Clinical trial NCT02834403
Conditions: Metastatic Triple Negative Breast Cancer
Keywords: breast cancer nitric oxide synthase docetaxel L-NMMA
Sponsor: The Methodist Hospital Research Institute
Start date: 2016-11-01; Completion date: 2021-01-01
Enrollment: 37 participants; Allocation: NON_RANDOMIZED; Intervention model: SEQUENTIAL

Brief summary:
This is a Phase Ib/II study assessing the maximum tolerated dose (MTD), dose-limiting toxicities (DLTs), recommended Phase 2 dose (RP2D), and efficacy of L-NMMA when combined with docetaxel in refractory locally advanced or metastatic triple negative breast cancer patients. The Phase Ib portion of the study is designed to investigate the combination at two dose levels of docetaxel (75 and 100 mg/m2) and 7 dose levels of L-NMMA (5, 7.5, 10, 12.5, 15, 17.5, and 20 mg/kg). The starting dose of L-NMMA will be 7.5 mg/kg. In the Phase II portion of the study, the starting dose will be the RP2D determined in the Phase Ib portion of the study.

Detailed description:
This is a Phase Ib/II study assessing the maximum tolerated dose (MTD), dose-limiting toxicities (DLTs), recommended Phase 2 dose (RP2D), and efficacy of L-NMMA when combined with docetaxel in refractory locally advanced or metastatic triple negative breast cancer patients. The Phase Ib portion of the study is designed to investigate the combination at two dose levels of docetaxel (75 and 100 mg/m2) and 7 dose levels of L-NMMA (5, 7.5, 10, 12.5, 15, 17.5, and 20 mg/kg). The starting dose of L-NMMA will be 7.5 mg/kg. L-NMMA dose will escalate/de-escalate based on DLT occurrence. For the 5, 7.5, 10, 12.5, and 15 mg/kg L-NMMA doses, docetaxel will be administered at 75 mg/m2. For the 17.5 and 20 mg/kg L-NMMA doses, docetaxel will be administered at 100 mg/m2. In the Phase II portion of the study, the starting dose will be the RP2D determined in the Phase Ib portion of the study. In the phase II portion of the study, patients will be treated with L-NMMA and taxane (docetaxel, paclitaxel, or nab-paclitaxel) per physician's choice. Patients will be treated with L-NMMA and taxane chemotherapy (docetaxel, paclitaxel, or nab-paclitaxel) per physician's choice. L-NMMA will be administered on Days 1-5 and taxane chemotherapy on Day 1 Q3W or Day 1 Q1W. L-NMMA and docetaxel will be administered at the RP2D determined in the phase Ib portion of the study. Paclitaxel at 175 mg/m2 will be IV infused over 3 hours or 80 mg/m2 will be IV infused over 1 hour, and nab-paclitaxel at 260 mg/m2 will be IV infused over 30 minutes. For L-NMMA-induced hypertension, amlodipine (10 mg) and enteric-coated low-dose aspirin (81 mg) will be orally administered. Amlodipine will be administered for 6 days at each cycle, starting 24 hours before the first dose of L-NMMA. Enteric-coated low-dose aspirin will be administered once daily during the 6 21-day cycles. For docetaxel-induced leukopenia, pegfilgrastim (6 mg) will be administered via subcutaneous injection approximately 24 hours after every dose of docetaxel.

Arms: arm 1: Phase Ib: L-NMMA and docetaxel will be given for 6 21-day cycles. L-NMMA at doses of 7.5 mg/kg (starting dose) will be administered IV on Days 1-5. Docetaxel will be administered at 75 mg/m2 as an IV 15 min after L-NMMA infusion Day 1. arm 2: Phase Ib: L-NMMA and docetaxel will be given for 6 21-day cycles. L-NMMA at doses of 10 mg/kg will be administered IV on Days 1-5. Docetaxel will be administered at 75 mg/m2 as an IV 15 min after L-NMMA infusion Day 1. arm 3: Phase Ib: L-NMMA and docetaxel will be given for 6 21-day cycles. L-NMMA at doses of 12.5 mg/kg will be administered IV on Days 1-5. Docetaxel will be administered at 75 mg/m2 as an IV 15 min after L-NMMA infusion Day 1. arm 4: Phase Ib: L-NMMA and docetaxel will be given for 6 21-day cycles. L-NMMA at doses of 15 mg/kg will be administered IV on Days 1-5. Docetaxel will be administered at 75 mg/m2 as an IV 15 min after L-NMMA infusion Day 1. arm 5: Phase Ib: L-NMMA and docetaxel will be given for 6 21-day cycles. L-NMMA at doses of 17.5 mg/kg will be administered IV on Days 1-5. Docetaxel will be administered at 100 mg/m2 as an IV 15 min after L-NMMA infusion Day 1. arm 6: Phase Ib: L-NMMA and docetaxel will be given for 6 21-day cycles. L-NMMA at doses of 20 mg/kg will be administered IV on Days 1-5. Docetaxel will be administered at 100 mg/m2 as an IV 15 min after L-NMMA infusion Day 1. arm 7: Phase II: L-NMMA starting dose will be the RP2D determined in the Phase Ib portion of the study.
Interventions: intervention 1: L-NMMA intervention 2: Docetaxel intervention 3: Amlodipine intervention 4: Pegfilgrastim intervention 5: Enteric-coated aspirin
Intervention descriptions: intervention 1: Nitric oxide synthase inhibitor intervention 2: Mitotic inhibitor, cytotoxic intervention 3: Long-acting calcium channel blocker intervention 4: Colony-stimulating factor intervention 5: non-steroidal anti-inflammatory drug

Protocol documents:
Prot_SAP_000.pdf: 
HMCC&HMRI   
   LNMMA Clinical Protocol  
 Page 1 
Version: 7 
CLINICAL PHASE Ib/II TRIAL OF L-NMMA PLUS TAXANE CHEMOTHERAPY IN THE 
TREATMENT OF REFRACTORY LOCALLY ADVANCED OR METASTATIC TRIPLE 
NEGATIVE BREAST CANCER PATIENTS. 
Authors 
Polly Niravath, M.D. 
Document type 
L-NMMA plus Taxane Chemotherapy Phase Ib/II Protocol
IRB number 
Pro00011685 
Development phase 
Ib/II 
Document status 
Final 
Release date 
07-29-2015
Version/Date:  
IND Number: 
V8; 11-19-2019 
127156 


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Table of contents 
Table of contents ..................................................................................................................................... 2 
Glossary of terms ..................................................................................... Error! Bookmark not defined. 
1. 
Background .............................................................................................. Error! Bookmark not defined. 
2. 
Significance .............................................................................................. Error! Bookmark not defined. 
2.1 
Approach And Preliminary Studies ............................................. Error! Bookmark not defined. 
2.2 
L-NMMA clinical experience ..................................................................................................... 12 
3. 
Hypothesis: ............................................................................................................................................ 18 
4. 
Objectives and endpoints ...................................................................................................................... 18 
5. 
Study design ............................................................................................. Error! Bookmark not defined. 
5.1 Phase Ib Dose Levels ...................................................................................................................... 21 
5.2 Phase II Dose Levels ....................................................................................................................... 21 
5.3 Biopsies ........................................................................................................................................... 21 
6. 
Population ............................................................................................................................................. 21 
7. 
Inclusion and Exclusion criteria ............................................................................................................. 22 
7.1 
Inclusion Criteria ...................................................................................................................... 22 
7.2 
Exclusion Criteria ..................................................................................................................... 23 
8. 
Treatment .............................................................................................................................................. 23 
9. 
Visit schedule and assessments ........................................................................................................... 26 
9.1 
Study Flow and Visit Schedule ................................................................................................. 26 
10 Clinical Trial Materials ........................................................................................................................... 33 
10.1 
L-NMMA ................................................................................................................................... 28 
10.2 
Docetaxel ................................................................................................................................. 37 
10.3 
Paclitaxel .................................................................................................................................. 37 
10.4 
Nab-paclitaxel .......................................................................................................................... 37 
10.4 
Amlodipine ............................................................................................................................... 41 
10.56 Enteric-Coated Aspirin 81 mg .................................................................................................. 50 
11 Prohibited Concomitat Therapy ............................................................................................................. 51 
11.1 Anti-Neoplastic Therapies ............................................................................................................. 52 
12 Assessment Types ................................................................................................................................ 52 
12.1 Criteria for response ...................................................................................................................... 52 
12.2 RECIST 1.1 cretiria. ...................................................................................................................... 52 
12.3 Withdrawal of Subjects from study ................................................................................................ 52 
12.4 Pregnancy and assessment of Fertility .......................................................................................... 53 
13. Safety monitoring and reporting ............................................................................................................ 54 
13.1 
Adverse events ........................................................................................................................ 54 
13.1.1 
Definitions and reporting ....................................................................................... 54 
13.1.2 
Laboratory test abnormalities ................................................................................ 55 
14 Serious Adverse Events ........................................................................................................................ 55 


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14.1 
Definitions ............................................................................................................. 55 
14.1.1 
Reporting ............................................................................................................... 55 
 
14.1.2 
Pregnancy ............................................................................................................. 56 
15 Statistical methods ................................................................................................................................ 57 
15.1 
Sample Size. ............................................................................................................................ 57 
16 Protocol amendments, or changes in study conduct ............................................................................. 60 
17 References: ........................................................................................................................................... 60 
18 Appendices ............................................................................................................................................ 66 
 
LIST OF FIGURES 
Fig. 1 Decrease in tumor volume in PDX models .................................................................................... 9 
Fig .2. Effect of RPL39 & MLF2 siRNA in lung metastasis .................................................................... 10 
Fig. 3 Significant worse survival in activating mutations RPL39&MLF2 ................................................ 10 
Fig. 4 iNOS & eNOS increased expression in overexpressen RPL39&MLF2 ....................................... 11 
Fig. 5  decrease in tumor volume with L-NMMA + Docetaxel ................................................................ 11 
Fig. 6 Significant decrease in volume in two PDX models (BCM 2147 and BCM 5898) ........................ 12 
Fig. 7 Systolic blood pressure change 15 minutes after administration of LNMMA. .............................. 15 
Fig. 8 Comparison between docetaxel/amlodipine vs. docetaxel .......................................................... 17 
Fig. 9 L-NMMA + Docetaxel Study Design ............................................................................................ 20 
Fig. 10 Structure of L-NMMA Drug Substance ...................................................................................... 28 
Fig. 13 Plasma concentration of L-NMMA ............................................................................................. 29 
Fig .14 Relationship between CO and plasma concentrations of L-NMMA ........................................... 30 
Fig. 15 Label for L-NMMA Injection for BOLUS Administration Vial ...................................................... 36 
Fig. 16 Label for L-NMMA Injection for INFUSION Administration Vial ................................................. 36 
Fig. 17 Label for L-NMMA Injection Kit Box  .......................................................................................... 36 
Fig. 18 Label Inside L-NMMA Injection Kit to Differentiate Between Bolus and Infusion Vials .............. 37 
Fig. 19 Amlodipine Molecular structure ................................................................................................. 41 
 
LIST OF TABLES 
Table. 1 Phase III L-NMMA cardiogenic-shock Patient Characteristics  ................................................ 14 
Table .2 Phase III L-NMMA cardiogenic-shock Baseline vasopresor use. ............................................. 15 
Table. 3 L-NMMA + Docetaxel Dose Levels .......................................................................................... 24 
Table. 4 L-NMMA + Docetaxel SOE ...................................................................................................... 26 
Table. 5 Pharmacokinetic parameters of 3 mg kg−1 L-NMMA  .............................................................. 29 
Table. 6 Physical Chemical Properties of L-NMMA Drug Substance ..................................................... 30 
Table. 7 Potential Impurities in L-NMMA Drug Substance . ................................................................... 31 
Table. 8 Potential Residual Solvents in L-NMMA Drug Substance  ....................................................... 31 
Table. 9 Stability Protocol for L-NMMA Drug Substance  ....................................................................... 32 


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Table. 10 Composition of L-NMMA Injection .......................................................................................... 32 
Table .11 Stability of L-NMMA Injection Stored at 121C and 30 psi for 1 Hour ..................................... 33 
Table. 12 Stability Protocol for L-NMMA Injection .................................................................................. 33 
Table. 13 Stability of L-NMMA Injection 7.5 mg/mL Diluted in Saline and Stored at Ambient 
Conditions for 24 Hours  ................................................................................................................. 34 
Table. 14 Stability of L-NMMA Injection 75 mg/mL Diluted into Saline and Stored at Ambient 
Conditions for 24 Hours .................................................................................................................. 35 
Table. 15 Specifications and Batch Analysis Results for L-NMMA Placebo . ......................................... 35 
Table. 16 Docetaxel pharmacokinetics .................................................................................................. 38 
Table. 17 Docetaxel side Effects  ........................................................................................................... 39 
Table. 18 Docetaxel drug Interactions ................................................................................................... 40 
Table. 19 Docetaxel Hem toxicities Dose Guidelines. ............................................................................ 40 
Table. 20 Docetaxel Liver toxicities Dose Guidelines ............................................................................ 41 
Table. 21 Docetaxel Cutaneous Toxicities & Neuropathy Dose Guidelines ........................................... 41 
Table. 22 Amlodipine Adverse Events ................................................................................................... 44 
Table .23 Amlodipine Adverse Events. .................................................................................................. 45 
Table. 24 Amlodipine Adverse Events  .................................................................................................. 45 
Table .25 Dose Escalation Table for L-NMMA + 75 mg/m2 of Docetaxel. .............................................. 57 
Table. 26 Phase I Design Operating Characteristics.  Optimal decisions are given in boldface type. .... 58 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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List of abbreviations 
Abbreviation 
Definition 
ALP 
alkaline phosphatase 
ALT 
alanine aminotransferase 
ANC 
absolute neutrophil count 
aPTT 
activated partial thromboplastin time 
ASCO 
American Society of Clinical Oncology 
AST 
aspartate aminotransferase 
ATA 
antitherapeutic antibody 
AUC 
area under the plasma concentration versus time curve 
C 
degrees Celsius 
M 
Micromolar 
AE 
adverse event 
BSA 
body surface area 
BUN 
blood urea nitrogen 
CBC 
complete blood count 
Cm 
CO 
Centimeter 
cardiac output 
CR 
complete response 
CTCAE 
(NCI) Common Terminology Criteria for Adverse Events 
CTEP 
Cancer Therapy Evaluation Program 
dL 
Deciliter 
CL 
clearance, IV dosing 
Cmax 
single-dose maximum (peak) concentration 
CO2 
carbon dioxide 
COPD 
chronic Obstructive Pulmonary Disease 
CSF 
CT 
cerebral spinal fluid 
computed tomography 
CSF 
CT 
cerebral spinal fluid 
computed tomography 
DLT 
dose-limiting toxicity 
DNA 
deoxyribonucleic acid 
ECT 
enteric-coated tablet 
FDA 
FPA 
Food and Drug Administration 
fundus pulsation amplitude 
GCP 
Good Clinical Practice 
G-CSF 
GEMM 
granulocyte colony stimulating factor 
genetically engineered mouse models 
GGT 
gamma glutamyl transferase 
GI 
Gastrointestinal 
GLP 
Good Laboratory Practice 
Hb 
Hemoglobin 
Hct 
Hematocrit 
ht 
Height 
HDPE 
high-density polyethylene 


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Abbreviation 
Definition 
HIV 
human immunodeficiency virus 
HMCC 
HMRI 
HNSTD 
Houston Methodist Cancer Center 
Houston Methodist Research Institute 
highest nonseverely toxic dose 
HDPE 
high-density polyethylene 
HR 
heart rate 
IEC 
independent ethics committee 
IND 
Investigational New Drug 
IRB 
institutional review board 
IV 
Intravenous 
kg 
Kilogram 
Ki 
inhibitory constant 
Lbs 
LDA 
L-NMMA 
Pounds 
limiting dilution assays 
NG-monomethyl-l-arginine 
m2 
square meters 
mg 
Milligram 
min 
Minute 
mL 
milliliter 
mm3 
cubic millimeters 
Mmol 
MSFE 
Millimole 
mammosphere formation efficiency 
MTD 
maximum tolerated dose 
NCI 
National Cancer Institute 
NF-B 
nuclear factor-B 
ng 
nanogram 
nM 
NO 
NYHA 
ORR 
Nanomole 
nitric oxide 
New York Heart Association 
Objective response rate 
OS 
oral solution 
p21 
p21(ras) farnesyl-protein transferase 
p27 
cyclin-dependent kinase inhibitor 
p53 
tumor suppressor protein with molecular weight of 53 kDa 
PIC 
RP2D 
RP56976 
powder-in-capsule 
recommended phase 2 dose 
Docetaxel 
SAE 
TCGA 
serious adverse event 
The Cancer Genome Atlas 
TGD 
tumor growth delay 
TGI 
tumor growth inhibition 
TK 
TNBC 
Toxicokinetics 
triple Négative Brest cancer 
TNF-R 
tumor necrosis factor-receptor 
US 
United States 
USP 
United States Pharmacopeia 
ULN 
upper limit of the normal range 
w/w 
weight-to-weight ratio 


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Abbreviation 
Definition 
wt 
Weight 
 
 
 


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Glossary of terms 
 


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1. 
Background 
Despite the significant advances in breast cancer biology, there has been limited progress in the treatment of 
advanced breast cancer, with little change in the overall survival for women with treatment resistant metastatic 
breast cancer over the last several decades1. It is notable that approximately 40,000 women with metastatic 
breast cancer die each year because of treatment resistance and failure of current therapies. Our research 
focuses on delineating mechanisms of treatment resistance to identify novel treatment approaches to improve 
patient outcome. Classical models of carcinogenesis can be described as random or “stochastic” in which any 
cell can be transformed by accumulating the right combination of mutations. An alternative model is that 
subpopulations or clones of cells retain key stem-like properties including the capacity for self-renewal that 
drives carcinogenesis, as well as differentiation which contributes to cellular heterogeneity.  Experimental 
evidence supporting this intratumoral clonal heterogeneity was first reported in human leukemia by John Dick 
et al2. These concepts were then extended to solid tumors by some groups demonstrating that human breast 
cancers were driven by stem-like cells characterized by cell surface expression of CD44+/CD24-/low3. Large-
scale sequencing analyses of solid cancers have provided further evidence of the extensive heterogeneity 
within individual tumors4. This intratumoral heterogeneity5-7 may be a major contributor to treatment resistance 
and treatment failure. Different subpopulations may be associated with heterogeneous protein function that 
may foster tumor adaptation and lead to therapeutic failure through Darwinian selection. Accordingly, the 
subpopulations of cells with stem-like properties within a heterogeneous bulk tumor have been shown to be 
responsible for tumor initiation and recurrence3,8-12. Three groups have recently and independently provided 
direct and functional evidence for the presence of cells with stem-like properties by lineage tracing experiments 
in glioblastomas (GBM), squamous skin tumors, and intestinal adenomas, further corroborating the 
hierarchical nature of cancer13-15.  These independent groups confirm that only a fraction of cells within the 
bulk tumor have clonogenic potential13-15 and that this fraction is intrinsically resistant to chemotherapy13. 
 
2. 
Significance  
We were one of the first groups to demonstrate that breast cancer cells with stem-like properties were 
intrinsically resistant to conventional therapy9,16. Since these initial observations, other groups have confirmed 
this view by establishing the resistance of these cells to conventional chemotherapy and radiation therapy17-
20. These and other studies have also supported our finding that an increase in the stem-like cell population is 
associated with a worse prognosis3,17,19-21. These findings have fundamental clinical implications. Current 
development of cancer therapeutics is largely based on identifying agents with the ability to cause bulk tumor 
regression in animal models or in clinical trials; however, an exclusive focus on drugs that elicit tumor 
regression by killing actively cycling or fully differentiated cells may spare the critical population of therapy-
resistant cells. We have recently extended these observations to breast cancer and have shown that 
subpopulations of chemoresistant cells within the bulk primary tumor had the propensity to metastasize 
through an array of different adaptive mechanisms. 
We have also identified a tumorigenic signature from patient breast cancer biopsies16 and, 
subsequently, used a functional approach to identify novel targets for treatment resistance from this gene set. 
Using shRNA knockdown of the 477 genes in our tumorigenic signature, we performed a high throughput 
mammosphere formation efficiency (MSFE) screen. This approach identified two target proteins,  RPL39 and 
MLF2. RPL39 was previously recognized as a component of the 60S ribosomal complex located on 
chromosome X (XQ24) with a proposed role in spermatogenesis and protein translation22,23. MLF2 is located 
on chromosome 12 and may participate in chromosomal aberrations and cellular defense responses24. While 
little is known about the role of RPL39 in cancer, there is even more limited knowledge available on MLF2. A 
series of amino acid modifications of MLF2 on Ser 144, 152 and 23825-27 and a somatic mutation (Phe80Cys) 
has been linked to colorectal cancer28. Notably, RPL39 and MLF2 overexpression increased cell migration, 
proliferation and mammosphere formation, suggesting a potentially important function for these two genes in 
cancer. Comprehensive understanding of the mechanisms of RPL39 and MLF2 is a salient prerequisite for the 
confirmation of these two genes as novel cancer targets. By mutual exclusivity analysis of RPL39 and MLF2 


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using The Cancer Genome Atlas (TCGA) database, RPL39 and MLF2 were found to exclusively co-occur 
(p<0.00001),  suggesting a shared mechanistic pathway for both genes. Using microarray analysis, we 
identified “cellular effects of sildenafil (Viagra)” i.e. nitric oxide (NO) signaling as the primary pathway that 
linked both RPL39 and MLF2. We then confirmed the role of NO signaling by inducing iNOS (inducible nitric 
oxide synthase) protein with overexpression of RPL39 and MLF2 and reducing iNOS protein levels with siRNA 
(small interfering ribonucleic acid) silencing of RPL39 and MLF2. In literature, the role of NOS signaling in 
breast cancer biology has not been extensively studied. Reports to date suggest that high NO concentrations 
are cytotoxic to cancer cells whereas lower NO concentrations can enhance tumor growth29-31. iNOS 
expression has been correlated with increased tumor grade and aggressiveness of breast tumor cells32,33, 
specifically in basal-like triple negative breast cancer (TNBC)32. Increased iNOS expression has been 
correlated with treatment resistance in GBM34,35. In vitro anti-tumor activity of iNOS inhibitors has been 
previously reported in epidermoid carcinoma36, oral37, hepatic38, glioblastoma39, and breast cancer35,40-42. 
Collectively, this lead us to ask whether inhibition of NO signaling could target TNBC therapeutically.  
Here, we hypothesize that targeting all cells within heterogeneous bulk tumor, particularly 
those cells that have metastatic propensity, will be essential for elimination of cancer. 
 
2.1 
Approach And Preliminary Studies 
We have previously identified two novel cancer genes (RPL39 and MLF2) that play a role in treatment 
resistance and lung metastases.  We determined that upregulation of NO signaling was a common mechanistic 
pathway for both genes. We next determined that inhibition of NO signaling with L-NMMA diminishes the 
number of treatment resistant cells, as well as lung metastases in human TNBC cell lines. Thus, we have 
identified a novel target for TNBC as well as a potential treatment, a re-purposed drug L-NMMA, to be further 
evaluated in the phase 1b clinical trial, as proposed here.  Secondly, we have developed one of the largest 
series of twenty-seven well-characterized patient-derived xenograft (PDX) models. Transcriptome and 
proteome analysis indicate that the serially transplanted xenografts are similar, thus confirming that we have 
generated a renewable source of clinically relevant patient-derived human breast cancers for in vivo 
experiments. Thirdly, we have genetically engineered mouse models (GEMM) with metastatic potential, with 
intact immune system. Fourthly, access to GMP grade L-NMMA from ArgiNox Pharmaceuticals Inc. will enable 
us to conduct a phase 1b clinical study. This investigation seeks to establish a novel approach in overcoming 
treatment-resistant triple negative breast cancer for which no targeted therapies exist and where most women 
will eventually die from their disease. 
APPROACH: Preliminary 
data:  
Derivation of a tumorigenic gene 
signature  
We previously reported that the 
proportion of treatment resistant cells 
with stem-like properties increases 
after chemotherapy9. In matched 
biopsies, the relative proportion of 
cells bearing stem cell marker 
CD44+/CD24-/low 
and 
MSFE 
increased 
significantly 
after 
chemotherapy 
(p<0.001)9. 
We 
isolated tumorigenic populations from 
patient biopsies by two selection criteria: expression of CD44+/CD24-/low or survival in MS assay. These 
populations were evaluated by comparative gene expression analysis: CD44+/CD24-/low and MS vs. non-
tumorigenic cells. The number of genes in the two separate “enrichment” comparisons greatly exceeded 
chance according to multiple statistical tests. Hence, we defined a “CD44+/CD24-/low/MS gene signature” of the 
477 genes present in the significant overlap between both enrichment methods16. 


