PEPTIDE AND PROTEIN DELIVERY 1ST EDITION CHRIS VAN

DER WALLE - IMMEDIATE DOWNLOAD

https://ebookultra.com/download/peptide-and-protein-
delivery-1st-edition-chris-van-der-walle/

★★★★★
4.7 out of 5.0 (47 reviews)

IMMEDIATE DOWNLOAD

Ebookultra.com
Peptide and Protein Delivery 1st Edition Chris Van Der Walle

EBOOK

Available Formats

■ PDF eBook Study Guide Ebook

EXCLUSIVE 2025 ACADEMIC EDITION – LIMITED RELEASE

Available Instantly Access Library

Featured Educational Resources

Peptide Folding Misfolding and Nonfolding Wiley Series in

Protein and Peptide Science 1st Edition Reinhard
Schweitzer-Stenner
https://ebookultra.com/download/peptide-folding-misfolding-and-
nonfolding-wiley-series-in-protein-and-peptide-science-1st-edition-
reinhard-schweitzer-stenner/

Protein Targeting Protocols 2nd Ed Methods in Molecular

Biology Vol 390 2nd Edition Mark Van Der Giezen

https://ebookultra.com/download/protein-targeting-protocols-2nd-ed-
methods-in-molecular-biology-vol-390-2nd-edition-mark-van-der-
giezen/

Lipobiology 1st Edition Van Der Vusse (Eds.)

https://ebookultra.com/download/lipobiology-1st-edition-van-der-
vusse-eds/

Elementary and Middle School Mathematics Teaching

Developmentally 6th edition John A. Van De Walle

https://ebookultra.com/download/elementary-and-middle-school-
mathematics-teaching-developmentally-6th-edition-john-a-van-de-
walle/
Elementary and Middle School Mathematics Teaching
Developmentally 8th Edition John A. Van De Walle

https://ebookultra.com/download/elementary-and-middle-school-
mathematics-teaching-developmentally-8th-edition-john-a-van-de-
walle/

Testing Code Security 1st Edition Maura A. Van Der Linden

https://ebookultra.com/download/testing-code-security-1st-edition-
maura-a-van-der-linden/

Mies Van Der Rohe in Berlin 1st Edition Terence Riley

https://ebookultra.com/download/mies-van-der-rohe-in-berlin-1st-
edition-terence-riley/

Modality in English Theory and Description 1st Edition

Johan Van Der Auwera

https://ebookultra.com/download/modality-in-english-theory-and-
description-1st-edition-johan-van-der-auwera/

Multicomponent Reactions towards Heterocycles Concepts and

Applications 1st Edition Erik Van Der Eycken

https://ebookultra.com/download/multicomponent-reactions-towards-
heterocycles-concepts-and-applications-1st-edition-erik-van-der-
eycken/
Peptide and Protein
Delivery

Chris Van der Walle

Strathclyde Institute of Pharmacy and Biomedical Sciences
University of Strathclyde
Glasgow, UK

AMSTERDAM l BOSTON l HEIDELBERG l LONDON

NEW YORK l OXFORD l PARIS l SAN DIEGO
SAN FRANCISCO l SINGAPORE l SYDNEY l TOKYO

Academic Press is an imprint of Elsevier

Academic Press is an imprint of Elsevier
32 Jamestown Road, London NW1 7BY, UK
30 Corporate Drive, Suite 400, Burlington, MA 01803, USA
525 B Street, Suite 1800, San Diego, CA 92101-4495, USA
First edition 2011
Copyright Ó 2011 Elsevier Inc. All rights reserved
Front cover image: The PyMOL Molecular Graphics System, Version 1.2, Schrödinger, LLC
No part of this publication may be reproduced, stored in a retrieval system or transmitted in
any form or by any means electronic, mechanical, photocopying, recording or otherwise without
the prior written permission of the publisher. Permissions may be sought directly from Elsevier’s
Science & Technology Rights Department in Oxford, UK: phone (+ 44) (0) 1865 843830;
fax (+44) (0) 1865 853333; email [email protected]. Alternatively, visit the Science and
Technology Books website at www.elsevierdirect.com/rights for further information
Notice
No responsibility is assumed by the publisher for any injury and/or damage to persons or property
as a matter of products liability, negligence or otherwise, or from any use or operation of any
methods, products, instructions or ideas contained in the material herein. Because of rapid advances
in the medical sciences, in particular, independent verification of diagnoses and drug dosages
should be made

