicologist 1988;8:1O. 4. Shapiro HM: Practical Flow Cytometry. New York, Alan Liss, 1988. 5. Van Dilla MA, Dean PN, Laerum OD, Melamed Cytometry: Instrumentation and Data Analysis. demic Press, 1985. 6. Stites DP: Clinical Laboratory Methods lar Immune Function, in Stites DP, Stobo and Clinical Immunology, Sixth Edition. and Lange, 1987; 285-303.
MR (eds): Orlando,
R.
Flow Aca-
for Detection of CelluJD, Wells JV (eds): Basic Norwalk, CT, Appleton
357
A. Eldon, E. Daigle,
PhD, PhD,
T Cell
Modulatory
turally
related
Peptide
FCP,
Richard
Darlene
V. Katz,
A. Smith,
PhD,
MD,
and
T. Leese,
Philip
Steven
P. Richieri,
MD, RPh
L-lysine-L-serine, is a synthetic dipeptide strucacid sequence in human immunoglobulin G. Based on in vitro and preclinical in vivo testing, TCMP-80 has immunomodulatory properties. This report describes the first administration of TCMP-80 to man in a randomized, double-blind, placebo-controlled, single rising-dose tolerability trial. Healthy male volunteers received TCMP-80 or placebo as a 10-minute intravenous infusion. At weekly intervals, two of four subjects were given TCMP-80; the remaining two received placebo. Each subject could receive only one dose during the study. Dosing started at 0.01 mg/kg and was increased to 0.03, 0.1, 0.3, 1, 3, 6.5, and 10 mg/kg. CBCs, blood chemistries, urinalyses, and lymphocyte subset populations were monitored predose and postdose on Days 1, 5, and 14. Three placebo and three TCMP-80 subjects reported adverse events. Adverse events reported after TCMP-80 administration were mild in nature (headache, dizziness, hematoma at injection site), appeared to be independent of dose, and resolved without medical intervention. No clinically significant alterations in vital signs, physical examination parameters, or clinical laboratory values were observed. Based on the results of this study, TCMP-80 is safe and well-tolerated within the dose range studied when administered as single intravenous infusions. Additionally, this study design represents an approach to assess the safety of an investigational immunomodulatory drug. to a selected
(TCMP-80),
amino
T
Cell Modulatory Peptide (TCMP-80), L-lysineL-serine, is a synthetic dipeptide composed of naturally occurring amino acids structurally related to a selected amino acid sequence in human immu-
incidence but not the severity of Experimental Allergic Encephalomyelitis (EAE) in SJL/J mice, a model of multiple sclerosis (MS).2 During preclinical evaluation, TCMP-80 adminis-
noglobulin
tered
C (IgG).
T Cell
Modulatory
Peptide
was
selected from a series of synthetic peptides based on its ability to suppress mixed lymphocyte response (MLR) in vitro. During in vitro evaluation, TCMP-80 was shown to modulate immunological responses in a manner which suggests that it influences Helper and/or Suppressor T cell activity.’ In vivo, TCMP-80 at subcutaneous doses of I to 10 mg/kg reduced the
From
Immunetech
Pharmaceuticals,
Inc.,
(Drs.
Eldon,
Daigle,
Katz,
and Richieri), San Diego, California, the Center for Neurologic Study (Dr. Smith), San Diego, California,and the Quincy Research Center Leese), Kansas City, Missouri. Address for reprints: Michael A. Eldon, PhD, Parke-Davis Pharmaceutical Research, Clinical Pharmacology Department, 2800 Plymouth Road, Ann Arbor, Ml 48105. An abstract of this work was presented at the American College of Clinical Pharmacology 1989 Annual Meeting, Baltimore, MD, October 12, 1989. (Dr.