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Identification of RPL39 and 
MLF2 By shRNAs knockdown of 
the ~477 genes45 using 1124 
shRNAs (~2-3 shRNA per gene) in 
high-throughput screening, two top 
candidate genes, RPL39 and 
MLF2 were identified. In PDX and 
other models,  animals treated with 
RPL39 and MLF2 siRNAs showed 
a significant decrease in tumor 
volume 
compared 
to 
chemotherapy (Fig 1), together 
with 
significant 
reduction 
in 
CD44+/CD24-/low and MSFE, as well as decreased tumor initiation in limiting dilution assays (LDA). Additionally, 
RPL39 and MLF2 siRNA significantly reduced lung metastasis (Fig 2). Importantly, we described potential 
activating mutations in RPL39 and MLF2 that had not been previously identified in TCGA in lung metastases 
from breast cancer patients. Primary tumors were sequenced in TCGA and were unlikely to be of sufficient 
depth of coverage to detect small subclones of these treatment resistant cells. Next, in paired patient primary 
and lung metastatic samples, we determined that these activating mutations were present only in metastatic 
lesions, thus confirming our hypothesis that small subpopulations of chemoresistant cells within the 
bulk primary had the propensity to metastasize, and that targeting this intra-tumoral heterogeneity is 
essential for the eradication of cancer.  
Importantly, 
patients 
with 
these 
activating mutations had a significantly 
worse survival (Fig 3). In summary, we 
have shown that silencing two novel 
genes (RPL39 and MLF2) in PDX and 
other xenograft models dramatically 
reduced primary tumor volume and 
lung metastases, thus improving 
overall survival. 
RPL39, MFL2, and nitric oxide 
signaling Mutual exclusivity analysis 
of MLF2 and RPL39 using TCGA 
database determined that RPL39 and 
MLF2 
co-occur 
exclusively 
with 
p<0.00001, thus indicating a common 
mechanistic pathway for both genes. To gain insight to this mechanistic pathway, siRNA-treated PDX tumors 
described above were analyzed by gene expression microarray profiling. Ingenuity Pathway Analysis revealed 
that the pathway associated with “cellular effects of sildenafil (Viagra)” or nitric oxide (NO) signaling was 
common to both RPL39 and MLF2. Consequently, we specifically addressed the role of NO signaling.  
A significant increase in inducible nitric oxide synthase (iNOS) was observed with overexpression of RPL39 
or MLF2, whereas a decrease in iNOS and eNOS was seen with knockdown of RPL39 and MFL2 (Fig 4). 
Fig 2. Effect of RPL39 and MLF2 siRNA on lung metastasis. (A) RPL39 and MLF2 
siRNA treated mice 6weeks after primary tumor injection. (B) Reduced luminescence 
observed with RPL39 and MLF2 siRNA treatment 


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Efficacy 
of 
the 
small 
molecule pan-NOS inhibitor 
LNMMA as a re-purposed 
cancer therapeutic  
As siRNA-based therapies are 
still 
in 
early 
clinical 
development, we sought to 
determine if the small molecule 
pan-NOS inhibitor LNMMA 
could be an effective targeted 
therapy for treatment resistant 
and 
metastatic 
TNBC. 
Specifically, this drug was 
selected as it has been tested 
in several thousand patients by 
Dr. 
Steven 
Gross 
and 
colleagues in a randomized 
phase 3 study for cardiogenic 
shock 
(reviewed 
from 
Kilbourn46), The study was 
conducted to established the 
safety of LNMMA, but failed to 
reach its efficacy goal for 
cardiogenic 
shock. 
Thus, 
LNMMA could be re-purposed 
as 
therapy 
for 
treatment 
resistant 
metastatic 
triple 
negative breast cancer, for 
which no effective targeted therapy currently exists.  In vitro, LNMMA potently and significantly decreased 
proliferation, MSFE, and migration.  
 LNMMA is associated with an acute blood pressure (BP) elevation through inhibition of constitutive endothelial 
nitric oxide synthase (eNOS)46. To overcome LNMMA-induced hypertension, we tested an attenuated 
treatment regimen of LNMMA with the anti-hypertensive amlodipine, for two cycles in two different mouse 
cancer models (MDA-MB-231 and SUM159 xenografts), together with docetaxel chemotherapy. LNMMA was 
given after chemotherapy for 5 days at 200 mg/kg, which is comparable to previous clinical doses47 . Our 


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results indicate that the increase in blood pressure (mean systolic pressure of 147 mmHg compared to basal 
levels of 120 mmHg) was reversed by the calcium channel blocker amlodipine (10 mg daily [QD] for 6 days, 
i.p). This elevation in BP was transient and disappeared within 24 hours after the last injection of LNMMA. Of 
note, this combination of LNMMA and docetaxel decreased tumor growth in a MDA-MB-231 orthotopic model 
(Fig 5). This dose regimen also showed improvement in survival.  Similar results were found in SUM159 
xenografts (data not shown). Overall, these data indicate that the dose regimen proposed here is effective and 
safe in reducing tumor growth and metastases (data not shown) without elevations in BP.  Limiting dilution 
data in both SUM 159 and MDA-MB-231 
showed a significant decrease in tumor 
initiating capacity with NOS inhibition. 
 
Preclinical in vivo results in patient derived 
xenografts 
We evaluated the effect of LNMMA in 
two basal-like TNBC PDX models (BCM 5898 
and BCM 2147). BCM 5898 had differential 
higher expression of iNOS pathways compared 
to BCM 2147 (Fig 8). LNMMA (200 mg/kg, i.p for 
5 days) every three-week cycle, compared to 
vehicle. This preliminary data in only two TNBC 
PDX models demonstrated a statistically 
significant reduction in tumor volume with 
LNMMA in both models (Fig 6).  This dose of 
LNMMA in mouse models is equivalent to 10-15 
mg/kg in humans, as is used in this proposal 
(FASEB J. 22, 659-661:2007). 
In summary, we describe LNMMA as 
an effective small molecule inhibitor targeted 
against treatment resistance and metastases. 
Our findings lay the foundation for developing 
new therapies for women with treatment-
refractory triple negative breast cancer for 
whom prognosis is extremely poor. 
 
 
2.2 LNMMA Clinical Experience 
Randomized phase 3 study in cardiogenic 
shock 
A Randomized Controlled Phase III Trial, testing the  effect of Tilarginine Acetate in patients with Acute 
Myocardial Infarction and Cardiogenic Shock was completed in 2007.67 TRIUMPH was a prospective, 
international, multicenter, randomized, double-blind, placebo-controlled trial that tested the hypothesis that 
Tilarginine compared with placebo would reduce by 25% 30-day all-cause mortality in patients with MI 
complicated by cardiogenic shock despite successful revascularization of the infarct artery. 
The study was conducted at 130 centers in 8 countries in North America and Europe. Participants, or 
their legally authorized representatives, provided written informed consent. Institutional review board or ethics 
committee approval was obtained at all sites. Inclusion required all of the following: (1) MI, confirmed by 
ischemic symptoms for at least 30 minutes with elevated cardiac markers and/or ST-segment elevation or left 
bundle-branch block; (2) patency ( <70% stenosis) of the infarct artery, either occurring spontaneously and 
confirmed at angiography or after percutaneous revascularization; (3) refractory cardiogenic shock of less than 
24 hours’ duration, confirmed by peripheral signs of tissue hypoperfusion and systolic blood pressure less than 
Fig 6. Significant decrease in volume in two PDX models (BCM 
2147 and BCM 5898) with LNMMA vs. vehicle. 


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100mmHg despite vasopressor therapy (dopamine≥7 μg/kg per minute or norepinephrine or epinephrine 
≥0.15 μg/kg per minute) continuing longer than 1 hour after infarct artery patency; (4) clinical or hemodynamic 
evidence of elevated left ventricular filling pressures; and (5) left ventricular ejection fraction of less than 40%. 
Hemodynamics and requirement for vasopressor treatment were reconfirmed after randomization just prior to 
study drug administration; patients with resolving shock were excluded. Between January 2005 and August 
2006, 398 met the study inclusion criteria and were enrolled. Baseline characteristics of the population are 
shown in TABLE 1. All baseline characteristics were well balanced between the treatment groups. More than 
one quarter of the population was older than 75 years; the majority were male and of white race. More than 
half of the patients had hypertension and one third had diabetes; 84 (21%) had a history of heart failure and 
almost a third of those had advanced heart failure symptoms in the 6 weeks prior to enrollment. A quarter of 
the patients had baseline creatinine levels of 1.7 mg/dL (150 mol/L) or higher. 


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Table 1. Baseline Patient Characteristics* 
Abbreviations: CABG, coronary artery bypass graft surgery; COPD, chronic obstructive pulmonary disease; ECG, electrocardiogram; 
IQR, interquartile range; MI, myocardial infarction; 
NYHA, New York Heart Association. 
SI conversion factor: To convert creatinine to μmol/L, multiply values by 88.4. 
*The 2 patients for whom treatment assignment is unknown were not included (Figure 1). 
†Blood pressure and ejection fraction were measured on support measures. 
‡Baseline creatinine available only for 158 of 396 (40%) patients. 


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The median supported blood pressure just prior to study drug administration was 88/52 mm Hg. Most patients 
were supported with a single vasopressor at the time of study drug administration (TABLE 2).  The majority of 
patients presented with anterior, ST-segment elevation MI with left anterior descending infarct artery location.  
Percutaneous coronary intervention was performed in nearly all patients to achieve the requirement for less 
than 70% infarct artery stenosis before study entry.  
No significant differences in safety in these hundreds of were found, and the study concludes that Tilarginine, 
had no effect on mortality in patients with MI complicated by refractory cardiogenic shock.  
 
Volunteer study 
As LNMMA has been administered to thousands of patients, including a phase 3 clinical trial for cardiogenic 
shock (see above), the anticipated serious adverse events are known and documented (reviewed by Kilbourn 
et al). In the cardiogenic shock trial, LNMMA proved to be safe and with few adverse events (AEs), 
other than transient reversible hypertension, i.e. the blood pressure reverted to normal within minutes of 
stopping LNMMA infusion (Fig 7). LNMMA was not 
associated with any abnormalities in hematologic, 
liver, or renal systems45. In normotensive patients, 
LNMMA was administered to metastatic renal cell 
carcinoma patient prior to infusion of interleukin-2 (IL-
2), to differentiate the side effects of LNMMA from IL-
245. Transient reversible hypertension was observed 
with LNMMA. Bolus injections of LNMMA at 3, 6, and 
12 mg/kg was administered to normotensive subjects 
(n=3 in each cohort). Doses of 3 and 6 mg/kg did not 
induce clinically apparent side effects, and blood 
pressure (BP) remained unchanged. At a dose level 
of 12 mg/kg, patients experienced increase in 
systolic BP up to 25 mmHg, without any clinical 
symptoms, which normalized rapidly within a few 
minutes on stopping the LNMMA infusion (Fig 10, 
personal communication from Dr. Kilbourn). In this 
phase 1 study, the initial dose level will be 7.5 mg/kg in patients with no prior cardiac history and systolic BP< 
150 mmHg.  Additionally, all patients will be fully evaluated by a cardiologist before entry into this phase 1 
study. This initial human dose of 7.5 mg/kg is comparable to the mouse equivalent does tested in PDX 
models in which LNMMA in combination with docetaxel resulted in significant tumor regression.  
These patients in these earlier clinical studies were not treated with anti-hypertensive like the calcium 
channel antagonist amlodipine, as is proposed here. Amlodipine is expected to decrease systolic BP by 
Fig 7. Systolic blood pressure change 15 minutes after 
administration of LNMMA in normotensive patients at 3, 6, 
12 mg/kg.  
 


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at least 15 mmHg within 24 hours of the first dose. This drug produces peak plasma concentrations 
between 6 and 12 hours, and the absolute bioavailability has been estimated to be between 64 and 90%. In 
the hundreds of patients (n=800 on amlodipine and 538 on placebo) who received amlodipine in 15 double-
blind, randomized trials, statistically significant reduction in systolic BP averaging 15 mmHg was observed 24 
hours post first dose54. Amlodipine has been evaluated for safety in more than 11,000 patients in U.S. and 
foreign clinical trials. In general, treatment with amlodipine was well-tolerated at doses up to 10 mg daily. Most 
adverse reactions reported during therapy with amlodipine were of mild or moderate severity. In controlled 
clinical trials directly comparing amlodipine (N=1730) at doses up to 10 mg to placebo (N=1250), 
discontinuation of amlodipine due to adverse reactions was required in only about 1.5% of patients and was 
not significantly different from placebo (about 1%). As seen in our preliminary data, as expected with one of 
the most commonly prescribed antihypertensive, there was significant difference in any laboratory 
measurements including renal, liver and hematologic values in patients receiving docetaxel/amlodipine vs. 
docetaxel alone. In our preclinical xenograft experiments, we administered amlodipine which effectively 
reversed any elevation in blood pressure. Additionally, given the short half-life of LNMMA, any elevation 
in BP will be reversed quickly by stopping the LNMMA infusion.  
LNMMA has been found to be a safe and tolerable in the thousands of patients studied. Nonetheless, 
additional measures to ensure the safety of the patients in this phase 1b/II study will be undertaken. First, only 
patients without any cardiac history or known hypertension (defined as a systolic BP >150 mm Hg) will be 
included. Second, all patients will be evaluated by a cardiologist before entry into this study. Third, at each L-
NMMA infusion, BP will be measured before infusion, 1 hour after infusion start, and at infusion completion. 
Automatic BP readings will be performed unless manual reading is needed. If systolic BP is >180 mmHg at 
the 1-hour check, recheck BP after 15−30 min. If BP is not <160 mmHg, recheck BP after another 15−30 min. 
If BP is still not <160 mmHg, the patient is to be discontinued from the study. Patients whose BP lowers to 
<160 mmHg at the first or second recheck will be rechallenged with the study drug. If systolic BP again 
increases beyond 180 mmHg, the patient is to be discontinued from the study. If systolic BP is >180 mmHg at 
infusion completion, recheck BP after 15−30 min. If BP is not <160 mmHg, recheck BP after another 15−30 
min. If BP is still not <160 mmHg, the patient is to be discontinued from the study. 
 
Safety of docetaxel and amlodipine 
Additionally, we investigated the safety of amlodipine and docetaxel, to ensure there is no unanticipated 
interaction with this duet. As amlodipine is a very common antihypertensive, as is the use of docetaxel in 
cancer patients, we investigated if there would be significant differences in safety profile with the combination 
of amlodipine to docetaxel.  
 
We identified 440 patients treated with docetaxel, and 65 patients treated with both 
docetaxel/amlodipine. Changes in total white cell count, urea, creatinine and bilirubin were examined in both 
groups at 3 weeks and 3 months after starting docetaxel chemotherapy. No statistically significant differences 
were identified in both groups (Fig 8A). In a smaller sample size of breast cancer patients, 40 in each group, 
again no statistically significant difference was noted in total white cell count, neutrophils, hemoglobin, platelet 
count, urea, creatinine, bilirubin, AST or ALT in docetaxel vs. docetaxel/amlodipine at 3 weeks and 3 months 
(Fig 8B).   Docetaxel/amlodipine is commonly administered in cancer patients who are hypertensive and who 
receive docetaxel chemotherapy, and no black box warnings have been issued with this combination. All 
parameters for this dual combination showed no statistical difference compared to docetaxel alone.  


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3.    Hypothesis  
L-NMMA plus taxane (docetaxel, paclitaxel, or nab-paclitaxel) will improve antitumor activity and drug 
tolerability in refractory locally advanced or metastatic TNBC patients.  
Rationale: Robust evidence indicates that treatment resistance and metastasis arise from a subpopulation of 
cells with stem-like properties within a heterogeneous primary cancer13-15. The persistence of this unique 
subpopulation of chemoresistant cells can serve to reinitiate tumor growth and seed metastases. We have 
demonstrated that residual tumors after chemotherapy exposure are enriched for these stem-like cells, and 
we have characterized several regulatory pathways involved in treatment resistance, in particular the iNOS 
Fig 8. Comparison between docetaxel/amlodipine vs. docetaxel in hematologic, renal and liver function tests in A) breast 
cancer 
patients 
and 
B) 
cancer 
patients 
at 
3 
weeks 
and 
3 
months 
of 
starting 
docetaxel
 
 
 
A.
B.


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signaling pathway. The overall scientific goal of this clinical trial is to target novel treatment resistance 
mechanisms in TNBC. 
 
4. 
Objectives and endpoints 
Primary Objectives: 
 
To assess the maximum tolerated dose (MTD) of L-NMMA when combined with docetaxel/amlodipine 
in the treatment of refractory locally advanced or metastatic TNBC patients. 
 
To determine the clinical benefit rate (CBR) of L-NMMA combined with taxane chemotherapy 
(docetaxel, paclitaxel, or nab-paclitaxel)/amlodipine, as assessed by the Response Evaluation Criteria 
in Solid Tumors (RECIST) 1.1. 
Secondary Objectives: 
 
To describe the dose-limiting toxicities (DLTs) and other toxicities associated with L-NMMA when 
combined with docetaxel/amlodipine, as assessed by the Common Terminology Criteria for Adverse 
Events (CTCAE) v4.03. 
 
To determine the recommended phase 2 dose (RP2D) of L-NMMA when combined with 
docetaxel/amlodipine. 
 
To assess the antitumor activity of L-NMMA when combined with taxane chemotherapy (docetaxel, 
paclitaxel, or nab-paclitaxel)/amlodipine, as assessed by the RECIST 1.1. 
Exploratory Objectives: 
 
To study the plasma pharmacokinetics (PK) and pharmacodynamics (PD) of L-NMMA when 
combined with docetaxel/amlodipine in patients with refractory locally advanced or metastatic TNBC. 
 
To determine potential predictive biomarkers such as iNOS (expression, phosphorylation), AKT, and 
the polycomb-regulated EZH2 pathway and markers related to treatment resistance, hypoxia and 
endoplasmic reticulum (ER) stress, survival, and autophagy. 
 
To measure serum levels of nitrite/nitrate and inflammatory biomarkers (interleukin [IL]-1, IL-6, IL-8, 
tumor necrosis factor- [TNF-], and circulating vascular endothelial growth factor [VEGF]). 
 
To explore changes in angiogenesis by IHC analysis of cluster of differentiation 31 (CD31; platelet 
endothelial cell adhesion molecule [PECAM-1]. 
 
To explore other blood-related biomarkers, including RPL39, MLF2, and phosphatidylinositol-4,5-
bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA) mutations in cell-free DNA. 
 
Primary Endpoints/Criteria for Evaluation 
Primary Endpoints: The primary endpoints will be the MTD of L-NMMA when combined with 
docetaxel/amlodipine and the CBR of L-NMMA combined with taxane chemotherapy (docetaxel, paclitaxel, or 
nab-paclitaxel)/amlodipine.  ORR will be defined as the percentage of patients with complete response (CR), 
partial response (PR), or stable disease (SD), as assessed by the RECIST 1.1. 
Secondary Endpoints: Secondary endpoints include 1) to describe the DLTs and other toxicities of L-NMMA 
when combined with docetaxel/amlodipine; 2) to determine the RP2D of L-NMMA when combined with 
docetaxel/amlodipine; 3) to assess the antitumor activity of L-NMMA when combined with taxane 
chemotherapy (docetaxel, paclitaxel, or nab-paclitaxel)/amlodipine; 4) to assess PK and PD of L-NMMA when 
combined with docetaxel/amlodipine; and 5) to explore tissue and blood-based markers of treatment response 
and toxicity. DLTs will be defined as any treatment-related death or any ≥ Grade 3 AE unless there is clear 
alternative evidence that the AE was not caused by the study treatment (See Section 8 for a detailed list of 
DLTs). The DLT assessment window is defined as the duration required to complete one full cycle of treatment 
with the L-NMMA and docetaxel combination. If treatment is delayed, the timelines for dose escalation and 
MTD determinations will be adjusted to allow for the completion of the DLT assessment window.  
Criteria for Evaluation of Subjects: Subjects will be considered evaluable for the purpose of establishing 
dose escalation/de-escalation decisions and the MTD if they satisfy one of the following criteria: receive at 
least one complete cycle of L-NMMA in combination with docetaxel and complete study assessments through 


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the DLT assessment window (through Day 21) without a DLT or experience a DLT within the DLT assessment 
window and are withdrawn from the study. 
Tumor Assessments: Computed tomography (CT) scan of the chest, abdomen, and pelvis will be performed 
during the screening period.  Repeat CT scans will be done at the end of cycles 2, 4 and 6 (+/- 5 days). Follow 
up scans will be done at the discretion of the physician. Disease status will be assessed using the RECIST 
1.1.  
Continuation of Treatment: Subjects will continue on study therapy (L-NMMA, docetaxel) if they have 
tolerated the study therapy without a DLT and their disease has not progressed. Subjects will be followed for 
approximately 3 months after the final treatment dose. The approximate length of the study from Cycle 1, Day 
1 will be approximately seven (7) months (approximately four [4] months of treatment plus three [3] months of 
follow-up). We expect to recruit up to 48 patients to complete the clinical trial within 24 to 36 months. 
 
Correlative Studies 
Experimental Methods for Secondary Endpoints:  
1). Blood samples 
a) PK and PD studies for L-NMMA plus docetaxel. 
Blood will be drawn to obtain plasma samples for PK and PD studies predose (10-30 minutes before L-NMMA 
infusion) on Days 1, 2, and 5 of Cycle 1 and Days 1 and 5 of Cycle 2. One 5-mL sample will be collected for 
PK assessment and one 5-mL sample wil be collected for PD assessment. NOTE: Patients should be fasted 
(nothing to eat or drink except water) for 12 hours prior to each collection time point. Patients should also keep 
a food diary the week before and the week of the L-NMMA infusion during Cycles 1 and 2. 
b) Blood will be drawn to obtain serum samples for the determination of nitrite and nitrate levels and cytokine 
levels and other inflammatory biomarkers (IL-1, IL-6, IL-8, TNF-, circulating VEGF) at baseline, 1 hr (± 30 
min) before every Day 1 L-NMMA infusion at each cycle, and 1 hr (± 30 min) after every Day 5 L-NMMA 
infusion at each cycle. 
c) Blood samples for RPL39/MLF2/PIK3CA mutational analysis of plasma cell-free DNA will be collected 
before each cycle and at end of treatment (at progression or completion of 6 cycles). 
 
2). Tissue biopsies  
For patients with tumors amenable to biopsy, tumor tissue will be collected at screening, the end of Cycle 1, 
and end of treatment (at progression or completion of 6 cycles). Tumor tissue obtained as part of the patient’s 
standard care and research biopsy tissues will be evaluated for biologic markers, including but not limited to 
polycomb-regulated EZH2 pathway, AKT, eNOS, and CD31.  
 
 
5. 
Study design 
This is a Phase Ib/II study assessing the MTD, DLTs, RP2D, and efficacy of L-NMMA when combined with 
taxane chemotherapy in refractory locally advanced or metastatic TNBC patients. The Phase Ib portion of the 
study is designed to investigate the combination at two dose levels of docetaxel (75 and 100 mg/m2) and 7 
dose levels of L-NMMA (5, 7.5, 10, 12.5, 15, 17.5, and 20 mg/kg). The starting dose will be L-NMMA at 7.5 


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mg/kg and docetaxel at 75 mg/m2. As patients are accrued, a standard Bayesian model averaging continual 
reassessment method (CRM) approach will be used to determine the appropriate L-NMMA dosage.   In the 
phase II portion of the study, patients will be treated with L-NMMA and taxane (docetaxel, paclitaxel, or nab-
paclitaxel) per physician’s choice. The L-NMMA and docetaxel combination will be administered at the RP2D 
determined in the phase Ib portion of the study. Paclitaxel will be administered per physician’s choice at 175 
mg/m2 IV every 3 weeks (Q3W) or at 80 mg/m2 IV weekly, and nab-paclitaxel will be administered at 260 
mg/m2 IV Q3W. All patients treated at the RP2D of L-NMMA and docetaxel in the phase Ib portion of the study 
will comprise the first patients enrolled into the phase II portion of the trial. The phase II portion of the trial will 
be conducted using a Simon’s optimal two-stage design. 
 