British Library Cataloguing-in-Publication Data

A catalogue record for this book is available from the British Library
Library of Congress Cataloging-in-Publication Data
A catalog record for this book is available from the Library of Congress
ISBN: 978-0-12-384935-9

For information on all Academic Press publications

visit our website at elsevierdirect.com

Typeset by TNQ Books and Journals Pvt Ltd.

www.tnq.co.in
Printed and bound in China
10 11 12 13 14 15 10 9 8 7 6 5 4 3 2 1
 Foreword

The discovery and first therapeutic use of insulin in Toronto in 1922 saved the
lives of countless people with type 1 diabetes e and in the course of doing so
provided Nobel-winning confirmation of the definitive hormonal pathophys-
iology of the condition. However, rather than bringing a “cure” for diabetes,
those days of discovery are now seen as heralding an epoch of devastating
diabetes-related complications e which we are still living through as the
centenary approaches.
There are lessons in this story for the contemporary race to discover new
treatments for the various other conditions that blight 21st century life. The
focus within both the pharmaceutical and academic sectors is firmly on
discovery of new (and ideally “blockbuster”) non-peptide molecules with
a prevailing assumption that non-peptide agents are preferred to avoid the
parenteral route of administration. The alternative approach of using devel-
oping technologies to refine drug formulation and better target existing mole-
cules using other routes is less glamorous, and possibly less lucrative, but may
ultimately lead to greater health gains.
It is almost certainly the case that if insulin could be administered thera-
peutically into the appropriate anatomical location in truly physiological
concentrations, which are finely-tuned in response to minute-to-minute
changes in blood glucose concentration, diabetes complications would be
obviated entirely. However, while such a “cure” remains elusive, intermittent
subcutaneous injection of insulin into the thighs or abdomen is by far the most
frequent approach worldwide. This provides the body with only a rough esti-
mate of the amount of the hormone that may actually be required over the next
few hours, delivered with a considerable degree of chronic discomfort, into the
wrong physiological compartment, at supra-physiological concentrations.
Perhaps it is little wonder that, as a result, over a period of months and years
adherence to insulin therapy can wax and wane in response to the challenges of
life. The clinical specialties around the nursing and medical care of people with
diabetes have grown up around supporting and motivating affected individuals
to engage with prescribed treatment and self-care in an effort to minimize the
adverse effects of therapy and the progression of complications over time. This
can be an uphill and unrewarding battle.
The chapter authors of this volume are those who are currently thinking
“outside the box” on these issues internationally, and grappling with the

vii
viii Foreword

challenges of drug delivery via less “conventional” routes. As clinicians we are

fortunate for the sake of those for whom we care that such work is progressing
and maturing, even while a clear “breakthrough” is not yet in sight. Any new
developments will of course require rigorous clinical trials for safety and
efficacy. However, within the field of diabetes e and far beyond e there is little
doubt that over the next decade or two alternative routes of drug delivery will
change the treatments we prescribe for our patients, and their concordance with
that treatment, beyond recognition. This volume is a forerunner of that new era.

John Petrie
Dr John R Petrie BSc PhD MD
Professor of Diabetic Medicine, University of Glasgow
 Preface