352
#{149} J Clin Pharmacoi
1990;30:352-357
to rats
in single
intravenous
doses
of 100
and
500 mg/kg did not cause acute toxicity but did result in increased T and B lymphocyte proliferation. In a subacute toxicity study, TCMP-80 was administered once daily for 14 days at intravenous doses of 1, 2, 10, or 100 mg/kg. After 14 days of dosing, proliferation of both T and doses of 10 mg/kg
B lymphocytes but was
was stimulated
suppressed at the
at 100
mg/kg dose level. No toxic effects attributable to TCMP-80 were observed at any dose. Other intravenous toxicity studies of TCMP-80 at doses up to 1.6 gm/kg have been performed in mice, rats, and dogs, with no drug-related toxicity observed. These results
suggest
that
TCMP-80
can
modulate
cellular
immune response in a dose dependent fashion but lacks acute toxicity at active doses.3 Based on its ability to suppress T cell activity, TCMP-80 is being developed as a putative therapy
IV TCMP-80
for autoimmune abnormal T cell lupus erythematosus first administration gle rising-dose IND 31,472.
MATERIALS Study This dose
tolerability
trial
conducted
AND
groups intravenous
mg/kg.
METHODS
of four to infusion
Subjects
receive doses
successively of TCMP-80
asin
saline (Immunetech PharmaCA) or placebo (matching vehieach dose group, two subjects and two subjects received 0.01, 0.03, 0.1, 0.3, 1.0, 3.0, 6.5, were
assigned
challenge
test
consisting
of 40 mcg
TCMP-80
or 0.1 mL placebo was administo detect potential hypersensibefore administration of the test challenges with histamine and also performed to serve as posicontrols, respectively. Test doses on the morning of each dosing
day as fusions
pump-controlled of 5 mL of
at the
of Study
rate
dosing
intravenous solution
inper
minute.
ScreenIng
lntradermal
Thirty-four healthy male subjects participated in this study conducted at Quincy Research Center, Kansas City, Missouri. The protocol was approved by the Quincy Research Center Institutional Review Committee and all subjects gave written informed consent before participating in the study. Before enrollment in the study, each subject was determined to be healthy and have normal immune status based on medical history and clinically normal hematology, blood chemistry, urinalysis, elec-
MATORY/IMMU
0
Day 14
x Xa
X
X x
X x
x X x
x X x
X
X
X
X
X
X
X X
challenge
x X
0.5. and 1 hour after hematology, chemistry,
X
dosing. and urinalysis.
See text for cell types analyzed. TCMP-80 or placebo administered randomization
Day 5
X X x
Study medicationd Symptom evaluation #{149} Performed Including
Day 1
X
Medical history Urine drug screen Physical examination Electrocardiogram Clinical laboratory’ Phenotype analysisc lgG. 1gM, and IgA analysis
as IV in fusion
according
to a blinded
code.
trocardiogram, lymphocyte analysis, and physical examination results. A urine drug screen was used to exclude subjects who might be substance abusers. Subjects were housed in the Quincy Research Center clinic overnight before and for approximately 36 hours after dosing. All medications were prohibited for 4 weeks before and 2 weeks after treatment administration. Safety Subjects
NOSUPPRESSIVES
and
Tolerability were
Criteria
monitored
for
adverse
events
and
changes in clinical status for the first 24 hours after dosing and at follow-up examinations 5 and 14 days after dosing as shown in Table I. Heart rate, respiration, 5-minute supine blood pressure, 2-minute erect blood pressure, and temperature were monitored every
Subjects
ANALGESIC/ANTI-INFLAM
Procedures
ascending
in 0.1 mL vehicle tered to each subject tivity to TCMP-80 dose. Intradermal normal saline were tive and negative were administered 10-minute
I
Day
identification numbers corresponding to their enrollment order, and were randomly assigned to receive TCMP-80 or placebo based on identification number according to a computer-generated randomization code which was blinded to the medical investigators and subjects. The next higher dose was administered to a new group of subjects provided that the previous dose was well tolerated. An intradermal
Schedule Procedure
phosphate-buffered ceuticals, San Diego, cle solution). Within received placebo TCMP-80 at doses of 10
TABLE
under
was a double-blind, placebo-controlled, singlestudy using healthy subjects randomly asin
VOLUNTEERS