 
 
 
 
 
 
 


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5.1 
Phase Ib 
L-NMMA and Docetaxel 
Dose Level 5: On Days 1 through 5 of each cycle, patients will receive a 20 mg/kg two-hour IV infusion of L-
NMMA. On Day 1 of each cycle, patients will receive a 100 mg/m2 one-hour IV infusion of docetaxel 
approximately 15 minutes after the L-NMMA infusion. 
Dose Level 4: On Days 1 through 5 of each cycle, patients will receive a 17.5 mg/kg two-hour IV infusion of 
L-NMMA. On Day 1 of each cycle, patients will receive a 100 mg/m2 one-hour IV infusion of docetaxel 
approximately 15 minutes after the L-NMMA infusion. 
Dose Level 3: On Days 1 through 5 of each cycle, patients will receive a 15 mg/kg two-hour IV infusion of L-
NMMA. On Day 1 of each cycle, patients will receive a 75 mg/m2 one-hour IV infusion of docetaxel 
approximately 15 minutes after the L-NMMA infusion. 
Dose Level 2: On Days 1 through 5 of each cycle, patients will receive a 12.5 mg/kg two-hour IV infusion of 
L-NMMA.  On Day 1 of each cycle, patients will receive a 75 mg/m2 one-hour IV infusion of docetaxel 
approximately 15 minutes after the L-NMMA infusion. 
Dose Level 1: On Days 1 through 5 of each cycle, patients will receive a 10 mg/kg two-hour IV infusion of L-
NMMA. On Day 1 of each cycle, patients will receive a 75 mg/m2 one-hour IV infusion of docetaxel 
approximately 15 minutes after the L-NMMA infusion. 


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Dose Level 0 (starting dose): On Days 1 through 5 of each cycle, patients will receive a 7.5 mg/kg two-hour 
IV infusion of L-NMMA. On Day 1 of each cycle, patients will receive a 75 mg/m2 one-hour IV infusion of 
docetaxel approximately 15 minutes after the L-NMMA infusion. 
Dose Level (-1): On Days 1 through 5 of each cycle, patients will receive a 5 mg/kg two-hour IV infusion of L-
NMMA. On Day 1 of each cycle, patients will receive a 75 mg/m2 one-hour IV infusion of docetaxel 
approximately 15 minutes after the L-NMMA infusion. 
 
 
L-NMMA dosage will de-escalate/escalate based on the CRM. 
 
 
Amlodipine will be administered at 10 mg orally (p.o.) QD for 6 days at each cycle. Amlodipine administration 
will start 24 hours before the first dose of L-NMMA. Dose may be adjusted by the treating physician if needed. 
Amlodipine dose will be held if systolic BP remains below 100 mmHg. 
 
Pegfilgrastim (a colony-stimulating factor) will be administered as 6 mg subcutaneous (SQ) injection 
approximately 24 hours after every dose of docetaxel. 
 
Enteric-coated aspirin will be administered as 81 mg QD during the 6 21-day cycles of L-NMMA and 
docetaxel. 
 
5.2 Phase II  
L-NMMA and Taxane Chemotherapy 
Patients will be treated with L-NMMA and taxane chemotherapy (docetaxel, paclitaxel, or nab-paclitaxel) per 
physician’s choice. L-NMMA will be administered on Days 1−5 and taxane chemotherapy on Day 1 Q3W or 
Day 1 Q1W. L-NMMA and docetaxel will be administered at the RP2D determined in the phase Ib portion of 
the study. Paclitaxel at 175 mg/m2 will be IV infused over 3 hours or 80 mg/m2 will be IV infused over 1 hour, 
and nab-paclitaxel at 260 mg/m2 will be IV infused over 30 minutes. 
 
Amlodipine, Pegfilgrastim, and Enteric-coated aspirin will be administered as described for the phase Ib 
portion of the study.  
 
5.3 
Biopsies 
Study patients with tumors amenable to biopsy will be consented for core biopsies at three time points: 
screening, after Cycle 1, and at treatment discontinuation or completion of 6 cycles. We anticipate that the 
majority of biopsy tissues will be obtained from patients with treatment-refractory locally advanced breast 
cancer. Pre-treatment and post-treatment tumor samples will be used to evaluate iNOS; apoptosis and survival 
pathways; hypoxia and ER stress by phosphorylated extracellular signal-regulated kinase (pERK) expression; 
and the percentage of treatment-resistant cells by flow cytometry and MSFE. Pre-treatment and post-treatment 
biopsies will also be snap frozen for transcriptome analysis. 
 
6. 
Population 
The study is designed for locally advanced or metastatic TNBC patients. The investigator or designee must 
ensure that the patient meets all the following inclusion and none of the exclusion criteria before being offered 
enrollment in the study. 
Study Duration:  The study duration will be approximately seven (7) months (four [4] months of treatment and 
three [3] months of follow-up). The maximum number of cycles will be six (6).  
Safety Criteria:  All participants will be assessed by a physician for pre-existing medical conditions and 
baseline physical abnormalities prior to the initiation of investigational therapy. Patients presenting with any 


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medical history, physical exam, or laboratory abnormality that, in the opinion of the treating physician, would 
put their safety at risk will be excluded. Baseline signs and symptoms are to be recorded and monitored 
throughout the trial. Any increase in the severity or frequency of baseline signs and symptoms that occurs 
during treatment or the follow-up period will be recorded. Participants will be assessed for AEs by a physician 
or designated midlevel provider prior to each infusion. Vital signs including blood pressure, heart rate, and 
temperature will be measured at each physical exam. At each L-NMMA infusion, BP will be measured before 
infusion, 1 hour after infusion start, and at infusion completion. Automatic BP readings will be performed unless 
manual reading is needed. If systolic BP is >180 mmHg at the 1-hour check, recheck BP after 15−30 min. If 
BP is not <160 mmHg, recheck BP after another 15−30 min. If BP is still not <160 mmHg, the patient is to be 
discontinued from the study. Patients whose BP lowers to <160 mmHg at the first or second recheck will be 
rechallenged with the study drug. If systolic BP again increases beyond 180 mmHg, the patient is to be 
discontinued from the study. If systolic BP is >180 mmHg at infusion completion, recheck BP after 15−30 min. 
If BP is not <160 mmHg, recheck BP after another 15−30 min. If BP is still not <160 mmHg, the patient is to 
be discontinued from the study. Assessments may be performed more frequently if clinically indicated. In 
addition, blood samples for determination of hematology and serum chemistry profiles will be drawn before 
treatment initiation and every infusion to determine whether the study drug combination affects hematologic 
values, electrolytes, or liver function tests. Laboratory assessments will be performed more frequently if 
clinically indicated. This clinical and laboratory data will be used to determine the symptoms or side effects 
associated with the use of the study medications (L-NMMA plus taxane chemotherapy). Patients with abnormal 
laboratory or clinical findings that are believed to be treatment related will be followed every four weeks until 
the condition resolves to baseline or at least Grade II or lower according to CTCAE v4.03.  CTCAE v4.03 will 
be used to grade toxicities. Laboratory tests may be done more frequently if medically indicated. After 
treatment initiation, for patients who experience a CTCAE v4.03 Grade 4 hematologic toxicity, a CBC with 
differential and platelets will be repeated in 7 days.   
At suspected recurrence, CT scan of the chest, abdomen, and pelvis or breast ultrasound and additional 
directed evaluation, as appropriate, will be performed. Recurrence should be documented by biopsy and/or 
evidence of disease progression on radiologic studies. Abnormal blood studies alone (e.g., elevated liver 
function tests, CA 15-3, CEA, etc.) are not sufficient evidence of relapse. 
 
7. 
Inclusion and Exclusion Criteria 
7.1    Inclusion criteria: 
Patient must meet all of the following criteria: 
 
Female patients with pathologically determined locally advanced TNBC refractory to at least 3 cycles 
of standard chemotherapy or metastatic (any line) TNBC. TNBC is defined as: 
 
Estrogen receptor (ER) negative and progesterone receptor (PR) negative (<10% staining by IHC). 
 
HER2 negative. HER2 negativity must be confirmed by one of the following: 
- 
Fluorescence in situ hybridization (FISH)-negative (FISH ratio <2), or 
-   IHC 0-1+, or 
- 
IHC 2+ AND FISH-negative (FISH ratio <2). 
 
Eastern Cooperative Oncology Group performance status of ≤ 2 
 
Age ≥ 18 years 
 
Laboratory values within the following ranges: 
- 
Hemoglobin ≥9.0 g/dL (transfusions permitted) 
- 
Absolute neutrophil count (ANC) ≥1500/mm3 (1.5 x 109/L) 
- 
Platelet count ≥100,000/mm3 (100 x 109/L) 
- 
Total bilirubin <2 X upper limit of normal (ULN) 
- 
Creatinine (Cr) <2 X ULN and Cr clearance (CrCl) ≥30 by Cockcroft and Gault   


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- 
ALT and AST <2 X ULN; if liver metastases are present then ALT and AST must be <5 X 
ULN 
 
Have adequate organ function (cardiac ejection fraction of ≥ 45%)  
 
Negative serum pregnancy test within 7 days of the administration of the first treatment dose for 
women of childbearing potential (WOCBP). For WOCBP, adequate contraception must be used 
throughout the study. For this study, acceptable contraception methods are defined in Section 12.4. 
 
Patients or their legally authorized representative (LAR) must be able to understand the requirements 
of the study, provide written informed consent and authorization of use and disclosure of protected 
health information, and agree to abide by the study restrictions and return for the required 
assessments. *Use of a LAR is permissible and at both the LAR and investigator’s discretion to 
determine if participation is in patient’s best interest and according to institutional, local, state, and 
federal guidelines. 
 
Patient must be willing to undergo biopsies as required by the study protocol. Biopsies will be based 
on acceptable clinical risks as judged by investigator. Tissue from a previous biopsy will be accepted 
in the form of tissue slides. 
 
7.2   Exclusion criteria: 
  History of poorly controlled hypertension (defined as systolic BP >150 mmHg at baseline) 
 Patients with metastatic disease who have received radiation therapy, chemotherapy, or non-cytotoxic 
investigational agents within 2 weeks of study treatment initiation. Patients may not receive any other 
antineoplastic treatment during the study or follow-up period.  
 Patients who received taxane chemotherapy (docetaxel, paclitaxel, or nab-paclitaxel) at any line of 
treatment within the past 12 months  
 
Evidence of New York Heart Association class III or greater cardiac disease 
 
History of myocardial infarction, stroke, ventricular arrhythmia, or symptomatic conduction 
abnormality within the past 12 months 
 
History of congenital QT prolongation 
 
Absolute corrected QT interval of >480 msec in the presence of potassium >4.0 mEq/L and 
magnesium >1.8 mg/dL 
 
Any medical or psychiatric condition that would prevent informed consent or limit expected survival 
to less than 4 weeks 
 
Symptomatic central nervous system metastases 
 
Pregnant or nursing women 
 
Hypersensitivity or intolerance to L-NMMA, taxane chemotherapy (docetaxel, paclitaxel, or nab-
paclitaxel), amlodipine, pegfilgrastim, or their components 
 
Use of amlodipine or another calcium channel blocker in the past 14 days 
 
Alcoholism or hepatic disease with the exception of liver metastases  
 
Severe renal insufficiency (CrCl <30 mL/min [Cockcroft and Gault]) 
 
History of gastrointestinal bleeding, ulceration, or perforation 
 
Concurrent use of potent cytochrome P450 (CYP)3A4 inhibitors, such as ketoconazole, itraconazole, 
clarithromycin, atanazir, indinavir, nefazodone, neflinavir, ritonavir, saquinavir, telithromycin, and 
voriconazole 
 
Concurrent use of potent CYP3A4 inducers, such as dexamethasone, phenytoin, carbamazepine, 
rifampin, rifabutin, rifapentin, phenobarbitol, and St. John’s wort. 
 
Concurrent use of substrates (e.g., repaglinide and rosiglitazone), inhibitors (e.g., gemfibrozil), or 
inducers (e.g., rifampin) of CYP2C8 
 
Concurrent use of medications that interact with nitrate/nitrites (See Appendix E) 
 
Use of an investigational drug within 14 days preceding the first dose of study medication. 


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 
Concurrent use of any complementary or alternative medicines   
 
Patients with > Grade 2 neuropathy  
 
Inability to take aspirin 
 
8. 
Treatment 
Intervention and Mode of Delivery:  
Based on the safety profile of L-NMMA, we do not anticipate major AEs from the drug combination. A standard 
Bayesian model averaging CRM is proposed to determine the MTD. There are two proposed doses of 
docetaxel (75 and 100 mg/m2). The starting dose of docetaxel will be 75 mg/m2.On Day 1 of each cycle, 
patients will receive a one-hour IV infusion of docetaxel approximately 15 minutes after the L-NMMA infusion. 
Premedications for docetaxel will be given based on institutional standards. On Days 1 through 5 of each cycle, 
patients will receive a two-hour IV infusion of L-NMMA. The dose of L-NMMA will be escalated or de-escalated 
based on the CRM model and will depend on the absence or presence of Grade 3 or higher 
AEs/toxicities. Dose levels are given in Section 5.1. Amlodipine will be given as 10 mg p.o. QD for 6 days at 
each cycle. Amlodipine will start 24 hours prior to the Day 1 L-NMMA infusion. Amlodipine will be given at least 
30 minutes before each L-NMMA infusion on Days 1 through 5. The dose will be adjusted by the treating 
physician if needed.  An initial cohort of two (2) patients will be treated at dose level 0 (DL0), where L-NMMA 
will be given Days 1 through 5 of each cycle at a dose of 7.5 mg/kg IV infused over two hours.  For the phase 
II portion of the trial, patients will be treated with L-NMMA and taxane chemotherapy (docetaxel, paclitaxel, or 
nab-paclitaxel per physician’s choice). L-NMMA will be administered on Days 1−5 and taxane chemotherapy 
on Day 1 Q3W. L-NMMA and docetaxel will be administered at the RP2D determined in the phase Ib portion 
of the trial. Paclitaxel at 175 mg/m2 will be IV infused over 3 hours or 80 mg/m2 will be IV infused over 1 hour, 
and nab-paclitaxel at 260 mg/m2 will be IV infused over 30 minutes. Amlodipine, pegfilgrastim, and enteric-
coated aspirin will be administered as described above for the phase Ib portion of the study.  
 
For a dose level to be chosen as the MTD, at least 4 patients must have received said dose. A maximum of 
34 subjects will be treated in the phase Ib portion of the trial. In deriving the RP2D, consideration will also be 
given to the rate and the nature of AEs observed beyond the first 3 weeks of treatment.  
DLTs will be defined as any treatment-related patient death or any ≥ Grade 3 AE (NCI CTCAE v4.03) unless 
there is clear alternative evidence that the AE was not caused by the study treatment: 
 
Grade 4 neutropenia (ANC <500/mm3) lasting ≥7 days 
 
Grade 3 febrile neutropenia lasting >24 hours (single temperature of >38.3ºC or a sustained 
temperature of  >38ºC for >1 hour [non-axillary] with ANC <1000/mm3) 
 
Grade 4 febrile neutropenia of any duration 
 
Grade 4 thrombocytopenia (platelets <25,000/mm3) or grade 3 thrombocytopenia with significant 
bleeding 
 
Hematological toxicities can be managed for a period that will not exceed more than 7 days; 
hematological toxicity that persists for more than 7 days will be considered a DLT 
 
Grade 3 AST or ALT elevation  
 
Grade 3 electrolyte abnormality that is considered clinically significant or lasts >72 hours 
 
Grade 3 infusion-related reactions that recur despite appropriate medical management  
 
Any other drug-related ≥ Grade 3 non-hematologic AE except 
- alopecia and constitutional symptoms  
- hyperlipidemia in subjects not receiving maximum medical management  
- electrolyte abnormalities that may be managed with supplements. 
The DLT assessment window is defined as the duration required for completing one full cycle (through Day 
21) of treatment with L-NMMA and docetaxel. The incidence of DLT(s) during the first 3 weeks of treatment 
will be used by the CRM in dose escalation/de-escalation decisions and to define the MTD. Subjects must 
complete one full cycle of treatment during the DLT evaluation period (unless DLT has occurred) to be 


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considered evaluable by CRM in terms of dose escalation/de-escalation decisions. If treatment is delayed, the 
timelines for MTD determinations will be adjusted to allow for the completion of the DLT assessment window.  
 
       
                                                                                                                                                                                                      Table 3 
Dose Level 
L-NMMA 2-Hour Infusion 
 Days 1 to 5 each cycle 
(mg/kg) 
Docetaxel (mg/m2) 1-Hour Infusion 
Day 1 each cycle 
 
DL5 
20 
100 
DL4 
17.5 
100 
DL3 
15 
75 
DL2 
12.5 
75 
DL1 
10 
75 
DL0* 
7.5 
75 
DL-1 
5 
75 
*Start dose level.   
 
Specific guidelines for management of potential L-NMMA toxicities 
L-NMMA has been administered to thousands of patients, including a Phase 3 clinical trial for cardiogenic 
shock, and the anticipated serious adverse events (SAEs) are known and documented. In the cardiogenic 
shock trial, L-NMMA proved to be safe with few AEs, other than transient reversible hypertension, i.e. the blood 
pressure reverted to normal within minutes of stopping L-NMMA infusion. L-NMMA was not associated with 
any hematologic, liver, or renal system abnormalities. In normotensive patients, L-NMMA was administered to 
metastatic renal cell carcinoma patients prior to IL-2 infusion to differentiate the side effects of L-NMMA and 
IL-2. Transient reversible hypertension was observed with L-NMMA. Bolus injections of L-NMMA at 3, 6, and 
12 mg/kg were administered to normotensive subjects (n=3 in each cohort). Doses of 3 and 6 mg/kg did not 
induce clinically apparent side effects, and BP remained essentially unchanged. At a dose level of 12 mg/kg, 
patients experienced an increase in systolic BP up to 25 mmHg, without any clinical symptoms, which 
normalized rapidly in less than 5 minutes on stopping the L-NMMA infusion. In this Phase Ib/II study, the initial 
L-NMMA dose level will be 7.5 mg/kg in patients with no prior cardiac history and systolic BP <150 mmHg. 
This human dose is equivalent to the 200 mg/kg dose used in preclinical xenograft mouse studies. Additionally, 
all patients will be fully evaluated by a cardiologist before entry into this Phase Ib/II study. Patients in the 
abovementioned clinical studies were not treated with antihypertensive agents such as the calcium channel 
antagonist amlodipine, as is proposed here. Amlodipine is expected to decrease systolic BP by at least 15 
mmHg within 24 hours of the first dose. As L-NMMA has a short half-life, any elevation in BP would be transient 
and reversed on stopping the infusion.   
 
L-NMMA has been found to be a safe and tolerable in the thousands of patients studied. Nonetheless, 
additional measures to ensure the safety of the patients in this Phase 1b/II study will be undertaken. First, only 
patients without any significant cardiac history or uncontrolled hypertension (defined as a systolic BP >150 
mm Hg) will be included. Second, all patients will be evaluated by a cardiologist before entry into this study. 
Third, at each L-NMMA infusion, BP will be measured before infusion start, 1 hour after infusion start, and at 
infusion completion. Automatic BP readings will be performed unless manual reading is needed. If systolic BP 
is >180 mmHg at the 1-hour check, recheck BP after 15−30 min. If BP is not <160 mmHg, recheck BP after 
another 15−30 min. If BP is still not <160 mmHg, the patient is to be discontinued from the study. Patients 
whose BP lowers to <160 mmHg at the first or second recheck will be rechallenged with the study drug. If  
systolic BP again increases beyond 180 mmHg, the patient is to be discontinued from the study. If systolic BP 


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is >180 mmHg at infusion completion, recheck BP after 15−30 min. If BP is not <160 mmHg, recheck BP after 
another 15−30 min. If BP is still not <160 mmHg, the patient is to be discontinued from the study. 
Taxane chemotherapy dose modifications are allowed and are based on toxicities (please refer to 
Section 10)  
 
L-NMMA and taxane chemotherapy (docetaxel, paclitaxel, or nab-paclitaxel) will be independently dose 
adjusted based on toxicity. For instance, if the docetaxel dose is modified, a subject can continue receiving 
the same dose of L-NMMA. 
Note: Amlodipine dose will be held if systolic BP remains below 100 mmHg. 
 
9. 
Visit schedule and assessments 
9.1 Study flow and visit schedule.  


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Procedure 
Screeninga    
Baselineb 
Prior to Each Cycle* 
Treatment Cycled* 
EOT 
Informed Consent 
X 
 
 
 
 
Inclusion/ Exclusion 
X 
 
 
 
 
Demographics 
X 
 
 
 
 
Medical History 
X 
Xb 
 
 
 
Symptom-directed Medical History and Physical Exame 
X 
Xb 
X 
 
X 
Height 
X 
X 
 
 
 
Weight 
X 
X 
X 
 
X 
ECOG Performance Status 
X 
Xb 
X 
 
X 
Vital Signsf, r 
X 
X 
X 
X 
X 
12-Lead ECG and  MUGA Scan or Echocardiogramg 
X 
 
 
 
X 
Concomitant Therapies 
Continuous from Screening period to study termination 
Hematologyh 
X 
Xc 
X 
 
X 
Clinical Chemistryi 
X 
Xc 
X 
 
X 
Serum Pregnancy (-hCG )j 
X 
 
 
 
 
Related Biomarkers & serum Nitrite/Nitrate Levels m 
X 
 
        X   
X 
 
Tumor Assessmentsk 
X 
 
 
X 
X 
Tumor  Biopsyl 
X 
 
 
X 
X 
Tumor Tissuem 
X 
 
 
 
X 
Adverse Events  and  Serious Adverse Events 
From informed consent signing up to and including 30 days after the last treatment doseq 
L-NMMA Administrationn 
 
 
Days 1-5 of each cycle 
 
Taxane Chemotherapy (Docetaxel, Paclitaxel, or Nab-
Paclitaxel) Administrationn 
 
 
X 
 
Amlodipine Administrationn 
 
X 
X 
 
Pegfilgrastim Administrationn 
 
 
X 
 
Enteric-coated aspirinn 
 
 
X 
 
PK AND PD STUDIES 
Blood Samples for  L-NMMA PKo 
X 
Blood Samples for  Docetaxel  PKo 
X 