Many pharmaceutical scientists like myself develop strong links with profes-
sionals working in the pharmaceutical industry and in clinical practice. This
forms a stimulating environment which gives good oversight of the issues
facing the introduction of a new drug, or drug delivery system, onto the market
for clinical use. Of course, part of that challenge is learning each others
“language” (or “what concerns us most”) in order to make a coherent plan
which addresses the particular need under discussion. An extra language has
been introduced with the emergence of the biotechnology industry. The bio-
pharmaceuticals produced within the biotechnology sector have proven to be
exciting with regard to their envisaged clinical potential, but also challenging
with regard to their (bio)processing and delivery.
Life science students, for example, quickly learn that “you can’t administer
proteins and peptides orally”; that is, there is the assumption that invasive
delivery by injection is required for biopharmaceuticals. They have, of course,
the appreciation that the stomach exposes drugs to very low pH, and the gastro-
intestinal tract as a whole secretes proteases. There is also an awareness of the
barriers to the absorption of macromolecules in the intestine, such as the cell
membrane, cellecell tight junctions, glycocalyx and efflux transporters.
However, it can be surprising to learn that research specifically setting out to
address the problems of non-invasive delivery of biopharmaceuticals has
continued apace. The large volume of work in this field has led to many
collaborative approaches, spin-out companies and mature, commercialized
technologies. Emergent delivery strategies have examined several routes in
addition to oral administration, and each has shown promise to some degree.
The level of interest at a life-science conference can always be piqued if
a presentation on “oral insulin delivery” (or other therapeutic protein) is
convincing. It is primarily a curiosity concerning the delivery strategies, and
how these can be applied to current therapeutic peptides and proteins, which
has driven the production of this book. I hope that students studying for higher
degrees in the life sciences, clinicians, scientists and industrialists in related
fields will find that the chapters herein provide grounding and an up-to-date
critical review of the advances made in overcoming the barriers to the non-
invasive delivery of peptides and proteins. Because bioprocessing, clinical
application and toxicology are so closely related to the issues of delivery, these
topics are also discussed in a concise manner. As biopharmaceuticals come to

ix
x Preface

play ever more important roles in modern medicine it is inevitable that the
concepts detailed in these chapters will form the focus of future research and
development.
I am in indebted to Ijeoma Uchegbu who first suggested this project to me
and to Ravi Kumar for his insight and guidance.

Chris van der Walle

Strathclyde, Glasgow, 2010
 Contributors

Ajay K. Banga College of Pharmacy and Health Sciences, Mercer University, Atlanta,
Georgia
Alex Berrill Department of Biochemical Engineering, University College London,
London, United Kingdom
Jamie Biddlecombe MedImmune Ltd., Granta Park, Cambridge, United Kingdom
Daniel Bracewell Department of Biochemical Engineering, University College
London, London, United Kingdom
Paolo Caliceti Department of Pharmaceutical Sciences, University of Padua, Padova,
Italy
Hak-Kim Chan Faculty of Pharmacy, The University of Sydney, New South Wales,
Australia
Woei Ping Cheng School of Pharmacy, University of Hertfordshire, Hatfield, United
Kingdom
Christine Dufès Strathclyde Institute of Pharmacy and Biomedical Sciences,
University of Strathclyde, Glasgow, Scotland
Anushree Herwadkar College of Pharmacy and Health Sciences, Mercer University,
Atlanta, Georgia
Philip Chi Lip Kwok Faculty of Pharmacy, The University of Sydney, New South
Wales, Australia
Orest Olejnik Allergan Inc., Dupont Drive, Irvine, California
Shivani Rai Paliwal Department of Pharmaceutical Sciences, Dr. Hari Singh Gour
University, Sagar, Madhya Pradesh, India
Rishi Paliwal Department of Pharmaceutical Sciences, Dr. Hari Singh Gour Univer-
sity, Sagar, Madhya Pradesh, India
Patrı́cia Pereira Faculdade de Farmácia, Universidade de Lisboa, Lisboa, Portugal
Margus Pooga Institute of Molecular and Cell Biology, University of Tartu, Tartu,
Estonia
Helin Räägel Institute of Molecular and Cell Biology, University of Tartu, Tartu,
Estonia
M. R. Rekha Biosurface Technology Division, Sree Chitra Tirunal Institute for
Medical Sciences & Technology, Thiruvananthapuram, Kerala, India
Garnpimol C. Ritthidej Department of Manufacturing Pharmacy, Chulalongkorn
University, Bangkok, Thailand

xi
xii Contributors

Stefano Salmaso Department of Pharmaceutical Sciences, University of Padua,

Padova, Italy
Elke Schmidt Strathclyde Institute of Pharmacy and Biomedical Sciences, University
of Strathclyde, Glasgow, Scotland
Chandra P. Sharma Biosurface Technology Division, Sree Chitra Tirunal Institute for
Medical Sciences & Technology, Thiruvananthapuram, Kerala, India
Colin Thompson School of Pharmacy and Life Sciences, The Robert Gordon
University, Schoolhill, Aberdeen, Scotland
Christopher F. van der Walle Strathclyde Institute of Pharmacy and Biomedical
Sciences, University of Strathclyde, Glasgow, Scotland
Suresh P. Vyas Department of Pharmaceutical Sciences, Dr. Hari Singh Gour
University, Sagar, Madhya Pradesh, India
 Chapter 1