diseases mediated by or displaying function such as MS and systemic (SLE). This report describes the of TCMP-80 to humans in a sin-
Design
signed cending
or
HEALTHY
IN
15 minutes
for
the
first
hour,
every
hour
for
the next 7 hours, and then every 4 hours for the next 16 hours. Physical examinations were performed 0.5, I, and 24 hours (day 1) after dosing, as well as on days 5 and 14. Electrocardiograms and samples for clinical laboratory tests and lymphocyte phenotype analysis
were
obtained
predose
and
on days
1, 5, and
14. The following clinical laboratory tests were performed on each occasion: hemoglobin, hematocrit, CBC with differential and reticulocyte count, platelet count, chemistry SMA-20 (fasting), and urinalysis. Lymphocyte phenotypes analyzed on each occasion were:
353
ELDON
TABLE Subject
Demographics
Characteristic
by Treatment Placebo
32 19 to 48 71.0
range (kg)
Mean height (cm) Height range (cm)
Cell
Presented TCMP.80
Mean age (yr) Age range (yr) Mean weight (kg) Weight
II
30 20 to 45 71.8
56.2 to 84.6
54.4 to 89.5
175 159 to 190
175 163 to 182
Type
Antibody
Human leukocytes Total T cells (E receptor) Mature T cells Helper T cells Suppressor T cells Activated T cells Mature B cells Natural killer cells Total lymphocytes (absolute count)
14. Lymphocyte
AL
lished for each method as values within ±2 standard deviations about the mean values obtained from healthy subjects included in the database for the clinical laboratory performing the analyses. The duration, severity, and clinical outcome of adverse events occurring during the study were recorded on case report forms. An assessment of the relationship of an adverse event to treatment was made by the medical investigators. The clinical status of each subject from the time of dosing through follow-up on day 5 was evaluated as the basis for deciding to advance to the next higher dose level. RESULTS
HLe1 TI 1
CD2
T3 T4 T8 Act BI
CD3 CD4 CD8 CD2O
populations
-
performed using concentrations of predose and on and
lin concentration values from each were compared to upper and lower each assay method. Normal limits
subject receiving TCMP-80 and one subject receiving placebo in the 1 mg/kg dose group did not return for follow-up on days 5 or 14. Both subjects were replaced to allow evaluation of the 1 mg/kg dose before administering the next higher dose. Thus, adequate data were available only from the 32 subjects completing the study. These subjects were well-matched between treatment groups for age, body weight, and height (Table II). Dose escalation proceeded as planned since each dose was found to be well tolerated. Three subjects receiving placebo and three subjects receiving TCMP-80 reported adverse events. In general, adverse events reported after TCMP-80 administration were mild in nature (headache, dizziness, hematoma at injection site), appeared to be independent of dose, and resolved without medical intervention. One
T
NKH1
All phenotype analyses were flow cytometry methods.45 Plasma IgG, 1gM, and IgA were determined day
Marker
ET
immunoglobu-
observation time normal limits for had been estab-
TABLE Adverse
Events
III
Presented
by Subject
and Dose
Dose Subject
Related
mg/kg
3 18
0.01 1.0
25
6.5
2
Placebo
16
Placebo
Hematoma at injection site Lightheaded, dizziness, slight fever, headache, myalgia Tightness in jaw and temple, dull
pain in temple Itching at injection site, loss of appetite, fatigue, runny nose, sneezing, tightness in abdomen, increased respiration rate Stiffness and soreness of head and neck,
28
354
Placebo
#{149} .1 Clin Pharmacol
Outcome
Symptoms
Tightness
1990;30:352-357
diplopia,
nystagmus
and ache in jaw and temple
To TCMP-80
Resolved Resolved
No Possibly
Resolved
Unlikely
Resolved
-
Discontinued, ultimately
preexisting Resolved
hospitalized, resolved as
-
conditions -
IV TCMP-80
IN
HEALTHY
After receiving placebo treatment, subject 16 was discontinued due to neurologic findings that led to hospitalization. The subject reported stiffness and soreness of the head and neck, diplopia, and nystagmus which were subsequently attributed to preexisting neurologic and psychiatric conditions. Adverse events and their outcomes are reported in Table III. No clinically significant changes in vital signs, physical examination results, electrocardiograms, clinical laboratory test results, or lymphocyte phenotype analyses were observed at any observation point after administration of any dose of TCMP-80, with the exception of one subject. After receiving 6.5 mg/kg of TCMP-80, subject 25 displayed mild elevation of serum transaminases without contributory clinical symptoms on days 5 and 14 as well as at an additional follow-up approximately 6 weeks after dosing. This finding was considered to be related to TCMP-80 administration since most other causes of transaminase elevation were ruled out. Other ab-