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Blood Samples for PDp  
X 


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Abbreviations: ALT = alanine transaminase; ANC = absolute neutrophil count; AST = aspartate transaminase; -hCG = beta-human chorionic 
gonadotropin; BP = blood pressure; BUN = blood urea nitrogen; CBC = complete blood count; CT = computed tomography; ECG = electrocardiogram; 
ECOG = Eastern Cooperative Oncology Group; eNOS = endothelial nitric oxide synthase; EOT = end of treatment; ER = endoplasmic reticulum; EZH2 
= enhancer of Zeste 2 polycomb repressive complex 2 subunit; ; IL = interleukin; iNOS = inducible nitric oxide synthase; LDH = lactate dehydrogenase; 
MUGA = multigated acquisition; p.o. = daily; PD = pharmacodynamics; PK = pharmacokinetics; PI = principal investigator; SAE = serious adverse 
event; SQ = subcutaneously; TNF = tumor necrosis factor; VEGF = vascular endothelial growth factor; WBC = white blood cell. Each treatment cycle 
is 21 days in length. Tests and procedures should be done on schedule, but visit windows of ± 5 days are allowed (except as otherwise specified) 
occasionally for holidays, vacations, and other administrative reasons. If extenuating circumstances prevent a patient from beginning treatment or 
completing a scheduled assessment within this time frame, the patient may continue in the study only with written permission of the PI. 
a Within 28 days prior to the Cycle 1, Day 1 (washout period of at least two [2] weeks before study treatment start)  
b Within 7 days prior to the Cycle 1, Day 1  
c 
Within 4 days prior to the Cycle 1, Day 1  
d Treatment cycles are to be repeated every 21 days.  The maximum number of cycles will be six (6).  
e  The baseline symptom-directed relevant medical history and physical examination are not required if the screening medical history and physical 
examination were conducted within 7 days prior to Cycle 1, Day 1. All patients will be evaluated by a cardiologist before entry into this study. 
f   Vital sign (blood pressure, heart rate, and oral temperature) measurements will be obtained during screening/baseline and before every infusion.  
g A 12-lead ECG will be performed at screening and EOT.  MUGA scan or echocardiogram will be performed at screening and when clinically 
indicated.  The same test (MUGA scan or echocardiogram) must be used thoughout the duration of the study.  
h A blood sample for CBC with platelet count and differential WBC count will be obtained during screening/baseline and before every cycle ( [± 5 
days).   After treatment initiation, for patients who experience a CTCAE v4.03 Grade 4 hematologic toxicity, a CBC with differential and platelets 
will be repeated in 7 days.  Patient will be followed until the ANC and platelet count have recovered to ≥1,000/mm3 and ≥50,000/mm3, respectively.  
i 
A blood sample for the clinical chemistry panel (glucose, albumin, sodium, potassium, carbon dioxide, chloride, BUN, creatinine, total bilirubin, uric 
acid, alkaline phosphatase, AST [SGOT], ALT [SGPT], LDH, and magnesium) will be obtained at screening/ baseline, before every cycle, at EOT, 
and when clinically indicated. Labs can be obtained ± 4 days from schedule. 
 j 
For women of childbearing potential, the results of a serum -hCG pregnancy test must be negative within 7 days of the administration of the first 
treatment dose. If the screening serum -hCG pregnancy test is performed more than 7 days before dosing, it must be repeated at baseline, with 
results known to be negative prior to the administration of the first dose of the study drugs. -hCG pregnancy testing is to be repeated as clinically 
indicated. 
k    CT scan of the chest, abdomen, and pelvis or breast ultrasound, as appropriate, is to be performed during screening.  If the patient has had an 
appropriate CT scan or breast ultrasound performed within 28 days of the Cycle 1, Day 1 treatment dose, then that scan may be used for screening. 
CT scan or breast ultrasound will be repeated every 6 weeks (± 5 days) until completion of the protocol-specified study treatment and 3 months 
after the last dose of study treatment. 
l  For patients with tumors amenable to biopsy, biopsy will be completed during screening (within 28 days of Cycle 1, Day 1 is acceptable), before 
Cycle 2, and at treatment discontinuation or completion of 6 cycles.  If the patient progresses while on treatment, a new tumor biopsy will be taken 
before the patient starts the new treatment. 
m   Tumor tissue obtained as part of the patient’s standard care and additional biopsy tissues will be evaluated for candidate biomarkers of response 
and toxicity of the L-NMMA + docetaxel combination, including but not limited to iNOS, eNOS, polycomb-regulated EZH2 pathway, and AKT; markers 
related to treatment resistance, hypoxia and ER stress, survival, autophagy, and angiogenesis (i.e., CD31); and inflammatory biomarkers. Ten 5-µm 
slides will be obtained. Blood samples for RPL39/MLF2/PIK3CA mutational analysis of plasma cell-free DNA will be collected before each cycle and 
at EOT (at progression or completion of 6 cycles). Blood samples (5 mL) will be collected into heparinized tubes and plasma prepared by centrifugation 
at 1000g for 15 minutes. Plasma samples must be frozen at -70ºC until analysis. Blood samples for determination of serum nitrite/nitrate levels and 
inflammatory biomarkers (IL-1β, IL-6, IL-8, TNF-α, and VEGF) will be obtained at baseline,  1 hr (± 30 min) before every Day 1 L-NMMA infusion at 
each cycle, and 1 hr (± 30 min) after every Day 5 L-NMMA infusion at each cycle.  
n  Phase Ib: L-NMMA at a dose of 7.5 mg/kg (dose level 0) will be intravenously infused over two hours on Day 1; patients will continue on this same 
L-NMMA dose from Days 2 to 5. L-NMMA dose will be escalated in 2.5 mg/kg increments up to 20 mg/kg or de-escalated to 5 mg/kg. Cohorts will be 
studied until the dose-limiting toxicity of the combination is seen (See Section 8). At each L-NMMA infusion, BP will be measured before infusion, 1 
hour after infusion start, and at infusion completion. Automatic BP readings will be performed unless manual reading is needed. If systolic BP is >180 
mmHg at the 1-hour check, recheck BP after 15-30 min. If BP is not <160 mmHg, recheck BP after another 15-30 min. If BP is still not <160 mmHg, 
the patient is to be discontinued from the study. Patients whose BP lowers to <160 mmHg at the first or second recheck will be rechallenged with the 
study drug. If systolic BP again increases beyond 180 mmHg, the patient is to be discontinued from the study. If systolic BP is >180 mmHg at 
infusion completion, recheck BP after 15-30 min. If BP is not <160 mmHg, recheck BP after another 15-30 min. If BP is still not <160 mmHg, the 
patient is to be discontinued from the study. 
Docetaxel at a dose of 75 mg/m2 will be infused over one hour every 21 days. Docetaxel dose modifications are allowed and are based on toxicities 
(See Section 10.2).  
 Amlodipine will be given as 10 mg p.o. daily x 6 days at each cycle. Amlodipine administration will start 24 hours before the first dose of L-NMMA.  
 Pegfilgrastim at a dose of 6 mg will be injected SQ approximately 24 hours after every dose of docetaxel. Enteric-coated aspirin will be administered 
as 81 mg once daily during the 6 21-day cycles of L-NMMA and docetaxel. 
Phase II: Patients will be treated with L-NMMA and taxane chemotherapy (docetaxel, paclitaxel, or nab-paclitaxel per physician’s choice). L-NMMA 
will be administered on Days 1−5 and taxane chemotherapy on Day 1 Q3W. L-NMMA and docetaxel will be administered at the RP2D determined in 
the phase Ib portion of the study. Paclitaxel at 175 mg/m2 will be IV infused over 3 hours or 80 mg/m2 will be IV infused over 1 hour, and nab-paclitaxel 
at 260 mg/m2 will be IV infused over 30 minutes. Taxane chemotherapy dose modifications are allowed and are based on toxicities (see Section 10).  
 o  Blood samples will be collected  predose (10-30 minutes before L-NMMA infusion) on Days 1, 2, and 5 of Cycle 1 and Days 1 and 5 of Cycle 2 for 
determination of L-NMMA plus docetaxel plasma PK. Blood (5 mL) will be drawn into heparin-containing green top tubes (See Section 4 and Appendix 
F).  
p For PD assessment, blood samples will be collected on Days 1, 2, and 5 of Cycle 1 and Days 1 and 5 of Cycle 2. Blood (5 mL) will be drawn into 
heparin-containing green top tubes. The 5-mL sample for PD assessment is in addition to the 5-mL sample for PK assessment. NOTE: For PK/PD 
assessment, patients should be fasted (nothing to eat or drink except for water) for 12 hours prior to each collection time point. Patients should keep 
a food diary the week before and the week of L-NMMA infusion during Cycles 1 and 2. 
q SAEs will be captured from the time of informed consent signing up to and including 30 days after the last treatment dose. SAEs will be followed 
until resolution to grade 1 or baseline. Treatment-related SAEs occurring beyond 30 days from the last treatment dose and any study patient death 


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should also be reported. All patients will be followed for 3 months after the final dose of treatment and will be evaluated accordingly with the 
required standard of care labs and tests.  
r At each L-NMMA infusion, BP will be measured before infusion, 1 hour after infusion start, and at infusion completion.  
*Patients should be on a low nitrite/nitrate diet including avoidance of processed meats such hot dogs, ham, bacon, and sausage 1 week before 
study treatment start and for the duration of the study. 
 
 
 


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10 Clinical Trial Materials 
10.1 
L-NMMA70 
L-NMMA Structure 
L-NMMA is a derivative of L-arginine, has one chiral center and is methylated on a terminal guanidino 
nitrogen.  
Molecular Formula:  C9H20N4O4 
Molecular Weight: 
248.28 
C
CH2
CH2
CH2
NH
C
NH
CH3
NH2
H
H2N
COOH
COO
H3C
 
 
Figure 10 
Structure of L-NMMA Drug Substance  
 
L-NMMA Pharmacokinetics & Pharmacodynamics  
Profile of systemic nitric oxidesynthase inhibition with L-NMMA in humans: It has been demonstrated that 
inhibition of endothelium derived nitric oxide with NG-monomethyl-l-arginine (L-NMMA) results in a 
different cardiac and peripheral vascular response. Pharmacokinetic-pharmacodynamic profile of L-
NMMA and pharmacokinetic interactions with l-arginine was studied in healthy subjects. Plasma 
pharmacokinetics were analyzed from two different studies: In study 1, 3 mg kg−1 L-NMMA was 
administered i.v. over 5 min and systemic hemodynamics, cardiac output (CO), fundus pulsation 
amplitude (FPA), and NO-exhalation (exhNO) were measured at baseline and 15, 65, 95, 155, and 305 
min after start of drug administration (n=7). In study 2, 17 mg kg−1 min−1 of the physiologic substrate for 
nitric oxide synthase, l-arginine, was coinfused i.v. over 30 min with a primed constant infusion of 50 mg 
kg−1 min−1 L-NMMA (n=8). Bolus infusion of L-NMMA resulted in a maximum plasma concentration of 
12.9±3.4 mg ml−1 (mean±s.d.) with elimination half-life of 63.5±14.5 min and clearance of 12.2±3.5 ml 
min−1 kg−1 and caused a small hypertensive response, decreased CO by 13%, FPA by 26%, exhNO by 
46% and increased systemic vascular resistance by 16% (P<0.05 each) 15 min after start of drug 
administration. Although only limited data points were available in the L-NMMA plasma concentration 
range between 0 and 4 mg ml−1, drug effects over time were in good agreement with an Emax model 
(r2>0.98 each), which also suggested that concentrations producing half-maximum effects were higher 
for FPA than for CO and exhNO. The coinfusion with l-arginine caused a nearly two-fold increase in 
plasma L-NMMA levels, indicating a pharmacokinetic interaction. 
 


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Table 5 Pharmacokinetic parameters of 3 mg kg−1 L-NMMA 
i.v. over 5 min obtained from non-compartmental analysis. Results are presented as means±s.d., n=7. 
Table 5 
t1/2 (min) 
63.5±14.5 
AUC (µg ml−1 min−1) 
260.4±62.4 
MRT (min) 
48.9±10.5 
Cmax (µg ml−1) 
12.9±3.4 
tmax (min) 
6 
CL (ml min−1 kg−1) 
12.2±3.5 
Vc (ml kg−1) 
257.6±116 
Vdss (ml kg−1) 
586.1±206.7 
t1/2, elimination half-life; AUC, area under the plasma concentration vs time curve; MRT, mean residence time; Cmax, maximal plasma 
concentration; tmax, time to reach Cmax, CL, total body clearance; Vc, initial volume of distribution; Vdss, volume of distribution at steady 
state. 
Plasma samples were available from seven 
subjects in study 1 and eight subjects in study 2. 
The pharmacokinetic parameters calculated from 
study 1 are given in Table 8, plasma 
concentrations are illustrated in Figure 13. In the 
primed constant infusion experiments in study 2, 
plasma levels of L-NMMA increased to 9.5±5.2 
µg ml−1 at the end of the 5 min bolus 
administration and to steady state plasma 
concentrations of #4.0±1.6 µg ml−1. During 
coinfusion with l-Arg, which resulted in a 66±6 fold 
increase in plasma l-Arg levels to 932±206 µg 
ml−1, a significant increase in mean plasma L-
NMMA to 7.1±1.6 µg ml−1 (P<0.001, anova) was 
noted.  
Plasma concentrations of L-NMMA remained 
elevated 25 min after the end of l-Arg infusion at 
7.5±1.7 µg ml−1. 
In study 1, L-NMMA had a transient and subtle 
effect on systemic hemodynamics: Mean arterial 
pressure increased slightly from 85.0±6.5–
87.5±7.0 mmHg, pulse rate decreased from 
69.5±9.6–65.2±10.6 beats min−1, and systemic 
vascular resistance significantly increased from 
1013±295 dyn s cm−5 by 16%  
(P<0.05) 15 min after start of drug administration. 
L-NMMA caused a maximum percentage 
reduction from the baseline value (Emax) in CO of 
13%, in FPA of 26%, and in exhaled NO of 46% 
(P<0.01 each, Figure 1). The presented PD model allowed very good fits of the experimentally measured 
data although only limited points were available and an EC50 for CO, FPA, and exhaled NO of 0.02 µg 
ml− 1, 0.66 µg ml− 1 and 0.02 µg ml−1 was calculated, respectively.  
 


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The continuous infusion of L-NMMA in study 2 
reduced exhaled NO by 69% (P<0.005), but no 
change in blood pressure and pulse rate was noted 
[10]. Figure 1 illustrates the effect of L-NMMA on the 
three dynamic parameters with respect to time. FPA 
and CO returned to near baseline values after 300 
min. The relationship between the percentage 
reduction from the baseline values and the total 
plasma concentrations of L-NMMA is presented in 
Figure 14. The effect predicted by model was close 
to experimental measures as indicated by the good 
fit for the effect of the drug (model selection criteria 
>2.00 each). 
 
 
 
 
 
 
 
 
 
 
 
General Properties 
Table 6 
Physical Chemical Properties of L-NMMA Drug Substance  
Property 
Result 
Appearance 
White to off-white powder 
Solubility in organic solvents 1 
 
Acetonitrile 
Slightly soluble 2  mg/mL 
Methanol 
Slightly soluble 2 mg/mL 
Acetone 
Insoluble 2 
2-Propanol 
Insoluble 2 
Aqueous Solubility  3 
 
pH 0.7 
At least freely soluble 2 
pH 2.4 
At least freely soluble 2 
pH 5.8 
At least freely soluble 2 
pH 6.7 
At least freely soluble 2 
pH 8.0 
At least freely soluble 2 
Hygroscopicity 
Extremely hygroscopic 
Optical rotation 
D25 = +9.0 to +13.0 
pH 0.01M in water 
5.5-7.5 
Solid-state form 
Under investigation 
1 
Determined by UV absorbance versus standard at 200 nm  
2 
USP definition:  
 
very soluble = < 1 part solvent for 1 part solute 
 
freely soluble = 1 to 10 parts solvent for 1 part solute 
 
slightly soluble = 100 to 1000 parts solvent for 1 part solute  
 
insoluble = >.10,000 parts solvent for 1 part solute 
3 
Determined visually. Aqueous solution adjusted with HCl or NaOH; solution at pH 6.7 was not adjusted 


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Other Characteristics 
The L-configuration of the amino group substituent at the α-carbon of L-NMMA is the only asymmetric 
center present. The configuration of the α-amino group is determined by the configuration of the L-
ornithine starting material and is unchanged during the synthesis of L-NMMA. 
Impurities 
Potential impurities in L-NMMA drug substance include organic impurities (process impurities and 
degradation products), residual solvents, and inorganic impurities, particularly copper, which is used in 
the synthesis. The potential organic impurities in L-NMMA, and their possible source(s), are outlined in 
Table 10 Potential residual solvents are listed in Table 11 
Table7 
Potential Impurities in L-NMMA Drug Substance 
Name 
Structure 
Source 
NG-Monomethyl-D-arginine acetate (D-NMMA) 
C CH2
NH2
H
HOOC
CH2 CH2 NH C
NH2
NH CH3
 
Process impurity (enantiomer) 
L-Ornithine 
C CH2
H
NH2
HOOC
CH2 CH2 NH2
 
Process impurity (starting material), 
degradation product 
1,2-Dimethyl-2-thiopseudourea  
C
H2N
NH
CH3
S
CH3
 
Process impurity (starting material) 
Flavianic acid 
OH
SO3H
NO2
O2N
Process impurity (auxiliary material) 
L-Arginine 
C CH2
H
NH2
HOOC
CH2 CH2 NH C
NH2
NH2
 
Process impurity 
-N-Methylamidino- 
L-ornithine  
(-NMMA) 
C CH2
H
NH
HOOC
CH2 CH2 NH2
C
H2N
NH CH3
 
Process impurity  
(substitution of -amino group)  
,δ-Di-(N-methylamidino)-L-ornithine (,δ-di 
NMMA)  
C CH2
H
NH
OOC
CH2 CH2 NH C
NH2
NH CH3
C
H2N
NH CH3
 
Process impurity  
(disubstitution product) 
Asymmetric NG-Dimethyl-L-Arginine (L-ADMA) 
C CH2
H
NH2
HOOC
CH2 CH2 NH C
NH2
N
CH3
CH3
 
Process impurity 
Symmetric NG-Dimethyl-L-Arginine(L-SDMS) 
C CH2
H
NH2
HOOC
CH2 CH2 NH C
NH
NH CH3
CH3
 
Process impurity 
Ammonia 
NH3 
Process impurity 
Methyl urea 
C
H2N
O
NH CH3
Degradation product 
L-Citrulline 
C CH2
H
NH2
HOOC
CH2 CH2 NH C NH2
O
 
Degradation product 
Methyl amine 
CH3 NH2 
Degradation product 
N-Methyl-L-citrulline  
C CH2
H
NH2
HOOC
CH2 CH2 NH C NH
O
CH3
 
Degradation product 
 
Table 8 
Potential Residual Solvents in L-NMMA Drug Substance 
Solvent 
Solvent Class 1 
2-Propanol 
3 
Methanol 
2 
1: 
ICH Q3C Impurities: Residual Solvents, December 1997 


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Stability 
Evaluation of the stability of L-NMMA drug substance batch 29020A, which was used to manufacture 
drug product for Phase 2 clinical studies, was initiated on October 8, 2003. The samples were evaluated 
under the ICH conditions recommended for product to be stored at room temperature, as shown in the 
protocol summarized in Table 9 
L-NMMA drug substance was used to formulate L-NMMA drug product within 15 days of its 
manufacturing date.  
Table 9  Stability Protocol for L-NMMA Drug Substance 
Storage  Condition1 
Time Point (months) 
 
Initial 
1 
3 
6 
9 
12 
18 
24 
25 ± 2oC/60 ± 5% RH 
X 
X 
X 
X 
X 
X 
X 
X 
30 ± 2oC/60 ± 5% RH2 
X 
X 
NT 
X 
X 
X 
NT 
NT 
40 ± 2oC/75 ± 5% RH 
X 
X 
X 
X 
NT 
NT 
NT 
NT 
1: 
Packaged in double LDPE bags with desiccant between the bags  
2: 
Tested only if there is a significant change at  40oC/75% RH 
X  =  
Description, Assay , Impurities , Water content 
NT = 
Not Tested 
Drug Product 
Description and Composition of the Drug Product 
L-NMMA Injection was administered as a bolus injection followed by an infusion of L-NMMA Injection 
diluted in saline infusion bags. Four doses ranging from 0.15 to 1.5 mg/kg bolus followed by the same 
dose were administered each hour by continuous infusion for 5 hours was evaluated in the Phase 2 
clinical study. For each patient, a kit was provided containing a vial of L-NMMA Injection for BOLUS 
Administration and a corresponding vial of L-NMMA Injection for INFUSION Administration. To allow for 
four doses, L-NMMA Injection was prepared in four bolus concentrations (1.2 to 12 mg/mL) and four 
infusion concentrations (7.5 to 75 mg/mL) that were diluted into 100-mL saline infusion bags prior to use. 
L-NMMA Injection is a clear colorless solution of NG-monomethyl-L-arginine acetate in saline (bolus 
administration vials) or water (infusion administration vials). It is supplied in 20-mL clear glass vials with 
rubber stoppers and white (bolus administration vials) or grey (infusion administration vials) caps. Vials 
are filled to 16.6 + 0.3 mL which represents a 0.6-mL overfill to allow withdrawal of up to 16 mL. An 
injection dosage form was chosen for ease of administration to the hospitalized patient population.  
The compositions of L-NMMA Injection are provided in Table 10. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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Table 10 Composition of L-NMMA Injection 
 
Injection for BOLUS Administration 
Injection for INFUSION Administration 
Dose (mg/kg): 
0.15 
0.50 
1.0 
1.5 
0.15 
0.50 
1.0 
1.5 
Formulation Number:  
001 
002 
003 
004 
005 
006 
007 
008 
Concentration (mg/mL): 
1.2 
4.0 
8.0 
12 
7.5 
25 
50 
75 
Component 
Quality 
Standard 
Function 
Composition 
L-NMMA 
In-house 
Drug 
Substance 
1.2 mg 
4.0 mg 
8.0 mg 
12 mg 
7.5 mg 
25 mg 
50 mg 
75 mg 
Sodium Chloride  
USP 
Osmolality 
adjustment 
8.7 mg 
8.0 mg 
7.0 mg 
5.9 mg 
none 
none 
none 
none 
Water for 
Injection, q.s. ad 
USP 
Diluent 
1 mL 
1 mL 
1 mL 
1 mL 
1 mL 
1 mL 
1 mL 
1 mL 
Acetic Acid NF and/or Sodium Hydroxide NF may be added for pH adjustment 
Stability Summary 
L-NMMA Injection is stable as demonstrated by the results of analysis after autoclaving for 1 hour 
(Section Stress Studies). The stability of L-NMMA Injection Lots MA046 (1.2 mg/mL) and MA060 
(75 mg/mL), the lowest and highest concentration of the product to be used in Phase 2 clinical studies, 
is currently being evaluated. 
L-NMMA Injection 1.2 to 75 mg/mL, packaged in clear Type 1 glass and sealed with rubber stoppers, is 
assigned an expiration dating period of 6 months based on the results of the autoclaving study. This 
expiration dating period may be extended based on the results of real-time long-term. 
Stress Studies 
L-NMMA Injection is stable when exposed to high heat. L-NMMA Injection Lots MA046 (1.2 mg/mL) and 
MA060 (75 mg/mL) packaged in clear Type 1 glass and sealed with rubber stoppers were autoclaved at 
121C and 30 psi for 1 hour. The results presented in Table 11 show that the amount of L-NMMA 
remaining is essentially unchanged. 
Table 11 Stability of L-NMMA Injection Stored at 121C and 30 psi for 1 Hour 
 
 
1.2 
mg/mL  
Lot MA046 
75 
mg/mL  
Lot MA060 
Test 
Acceptance Criteria 
Initial 
1 hour 
Initial 
1 hour 
Description 
Clear, 
colorless 
solution 
free 
of 
foreign matter 
 
 
 
 
Assay anhydrous basis (% w/w) 
90.0 – 110.0 
 
 
 
 
Impurities 
 
 
 
 
 
Each specified impurity 
 
 
 
 
 
L-Ornithine (% w/w) 
NMT 0.5 
 
 
 
 
L-Citrulline (% w/w) 
NMT 0.5 
 
 
 
 
Each unspecified impurity (% w/w) 
NMT 0.2 
 
 
 
 
Total impurities (% w/w) 
NMT 3.0 
 
 
 
 
pH 
5.5 – 7.5 
 
 
 
 
NMT = Not more than 
Accelerated and Long-term Stability 
The stability of L-NMMA Injection Lots MA046 (1.2 mg/mL) and MA060 (75 mg/mL), packaged in clear 
Type 1 glass and sealed with rubber stoppers, is being evaluated under the ICH conditions recommended 


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for product to be stored at room temperature. These lots are the lowest and highest concentration of the 
lots used in the Phase 2 clinical studies. The protocol is summarized in table 12.  
Table 12 Stability Protocol for L-NMMA Injection 
Storage Condition1 
Time Point (months) 
 
Initial 
3 
6 
9 
12 
18 
24 
25 ± 2oC/60 ± 5% RH 
XYZ 
X 
X 
X 
XY 
X 
XYZ 
30 ± 2oC/60 ± 5% RH2 
XYZ 
NT 
X 
X 
X 
NT 
NT 
40 ± 2oC/75 ± 5% RH 
XYZ 
X 
X 
NT 
NT 
NT 
NT 
1: 
Samples are to be stored in both upright and inverted positions. Upright samples are tested only if there is a significant change 
in the inverted samples. 
2: 
Tested only if there is a significant change at  40oC/75% RH 
X  =  
Description, Assay, Impurities , pH 
Y =   
Sterility, Container closure integrity 
Z = 
Endotoxins, Particulate matter 
NT = 
Not Tested 
Infusion Solution Preparation  
L-NMMA Injection for INFUSION Administration is administered by withdrawing 16 mL from the 
appropriate INFUSION vial and adding that volume to a 100-mL (nominal volume) IV infusion bag of 
saline infusion solution (actual volume 105 to 115 ml). The concentrations of L-NMMA after mixing with 
infusion solution (average final volume 126 mL) are: 
 