An Overview of the Field of

Peptide and Protein Delivery
Christopher F. van der Walle 1 and Orest Olejnik 2
1
University of Strathclyde, Strathclyde Institute of Pharmacy and Biomedical Sciences,
161 Cathedral Street, Glasgow, G4 0RE, Scotland, 2 Visiting Professor, University of Strathclyde,
Allergan Inc., 2525 Dupont Drive, Irvine, California 92623, USA

Chapter Outline
1.1. Introduction 1 1.4. Controlled Delivery 11
1.2. Current Formulation 1.5. Future Trends 13
Development 5 1.6. Toxicity Profiles 14
1.3. Case Studies of Delivery 1.7. Regulatory Matters 16
Approaches: Cyclosporine A 1.8. Commercialization
and Insulin 8 Considerations 18

1.1. INTRODUCTION
The so called “biologics portfolio” of biotechnology and pharmaceutical
companies represents an area of strong market growth. Biologics may include
a variety of therapeutics, such as vaccines, recombinant proteins, genes,
synthetic tissues and viruses, which have emerged from research in molecular
and cell biology, in response to unmet clinical needs and expanding indications.
While there is keen interest in areas such as gene therapy and tissue engi-
neering, the recombinant protein sector, particularly that of the monoclonal
antibodies (mAbs), provides much of the focus as regards protein delivery. The
therapeutic antibodies market is complex and continually under review [1], but
the sample of approved antibodies that is shown in Table 1.1 gives some
indication of the rate of growth in this area.
It is important at this stage to outline some of the key events that have led
to the current level of interest in peptide and protein drugs, and to define the
terms “peptide” and “protein”. A consensus has not been properly reached
Peptide and Protein Delivery. DOI: 10.1016/B978-0-12-384935-9.10001-X
Copyright Ó 2011 Elsevier Inc. All rights reserved. 1
 2
TABLE 1.1 Examples of therapeutic monoclonal antibodies
Generic name Brand name Year of approval Pharmacological target Clinical indication Type

Muromonab-CD3 Orthoclone OKT3 1986 T cell CD3 Receptor Immune disorders, murine
transplant rejection

Abciximab ReoPro 1994 Glycoprotein IIb/IIIa Cardiovascular disease chimera

Rituximab Rituxan 1997 B-lymphocyte antigen CD20 Oncology (Non-Hodgkin chimera

lymphoma)

Infliximab Remicade 1998 Tumor necrosis factor Immune disorders chimera

(TNF)-a signaling

Cetuximab Erbitux 2004 Epidermal growth factor Colorectal cancer, head and chimera
(EGF) receptor neck cancer

Peptide and Protein Delivery

Trastuzumab Herceptin 1998 Human epidermal growth Breast cancer humanized
factor receptor 2 (HER2/neu)

Palivizumab Synagis 1998 Respiratory syncytial virus RSV infection humanized

(RSV) protein F

Gemtuzumab Mylotarg 2000 Transmembrane receptor Acute myelogenous humanized

CD33 leukemia

Efalizumab Raptiva 2002 Integrin, alpha L subunit Psoriasis humanized

(CD11a)
 Chapter j 1
Omalizumab Xolair 2004 Immunoglobulin E (IgE) Allergy-related asthma humanized

Natalizumab Tysabri 2006 Integrin alpha-4 (a4) subunit Multiple sclerosis, Crohn’s humanized
disease

Ranibizumab Lucentis 2006 Vascular endothelial growth Macular degeneration humanized

Introduction
factor A (VEGF-A)

Eculizumab Soliris 2007 Complement protein C5 Paroxysmal nocturnal humanized

hemoglobinuria

Certolizumab pegol Cimzia 2008 Tumor necrosis factor (TNF)-a Crohn’s disease humanized
inhibitor

Ustekinumab Stelara 2009 Cytokines IL-12 and IL-23 Psoriasis human

Canakinumab Ilaris 2009 Activated cytokine IL-1b Cryopyrin-associated human

(catabolin) periodic syndrome
(autoimmune disorder)

Golimumab Simponi 2009 Tumor necrosis factor (TNF)-a Rheumatoid arthritis, human
ankylosing spondylitis