VOLUNTEERS
HELPER
-
UPPER
LIMIT
S
1500 1000
500
LOWER
LIMIT
0 -1
0
1
2
3
4
5
6
7
8
9
101112131415
DAY
HELPER
ALL DOSE
2500
T CELLS GROUPS
-
PLACEBO UPPER
LIMIT
LOWER
LIMIT
2000 1500
500
UPPER
LIMIT
0 -10
6000
B
1
2
3
4
5
6
7
8
9101112131415
DAY
4000 2000 --
LOWER
-
LIMIT
0 1
0
1
2
3
4
5
6
7
8
9
10
11 12131415
DAY
TOTAL LYMPHOCYTE ALL DOSE GROUPS
8000
-
COUNT PLACEBO UPPER
-
UMIT
6000
x
TCMP-80
1000
COUNT TCMP-80
-
X
-
E 2000
-
TOTAL LYMPHOCYTE ALL DOSE GROUPS
T CELLS GROUPS
-
S
-
ALL DOSE
2500
2000 LOWER
-1
0
1
2
3
4
5
6
7
8
LIMIT
9101112131415
DAY
Figure 1. Total lymphocyte count as a function ing. Data from all subjects receiving TCMP-80 from all subjects receiving placebo have been upper and lower plots, respectively, tipper and cate range of normal values for healthy subjects.
ANALGESIC/ANTI-INFLAM
MATORY/IMM
of time after dosat any dose and combined on the lower limits indi-
UNOSU
PPRESSIVES
Figure 2. Helper T cells count as a function of time after dosing. Data from all subjects receiving TCMP-8O at any dose and from all subjects receiving placebo have been combined on the upper and lower plots, respectively. Upper and lower limits indicate range of normal values for healthy subjects.
normal clinical laboratory values were sporadic and felt to be unrelated to treatment when viewed in relation to associated laboratory values collected over the course of the trial. Values for all clinical parameters were similar between TCMP-80 and placebo treatments when compared within or among dose groups. No clinically significant changes in lymphocyte populations or in plasma immunoglobulin concentrations were observed in any subject at any dose level. Populations of lymphocyte subtypes were similar within and among subjects before and after administration of both treatments. Since phenotype analysis results were similar for all TCMP-80 treated subjects, the data were pooled for graphic comparison to pooled data from placebo treated subjects. Figure 1 shows pooled total lymphocyte data for both treatments. Phenotype values for both treatments were generally within normal limits at all
355
ELDON
SUPPRESSOR 1600
UPPER
LIMIT
LOWER
LIMIT
1200 -1000 800 600
400 200 -1
0
1
2
3
4
5
6
7
8
9101112131415
DAY
SUPPRESSOR ALL DOSE
I CELLS
GROUPS
and longer status, with
PLACEBO
-
1600 UPPER
AL
components of immune response by quantitating selected T and B cell subsets in peripheral blood and serum immunoglobulin concentrations, respectively. The T and B cell markers chosen are suitable for monitoring the type of immunomodulation observed during preclinical development of TCMP-80, plus they have clinical applications in monitoring diseases that could be treated using immunosuppression therapy.6 The immune system tests used in this study monitor late indicators of immunomodulation and often take days after exposure before changes are observable. Since the effect of TCMP-80 on the immune system of healthy subjects was unknown, the tests were expected to indicate gross changes in immune status such as generalized stimulation or suppression. Quantitation of lymphocyte phenotypes was scheduled to monitor potential acute (days I and 5)
T CELLS
1400
ET
LIMIT
term the
(up to day 14) intent to monitor
changes in for longer
immune periods
1400 1200 -
1000
800
MATURE B CELLS ALL DOSE GROUPS - TCMP-80
600
400
1000
200
LOWER
LIMIT 800
-1
0
1
2
3
4
5
6
7
8
S
9101112131415
600
DAY
UPPER
-.-_
LIMIT
51
400 U
Figure 3. Suppressor T cells count as a function ing. Data from all subjects receiving TCMP-80 from all subjects receiving placebo have been upper and lower plots, respectively. Upper and cate range of normal values for healthy subjects.
observation points as shown Helper T cells, Suppressor cells, respectively. Occasional mal limits were judged to be did not appear to represent group trend.