0.952 mg/mL for the 0.15 mg/kg/hr dose 
 
3.17 mg/mL for the 0.50 mg/kg/hr dose 
 
6.35 mg/mL for the 1.0 mg/kg/hr dose 
 
9.52 mg/mL for the 1.5 mg/kg/hr dose 
The stability of the highest and lowest of these solutions in the infusions bags to be used in the clinical 
studies and stored at 25C for 24 hours, which is the maximum time the infusion solution may be used 
in the clinical study, has been evaluated. Results presented in Table 13 and table 14, respectively, show 
the product is essentially unchanged. 
Table 13 
Stability of L-NMMA Injection 7.5 mg/mL Diluted in Saline and Stored at Ambient 
Conditions for 24 Hours 
Test 
Acceptance Criteria 
Time Point (hours) 
Initial 
3 
6 
9 
12 
24 
Description 
Clear, 
colorless 
solution free of foreign 
matter 
 
 
 
 
 
 
Assay (% w/w) 
90.0 – 110.0 
 
 
 
 
 
 
Impurities 
 
 
 
 
 
 
 
Each specified impurity 
 
 
 
 
 
 
 
L-Ornithine (% w/w) 
NMT 0.5 
 
 
 
 
 
 
L-Citrulline (% w/w) 
NMT 0.5 
 
 
 
 
 
 
Each unspecified impurity (% w/w) 
NMT 0.2 
 
 
 
 
 
 
Total impurities (% w/w) 
NMT 3.0 
 
 
 
 
 
 
pH 
5.5 - 7.5 
 
 
 
 
 
 
Particulate matter 
 
 
 
 
 
 
 
Particles >10 μm/vial 
NMT 6,000 
 
 
 
 
 
 
Particles >25 μm/vial 
NMT 600 
 
 
 
 
 
 
Lot MA054 
NMT = Not more than 
 
 
 


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L-NMMA Previously Data Compared to Placebo  
L-NMMA Placebo was 0.9% Sodium Chloride USP. It was filtered, filled into vials and sterilized at the 
same facility, using the same conditions and packaged in the same container closure system as L-NMMA 
Injection.  
The specifications for L-NMMA Placebo and the analytical results for Lots XXX (white cap for bolus 
administration) and XXX (grey cap for infusion administration), which were used in the Phase 2 clinical 
studies Testing for endotoxins, particulate matter and sterility are in-progress. No material was used in 
any clinical study until testing is complete and results indicate that the lot has passed all acceptance 
criteria. 
Table 14 
Stability of L-NMMA Injection 75 mg/mL Diluted into Saline and Stored at Ambient 
Conditions for 24 Hours 
Test 
Acceptance Criteria 
Time Point (hours) 
Initial 
3 
6 
9 
12 
24 
Description 
Clear, 
colorless 
solution free of foreign 
matter 
 
 
 
 
 
 
Assay (% w/w) 
90.0 – 110.0 
 
 
 
 
 
 
Impurities 
 
 
 
 
 
 
 
Each specified impurity 
 
 
 
 
 
 
 
L-Ornithine (% w/w) 
NMT 0.5 
 
 
 
 
 
 
L-Citrulline (% w/w) 
NMT 0.5 
 
 
 
 
 
 
Each unspecified impurity (% w/w) 
NMT 0.2 
 
 
 
 
 
 
Total impurities (% w/w) 
NMT 3.0 
 
 
 
 
 
 
pH 
5.5 - 7.5 
 
 
 
 
 
 
Particulate matter 
 
 
 
 
 
 
 
Particles >10 μm/vial 
NMT 6,000 
 
 
 
 
 
 
Particles >25 μm/vial 
NMT 600 
 
 
 
 
 
 
Lot MA060 
NMT = Not more than 
Table 15 
Specifications and Batch Analysis Results for L-NMMA Placebo 
Lot No.: 
DALTON 
DALTON 
Batch Size: 
DALTON 
DALTON 
Manufacturing Site 1: 
Dalton 
Dalton 
Date of Manufacture: 
DALTON 
DALTON 
Test 
Method 
Acceptance Criteria 
Result 
Result 
Description 
Visual 
Clear, colorless solution free of 
foreign matter 
 
 
Identification 
HPLC 
Absence of L-NMMA 
 
 
pH 
Potentiometer 
5.5 to 7.5 
 
 
Fill volume (mL) 
Gravimetric 
NLT 16.3 
 
 
Particulate matter 
USP <788> Light 
Obscuration 
USP 
 
 
Particles >10 μm/vial 
NMT 6000 
In-prog 
In-prog 
Particles >25 μm/vial 
NMT 600 
In-prog 
In-prog 
Endotoxins (EU/mL) 
USP 
NMT 0.5 
In-prog 
In-prog 
Sterility 
Sterile 
Sterile 
In-prog 
In-prog 
1: Dalton = Dalton Chemical Laboratories, Inc., Toronto, Ontario, Canada 
NMT 
= Not more than 
NLT 
= Not less than 
In-prog =  
Testing is in-progress. No material will be used in any clinical study until testing is complete and results indicate that 
 the lot has passed all acceptance criteria. 


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Labeling 
Copies of the following labels are presented in Fig 15 through Fig 18: 
 
Bolus vial label  
 
Infusion vial label  
 
Kit box label 
 
Label inside kit to differentiate between bolus and infusion vials 
 
Figure 15 
Label for L-NMMA Injection for BOLUS Administration Vial 
 
 
 
Figure 16 
Label for L-NMMA Injection for INFUSION Administration Vial 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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Figure 17  
Label for L-NMMA Injection Kit Box 
 
 
Figure 18 Label Inside L-NMMA Injection Kit to    
     Differentiate Between Bolus and Infusion Vials 
 
10.2 Docetaxel 
DRUG NAME: Docetaxel  
SYNONYM(S): RP56976  
COMMON TRADE NAME(S): TAXOTERE®  
CLASSIFICATION: Mitotic inhibitor, cytotoxic  
Special pediatric considerations are noted when applicable, 
otherwise adult provisions apply.  
MECHANISM OF ACTION:  
Docetaxel is a semi-synthetic drug derived from precursor extracted from the needles of the European 
yew tree, Taxus baccata. It acts by disrupting the microtubular network that is essential for mitotic and 
interphase cellular functions. It promotes the assembly of tubulin into stable microtubules and inhibits 
their disassembly, causing inhibition of cell division and eventual cell death. Both docetaxel and paclitaxel 
bind to the same microtubule site, although the affinity of docetaxel is 1.9-fold higher. Cross-resistance 
between docetaxel and paclitaxel does not occur consistently. Docetaxel is a radiation-sensitizing agent. 
It 
is 
cell 
cycle 
phase-specific 
(G2/M 
phase). 
71                                                     
Table 16 
PHARMACOKINETICS:  
Interpatient variability  
wide interpatient variability, probably due  
to interpatient differences in cytochrome  
P450 3A4 (CYP3A4) activity.  
Distribution  
distributed to all tissues and organs except 
 the brain in animal studies  
Cross blood brain barrier?  
Very low levels were found in the brain in animal 
studies. In a single patient with leptomeningeal 
carcinomatosis, docetaxel was detected in CSF 2 
hours after cessation of docetaxel infusion.  
volume of distribution  
113 L  
plasma protein binding  
> 95%  
Metabolism  
CYP3A involved in docetaxel metabolism 
 in vitro  
active metabolite(s)  
none  
inactive metabolite(s)  
one major and 3 minor  
Excretion  
primarily biliary/fecal elimination 
urine  
6% recovered in urine over 7 days 
feces  
75% recovered in feces over 7 days, 80% of which 
was excreted during the first 2 days; < 8% was 
unchanged docetaxel. 
terminal half life  
11.1 h  
clearance  
21 L/h/m2  
Gender  
no clinically significant difference  
Elderly  
no clinically significant difference  
SPECIAL PRECAUTIONS:  
Contraindicated in patients with a history of hypersensitivity reaction to docetaxel or to drugs formulated 
with polysorbate 80. Patients with prior severe hypersensitivity reactions should generally not be 
rechallenged with docetaxel. However, in patients with objective tumor responses and without other 
options to docetaxel therapy, re-treatment may be attempted with extreme caution and aggressive 
premedication by experienced practitioners. Contraindicated in patients with severe liver impairment. 
Patients hypersensitive to paclitaxel may also react to docetaxel. 


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Preexisting effusions: Patients with preexisting effusion should be closely monitored from the first dose 
for the possible exacerbation of the effusions.  
Liver impairment: Patients treated with docetaxel 100 mg/m2 are at a higher risk of developing severe 
adverse reactions if they have elevated transaminase (ALT and/or AST greater than 2 times the upper 
limit of normal [ULN]) and alkaline phosphatase (greater than 2 times ULN). Liver impairment reduces 
clearance and increases systemic exposure to docetaxel. Docetaxel should not be administered if 
AST/ALT >2x ULN or bilirubin >5x ULN unless liver metastases are present. Adverse reactions include 
life-threatening 
sepsis 
and 
gastrointestinal 
hemorrhage, 
febrile 
neutropenia, 
infections, 
thrombocytopenia, stomatitis and asthenia. 
Alcohol abuse: When docetaxel is used in patients who abuse alcohol, or have abused alcohol, the risk 
of severe neurotoxic reactions may be increased. 
Mutagenicity: Not mutagenic in Ames test or mammalian in vitro mutation test.  
Clastogenic in mammalian in vitro and in vivo chromosome tests.  
Pregnancy: FDA Pregnancy Category D. There is positive evidence of human fetal risk, but the benefits 
from use in pregnant women may be acceptable despite the risk (e.g., if the drug is needed in a life-
threatening situation or for a serious disease for which safer drugs cannot be used or are ineffective).  
Breastfeeding is not recommended due to the potential secretion into breast milk. 71                             Table 
17 
SIDE EFFECTS: ORGAN SITE  
9.1.1 
SIDE EFFECT  
allergy/immunology  
hypersensitivity reaction  
(21%, severe 4%);  
moderate immunosuppression  
blood/bone marrow febrile neutropenia  
anemia (90%, severe 9%) , death, septic (1.7%) , death, non-septic (0.3%) , febrile 
neutropenia (11%) , infection with neutropenia (severe 6%) , leukocytopenia (96%, 
severe 75%) , neutropenia (96%, severe 32%) , nadir 8 days, duration of severe 
neutropenia 7 days , thrombocytopenia (8%)  
cardiovascular (arrhythmia)  
dysrhythmia (2%, severe 0.4%) , tachycardia (1%)  
cardiovascular (general)  
fluid retention, with premedication (52%), severe  fluid retention, without 
premedication (82%, severe 22%); see paragraph , heart failure (0.3%) , 
hypertension (2%) , hypotension (3%, severe 0.5%)  
constitutional symptoms  
fatigue, 3-weekly schedule (62%, severe 13%) , fatigue, weekly schedule (72%, 
severe 14%), fever (32%, severe 2%)  
dermatology/skin  
extravasation hazard: irritant , alopecia (56-85%, severe 0.5%), injection site 
reactions (6%); , nail changes (19-51%, severe 2-22%) , hand-foot skin reaction 
(rare),  rash/pruritus (48%, severe 5%) 
gastrointestinal  
emetogenic potential: low moderate, diarrhea (39%, severe <5%) , nausea (39%, 
severe <5%) , perforation (rare) , stomatitis (42%, severe 6%) , vomiting (22%, 
severe <5%)  
hemorrhage  
bleeding episode (1%) , bleeding episode with thrombocytopenia (0.1%)  
infection  
infection, including sepsis and pneumonia (22%)  
metabolic/laboratory  
elevated AST, ALT, bilirubin, and alkaline phosphatase (<2%)  
neurology  
neuropathy, motor (14%%, severe 4%), neuropathy, sensory (49%, severe 4%) 
ocular/visual  
tearing/watery eyes, weekly schedule (52%)  
pain  
arthralgia (9%, severe 0.6%) , myalgia (19%, severe 2%)  
 
Pretreatment administration of dexamethasone is recommended to decrease the frequency and 
severity, and to delay the onset of docetaxel-induced fluid retention. Dexamethasone also reduces the 
severity of docetaxel-induced hypersensitivity reactions and cutaneous toxicity.  
3-weekly regimen: A commonly used regimen consists of dexamethasone 8 mg PO twice a day for 3 
consecutive days starting one day prior to each docetaxel infusion. This regimen has also been shown 


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to decrease the occurrence of severe stomatitis and infection.2 Patients must receive a minimum of 3 
doses of dexamethasone prior to docetaxel treatment. If dexamethasone has not been taken prior to 
treatment, it should be started and the docetaxel infusion delayed until the following day. If treatment 
delay is not possible, diphenhydramine 50 mg IV and dexamethasone 10 mg IV may be given 30 minutes 
before starting docetaxel.35 Note that this premedication regimen has not been shown to reduce the 
incidence and severity of fluid retention, but is only an attempt to ameliorate hypersensitivity reactions. 
The patient should then be instructed to take dexamethasone 8 mg PO twice a day for two days.  
Hypersensitivity reactions are most likely to occur during the first two cycles of docetaxel treatment, 
generally within the first few minutes after the infusion is started. Signs and symptoms usually abate 
within 15 minutes after the infusion is stopped. The most frequent minor manifestations were flushing, 
rash with or without pruritus, chest tightness, back pain, dyspnea, drug fever or chills. If minor reactions 
occur, therapy with docetaxel does not have to be discontinued. For severe reactions such as 
hypotension requiring treatment, bronchospasm, and generalized rash/erythema; stop the docetaxel, and 
have a physician assess the patient and order appropriate treatment.  
Re-challenge after severe hypersensitivity reaction: Patients who have developed severe 
hypersensitivity reactions should generally not be rechallenged with docetaxel. However, in patients with 
objective tumor responses and without other options to docetaxel therapy, re-treatment may be attempted 
with extreme caution and aggressive premedication by experienced practitioners. It is recommended that 
a slower rate of infusion be used. One patient experienced major hypersensitivity symptoms during the 
first two cycles of docetaxel therapy despite prophylaxis with a corticosteroid and a histamine H1 blocking 
antagonist. Treatment was continued without further difficulty after sodium cromoglycate (400 mg PO 
four times a day, starting immediately after the second cycle) was added to the prophylactic regimen. 
Fluid retention includes edema, and less frequently, pleural effusion, ascites, pericardial effusion and 
weight gain. Fluid retention occurs in 52% of patients receiving dexamethasone premedication, and in 
82% of patients without premedication. It usually begins at the lower extremities and may become 
generalized with a weight gain of 3 kg or more. Fluid retention is not accompanied by acute episodes of 
dehydration, oliguria or hypotension. Fluid accumulation is due to increased capillary permeability rather 
than hypoalbuminemia or cardiac, hepatic or renal damage. It is slowly reversible after treatment is 
discontinued (median 29 [range, 0 to > 42] weeks). However, early, aggressive diuretic treatment may 
occasionally be required. Antihistamines have not been shown to be useful in controlling fluid retention. 
Neuropathy: Rarely, neurologic effects result in moderate to severe neuropathy, leading to decreased 
dexterity and/or disturbances in gait, usually after cumulative doses of 600 mg/m2.  
Alopecia: Loss of hair, including on the head, eyebrows, eyelashes, pubic area, and underarm, occurs 
in most patients. Alopecia has a sudden onset, and occurs 14-21 days after treatment has begun. Hair 
should grow back once treatment has been completed; however cases of poor hair regrowth and/or 
persistent hair loss have been reported. Reports suggest some patients may experience prolonged hair 
loss lasting beyond 24 months, and possibly irreversibly. 
Rash/pruritus: Cutaneous reactions are characterized by a rash, including localized eruptions mainly 
on feet and hands, but also on arms, face or thorax. These reactions are observed in 48% of patients. 
They are occasionally associated with pruritus. Eruptions generally occur within one week following the 
docetaxel infusion, resolve before the next infusion, and are not disabling.  
Injection site reactions include skin sensitivities such as hyperpigmentation, inflammation, local 
erythema, dryness of the skin, or swelling of the vein. Injection site reactions occur in 6% of patients and 
are generally mild. Phlebitis or extravasations are observed less frequently. Leakage into surrounding 
tissue during intravenous administration (i.e., extravasation) may cause irritation, local tissue necrosis 
and/or thrombophlebitis.  
Nail changes are characterized by hyperpigmentation, splinter hemorrhage, subungual hematoma and 
hyperkeratosis, orange discoloration, Beau-Reil lines (which indicate the cessation of nail growth), and 
acute paronychia. Some changes are cosmetic and asymptomatic, whereas others can be accompanied 


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by discomfort or pain. Changes are usually transient and disappear with treatment withdrawal, although 
there are reports of persistent changes. Loosening or loss of nails (onycholysis) occurs in 2-22%. Nail 
bed infections may be a complication and the application of topical antibiotics or antifungals may be 
necessary. Applying the principle of cold-induced vasoconstriction by wearing frozen gloves on the hands 
during treatment may reduce the incidence of nail and skin toxicity. In one study, overall incidence of nail 
toxicity was reduced with frozen glove treatment from 51% to 11%, and the incidence of grade 2 toxicity 
(onycholysis) was reduced from 22% to 0%. Additionally, median time of occurrence of nail toxicity was 
delayed to 106 days with frozen glove treatment as opposed to 58 days without.  
Hand-foot skin reaction that occurs despite dexamethasone prophylaxis may respond to administration 
of pyridoxine 50 mg p.o. three times a day. 
Tearing/watery eyes: An unexpected toxicity with the weekly schedule is excessive tearing, which was 
reported by half of the patients in a phase II study in women with metastatic breast cancer. Patients 
reported increased tearing and, in some cases, mild conjunctivitis or eye irritation. Formal ophthalmologic 
examination of several patients revealed no abnormalities. As with fluid retention, the onset of tearing 
seems to be related to cumulative dose and occurs after a median of 400 mg/m² (range, 120-960 mg/m²). 
Ten of 15 patients with tearing also developed some degree of fluid retention. Treatment with artificial 
tears or other ocular moisturizers ameliorated symptoms in some patients. Eye irritation led to a dose 
reduction in one patient. 
INTERACTIONS:                                                                                                                                                                           Table 
18 
AGENT 
EFFECT 
MECHANISM 
MANAGEMENT 
dexamethasone  
does not affect protein  
binding of docetaxel  
 
 
 
 
Doxorubicin 
AUC of docetaxel increased, one 
hour interval between drugs does 
not change the results  
doxorubicin may interfere at 
hepatic microsomal enzyme 
level  
avoid concurrent therapy outside of 
clinical trial  
Epirubicin 
increased exposure to active 
metabolite of epirubicin  
altered metabolism of 
epirubicin  
avoid concurrent therapy outside of 
clinical trial  
Etoposide 
clearance of docetaxel decreased  
 
avoid concurrent therapy outside of 
clinical trial  
ifosfamide  
when docetaxel given first it is 
associated with increased 
ifosfamide plasma clearance and 
decreased AUC  
may be due to increased 
ifosfamide metabolism caused 
by corticosteroid premedication 
for docetaxel  
avoid concurrent therapy outside of 
clinical trial  
 
There have been no formal clinical studies to evaluate the drug interactions of docetaxel with other medications. In vitro studies have 
shown that the metabolism of docetaxel may be modified by the concomitant administration of compounds which induce, inhibit or are 
metabolized by (and thus may inhibit the enzyme competitively) cytochrome P450-3A such as cyclosporine, ketoconazole and 
erythromycin. 
DOSAGE GUIDELINES:                        
 
 
 
 
 
 
           
Table 19 
PLATELET COUNT (x 109/L) 
ABSOLUTE NEUTROPHIL (ANC)* (x 109/L)  
   
> 1.8 
1.5-1.8 
1.0-1.5 
<1.0 
> 100 
70-100 
50-70 
<50 
75mg/m2 
60mg/m2 
50mg/m2 
0% 
60mg/m2 
60mg/m2 
50mg/m2 
0% 
50 mg/m2 
50 mg/m2 
50 mg/m2 
0% 
0% 
0% 
0% 
0% 
* ANC = WBC x (% polys + % stabs) 
** 0% Indicates treatment should be postponed a week until the counts return to a level at which drugs may be given. 
 
Guidelines for dosing also include consideration of ANC. Dosage may be reduced, delayed or 
discontinued in patients with bone marrow depression due to cytotoxic/radiation therapy or with other 


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toxicities. 
Dosage in hepatic failure:  
 
 
 
 
 
 
 
 
             
Table 20 
Cycle length 
Alkaline Phosphatase 
AST +/or ALT 
Dose 
3 weeks: 
< 2.5 x ULN 
and 
<1.5 x ULN 
75mg/m2 
 
2.5 – 5 x ULN 
and 
1.6 – 5 x ULN 
60mg/m2 
 
> 5 x ULN 
or 
> 5 ULN 
discuss with contact physician 
*Liver enzymes are recommended before cycle 1 and then prior to each treatment if clinically indicated (e.g., if liver enzymes are 
elevated, liver metastases are present or there is severe toxicity such as neutropenia). If liver enzymes are normal and there is no 
evidence of liver metastases or severe toxicity, check liver enzymes after 3 cycles (ie, at cycle 4). 
            
Table 21 
Dosage in severe peripheral 
neuropathy:  
Cycle length 3 
weeks:  
reduce by 25% to 60mg/m2; further reduce by 15% to 50mg/m2 if 
reactions continue; discontinue if patient experiences ≥ grade 3 
peripheral neuropathy 
Dosage in severe or 
cumulative cutaneous 
reactions:  
Cycle length 3 
weeks:  
reduce by 25% to 60mg/m2; further reduce by 15% to 50mg/m2 if 
reactions continue ; discontinue if patient experiences ≥ grade 3 
cutaneous reactions 
Dosage in dialysis: Hemodialysis: no significant removal; no dose adjustment required, may be administered before or after 
hemodialysis 
 
10.3 Paclitaxel 
 
DRUG NAME:  
Paclitaxel 
 
COMMON TRADE NAME 
Taxol 
 
SIDE EFFECTS 
Hypersensitivity reactions: Anaphylaxis and severe hypersensitivity reactions characterized by 
dyspnea and hypotension requiring treatment, angioedema, and generalized urticaria have occurred in 
2 to 4% of patients receiving paclitaxel in clinical trials. Fatal reactions have occurred in patients despite 
premedication. All patients should be pretreated with corticosteroids, diphenhydramine, and H2 
antagonists. Such premedication may consist of dexamethasone 20 mg p.o. administered approximately 
12 and 6 hours before paclitaxel, diphenhydramine (or its equivalent) 50 mg IV 30 to 60 minutes before 
paclitaxel, and cimetidine (300 mg) or ranitidine (50 mg) IV 30 to 60 minutes before paclitaxel. Minor 
symptoms such as flushing, skin reactions, dyspnea, hypotension, or tachycardia do not require 
interruption of therapy. However, severe reactions, such as hypotension requiring treatment, dyspnea 
requiring bronchodilators, angioedema, or generalized urticaria require immediate discontinuation of 
paclitaxel and aggressive symptomatic therapy. Patients who have developed severe hypersensitivity 
reactions should not be re-challenged with paclitaxel. 
 
Hematologic effects: Bone marrow suppression (primarily neutropenia) is dose-dependent and is the 
DLT. Neutrophil nadirs occur at a median of 11 days. Paclitaxel should not be administered to patients 
with baseline neutrophil counts of less than 1500 cells/mm3. Patients should not be re-treated with 
subsequent cycles of paclitaxel until neutrophils recover to a level >1500 cells/mm3
 and platelets recover 
to a level >100,000 cells/mm3.  
 
Cardiovascular effects: Hypotension, bradycardia, and hypertension have been observed during 
administration of paclitaxel but generally do not require treatment. Occasionally paclitaxel infusions must 


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be interrupted or discontinued because of initial or recurrent hypertension. Frequent vital sign monitoring, 
particularly during the first hour of paclitaxel infusion, is recommended.  
 
Nervous system: Although the occurrence of peripheral neuropathy is frequent, the development of 
severe symptomatology is unusual and requires a dose reduction of 20% for all subsequent courses of 
paclitaxel. 
 
Injection site reaction: Injection site reactions, including reactions secondary to extravasation, are 
usually mild and consist of erythema, tenderness, skin discoloration, or swelling at the injection site. More 
severe events such as phlebitis, cellulitis, induration, skin exfoliation, necrosis, and fibrosis have been 
reported. In some cases the onset of the injection site reaction either occurred during a prolonged infusion 
or was delayed by a 7 to 10 days. 
 