3
4 Peptide and Protein Delivery

with respect to the use of these latter two terms, with the 51 amino acid,
mature human insulin being a good example of the ambiguity, since it is
generally described as a peptide but also as a protein by some. As a guide,
peptides can be considered to be up to 50 amino acids in length, with proteins
being larger than this. This boundary corresponds approximately to the upper
limit of routine peptide synthesis in the solid phase, i.e. that which is
achievable using iterative cycles of “activation” and “deprotection” for the
growing peptide chain attached to functionalized resin beads (though much
longer chains can be synthesized) [2]. There are competing recombinant
technologies for the heterologous expression of peptides, employing a DNA
cassette encoding a series of concatenated peptides linked by methionine
residues, which are then cleaved by cyanogen bromide [3]. However, solid
phase peptide synthesis remains the production method of choice for many
small peptide hormones of around 20 amino acids in length.
The first peptide-based drugs approved for clinical use were insulin, thyroid
hormone and factor VIII, introduced from the 1920s onwards. However, the
trajectory of the field changed dramatically as molecular biology techniques
developed in the 1960s. These techniques allowed proteins to be engineered
through recombinant means at the level of the gene, doing away with the need for
extraction and purification from animal or human tissue. The best example of this
is human insulin, which in the late 1970s and early 1980s was synthesized
through heterologous expression in Escherichia coli by Genentech and Eli Lilly.
The rise of molecular biology as a tool by which to generate biopharmaceutical
drugs e those that include proteins, DNA, conjugates, viruses, etc., initially far
outpaced the development of delivery technologies. These biomacromolecules
almost inevitably do not survive the stomach and intestinal environment due to
pH and the presence of proteases. Nor do they readily transit the epithelial barrier
due to the presence of cellecell tight junctions, the semi-permeable cell
membrane and, for intestinal epithelia, efflux proteins such as P-glycoprotein and
the cell glycocalyx. Furthermore, biomacromolecules are very much larger than
organic drugs, have short plasma half-lives, are involved in active transport
processes (e.g. receptor mediated endocytosis), are susceptible to chemical and
physical degradation (including aggregation) and are very potent. The need to
overcome these challenges to their delivery has resulted in huge volumes of
research, drawn from many disciplines including pharmaceutical materials,
chemical engineering, biophysics, analytical methodology, cell and molecular
biology and in vivo studies. Recently however, there is evidence for optimism in
the general field of peptide and protein delivery, which hopefully is reflected in
the various chapters of this book.
Following on from the difference in the number of amino acids that
differentiate a peptide and a protein, it is necessary to consider differences in
structure beyond the primary sequence. Secondary structure, which typically
involves b-sheets, b-turns and a-helices is not necessarily found in peptides
around 20 amino acids in length. When we reach chains of about 50 amino
Chapter j 1 Current Formulation Development 5

acids, secondary structure is likely, particularly if stabilized by disulphide

bonds, as is the case for insulin. Tertiary structure, however, remains the
preserve of proteins, since it involves the precise organization of secondary
structural elements into 3-dimensional (3-D) domains. Quaternary structure,
similarly, will involve non-covalent bonding between domains and amino acids
which may be very distant in their primary structure but are brought together
through the intricate folding of the peptide backbone. There is of course much
information on protein structure, and the reader is referred to other key texts
[4]. It is important to realize that, whereas the function of a small peptide will
be dependent only on the functional groups of various amino acids, the function
of a protein will additionally be dependent on maintenance of a precise 3-D
structure. One can therefore appreciate that delivery technologies relating to
proteins have the harder task of maintaining the non-covalent bonding pattern
of this 3-D structure. This may be one reason that the encapsulation of peptides
into polyester microspheres for controlled delivery is an established tech-
nology, but encapsulation of proteins remains largely at the research stage,
albeit promising [5].