of time after dosat any dose and combined on the lower limits indi-
200 LOWER
--..-.--------.---------
LIMIT
0 ‘1
in Figures 2, 3, and 4 for T cells, and Mature B values outside norepisodic in nature and a clinically significant
0
1
2
3
4
5
MATURE 1000
ALL DOSE
6
7 DAY
8
9101112131415
B CELLS GROUPS - PLACEBO
-
600
UPPER
LIMIT
DISCUSSION Designing
a safety
and
tolerability
trial
for
the
_________
first
LOWER
administration of a potential immunomodulator to humans proved to be challenging. The study was designed to identify potential side effects of TCMP-80 by combining practical monitoring of immune
system
parameters
with
conventional
monitoring, while keeping subject foremost concern. The panel of immune was assembled to monitor the cellular
356
#{149} J ClIn Pharmacol
1990;30:352-357
safety
safety as the system tests and humoral
LIMIT
-10123456789101112131415 DAY
4. Mature B cells count as a function of time after dosing. Data from all subjects receiving TCMP-8O at any dose and from all subjects receiving placebo have been combined on the upper and lower plots, respectively. Upper and lower limits indicate range of normal values for healthy subjects. Figure
IV
TCMP-80
IN
HEALTHY
if required. Serum immunoglobulins were determined predose and at the last follow-up visit since they have relatively long intrinsic half-lives and thus would change more slowly in response to stimuli. We elected to limit exposure to active drug to two subjects per dose level in case significant adverse events, especially those involving the immune system, occurred. For the same reasons, each subject could receive only one dose rather than administering a series of single doses to one subject. The protocol was designed to allow repetition of a given dose or inclusion of doses intermediate to those originally planned should clinically significant adverse effects be observed. Dose escalation was not to be resumed until the relationship between adverse events and TCMP-80 dose was resolved, and the drug was determined to be well tolerated at the dose level in question. It was not necessary to invoke any of these options during the course of the study since all dose levels were well tolerated. Placebo treatment was included in the study design to provide an unbiased control for evaluation of the safety and tolerability of TCMP-80. In particular, the most serious adverse events were reported by a subject receiving placebo treatment. Without a placebo control treatment, this occurrence as well as other nondrug-related random and systematic changes in subject status could have been attributed to TCMP-80 treatment. Based on the results of this study, TCMP-80 is safe and well tolerated in healthy human subjects over the dose range studied when administered as single
ANALGESIC/ANTI-INFLAMMATORY/IMMUNOSUPPRESSIVES
VOLUNTEERS
intravenous infusions. Since no limiting adverse events were observed, the maximum tolerated dose (MTD) was determined to be 10 mg/kg, the highest dose studied. Further safety evaluation of TCMP-80 in healthy subjects, including administration of multiple doses, is desirable before initiating testing in target patient populations. It is likely that potent immunomodulatory drugs for the treatment of a variety of medical conditions will be discovered. As these drugs are developed for clinical use, protocols must be designed to meet the special requirements of drugs which have the potential to effect the immune system. This study represents an approach for initial safety and tolerability assessment of this class of drugs. REFERENCES 1. Unpublished Inc., San Diego,
research CA 92121,
data, Immunetech submitted under
Pharmaceuticals, IND 31,472.
2. Hahn CS, Moore CX, Plummer JM: Suppression tal allergic encephalomyelitis (EAE) by a synthetic peptide. Fed Proc 1987;46:1376.
of experimenIgG Fc-derived
3. Atkinson JE, McCoy JL, Richieri SP: A two-week intravenous safety study of T Cell Modulatory Peptide in rats. Toxicologist 1988;8:1O. 4. Shapiro HM: Practical Flow Cytometry. New York, Alan Liss, 1988. 5. Van Dilla MA, Dean PN, Laerum OD, Melamed Cytometry: Instrumentation and Data Analysis. demic Press, 1985. 6. Stites DP: Clinical Laboratory Methods lar Immune Function, in Stites DP, Stobo and Clinical Immunology, Sixth Edition. and Lange, 1987; 285-303.
MR (eds): Orlando,
R.
Flow Aca-
for Detection of CelluJD, Wells JV (eds): Basic Norwalk, CT, Appleton
357