DRUG INTERACTIONS 
The metabolism of paclitaxel is catalyzed by CYP isoenzymes CYP2C8 and CYP3A4. Caution should be 
exercised when paclitaxel is concomitantly administered with known substrates (e.g., midazolam, 
buspirone, felodipine, lovastatin, eletriptan, sildenafil, simvastatin, and triazolam), inhibitors (e.g., 
atazanavir, clarithromycin, indinavir, itraconazole, ketoconazole, nefazodone, nelfinavir, ritonavir, 
saquinavir, and telithromycin), or inducers (e.g., rifampin and carbamazepine) of CYP3A4. Caution 
should also be exercised when paclitaxel is concomitantly administered with known substrates (e.g., 
repaglinide and rosiglitazone), inhibitors (e.g., gemfibrozil), or inducers (e.g., rifampin) of CYP2C8. 
 
DOSAGE AND ADMINISTRATION 
Paclitaxel at 175 mg/m2 will be IV infused over 3 hours or 80 mg/m2 will be IV infused weekly over 1 hour 
on Day 1 at each cycle. Paclitaxel will be administered 15 min after L-NMMA infusion on Day 1. 
 
10.4 Nab-Paclitaxel 
 
DRUG NAME 
Nab-paclitaxel 
 
COMMON TRADE NAME 
Abraxane 
 
SIDE EFFECTS 
 
Hematologic effects: Bone marrow suppression (primarily neutropenia) is dose-dependent and a DLT 
of nab-paclitaxel. In clinical studies, Grade 3-4 neutropenia occurred in 34% of patients with metastatic 
breast cancer. Do not administer nab-paclitaxel to patients with baseline ANC of less than 1,500 
cells/mm3. In the case of severe neutropenia (<500 cells/mm3 for seven days or more) during a course 
of nab-paclitaxel therapy, reduce the dose of nab-paclitaxel in subsequent courses. Resume treatment 
with nab-paclitaxel after ANC recovers to a level >1,500 cells/mm3 and platelets recover to a level 
>100,000 cells/mm3. 
 
Nervous system: Sensory neuropathy is dose- and schedule-dependent. The occurrence of Grade 1 or 
2 sensory neuropathy does not generally require dose modification. If ≥ Grade 3 sensory neuropathy 
develops, withhold nab-paclitaxel treatment until resolution to Grade 1 or 2. 
 


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Hypersensitivity reactions: Grade 1 or 2 hypersensitivity reactions consisting of dyspnea and flushing, 
hypotension, chest pain, and arrhythmia can occur on the day of nab-paclitaxel administration. Severe 
and sometimes fatal hypersensitivity reactions, including anaphylactic reactions, have also been 
reported. Patients who experience a severe hypersensitivity reaction to nab-paclitaxel should not be re-
challenged with this drug.  
 
Albumin (Human): Nab-paclitaxel contains albumin (human), a derivative of human blood. Based on 
effective donor screening and product manufacturing processes, it carries a remote risk for transmission 
of viral diseases. A theoretical risk for transmission of Creutzfeldt-Jakob disease also is considered 
extremely remote.  
 
Infections: Infectious episodes were reported in 24% of patients treated with nab-paclitaxel. Oral 
candidiasis, respiratory tract infections, and pneumonia were the most frequently reported infectious 
complications. 
 
Cardiovascular effects: Hypotension, during the 30-minute infusion, has occurred in 5% of patients. 
Bradycardia, during the 30-minute infusion, occurred in <1% of patients. These vital sign changes most 
often caused no symptoms and required neither specific therapy nor treatment discontinuation. Severe 
cardiovascular events possibly related to single-agent nab-paclitaxel have occurred in approximately 3% 
of patients. These events included cardiac ischemia/infarction, chest pain, cardiac arrest, 
supraventricular tachycardia, edema, thrombosis, pulmonary thromboembolism, pulmonary emboli, and 
hypertension. Cases of cerebrovascular attacks (strokes) and transient ischemic attacks have been 
reported. Electrocardiogram (ECG) abnormalities were common among patients at baseline. ECG 
abnormalities on study did not usually result in symptoms, were not dose-limiting, and required no 
intervention. ECG abnormalities were noted in 60% of patients. Among patients with a normal ECG prior 
to study entry, 35% of all patients developed an abnormal tracing while on study. The most frequently 
reported ECG modifications were non-specific repolarization abnormalities, sinus bradycardia, and sinus 
tachycardia. 
 
Respiratory effects: Dyspnea (12%), cough (7%), and pneumothorax (<1%) have been reported after 
treatment with nab-paclitaxel. 
 
Vision disorders: Ocular/visual disturbances have occurred in 13% of all patients treated with nab-
paclitaxel and 1% were severe. The severe cases (keratitis and blurred vision) were reported in patients 
who received higher doses than those recommended (300 or 375 mg/m2). These effects generally have 
been reversible. 
 
Arthralgia/Myalgia: The symptoms were usually transient, occurred two or three days after nab-
paclitaxel administration, and resolved within a few days. 
 
Renal effects: Overall 11% of patients experienced creatinine elevation (1% severe). No 
discontinuations, dose reductions, or dose delays were caused by renal toxicities. 
 
Other clinical events: Nail changes (changes in pigmentation or discoloration of the nail bed) have been 
reported. Edema occurred in 10% of patients; no patients had severe edema. Dehydration and pyrexia 
were also reported. 
 
DRUG INTERACTIONS 
The metabolism of paclitaxel is catalyzed by CYP2C8 and CYP3A4. Caution should be exercised when 
administering nab-paclitaxel concomitantly with medicines known to inhibit (e.g., ketoconazole and other 


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imidazole antifungals, erythromycin, fluoxetine, gemfibrozil, cimetidine, ritonavir, saquinavir, indinavir, 
and nelfinavir) or induce (e.g., rifampicin, carbamazepine, phenytoin, efavirenz, and nevirapine) either 
CYP2C8 or CYP3A4. 
 
DOSAGE AND ADMINISTRATION 
Nab-paclitaxel at 260 mg/m2 will be IV infused over 30 minutes on Day 1 of each cycle. Nab-paclitaxel 
will be administered 15 min after L-NMMA infusion on Day 1. 
 
 
10.5  Amlodipine72 
DRUG NAME: Amlodipine 
SYNONYM(S): besylate salt of amlodipine 
COMMON TRADE NAME(S): NORVASC®  
CLASSIFICATION: long-acting calcium channel blocker 
Amlodipine besylate is chemically described as 3-Ethyl-5-methyl (±)-2-[(2-aminoethoxy)methyl]4-(2-
chlorophenyl)-1,4-dihydro-6-methyl-3,5-pyridinedicarboxylate, monobenzenesulphonate. Its empirical 
formula is C20H25CIN2O5•C6H6O3S, and its structural formula is: 
 
Figure 19: Amlodipine Molecular structure 
Amlodipine besylate is a white crystalline powder with a molecular weight of 567.1. It is slightly soluble 
in water and sparingly soluble in ethanol. AMLODIPINE (amlodipine besylate) Tablets are formulated as 
white tablets equivalent to 2.5, 5, and 10 mg of amlodipine for oral administration. In addition to the active 
ingredient, amlodipine besylate, each tablet contains the following inactive ingredients: microcrystalline 
cellulose, dibasic calcium phosphate anhydrous, sodium starch glycolate, and magnesium stearate. 
MECHANISM OF ACTION:  
Amlodipine is a dihydropyridine calcium antagonist (calcium ion antagonist or slow-channel blocker) that 
inhibits the transmembrane influx of calcium ions into vascular smooth muscle and cardiac muscle. 
Experimental data suggest that amlodipine binds to both dihydropyridine and nondihydropyridine binding 
sites. The contractile processes of cardiac muscle and vascular smooth muscle are dependent upon the 
movement of extracellular calcium ions into these cells through specific ion channels. Amlodipine inhibits 
calcium ion influx across cell membranes selectively, with a greater effect on vascular been seen in intact 
animals at therapeutic doses. Serum calcium concentration is not affected by amlodipine. Within the 
physiologic pH range, amlodipine is an ionized compound (pKa=8.6), and its kinetic interaction with the 
calcium channel receptor is characterized by a gradual rate of association and dissociation with the 
receptor binding site, resulting in a gradual onset of effect. 


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Amlodipine is a peripheral arterial vasodilator that acts directly on vascular smooth muscle to cause a 
reduction in peripheral vascular resistance and reduction in blood pressure. The precise mechanisms by 
which amlodipine relieves angina have not been fully delineated, but are thought to include the following: 
Exertional Angina: In patients with exertional angina, AMLODIPINE reduces the total peripheral 
resistance (afterload) against which the heart works and reduces the rate pressure product, and thus 
myocardial oxygen demand, at any given level of exercise. 
Vasospastic Angina: AMLODIPINE has been demonstrated to block constriction and restore blood flow 
in coronary arteries and arterioles in response to calcium, potassium epinephrine, serotonin, and 
thromboxane A2 analog in experimental animal models and in human coronary vessels in vitro. This 
inhibition of coronary spasm is responsible for the effectiveness of AMLODIPINE in vasospastic 
(Prinzmetal’s or variant) angina. 
PHARMACODYNAMICS 
Hemodynamics: Following administration of therapeutic doses to patients with hypertension, 
AMLODIPINE produces vasodilation resulting in a reduction of supine and standing blood pressures. 
These decreases in blood pressure are not accompanied by a significant change in heart rate or plasma 
catecholamine levels with chronic dosing. Although the acute intravenous administration of amlodipine 
decreases arterial blood pressure and increases heart rate in hemodynamic studies of patients with 
chronic stable angina, chronic oral administration of amlodipine in clinical trials did not lead to clinically 
significant changes in heart rate or blood pressures in normotensive patients with angina.   With chronic 
once daily oral administration, antihypertensive effectiveness is maintained for at least 24 hours. Plasma 
concentrations correlate with effect in both young and elderly patients. The magnitude of reduction in 
blood pressure with AMLODIPINE is also correlated with the height of pretreatment elevation; thus, 
individuals with moderate hypertension (diastolic pressure 105–114 mmHg) had about a 50% greater 
response than patients with mild hypertension (diastolic pressure 90–104 mmHg). Normotensive subjects 
experienced no clinically significant change in blood pressures (+1/–2 mmHg). 
In hypertensive patients with normal renal function, therapeutic doses of AMLODIPINE resulted in a 
decrease in renal vascular resistance and an increase in glomerular filtration rate and effective renal 
plasma flow without change in filtration fraction or proteinuria.  As with other calcium channel blockers, 
hemodynamic measurements of cardiac function at rest and during exercise (or pacing) in patients with 
normal ventricular function treated with AMLODIPINE have generally demonstrated a small increase in 
cardiac index without significant influence on dP/dt or on left ventricular end diastolic pressure or volume. 
In hemodynamic studies, AMLODIPINE has not been associated with a negative inotropic effect when 
administered in the therapeutic dose range to intact animals and man, even when co-administered with 
beta-blockers to man. Similar findings, however, have been observed in normal or well-compensated 
patients with heart failure with agents possessing significant negative inotropic effects. 
Electrophysiologic Effects: AMLODIPINE does not change sinoatrial nodal function or atrioventricular 
conduction in intact animals or man. In patients with chronic stable angina, intravenous administration of 
10 mg did not significantly alter A-H and H-V conduction and sinus node recovery time after pacing. 
Similar results were obtained in patients receiving AMLODIPINE and concomitant beta-blockers. In 
clinical studies in which AMLODIPINE was administered in combination with beta-blockers to patients 
with either hypertension or angina, no adverse effects on electrocardiographic parameters were observed. 
In clinical trials with angina patients alone, AMLODIPINE therapy did not alter electrocardiographic 
intervals or produce higher degrees of AV blocks. 
 
PHARMACOKINETICS AND METABOLISM 
After oral administration of therapeutic doses of AMLODIPINE, absorption produces peak plasma 
concentrations between 6 and 12 hours. Absolute bioavailability has been estimated to be between 64 
and 90%. The bioavailability of AMLODIPINE is not altered by the presence of food.  Amlodipine is 
extensively (about 90%) converted to inactive metabolites via hepatic metabolism with 10% of the parent 


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compound and 60% of the metabolites excreted in the urine. Ex vivo studies have shown that 
approximately 93% of the circulating drug is bound to plasma proteins in hypertensive patients. 
Elimination from the plasma is biphasic with a terminal elimination half-life of about 30–50 hours. Steady-
state plasma levels of amlodipine are reached after 7 to 8 days of consecutive daily dosing.  The 
pharmacokinetics of amlodipine are not significantly influenced by renal impairment. Patients with renal 
failure may therefore receive the usual initial dose. 
Elderly patients and patients with hepatic insufficiency have decreased clearance of amlodipine with a 
resulting increase in AUC of approximately 40–60%, and a lower initial dose may be required. A similar 
increase in AUC was observed in patients with moderate to severe heart failure. 
CLINICAL STUDIES 
The antihypertensive efficacy of AMLODIPINE has been demonstrated in a total of 15 double-blind, 
placebo-controlled, randomized studies involving 800 patients on AMLODIPINE and 538 on placebo. 
Once daily administration produced statistically significant placebo-corrected reductions in supine and 
standing blood pressures at 24 hours postdose, averaging about 12/6 mmHg in the standing position and 
13/7 mmHg in the supine position in patients with mild to moderate hypertension. Maintenance of the 
blood pressure effect over the 24-hour dosing interval was observed, with little difference in peak and 
trough effect. Tolerance was not demonstrated in patients studied for up to 1 year. The 3 parallel, fixed 
doses, dose response studies showed that the reduction in supine and standing blood pressures was 
dose-related within the recommended dosing range. Effects on diastolic pressure were similar in young 
and older patients. The effect on systolic pressure was greater in older patients, perhaps because of 
greater baseline systolic pressure. Effects were similar in black patients and in white patients. 
Effects in Chronic Stable Angina 
The effectiveness of 5–10 mg/day of AMLODIPINE in exercise-induced angina has been evaluated in 8 
placebo-controlled, double-blind clinical trials of up to 6 weeks duration involving 1038 patients (684 
AMLODIPINE, 354 placebo) with chronic stable angina. In 5 of the 8 studies, significant increases in 
exercise time (bicycle or treadmill) were seen with the 10 mg dose. Increases in symptom-limited exercise 
time averaged 12.8% (63 sec) for AMLODIPINE 10 mg, and averaged 7.9% (38 sec) for AMLODIPINE 
5 mg. AMLODIPINE 10 mg also increased time to 1 mm ST segment deviation in several studies and 
decreased angina attack rate. The sustained efficacy of AMLODIPINE in angina patients has been 
demonstrated over long-term dosing. In patients with angina, there were no clinically significant 
reductions in blood pressures (4/1 mmHg) or changes in heart rate (+0.3 bpm). 
Effects in Vasospastic Angina 
In a double-blind, placebo-controlled clinical trial of 4 weeks duration in 50 patients, AMLODIPINE 
therapy decreased attacks by approximately 4/week compared with a placebo decrease of approximately 
1/week (p<0.01). Two of 23 AMLODIPINE and 7 of 27 placebo patients discontinued from the study due 
to lack of clinical improvement. 
Effects in Documented Coronary Artery Disease 
In PREVENT, 825 patients with angiographically documented coronary artery disease were randomized 
to AMLODIPINE (5–10 mg once daily) or placebo and followed for 3 years. Although the study did not 
show significance on the primary objective of change in coronary luminal diameter as assessed by 
quantitative coronary angiography, the data suggested a favorable outcome with respect to fewer 
hospitalizations for angina and revascularization procedures in patients with CAD.    CAMELOT enrolled 
1318 patients with CAD recently documented by angiography, without left main coronary disease and 
without heart failure or an ejection fraction <40%. Patients (76% males, 89% Caucasian, 93% enrolled 
at US sites, 89% with a history of angina, 52% without PCI, 4% with PCI and no stent, and 44% with a 
stent) were randomized to double-blind treatment with either AMLODIPINE (5–10 mg once daily) or 
placebo in addition to standard care that included aspirin (89%), statins (83%), beta-blockers (74%), 
nitroglycerin (50%), anti-coagulants (40%), and diuretics (32%), but excluded other calcium channel 
blockers. The mean duration of follow-up was 19 months. The primary endpoint was the time to first 


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occurrence of one of the following events: hospitalization for angina pectoris, coronary revascularization, 
myocardial infarction, cardiovascular death, resuscitated cardiac arrest, hospitalization for heart failure, 
stroke/TIA, or peripheral vascular disease. A total of 110 (16.6%) and 151 (23.1%) first events occurred 
in the AMLODIPINE and placebo groups, respectively, for a hazard ratio of 0.691 (95% CI: 0.540–0.884, 
p = 0.003). The outcome of this study was largely derived from the prevention of hospitalizations for 
angina and the prevention of revascularization procedures. In an angiographic substudy (n=274) 
conducted within CAMELOT, there was no significant difference between amlodipine and placebo on the 
change of atheroma volume in the coronary artery as assessed by intravascular ultrasound. 
Studies in Patients with Heart Failure 
AMLODIPINE has been compared to placebo in four 8–12 week studies of patients with NYHA Class 
II/III heart failure, involving a total of 697 patients. In these studies, there was no evidence of worsened 
heart failure based on measures of exercise tolerance, NYHA classification, symptoms, or left ventricular 
ejection fraction. In a long-term (follow-up at least 6 months, mean 13.8 months) placebo-controlled 
mortality/morbidity study of AMLODIPINE 5–10 mg in 1153 patients with NYHA Classes III (n=931) or IV 
(n=222) heart failure on stable doses of diuretics, digoxin, and ACE inhibitors, AMLODIPINE had no 
effect on the primary endpoint of the study which was the combined endpoint of all-cause mortality and 
cardiac morbidity (as defined by life-threatening arrhythmia, acute myocardial infarction, or hospitalization 
for worsened heart failure), or on NYHA classification, or symptoms of heart failure. Total combined all-
cause mortality and cardiac morbidity events were 222/571 (39%) for patients on AMLODIPINE and 
246/583 (42%) for patients on placebo; the cardiac morbid events represented about 25% of the 
endpoints in the study. Another study (PRAISE-2) randomized patients with NYHA Class III (80%) or IV 
(20%) heart failure without clinical symptoms or objective evidence of underlying ischemic disease, on 
stable doses of ACE inhibitors (99%), digitalis (99%), and diuretics (99%), to placebo (n=827) or 
AMLODIPINE (n=827) and followed them for a mean of 33 months. There was no statistically significant 
difference between AMLODIPINE and placebo in the primary endpoint of all-cause mortality (95% 
confidence limits from 8% reduction to 29% increase on AMLODIPINE). With AMLODIPINE there were 
more reports of pulmonary edema. 
Clinical Trials Experience 
Because clinical trials are conducted under widely varying conditions, adverse reaction rates observed 
in the clinical trials of a drug cannot be directly compared to rates in the clinical trials of another drug and 
may not reflect the rates observed in practice. AMLODIPINE has been evaluated for safety in more than 
11,000 patients in U.S. and foreign clinical trials. In general, treatment with AMLODIPINE was well-
tolerated at doses up to 10 mg daily. Most adverse reactions reported during therapy with AMLODIPINE 
were of mild or moderate severity. In controlled clinical trials directly comparing AMLODIPINE (N=1730) 
at doses up to 10 mg to placebo (N=1250), discontinuation of AMLODIPINE due to adverse reactions 
was required in only about 1.5% of patients and was not significantly different from placebo (about 1%).  
The most common side effects are headache and edema. The incidence (%) of side effects that occurred 
in a dose related manners are as follows:                                                                                                    Table 
22 
Adverse Event 
2.5 mg 
5.0 mg 
10.0 mg 
Placebo 
 
N=275 
N=296 
N=268 
N=520 
Edema 
1.8 
3.0 
10.8 
0.6 
Dizziness 
1.1 
3.4 
3.4 
1.5 
Flushing 
0.7 
1.4 
2.6 
0.0 
Palpitation 
0.7 
1.4 
4.5 
0.6 
                                                                                                                                                       
Other adverse experiences that were not clearly dose related but were reported with an incidence greater 
than 1.0% in placebo-controlled clinical trials include the following:                                                          
Table 23 
Placebo-Controlled Studies 
 
 
 


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AMLODIPINE (%) 
Placebo (%) 
 
 
 
 
(N=1730) 
(N=1250) 
 
 
 
Headache 
7.3 
7.8 
 
 
 
Fatigue 
4.5 
2.8 
 
 
 
Nausea 
2.9 
1.9 
 
 
 
Abdominal Pain 
1.6 
0.3 
 
 
 
Somnolence 
1.4 
0.6 
 
 
 
 
For several adverse experiences that appear to be drug and dose related, there was a greater incidence 
in women than men associated with amlodipine treatment as shown in the following table:                        
Table 24 
 
AMLODIPINE 
Placebo 
Adverse Event 
Male=% 
Female=% 
Male=% 
Female=% 
 
(N=1218) 
(N=512) 
(N=914) 
(N=336) 
 
Edema 
5.6 
14.6 
1.4 
5.1 
Flushing 
1.5 
4.5 
0.3 
0.9 
Palpitations 
1.4 
3.3 
0.9 
0.9 
Somnolence 
1.3 
1.6 
0.8 
0.3 
 
The following events occurred in <1% but >0.1% of patients in controlled clinical trials or under conditions 
of open trials or marketing experience where a causal relationship is uncertain; they are listed to alert the 
physician to a possible relationship: 
Cardiovascular: arrhythmia (including ventricular tachycardia and atrial fibrillation), bradycardia, chest 
pain, hypotension, peripheral ischemia, syncope, tachycardia, postural dizziness, postural hypotension, 
vasculitis. 
Central and Peripheral Nervous System: hypoesthesia, neuropathy peripheral, paresthesia, tremor, 
vertigo. 
Gastrointestinal: anorexia, constipation, dyspepsia, dysphagia, diarrhea, flatulence, pancreatitis, 
vomiting, gingival hyperplasia. 
General: allergic reaction, asthenia, back pain, hot flushes, malaise, pain, rigors, weight gain, weight 
decrease. 
Musculoskeletal System: arthralgia, arthrosis, muscle cramps, myalgia. 
Psychiatric: sexual dysfunction (male and female), insomnia, nervousness, depression, abnormal 
dreams, anxiety, depersonalization. 
Respiratory System: dyspnea, epistaxis. 
Skin and Appendages: angioedema, erythema multiforme, pruritus, rash, rash erythematous, rash 
maculopapular. 
Special Senses: abnormal vision, conjunctivitis, diplopia, eye pain, tinnitus. 
Urinary System: micturition frequency, micturition disorder, nocturia. 
Autonomic Nervous System: dry mouth, sweating increased. 
Metabolic and Nutritional: hyperglycemia, thirst. 
Hemopoietic: leukopenia, purpura, thrombocytopenia. 
These events occurred in less than 1% in placebo-controlled trials, but the incidence of these side effects 
was between 1% and 2% in all multiple dose studies.  The following events occurred in <0.1% of patients: 
cardiac failure, pulse irregularity, extrasystoles, skin discoloration, urticaria, skin dryness, alopecia, 
dermatitis, muscle weakness, twitching, ataxia, hypertonia, migraine, cold and clammy skin, apathy, 
agitation, amnesia, gastritis, increased appetite, loose stools, coughing, rhinitis, dysuria, polyuria, 
parosmia, taste perversion, abnormal visual accommodation, and xerophthalmia. Other reactions 
occurred sporadically and cannot be distinguished from medications or concurrent disease states such 
as myocardial infarction and angina. 