1.2. CURRENT FORMULATION DEVELOPMENT

Proteins such as monoclonal antibodies (mAbs) are most commonly
administered by subcutaneous injection. It is often suggested that patient
compliance would improve if a non-invasive route of administration were
available. However, this need not always be the case. For example, a depot
injection of a particular proteinaceous drug, administered once every month
or less, may be more convenient than the requirement for repeated, daily, oral
or pulmonary doses. Thus, on the consideration of patient compliance alone,
it is likely that invasive delivery will remain one route of administration. Of
course, the need for daily injections of the drug insulin has in itself stimu-
lated much of the research regarding non-invasive protein delivery, partic-
ularly oral and pulmonary delivery. The story of inhalable insulin, marketed
by Pfizer Inc. as Exubera, is now widely known: although Exubera was
greeted with much enthusiasm in 2006, around a year later it was removed
from the market due to “lack of acceptance”, and considerable uncertainties
over patient compliance, long-term safety and cost remain [6]. Nevertheless,
research is still ongoing in these areas of protein delivery and many others e
such as ocular, dermal, central nervous system and nasal. It is these topics
that form the initial focus of this book, in each chapter the case is made for
approaches which have shown promise in overcoming the inherent barriers to
delivery mentioned above. The same approaches must of course also
consider stabilization and the overall immunogenicity of the therapeutic
product. For this reason, this book finishes with chapters on bioconjugation,
then moves to formulation for medical devices, and finally manufacture and
supply.
6 Peptide and Protein Delivery

In addition to developing a stable formulation that ensures the desired safety

and efficacy, there are clinical, commercial and regulatory requirements. The
preformulation studies for purified peptides and proteins must take into account
the unique attributes of these molecules. That is:
(i) high molecular weight, e.g. the cyclic peptide cyclosporine A may be
only 1.2 kDa but a typical mAb will be 144 kDa;
(ii) multiple functional groups with a wide range of pKa values, leading to
chemical and physical instability, in turn effecting the shelf life and
the decision to make liquid versus solid state (lyophilized) formulations;
(iii) maintenance of the 3-D structure: Figure 1.1 of an example mAb [7]
shows how intricate the folding of the peptide backbone and disulphide
bridging can be;
(iv) high water solubility but increase in viscosity at concentrations above
50 mg/ml, particularly with regard to subcutaneous injection;

FIGURE 1.1 Crystal structure of a murine immunoglobulin (IgG1) monoclonal antibody

(described in [7]), rendered in cartoon form showing b-strands (arrows) and a-helices (generated
using the PyMOL Molecular Graphics System, Version 1.2, Schrödinger, LLC). The “heavy chains”
are coloured blue and yellow, the “light chains” green and magenta, and the disulphides bridging
heavy/light chains as red spheres. Fv, Fragment variable; Fab, Fragment antigen binding; Fc,
Fragment crystallization.
Chapter j 1 Current Formulation Development 7

(v) interactions with packaging, especially silicone-coated glass syringes; and

(vi) adsorption to surfaces.
Stabilization of the product must be compatible with aseptic processing and
manufacture, which may use non-conventional technologies such as freeze/
spray drying. Various analytical techniques are central in preformulation and
will include methods such as:

(i) chromatography and light scattering, to determine purity and

aggregation;
(ii) microscopy, to observe particulates 2 to  10 mm [8,9];
(iii) circular dichroism and infra-red spectroscopy, for conformational
changes;
(iv) iso-electric focusing, for determination of the iso-electric point (Pi);
(v) mass spectrometry, for chemical degradation;
(vi) calorimetry and fluorescence, for determination of unfolding/stabiliza-
tion; and
(vii) rheometry.

The various formulation parameters must be closely monitored since

degradation may occur in response to changes in buffer (pH and ionic strength),
metal impurities, temperature, irradiation, gases (particularly oxygen and
moisture), shear and surface adsorption.
The excipients added to stabilize proteins during formulation, processing
and to increase shelf life typically are non-reducing sugars (especially sucrose
and trehalose) and polyethylene glycol (PEG); these excipients are represen-
tatives of lyoprotectants and cryoprotectants. Lyo- and cryo-protection afforded
by sucrose is thought to occur through its preferential exclusion from the
protein/polypeptide surface [10], thermodynamically favoring the folded state
[11], or via hydrogen bonding with polar groups on the protein surface [12].
The level of stabilization afforded by sugars is usually dependent on their bulk
concentration. In contrast, the stabilization mechanism of PEG on proteins is
less well understood and may occur through steric hindrance of proteine
protein interactions, increased solution viscosity and/or decreased protein
structural mobility [13]. There are several other categories of excipient,
including non-ionic surfactants (to reduce adsorption and aggregation), amino
acids and various salts and buffers (e.g. phosphate salts may be avoided due to
differential crystallization of the acid/base). Screening programs are then
required to determine changes in protein stability, conformation, aggregation,
degradation, etc., using the analytical techniques mentioned above. Depending
on the desired product, excipient compatibility must be screened for in the
liquid and/or solid state. For solid state formulations, careful optimization of
the lyophilization cycle is required [14]. Although the primary goal is product
stability, the cost of the freezeedry cycle is also a consideration, particularly on
scale-up [15,16]. The glass transition temperature of the frozen solution (Tg0 )
8 Peptide and Protein Delivery