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AMLODIPINE therapy has not been associated with clinically significant changes in routine laboratory 
tests. No clinically relevant changes were noted in serum potassium, serum glucose, total triglycerides, 
total cholesterol, HDL cholesterol, uric acid, blood urea nitrogen, or creatinine. 
DRUG INTERACTIONS 
In Vitro Data, indicate that AMLODIPINE has no effect on the human plasma protein binding of digoxin, 
phenytoin, warfarin, and indomethacin. 
Co-administration of AMLODIPINE with cimetidine did not alter the pharmacokinetics of AMLODIPINE. 
Co-administration of 240 mL of grapefruit juice with a single oral dose of amlodipine 10 mg in 20 healthy 
volunteers had no significant effect on the pharmacokinetics of amlodipine. 
Co-administration of a magnesium and aluminum hydroxide antacid with a single dose of AMLODIPINE 
had no significant effect on the pharmacokinetics of AMLODIPINE. 
A single 100 mg dose of sildenafil in subjects with essential hypertension had no effect on the 
pharmacokinetic parameters of AMLODIPINE. When AMLODIPINE and sildenafil were used in 
combination, each agent independently exerted its own blood pressure lowering effect. 
Co-administration of multiple 10 mg doses of AMLODIPINE with 80 mg of atorvastatin resulted in no 
significant change in the steady-state pharmacokinetic parameters of atorvastatin. 
Co-administration of AMLODIPINE with digoxin did not change serum digoxin levels or digoxin renal 
clearance in normal volunteers. 
Single and multiple 10 mg doses of AMLODIPINE had no significant effect on the pharmacokinetics of 
ethanol. 
Co-administration of AMLODIPINE with warfarin did not change the warfarin prothrombin response time. 
Co-administration of a 180 mg daily dose of diltiazem with 5 mg amlodipine in elderly hypertensive 
patients resulted in a 60% increase in amlodipine systemic exposure. Erythromycin co-administration in 
healthy volunteers did not significantly change amlodipine systemic exposure. However, strong inhibitors 
of CYP3A4 (e.g., ketoconazole, itraconazole, ritonavir) may increase the plasma concentrations of 
amlodipine to a greater extent. Monitor for symptoms of hypotension and edema when amlodipine is co-
administered with CYP3A4 inhibitors. 
No information is available on the quantitative effects of CYP3A4 inducers on amlodipine. Blood pressure 
should be closely monitored when Amlodipine is co-administered with CYP3A4 inducers. 
OVERDOSAGE 
Overdosage might be expected to cause excessive peripheral vasodilation with marked hypotension and 
possibly a reflex tachycardia. In humans, experience with intentional overdosage of AMLODIPINE is 
limited. 
Single oral doses of amlodipine maleate equivalent to 40 mg amlodipine/kg and 100 mg amlodipine/kg 
in mice and rats, respectively, caused deaths. Single oral amlodipine maleate doses equivalent to 4 or 
more mg amlodipine/kg or higher in dogs (11 or more times the maximum recommended human dose 
on a mg/m2 basis) caused a marked peripheral vasodilation and hypotension. 
If massive overdose should occur, initiate active cardiac and respiratory monitoring. Frequent blood 
pressure measurements are essential. Should hypotension occur, provide cardiovascular support 
including elevation of the extremities and the judicious administration of fluids. If hypotension remains 
unresponsive to these conservative measures, consider administration of vasopressors (such as 
phenylephrine) with attention to circulating volume and urine output. As AMLODIPINE is highly protein 
bound, hemodialysis is not likely to be of benefit. 
DOSAGE AND ADMINISTRATION 72 
The initial antihypertensive oral dose of AMLODIPINE is 10 mg once daily starting the day before of 
C1D1.  
Dose may be adjusted by the treating physician if needed. Amlodipine dose will be held if systolic BP 
remains below 100 mmHg.  
 


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10.6 Enteric-Coated Aspirin 81 mg 
DRUG NAME: Acetylsalicylic acid 
 
SIDE EFFECTS 
Many adverse reactions due to acetylsalicylic acid ingestion are dose-related. 
 
Gastrointestinal (the frequency and severity of these adverse effects are dose-related): nausea, vomiting, 
diarrhea, gastrointestinal bleeding and/or ulceration, dyspepsia, heartburn, hematemesis, melena, 
abdominal pain, and rarely gastrointestinal inflammation. 
 
Bleeding: Due to platelet inhibition, bleedings e.g. perioperative hemorrhage, hematomas, epistaxis, 
urogenital bleedings, and gingival bleedings may occur. Serious bleedings, such as gastrointestinal tract 
hemorrhages, and cerebral hemorrhages are rare. Isolated cases of potentially life-threatening bleedings 
have been reported, especially in patients with uncontrolled hypertension and/or concomitant 
antihemostatic agents. 
 
Ear: dizziness, tinnitus, vertigo, hearing loss. Dizziness and tinnitus have been reported, which may be 
indicative of an overdose. 
 
Hematologic: leukopenia, thrombocytopenia, purpura, anemia. Anemia with respective laboratory and 
clinical signs and symptoms, such as asthenia, pallor, and hypoperfusion is generally caused by bleeding 
(e.g., occult microbleeding, acute or chronic bleeding). Hemolysis and hemolytic anemia in patients with 
severe forms of glucose-6-phosphate dehydrogenase deficiency have been reported. 
 
Dermatologic and hypersensitivity: urticaria, pruritus, skin eruptions, asthma, anaphylaxis, edema, nasal 
congestion, and rhinitus. Severe allergic reactions, including anaphylactic shock are very rarely reported. 
 
DRUG INTERACTIONS 
Methotrexate, used at 15 mg/week or less: Salicylates may retard the elimination of methotrexate by 
decreasing renal clearance of methotrexate, displacing methotrexate from protein binding sites, and 
thereby increasing its hematological toxicity. 
 
Anticoagulants, thrombolytics / other inhibitors of platelet aggregation/hemostasis, e.g. warfarin, heparin: 
Caution is necessary when salicylates and anticoagulants, thrombolytics /other inhibitors of platelet 
aggregation / hemostasis prescribed concurrently, as salicylates can depress the concentration of 
prothrombin in the plasma, leading to an increased risk of bleeding. 
 
Oral hypoglycemics, e.g. insulin, sulfonylureas: Large doses of salicylates have a hypoglycemic action 
and may enhance the effect of oral hypoglycemic agents. Diabetics receiving concurrent salicylate and 
hypoglycemic therapy should be monitored closely: reduction of the sulfonylurea hypoglycemic drug 
dosage may be necessary. 
 
Diuretics: Diuretics in combination with acetylsalicylic acid at higher doses leads to decreased glomerular 
filtration via decreased prostaglandin synthesis. As a result, sodium excretion may be decreased by 
salicylate administration. 
 
Uricosuric Agents: Salicylates in large doses are uricosuric agents; smaller amounts may depress uric 
acid clearance and thus decrease the uricosuric effects of other drugs. 
 


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Valproic Acid: Salicylates may alter valproic acid (VPA) metabolism and may displace VPA from protein 
binding sites, possibly intensifying the effects of VPA. Caution is recommended when VPA is 
administered concomitantly with salicylates. 
 
Glucocorticoids (systemic), except hydrocortisone used as replacement therapy in Addison’s disease: 
Decreased blood salicylate levels during corticosteroid treatment and risk of salicylate overdose after this 
treatment is stopped via increased elimination of salicylates by corticosteroids. 
 
Angiotensin Converting Enzyme (ACE) Inhibitors: The hyponatremic and hypotensive effects of ACE 
inhibitors may be diminished by the concomitant administration of acetylsalicylic acid due to its indirect 
effect on the renin-angiotensin conversion pathway (i.e., inhibition of vasodilatory prostaglandins leading 
to decreased glomerular filtration). The potential interaction may be related to the dose of acetylsalicylic 
acid (≥3 g/day). 
 
Selective Serotonin Re-uptake Inhibitors: Increased risk of upper gastrointestinal bleeding due to possibly 
synergistic effect. 
 
Digoxin: Plasma concentrations of digoxin are increased due to a decrease in renal excretion. 
 
Non-steroidal anti-inflammatory drugs (NSAIDs): The use of other NSAIDs with salicylates at high doses 
(≥3 g/day) may increase the risk of ulcers and gastrointestinal bleeding due to a synergistic effect. 
Ibuprofen can interfere with the anti-platelet effect of low-dose acetylsalicylic acid (81-325 mg QD). Long-
term daily use of ibuprofen may render acetylsalicylic acid less effective when used for cardioprotection 
and stroke prevention. To minimize this interaction, regular users of ibuprofen and of low-dose, 
immediate-release acetylsalicylic acid should take the ibuprofen at least one hour after and 11 hours 
before the daily acetylsalicylic acid dose. The use of delayed-release (e.g. enteric-coated) acetylsalicylic 
acid is not recommended when using ibuprofen regularly. Naproxen may attenuate the irreversible 
platelet inhibition induced by acetylsalicylic acid. Clinical pharmacodynamic data suggest that concurrent 
(same day) naproxen sodium usage for more than one day consecutively inhibits the effect of low-dose 
acetylsalicylic acid on platelet activity and this inhibition may persist for up to several days after stopping 
naproxen sodium therapy. The clinical relevance of this interaction is not known. Treatment with 
naproxen, in patients with increased cardiovascular risk may limit the cardiovascular protection of 
acetylsalicylic acid. 
 
DOSAGE AND ADMINISTRATION 
Enteric-coated daily low-dose aspirin should preferably be taken after meals, with plenty of liquid. Enteric-
coated aspirin will be administered as 81 mg QD during the 6 21-day cycles of L-NMMA and docetaxel. 
 
11 Prohibited concomitant therapy and dietary restrictions 
The following medications and procedures are prohibited during the study: 
 Alternative therapy, including palliative radiotherapy, for treatment of the patient’s malignancy  
 Any investigational therapy other than L-NMMA, amlodipine, and taxane chemotherapy. 
 Medications Listed in Appendix E  
 Any complementary or alternative medications 
 Enzyme inducers, such as the enzyme-inducing antiepileptic drugs phenytoin, carbamazepine 
or phenobarbital, or rifampin, rifabutin, rifapentine or St. John's Wort within 14 days prior to the 
first dose of the study drugs and during the period of treatment with L-NMMA in this study. 
 Use of the following medications and procedures should be considered with caution: 


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 Interaction with medicinal products metabolized through CYP3A4 and CYP2C8 route:  
o Docetaxel is a CYP3A4 substrate. In vitro studies have shown that the metabolism of 
docetaxel may be modified by the concomitant administration of compounds that 
induce, inhibit, or are metabolized by cytochrome P450 3A4. 
o In vivo studies showed that the exposure of docetaxel increased 2.2-fold when it was 
coadministered with ketoconazole, a potent inhibitor of CYP3A4. Protease inhibitors, 
particularly ritonavir, may increase the exposure of docetaxel. Concomitant use of 
docetaxel and drugs that inhibit CYP3A4 may increase exposure to docetaxel and 
should be avoided. 
o The metabolism of paclitaxel is catalyzed by CYP2C8 and CYP3A4. Caution should be 
exercised when administering paclitaxel and nab-paclitaxel concomitantly with 
medicines known to inhibit (e.g., ketoconazole and other imidazole antifungals, 
erythromycin, fluoxetine, gemfibrozil, cimetidine, ritonavir, saquinavir, indinavir, and 
nelfinavir) or induce (e.g., rifampicin, carbamazepine, phenytoin, efavirenz, and 
nevirapine) either CYP2C8 or CYP3A4. 
 
 
11.1 
Anti-neoplastic therapies 
Other anti-neoplastic therapies must be avoided during the length of the trial 
11.2 
Dietary restrictions 
Patients should be on a low nitrite/nitrate diet including avoidance of processed meats such as hot 
dogs, ham, bacon, and sausage 1 week before study treatment start and for the duration of the study. 
Patients should also keep a food diary the week before and the week of the L-NMMA infusion during 
Cycles 1 and 2. 
12 Assessment types 
12.1 
CRITERIA FOR RESPONSE 
The main endpoint in this study is to determine the MTD of the L-NMMA plus docetaxel combination and 
the CBR of the L-NMMA plus taxane chemotherapy (docetaxel, paclitaxel, or nab-paclitaxel) combination 
in patients with refractory locally advanced or metastatic triple negative breast cancer. Tumor response 
will be evaluated using the RECIST 1.1 
 
12.2 
RECIST 1.1 Criteria 
The clinical tumor response will be assessed using RECIST criteria at baseline and before surgery.  
RECIST criteria offer a simplified, conservative, extraction of imaging data for wide application in clinical 
trials. They presume that linear measures are an adequate substitute for 2-D methods and register four 
response categories73  
 
CR (complete response) = disappearance of all target lesions  
 
PR (partial response) = 30% decrease in the sum of the longest diameter of target lesions  
 
PD (progressive disease) = 20% increase in the sum of the longest  diameter of target lesions  
 
SD (stable disease) = small changes that do not meet above criteria  
 
 
12.3 
Withdrawal of subjects from study 
Withdrawal 
Subjects must be withdrawn from the trial (treatment and procedures) for the following reasons:  
 
Subject withdraws consent from study treatment and study procedures.  A subject must be 
removed from the trial at her own request or at the request of her legally acceptable 


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representative.  At any time during the trial and without giving reasons, a subject may decline 
to participate further.  The subject will not suffer any disadvantage as a result. 
 
Blood pressure increases beyond 180 mmHg after L-NMMA infusion and does not return to 
<160 mmHg after two rechecks spaced 15-30 min apart. 
 
Subject is lost to follow-up. 
 
Death. 
Subjects may be withdrawn from the study for the following reasons: 
 
The subject is non-compliant with study drug, trial procedures, or both; including the use of anti-
cancer therapy not prescribed by the study protocol. Non-compliant will be considered if a 
subject takes ≤ 75% of study drug (L-NMMA or taxane chemotherapy). 
 
Pregnancy.  Pregnancy will be reported as an SAE.  (Note: subjects who have been withdrawn 
from treatment with study drug because of pregnancy should not undergo any scans while 
pregnant, except ultrasound). 
 
If, in the investigator's opinion, continuation of the trial would be harmful to the subject's well-
being. 
 
Severe allergic reaction to study drug (L-NMMA, taxane chemotherapy) (such as stomatitis or 
Grade 3 or 4 hypersensitivity reaction). 
 
The development of a second cancer.  
 
Development of an intercurrent illness or situation which would, in the judgment of the 
investigator, significantly affect assessments of clinical status and trial endpoints. 
 
Deterioration of ECOG performance status to 4. 
 
Use of illicit drugs or other substances that may, in the opinion of the investigator, have a 
reasonable chance of contributing to toxicity or otherwise skewing trial result. 
Any subject removed from the trial will remain under medical supervision until discharge or transfer is 
medically acceptable and for 30 days following last dose of study treatment. In all cases, the reason for 
withdrawal must be recorded in the CRF and in the subject's medical records. 
 
12.4 
Pregnancy and assessments of fertility 
Pregnancy testing is required at screening or whenever pregnancy is suspected.  Serum pregnancy 
testing should be performed at screening and at the end of the study or if clinically indicated.  
Women of child-bearing potential, defined as all women physiologically capable of becoming pregnant 
as defined in Appendix D, must use highly effective contraception during the study and for 60 days after 
stopping L-NMMA plus taxane chemotherapy. Highly effective contraception is defined as either: 
 
Total abstinence: When this is in line with the preferred and usual lifestyle of the subject. [Periodic 
abstinence (e.g., calendar, ovulation, symptothermal, post-ovulation methods) and withdrawal are 
not acceptable methods of contraception] 
 
Sterilization: have had surgical bilateral oophorectomy (with or without hysterectomy) or tubal ligation 
at least six weeks before taking study treatment. In case of oophorectomy alone, only when the 
reproductive status of the woman has been confirmed by follow up hormone level assessment 
 
Male partner sterilization (with the appropriate post-vasectomy documentation of the absence of 
sperm in the ejaculate). [For female subjects on the study, the vasectomised male partner should be 
the sole partner for that subject]. 
or 
 
Use of a combination of any two of the following (a+b or a+c or b+c): 
a. Use of oral, injected, implanted or other hormonal methods of contraception 
b. Placement of an intrauterine device (IUD) or intrauterine system (IUS) 
c. Barrier methods of contraception: Condom or Occlusive cap (diaphragm or cervical/vault caps) 
with spermicidal foam/gel/film/cream/vaginal suppository 


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 
In case of use of oral contraception, women should have been stable on the oral agent before taking 
study treatment. 
 
13. 
Safety monitoring and reporting 
Information about all adverse events, whether volunteered by the subject, discovered by investigator 
questioning, or detected through physical examination, laboratory test or other means, will be collected 
and recorded and followed as appropriate. 
 
13.1 
Adverse events 
13.1.1 Definitions and reporting 
Adverse events that begin or worsen after informed consent should be recorded in the Adverse Events 
CRF. Conditions that were already present at the time of informed consent should be recorded in the 
Medical History page of the patient’s CRF. Adverse event monitoring should be continued for at least 30 
days or until adverse event is resolved to baseline or at least Grade II according to CTCAE v4.03 following 
the last dose of study treatment. Adverse events (including lab abnormalities that constitute AEs) should 
be described using a diagnosis whenever possible, rather than individual underlying signs and symptoms. 
When a clear diagnosis cannot be identified, each sign or symptom should be reported as a separate 
Adverse Event. 
The occurrence of adverse events should be sought by non-directive questioning of the patient at each 
visit during the study. Adverse events also may be detected when they are volunteered by the patient 
during or between visits or through physical examination, laboratory test, or other assessments. As far 
as possible, each adverse event should be evaluated to determine: 
1. The severity grade (CTCAE Grade 1-4) 
2. Its duration (Start and end dates or if continuing at the Safety Follow-up Visit) 
3. Its relationship to the study treatment (Reasonable possibility that AE is related: No, Yes)  
4. Action taken with respect to study or investigational treatment (none, dose adjusted, temporarily 
interrupted, permanently discontinued, hospitalized, unknown, not applicable) 
5. Whether medication or therapy was given (no concomitant medication/non-drug therapy, 
concomitant medication/non-drug therapy) 
6. Outcome (not recovered/not resolved, recovered/resolved, recovering/resolving, recovered/resolved 
with sequelae, fatal, unknown) 
7. Whether it is serious, where a serious adverse event (SAE) is defined as in Section 14. 
All adverse events should be treated appropriately. Such treatment may include changes in study drug 
treatment including possible interruption or discontinuation, starting or stopping concomitant treatments, 
changes in the frequency or nature of assessments, hospitalization, or any other medically required 
intervention. Once an adverse event is detected, it should be followed until its resolution to baseline or 
at least Grade II according to CTCAE v4.03, and assessment should be made at each visit (or more 
frequently, if necessary) of any changes in severity, the suspected relationship to the study drug, the 
interventions required to treat it, and the outcome. Information about common side effects already known 
about the investigational drug can be found in the Investigators’ Brochure. This information should be 
included in the patient informed consent and should be discussed with the patient during the study as 
needed. Adverse event monitoring should be continued for at least 30 days following the last dose of 
study treatment 
 
13.1.2 Laboratory test abnormalities 
Laboratory abnormalities that constitute an Adverse event in their own right (are considered clinically 
significant, induce clinical signs or symptoms, require concomitant therapy or require changes in study 
treatment), should be recorded on the Adverse Events CRF. Whenever possible, a diagnosis, rather than 


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a symptom should be provided (e.g. anemia instead of low hemoglobin). Laboratory abnormalities that 
meet the criteria for Adverse Events should be followed until they have returned to normal or an adequate 
explanation of the abnormality is found. When an abnormal laboratory or test result corresponds to a 
sign/symptom of an already reported adverse event, it is not necessary to separately record the lab/test 
result as an additional event. Laboratory abnormalities, that do not meet the definition of an adverse 
event, should not be reported as adverse events. A Grade 3 or 4 event (severe) as per CTCAE does not 
automatically indicate a SAE unless it meets the definition of serious as defined below and/or as per 
investigator’s discretion. A dose hold or medication for the lab abnormality may be required by the 
protocol and is still, by definition, an adverse event. 
 
14 Serious Adverse Events 
14.1 
Definitions 
A serious adverse event is an undesirable sign, symptom or medical condition which: 
 
is fatal or life-threatening 
 
results in persistent or significant disability/incapacity 
 
constitutes a congenital anomaly/birth defect 
 
requires inpatient hospitalization or prolongation of existing hospitalization, unless hospitalization is 
for: 
 
routine treatment or monitoring of the studied indication, not associated with any deterioration 
in condition (specify what this includes) 
 
elective or pre-planned treatment for a pre-existing condition that is unrelated to the indication 
under study and has not worsened since the start of study drug 
 
treatment on an emergency outpatient basis for an event not fulfilling any of the definitions of a 
SAE given above and not resulting in hospital admission 
 
social reasons and respite care in the absence of any deterioration in the patient’s general 
condition 
 
is medically significant, i.e., defined as an event that jeopardizes the patient or may require medical 
or surgical intervention to prevent one of the outcomes listed above 
 
14.1.1 Reporting 
The principal investigator has the obligation to report all related and unexpected adverse events to the 
local IRB.  To ensure patient safety, every SAE, regardless of suspected causality, following local IRB 
policy, occurring: 
 
after the patient has provided informed consent and until at least 30 days after the patient has 
stopped study treatment/participation 
 
after protocol-specified procedures begin (e.g., placebo run-in, washout period, double-blind 
treatment, etc.) and 30 days after the patient has stopped study treatment 
 
after the start of any period in which the study protocol interferes with the standard medical treatment 
given to a patient (e.g., treatment withdrawal during washout period, change in treatment to a fixed 
dose of concomitant medication) and until 30 days after the patient has stopped study treatment 
must be reported to the institutional IRB  within 24 hours of learning of its occurrence ; This includes 
serious, related, labeled (expected) and serious, related, unlabeled (unexpected) adverse 
experiences. All deaths during treatment or within 30 days following completion of active protocol 
therapy must be reported within 5 working days. 
Recurrent episodes, complications, or progression of the initial SAE must be reported as follow-up to the 
original episode within 24 hours of the investigator receiving the follow-up information. A SAE occurring 
at a different time interval or otherwise considered completely unrelated to a previously reported one 
should be reported separately as a new event.  The end date of the first event must be provided. 


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Each re-occurrence, complication, or progression of the original event should be reported as a follow-up 
to that event regardless of when it occurs. The follow-up information should describe whether the event 
has resolved or continues, if and how it was treated, whether the blind was broken or not (if applicable), 
and whether the patient continued or withdrew from study participation. 
If the SAE is not previously documented in the Investigator Brochure or Package Insert (new occurrence) 
and is thought to be related to the study drug, may urgently require further information from the 
investigator to the local IRB. The Investigator and the local IRB may need to issue an Investigator 
Notification (IN), to inform all investigators involved in any study with the same drug that this SAE has 
been reported. Suspected Unexpected Serious Adverse Reactions (SUSARs) will be collected and 
reported to the competent authorities.  For Comparator Drugs/Secondary Suspects (Concomitant 
Medications), all serious adverse experiences will be forwarded to Investigational Drug Services 
Department by the investigator. The following categories and definitions of causal relationship to study 
drug should be considered for use for this clinical study. 
 
Certain: There is a known causal relationship between the study drug and the SAE. The event 
responds to withdrawal of study drug (dechallenge), and recurs with rechallenge when clinically 
feasible. (>95% certainty) 
   Probable: There is reasonable causal relationship between the study drug and the SAE. The event 
responds to dechallenge. Rechallenge is not required. (65%-95% probability) 
   Possible: There is reasonable causal relationship between the study drug and the SAE. Dechallenge 
information is lacking or unclear. (35%-65% probability of relatedness) 
   Not likely: There is temporal relationship to study drug administration, but there is not a reasonable 
causal relationship between the study drug and the SAE. (5-35% probability of relatedness) 
   Not related: There is not a temporal relationship to study drug administration (too early, or late, or 
study drug not taken), or there is known causal relationship between the SAE and another drug, 
concurrent disease, or other circumstance. (<5% chance of relatedness) 
 
Adverse events classified as “serious” require expeditious handling and reporting to Houston  
Methodist Cancer Center (HMCC) and local IRB to comply with regulatory requirements.  
 
All related or unexpected AEs of any of the study drug must be immediately reported to local IRB  
by the investigator or designee within 24 hours of becoming aware of the event. If only limited 
information is initially available, follow-up reports are required.  The original SAE form must be kept 
on file at the study site. 
All SAEs should be faxed or emailed to  
Houston Methodist Cancer Center at: 
hmccsaereports@houstonmethodist.org   
Fax Number: 713-790-5106 
 
For studies conducted under an Investigator IND, any event that is both related and  unexpected 
must be reported to the local IRB as soon as possible and, in no event, later than 7  days (death or 
life-threatening event) or 15 days (all other SAEs) after the investigator’s or  institution’s initial receipt 
of the information.  
14.1.2 Pregnancy 
Preclinical data regarding reproductive toxicity is described in the most recent Investigator Brochure. The 
potential reproductive risk for humans is unknown.  Women of childbearing potential should be advised 
to use highly effective contraception methods while they are receiving L-NMMA plus Docetaxel 
combination and up to 8 weeks after treatment has been stopped. 
To ensure patient safety, each pregnancy occurring while the patient is on study treatment must be 
reported to the local HMRI&HMCC IRB within 24 hours of learning of its occurrence. The pregnancy 
should be followed up to determine outcome, including spontaneous or voluntary termination, details of 


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the birth, and the presence or absence of any birth defects, congenital abnormalities, or maternal and/or 
newborn complications. The newborn will be followed for at least 12 months. 
Pregnancy should be recorded on a Clinical Trial Pregnancy Form and reported by the investigator to the 
local HMRI&HMCC IRB Medication Safety office. Pregnancy follow-up should be recorded on the same 
form and should include an assessment of the possible relationship to the study treatment and any 
pregnancy outcome. Any SAE experienced during pregnancy must be reported on the SAE Report Form. 
 
 
 
15 Statistical methods 
15.1 
Sample Size. 
Statistical Considerations  
This phase Ib/II study is designed to determine the DLTs, MTD, and RP2D of an L-NMMA plus docetaxel 
combination as well as to investigate the efficacy of the L-NMMA and taxane chemotherapy (docetaxel, 
paclitaxel, or nab-paclitaxel) combination.  The phase Ib portion of the study will utilize the standard 
Bayesian CRM. A method based entirely on a Bayesian decision framework.  This portion of the trial is 
designed to investigate the combination at two dose levels of docetaxel (75 and 100 mg/m2) and 7 dose 
levels of L-NMMA (5, 7.5, 10, 12.5, 15, 17.5, and 100 mg/kg).  Table 25 presents the proposed dose 
levels of the combination. The starting dose will be docetaxel at 75 mg/m2 and L-NMMA at 7.5 mg/kg.  
The phase Ib portion of the trial will be comprised of 34 patients.  Once the MTD and RP2D for L-NMMA 
in combination with docetaxel has been determined, the phase II portion of the trial will proceed.   
 
The phase Ib portion of the study was designed and will be conducted using Bayesian Model Averaging 
Continual Reassessment Method software developed by the Biostatistics Department of the University 
of Texas M. D. Anderson Cancer Center (https://biostatistics.mdanderson.org/SoftwareDownload/) 74, 75.  
The software assumes the probability of toxicity at dose i (πi) is modeled as πi = pi×exp(α) where pi is a 
constant and α is distributed a priori as a normal random variable with mean 0 and variance 2. The 
software requires the investigator to specify the prior median probability of toxicity at each of the 6 doses 
of L-NMMA under consideration (si, i = -1, 0, 1, 2, 3, 4).  The CRM model assumes that toxicity is a 
monotonic increasing function with dose, and thus, the si values may not decrease as i increases. The 
values pi in the probability model are selected so that E[pi×exp(α)] = si.  After the first cohort, each 
successive cohort is given the dose whose posterior probability of toxicity given the data collected thus 
far is closest to the target toxicity (the software does not allow an untried dose to be skipped).  Thus as 
information accumulates, the model is continually updated posteriorly and decisions are made on the 
posterior distribution.  As a result, no data is lost and there is the assurance that the decision to increase 
or decrease the dose level is consistently reassessed, as more information becomes available. For a 
dose level to be chosen as the MTD, at least 4 patients must have received said dose. 
 
Table 25. Phase Ib Dose Levels   
Dose Level  
L-NMMA (mg/Kg) 
Docetaxel (mg/m2) 
-1 
5 
75 
0 
7.5 
75 
1 
10 
75 
2 
12.5 
75 
3 
15 
75 
4 
17.5 
100 
5 
20 
100 


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The target toxicity probability will be 0.25 to 0.30 and a maximum of 34 patients will be treated in the 
phase Ib portion of the trial. The first cohort of patients will be treated at dose level 0. As an extra measure 
of safety, the trial will be stopped early if the lowest dose level is unacceptably toxic, formally if  
𝑃𝑟{𝑝−1 × 𝑒𝛼> 0.27|𝑑𝑎𝑡𝑎} > 0.85 
The Bayesian model averaging CRM will be implemented using the following three toxicity probability 
sets: 1) (p-1, p0, p1, p2, p3, p4, p5) = (0.01, 0.04, 0.07, 0.10, 0.13, 0.16, 0.19); 2) (p-1, p0, p1, p2, p3, p4, p5) = 
(0.96, 0.96, 0.96, 0.96, 0.96, 0.96, 0.96); and 3) (p-1, p0, p1, p2, p3, p4, p5) = (0.30, 0.30, 0.30, 0.30, 0.30, 
0.30, 0.30). Operating characteristics of the design under six dose-toxicity scenarios are tabulated 
below.  
 
Table 26.  Phase Ib Design Operating Characteristics.  Optimal decisions are given in boldface type. 
 
Scenario 
Study 
Parameter 
Dose Level 
None 
-1 
0 
1 
2 
3 
4 
5 
 
1 
High Toxicity 
Prob[toxicity] 
0.45 
0.55 
0.65 
0.75 
0.85 
0.90 
0.95 
 
Prob[Selected] 
0.03 
0.00 
0.00 
0.00 
0.00 
0.00 
0.00 
0.96 
# treated 
5.3 
2.7 
0.6 
0.1 
0.00 
0.00 
0.00 
 
2 
Target @ 0 
Prob[toxicity] 
0.14 
0.28 
0.50 
0.60 
0.70 
0.80 
0.90 
 
Prob[Selected] 
0.20 
0.54 
0.09 
0.00 
0.00 
0.00 
0.00 
0.17 
# treated 
9.7 
13.1 
5.0 
1.1 
0.2 
0.0 
0.00 
 
3 
Target @ 1 
Prob[toxicity] 
0.08 
0.16 
0.28 
0.50 
0.65 
0.80 
0.95 
 
Prob[Selected] 
0.02 
0.33 
0.48 
0.12 
0.01 
0.00 
0.00 
0.04 
# treated 
4.0 
10.8 
11.1 
5.3 
1.2 
0.2 
0.0 
 
4 
Target @ 2 
Prob[toxicity] 
0.04 
0.08 
0.16 
0.28 
0.50 
0.70 
0.90 
Prob[Selected] 
0.00 
0.06 
0.33 
0.47 
0.13 
0.01 
0.00 
0.01 
# treated 
1.4 
5.3 
9.5 
10.4 
5.7 
1.3 
0.1 
 
5 
Target @ 3 
Prob[toxicity] 
0.02 
0.04 
0.08 
0.16 
0.28 
0.50 
0.75 
Prob[Selected] 
0.00 
0.00 
0.06 
0.33 
0.45 
0.15 
0.01 
0.00 
# treated 
0.5 
2.9 
4.7 
8.5 
10.1 
5.8 
1.5 
 
6 
Target @ 4 
Prob[toxicity] 
0.01 
0.02 
0.04 
0.08 
0.16 
0.28 
0.50 
 
Prob[Selected] 
0.00 
0.00 
0.01 
0.07 
0.30 
0.40 
0.22 
0.00 
# treated 
0.2 
2.3 
2.5 
4.0 
7.3 
8.9 
8.7 
 
7 
Target @ 5 
Prob[toxicity] 
0.01 
0.02 
0.04 
0.08 
0.12 
0.16 
0.28 
 
Prob[Selected] 
0.00 
0.00 
0.00 
0.01 
0.05 
0.13 
0.80 
0.00 
# treated 
0.2 
2.3 
2.3 
2.7 
3.7 
4.7 
18.1 
 
8 
Low Toxicity 
Prob[toxicity] 
0.01 
0.02 
0.03 
0.04 
0.06 
0.08 
0.10 
 
Prob[Selected] 
0.00 
0.00 
0.00 
0.00 
0.00 
0.00 
1.00 
0.00 
# treated 
0.2 
2.2 
2.2 
2.2 
2.4 
2.6 
22.2 
 
 
Under Scenario 7 in none of the dose levels are too toxic, the estimated probability of finding the highest 
dose as the RP2D is 1.00.  Scenario 1 represents a setting in all doses are too toxic.  For this scenario, 
the probability of finding no acceptable doses is 0.96.  If 15 patients are treated at an individual dose, the 
phase Ib portion of the trial will end and these 15 patients will be considered the first patients of the phase 
II portion of the trial. 
 


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The primary endpoint of the phase II portion of this study will be CBR (CR + PR + SD).  All patients 
treated at the RP2D in the phase Ib portion of the study will comprise the first patients enrolled into the 
phase II portion of the trial, which will be conducted using a Simon’s optimal two-stage design.  A null 
response rate of 15% is assumed and the target response rate for the combination will be 35%.  When 
the probability of accepting a “bad” regimen (i.e. response rate  15%) is 0.10 (i.e., alpha) and the 
probability of rejecting a “good” regimen (i.e. response rate  35%) is 0.20 (i.e., beta or 1 – power), 
Simon’s optimal design requires 15 patients in the first stage.  If three or fewer patients respond to the 
combination, the trial will be stopped and the regimen will be declared as ineffective.  If at least four of 
the first 15 patients respond, 9 additional patients will be entered on the study to reach a total of 24 
patients.  The new regimen will be rejected if the response rate is less than or equal to 5 out of 24 patients 
and will be accepted otherwise.  This design yields a type I error rate of a = 0.098 (targeted rate of 0.10) 
and power of 80.1% (targeted power of 80%) when the true response rate is 0.35.  The operating 
characteristics of the phase II portion of the trial are as follows: 1) When the true response rate is 15% 
(i.e., null hypothesis is true), the probability of stopping the trial early (i.e., after the first stage) is 82.3%.  
On the other hand, if the true response rate is 35%, the probability of stopping the trial early is only 17.3%.  
The expected sample size is 16.6 if the response rate is 15% and 22.5 if the true response rate is 35%.  
If there are 3 or fewer responders in the first 15 patients the probability of achieving a significant result at 
the end of stage 2 is only 0.046 (the futility rate) assuming the observed data in stage 1 provides a 
reasonable estimate of the response rate.  At the end of the study, the CBR and its associated 95% 
Wilson score confidence interval will be reported.  With 24 patients, the CBR confidence interval will be 
no wider than 37.1 percentage points.  If a total of 6 patients out of 24 respond such that the combination 
will be considered successful and warrant further investigation, the confidence the estimated CBR will be 
25% with a confidence interval of (12.0%, 44.9%).  This trial was designed using PASS 13.0.8.  Based 
on the Phase Ib operating characteristics given in Table 26, it is anticipated that total accrual for this 
phase Ib/II trial will be 36 patients with a maximum of 48 patients. 
 
 
Statistical Analysis Plan 
Safety Analysis of DLT and Adverse Event Data: All patients who received study drug will be included in 
the safety analysis of this combination regimen.  Dose-limiting toxicity rate, adverse event data and 
corresponding toxicity grades will be summarized in each dose level and in the overall patient 
population.  Incidence tables will be generated to summarize incidence of patients reporting at least one 
episode of each specific adverse event, incidence of adverse events causing withdrawal and incidence 
of serious adverse events. The total number of episodes for each event reported (Frequency Table), the 
severity and attribution to study therapy of each episode reported (Severity Table and Attribution Table) 
will also be displayed.    
Listings of adverse events by patients will include the time to onset, the duration of each event, the 
severity of each event, and the relationship of the event to study therapy, whether it was a serious event, 
and whether it caused withdrawal. Safety data will be summarized for the overall patient group and by 
dose levels.  Toxicities will be graded according to Common Toxicity Criteria (CTCAE) v4.03 .      
The safety profiles of the study will be assessed through summaries of adverse events (AEs), serious 
adverse events (SAEs), AEs leading to treatment discontinuation, and treatment-related death. All 
patients who receive at least 1 dose of treatment will be included in the analysis for safety. The safety 
analysis will report the frequency of all AEs and laboratory abnormalities, as well as the frequency of 
dose interruptions, dose reductions, and treatment discontinuation for toxicity. Toxicity rates will be 
presented using the worst NCI CTCAE V4.03 grade per patient. 
Several exploratory analyses of biomarkers will be perfomed in this aim. Analysis will primarily be based 
on correlations between biomarkers using correlation coefficients and comparison of changes in 
biomarker levels from baseline to post-treatment levels using paired t-test or nonparametric analogs as 
well as linear mixed models for repeated measurements.  Although the correlative results will be largely 


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hypothesis generating, they will likely provide useful information for the design of future studies. We would 
be able to evaluate impact of L-NMMA on iNOS levels, as a measurement of target engagement.  
For transcriptional analysis, we will align RNA-Seq short reads to the mm9 genome using STAR and will 
extract read counts mapping to ENSEMBL genes using HTseq. Then DEseq2 will be used in pre/post 
paired mode to identify a gene expression signature of treatment response using FDR=5% and fold-
change >2. Heat maps will be generated for visual exploration. We will perform pathway analyses of the 
treatment response signature using DAVID, network analysis using IPA Ingenuity and rank-based 
pathway analysis using GSEA and gene sets in MSigDB. The goal of these analyses is to identify 
mechanisms and pathways that are mobilized by treatment. In parallel, we will perform mutational 
analysis as described in to identify point mutations and indels in expressed genes whose abundance 
differs between pre and post treatment. 75-77 These analyses will only be performed in the context of well-
expressed genes in both conditions, using Fisher exact test p-values adjusted for multiple testing. The 
objective of these analyses is to nominate genes and pathways where mutations may be either 
associated with sensitivity (mutation abundance decreases in post vs. pre) or resistance (mutation 
abundance decreases in post vs. pre). These analyses will be performed tumor by tumor and the gene 
lists will be pooled and frequently altered genes will be identified as described in 77.78.  Dr. Elemento and 
his group including Ms. Verma have extensive experience performing these analyses as demonstrated 
by their extensive publication record. 
 
16 Protocol amendments, or changes in study conduct 
Any change or addition to this protocol requires a written protocol amendment that must be reviewed by 
the local IRB and the investigator before implementation. Amendments significantly affecting the safety 
of subjects, the scope of the investigation or the scientific quality of the study require additional approval 
by the IRB at each study center.  Examples of amendments requiring such approval are: 
1. increases in drug dose or duration of exposure of subjects, 
2. significant changes in the study design (e.g. addition or deletion of a control group), 
3. increases in the number of invasive procedures, 
4. addition or deletions of a test procedure required for monitoring of safety. 
These requirements for approval should in no way prevent any immediate action from being taken by the 
investigator in the interests of preserving the safety of all patients included in the trial.  If an immediate 
change to the protocol is felt to be necessary by the investigator and is implemented for safety reasons 
must be notified to the IRB and other centers must be informed immediately.   Amendments affecting 
administrative aspects of the study do not require formal protocol amendments. Amendments affecting 
administrative aspects of the study will require IRB approval.  
 
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34. Eyler CE, Wu Q, Yan K, et al: Glioma stem cell proliferation and tumor growth are promoted by 
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38. Tang CH, Wei W, Hanes MA, et al: Hepatocarcinogenesis driven by GSNOR deficiency is 
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39. Curtis C, Shah SP, Chin SF, et al: The genomic and transcriptomic architecture of 2,000 breast 
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43. Murohara T, Asahara T, Silver M, et al: Nitric oxide synthase modulates angiogenesis in 
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44. Vakkala M, Kahlos K, Lakari E, et al: Inducible nitric oxide synthase expression, apoptosis, and 
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45. Dave B, Landis MD, Dobrolecki LE, et al: Selective small molecule stat3 inhibitor reduces breast 
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48. Matrone C, Pignataro G, Molinaro P, et al: HIF-1alpha reveals a binding activity to the promoter 
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49. Townsend DM, Manevich Y, He L, et al: Nitrosative stress-induced s-glutathionylation of protein 
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53. Landis MD, Lehmann BD, Pietenpol JA, et al: Patient-derived breast tumor xenografts facilitating 
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54. Schott AF, Landis MD, Dontu G, et al: Preclinical and clinical studies of gamma secretase 
inhibitors with docetaxel on human breast tumors. Clin Cancer Res 19:1512-24, 2013 
 
55. Korkaya H, Liu S, Wicha MS: Breast cancer stem cells, cytokine networks, and the tumor 
microenvironment. J Clin Invest 121:3804-9, 2011 
 
56. Chang JC, Makris A, Gutierrez MC, et al: Gene expression patterns in formalin-fixed, paraffin-
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57. Chang JC, Wooten EC, Tsimelzon A, et al: Gene expression profiling for the prediction of 
therapeutic response to docetaxel in patients with breast cancer. Lancet 362:362-9, 2003 
 
58. Chang JC, Wooten EC, Tsimelzon A, et al: Patterns of resistance and incomplete response to 
docetaxel by gene expression profiling in breast cancer patients. J Clin Oncol 23:1169-77, 2005 
 
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60. Tham YL, Gomez LF, Mohsin S, et al: Clinical response to neoadjuvant docetaxel predicts 
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62. Storer BE: Small-sample confidence sets for the MTD in a phase I clinical trial. Biometrics 
49:1117-25, 1993 
 
63. Suda O, Tsutsui M, Morishita T, et al: Long-term treatment with N(omega)-nitro-L-arginine 
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64. Vermeulen PB, Gasparini G, Fox SB, et al: Quantification of angiogenesis in solid human 
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65. Allred DC, Harvey JM, Berardo M, et al: Prognostic and predictive factors in breast cancer by 
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66. Brown RW, Allred CD, Clark GM, et al: Prognostic value of Ki-67 compared to S-phase fraction 
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             67. John H. Alexander,MD. Harmony R. Reynolds,M.D. AmandaL. Stebbins, MS. VladimirDzavik, 
MD. Robert A. Harrington,MD. Frans Van de Werf, MD, and Judith S. Hochman, MD. Effect of 
tilarginine acetate in patients with acute myocardial infarction and cardiogenic shock. The TRIUMPH 
Randomized Controlled Trial. JAMA, April 18, 2007—Vol 297, No. 15 
 
68. Cotter G, Kaluski E, Milo O, et al. LINCS: LNAME (a NO synthase inhibitor) in the treatment of 
refractory cardiogenic shock. Eur Heart J. 2003;24:1287-1295. 
 
69. Cotter G, Kaluski E, Blatt A, et al. L-NMMA (a nitric oxide synthase inhibitor) is effective in the 
treatment of cardiogenic shock. Circulation. 2000;101:1358-1361. 
 
70. L-NMMA IB 
 
71. Docetaxel IB 
             72. Amlodipine IB  
 
73. RECIST 1.1 http://www.recist.com/recist-in-practice 
      
 
74. Daimon T, Zohar S, O'Quigley J. Posterior maximization and averaging for Bayesian working 
model choice in the continual reassessment method. Stat Med. 2011; 30(13):1563-1573. 
 
75. Asakawa T, Hirakawa A, Hamada C. Bayesian model averaging continual reassessment method 
for bivariate binary efficacy and toxicity outcomes in phase I oncology trials. J Biopharm Stat. 2014; 
24(2):310-25. 
 
76. Wacker SA, Houghtaling BR, Elemento O, et al: Using transcriptome sequencing to identify 
mechanisms of drug action and resistance. Nat Chem Biol 8:235-7, 2012 
 
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method to analyze drug targets. Nat Chem Biol 10:626-8, 2014. 
 
 
 


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18 Appendices  
Appendix A: ECOG/ Karnofsky Performance Status Criteria 
 
 
 
 


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Appendix B: New York Heart Association (NYHA) Classifications 
 
 
 


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Appendix C: Version 4.03 (dated June-14-2010) 
CTCAE Files: 
NCI Common Terminology Criteria for Adverse Events (CTCAE) v.4 data files and related 
documents are published here. The most current release files appear in this directory:  
Files: Booklet 
  
Content 
CTCAE_4.03_2010-06-
14_QuickReference_5x7.pdf 
Most recent release of core terminology: PDF document, 
traditional small booklet format. 
 
 
http://evs.nci.nih.gov/ftp1/CTCAE/About.html 
 
 


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WOCBP 
Women of childbearing potential and men must agree to use adequate contraception (one of the following 
listed below) prior to study entry, for the duration of study participation and up to 60 days following 
completion of therapy.  Additionally, male subjects (including those who are vasectomized) whose 
partners are pregnant or might be pregnant must agree to use condoms for the duration of the study and 
for 90 days following completion of therapy. 
● Total abstinence from sexual intercourse (minimum one complete menstrual cycle). 
● Vasectomized male subjects or vasectomized partner of female subjects. 
● Hormonal contraceptives (oral, parenteral or transdermal) for at least 3 months prior to study drug 
administration. 
● Double-barrier method (condoms, contraceptive sponge, diaphragm or vaginal ring with 
spermicidal jellies or cream). 
● Intrauterine device (IUD). 
 
The following criteria will be used to define postmenopausal: 
 
Age 56 or older with no spontaneous menses for at least 12 months prior to study entry;  
Or 
 
Age 55 or younger with no spontaneous menses for at least 12 months prior to study entry (e.g., 
spontaneous or secondary to hysterectomy) and with a documented estradiol level in the 
postmenopausal range according to local institutional/laboratory standard;  
Or 
 
Documented bilateral oophorectomy. 
 
Women failing to meet one of these criteria will be classified as pre-menopausal. 
 
 


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Appendix E : Nitrate/Nitrite Drug Interactions 
 
Drug Class 
Medications 
Phosphodiesterase inhibitors 
sildenafil, tadalafil, vardenafil, avanafil 
Nitrates 
isosorbide 
dinitrate, 
isosorbide 
mononitrate, 
nitroglycerin,  
Nitrites 
amyl nitrite, sodium nitrite 
General anesthetics 
Nitrous oxide 
** Patients should be on a low nitrite/nitrate diet including avoidance of processed meats such hot dogs, ham, bacon, and sausage.  
 
 


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Appendix F: Pharmacokinetic (PK) and Pharmacodynamic (PD) Sample Collection and Processing Guidelines 
 
 
Sampling Schedule for L-NMMA and Docetaxel PK and PD Evaluation 
Patients will have PK and PD sampling (plasma) performed predose (10-30 minutes before L-NMMA 
infusion) on Days 1, 2, and 5 of Cycle 1 and Days 1 and 5 of Cycle 2. 
One 5-mL blood sample is required for each PK sample time point and each PD sample time point. 
 
 
*The actual times and date of samples drawn must be recorded. 
 
Sample Preparation Guidelines 
Samples should be labeled with the protocol number, patient identification (ID) number, date, time of collection (recorded 
as 24-hour clock time) and sample type (PK or PD) 
 
PLASMA SAMPLE PREPARATION FOR L-NMMA & DOCETAXEL 
1. Draw at least 5mL blood into one 6-mL green top heparinized (Na or Lithium) tube.  Mix the tube by gently inverting 
the tube 2-3 times. To avoid hemolysis, do not mix vigorously. 
2. Centrifuge the blood using 1,100 to 1,300 relative centrifugal force (RCF) (g) for 10 minutes for a swinging bucket rotor 
or for 15 minutes for a fixed-angle bucket rotor.  If ideal equipment not available, the minimum requirement are 1,000 
RCF at room temperature for 15 minutes. 
3. Using a transfer pipet, withdrawn the plasma from the vacutainer and dispense the plasma into a cryovial.  Securely 
cap the cryovial. 
4. Affix label designated as L-NMMA onto the vials.  Document the patient ID #, date, time of blood collection on the label.  
5. Freeze and store the plasma in an upright position at -200 C or colder and ship when directed by the principle 
investigator. 
 
BIOMARKER SAMPLE PREPARATION 
1. Draw at least 5 mL blood into one 6 mL Lavender EDTA tube. Mix the tube by gently inverting the tube 2-3 times. To 
avoid hemolysis, do not mix vigorously 
2. Centrifuge the blood using 1,100 to 1,300 relative centrifugal force (RCF) (g) for 10 minutes for a swinging bucket rotor 
or for 15 minutes for a fixed-angle bucket rotor.  If ideal equipment not available, the minimum requirement are 1,000 
RCF at room temperature for 15 minutes. 
3. Using a transfer pipet, withdrawn the plasma from the vacutainer and dispense the plasma into a cryovial.  Securely 
cap the cryovial. 
4. Affix label designated as L-NMMA onto the vials.  Document the patient ID #, date, time of blood collection on the label. 
5. Freeze and store the plasma in an upright position at -200 C or colder and ship when directed by the principle 
investigator. 
 
PK and PD Sample Shipping Guidelines 
1.   Shipments should be performed Monday through Thursday ONLY 
2.   Place the frozen samples into a biohazard bag containing absorbent material, seal the bag and place it into  
      Styrofoam container. 
3.   Add a minimum of 2 Kg of Dry-Ice into the Styrofoam shipping container. 
4.  Affix IATA labels (UN33733, Biohazard and Dry-Ice Label) to the cardboard box 
5.  Seal the cardboard box. 
6.  Notify the laboratory by email of the shipment prior to shipping.  Include the air bill number, the name of the site  
     Shipping the package, and a contact person at the site.  
 


HMCC&HMRI                                           LNMMA Clinical Protocol                                                     Page 76 
          
Version: 7 
Ship to:  TBD