and the collapse temperature of the lyophilized cake (Tc) are important
parameters. A detailed overview of lyophilization is outside the scope of this
book and the reader is referred to some excellent reviews [17,18].

1.3. CASE STUDIES OF DELIVERY APPROACHES:

CYCLOSPORINE A AND INSULIN
It should be pointed out that these examples have been selectively chosen.
Cyclosporine A (CsA, also written as cyclosporin and ciclosporin) is a small,
cyclic peptide with a molecular weight of the same order of magnitude as many
organic drugs. Its solubility and bioavailability are poor and so it would be
a Class IV drug according to the Biopharmaceutics Classification System
(BCS) (Table 1.2) [19]. In both respects CsA is perhaps atypical of many
proteins, which may be three orders of magnitude larger in size but of high
aqueous solubility and low permeability (Class III).
Nevertheless, CsA is an interesting case, since its numerous formulations
illustrate what can be achieved, and their development provides inspiration for
other peptide delivery systems. CsA is very hydrophobic and was first
formulated with the oil cremophor EL as an irregular oil/water mixture which
undergoes emulsification and digestion in situ with bile salts and pancreatic
enzymes, prior to absorption (SandimmuneÒ, Novartis, Switzerland). Patient
variability does affect the therapeutic outcome of CsA, which increases the risk
of graft rejection. A later dosage form (NeoralÒ, Novartis), available as a soft
gelatin capsule or oral solution containing micro-emulsion pre-concentrate,
was designed to rapidly emulsify in the presence of aqueous fluid, rather than
depend on the action of bile salts and enzymes. This reduced interpatient
variability and improved bioavailability. Thus, to the field of drug delivery, CsA
represents an irresistible challenge, the overall aim being the development of
non-invasive delivery strategies for doses targeting its narrow therapeutic
index. Table 1.3 shows the depth of research and outlines the potential for
alternative routes of administration [20], each of which are subsequently dealt

TABLE 1.2 The classification of drugs according to the Biopharmaceutics

Classification System (BCS)

Class Aqueous solubility Permeability Example

I High High

II Low High

III High Low Immunoglobulins

IV Low Low Cyclosporin A

Chapter j 1 Case Studies of Delivery Approaches 9

TABLE 1.3 An overview of cyclosporin A (CsA) delivery systems. Reprinted

from reference [20] with permission of Elsevier

Route Formulation/Delivery system Summary

Oral Emulsion pre-concentrate for soft Higher solubilizing capacity

gelatin capsule and oral solution

Microemulsion pre-concentrate Improved bioavailability, reduced

for soft gelatin capsule and oral variability of absorption, marketed
solution by Novartis as NeoralÒ

Nanoparticles of polycaprolactone Polymer matrix controlled release,

(PCL) and polylactic acid- enhanced lymphocytic uptake,
polyethylene glycol (PLA-PEG) improved bioavailability without
increase in toxicity

pH sensitive nanoparticles of pH dependent release and

EudragitÒ S100 and hydroxypropyl improved bioavailability
methylcellulose phthalate

Positively charged nanoparticles Improved bioavailability with

of chitosan lower variability

Solid dispersions containing Improved CsA dissolution and

polyoxyethylene stearate bioavailability comparable to that
of NeoralÒ

Cyclodextrins Improved bioavailability with

reduced variability in absorption

Parenteral Solution of CsA in cremophor EL Improved solubilization, marketed

(polyoxyl 35 castor oil) and by Novartis as SandimmunÒ
ethanol

PLA and PLA-PCL microspheres Sustained drug release and

for intramuscular and improved immunosupression
subcutaneous injection

Pulmonary Propylene glycol aerosols by Undergoing clinical trials for

nebulization and dry powder prevention of graft lung transplant
formulations rejection

Liposomal aerosol Higher CsA retention in the lungs

Dermal Topical penetration enhancer Improved dermal delivery

monoolein

Iontophoresis with lecithin Enhanced transdermal delivery

vesicles, or electroporation
combined with ethanol

(Continued )
10 Peptide and Protein Delivery

TABLE 1.3 An overview of cyclosporin A (CsA) delivery systems. Reprinted

from reference [20] with permission of Elsevierdcont’d

Route Formulation/Delivery system Summary

Ocular Topical anionic microemulsion Marketed by Allergan

Pharmaceuticals as RestasisÒ
Ophthalmic Emulsion for treating
dry eye syndrome

Topical micelles of polyoxyl 40 Higher corneal permeation

stearate

Topical chitosan nanoparticles Longer retention with higher CsA

levels in cornea and conjunctiva

Poly(lactic-co-glycolic acid) Higher corneal levels and CsA and

(PLGA) microspheres and implants improved corneal graft suvival,
for intraocular and subconjunctival controlled release
injection

with in detail in this book. The associated formulations of CsA that remain
under investigation for potential translation to clinical use/the market range
from emulsion systems and liposomes to biodegradable particulates and
penetration enhancers [21]. There is no single ideal formulation, which
underscores the need for delivery strategies tailored to the individual molecule,
i.e. it is unlikely that a generic platform technology will be applicable to all
peptides and proteins. For instance, emulsion systems are clearly suitable for
CsA but are not conducive to proteins because of unfolding and aggregation at
the oil/water interface.
Insulin is usually administered by subcutaneous injection or a pump and
cannula, the latter affording greater control when needed. The subcutaneous
route is more convenient, and is inevitably used for control of type I diabetes.
However, as discussed above, the drive remains to provide non-invasive routes
for insulin dosing and, despite the set-back with Exubera, pulmonary delivery
systems are the subject of ongoing clinical studies, albeit with mixed results
[22]. An added challenge to insulin delivery is the need for a pulsatile release
system which would follow the normal post-prandial response of a sudden rise
in blood glucose levels. Oral insulin delivery is perhaps the primary target,
which must overcome enzymatic degradation and poor permeability in the
gastrointestinal tract. Although these obstacles were once considered insur-
mountable, there are currently several companies pursuing oral insulin
delivery technologies which are in various stages of clinical trials. For
example, one Phase III trial due for completion 2014 will report on the
potential role of oral insulin to delay or prevent type 1 diabetes in patients who
Chapter j 1 Controlled Delivery 11

have high levels of insulin autoantibodies [23]. Beyond current commercial-

ization activities, there lies a large volume of research employing micro-
emulsions and nanoparticulates composed of polysaccharides/polyesters/
Eudragit to achieve oral insulin delivery; though it should be noted that oral
bioavailability remains stubbornly around 10% or less [24e27]. Nasal, buccal,
rectal and vaginal delivery systems have also been explored for insulin
delivery. Of these, nasal and buccal delivery have generated the most attention
[22]. The nasal cavity is highly vascularized and bypasses the hepatic
metabolism but suffers from a low surface area (compared to the lung),
mucociliary clearance and the presence of proteolytic enzymes. Chitosan
derivatives are commonly used materials for these routes [28], in part because
chitosan acts as a permeation enhancer by transiently loosening the epithelial
tight junctions. Sublingual delivery has the advantage of ease of administra-
tion, but the challenge includes the multilayered structure of the buccal
mucosa and the constant flow of saliva. However, there remains scope in
typical insulin treatment regimes to include these novel delivery systems as
adjuvants. For example, following a regular insulin regime in type I diabetics,
oral insulin spray caused similar changes in blood glucose compared to
subcutaneous insulin injection [29]. Note that much of the research involves
the use of animal models (notably streptozotocin-induced diabetic rats), and
scaling factors and the extrapolation of results to human subjects are important
topics for examination. Because of this, the promise of earlier research has not
been fully realized and much work still remains before non-invasive insulin
delivery becomes a reality.

1.4. CONTROLLED DELIVERY

It is important to distinguish between non-invasive delivery and controlled
delivery, since the development of both may draw from polymeric materials
technologies. The use of biodegradable polyester microspheres for controlled
delivery is not limited to a specific route of administration, which may be
either invasive (e.g. subcutaneous/depot injection) or non-invasive (e.g. oral,
inhaled). Conversely, polymers used in non-invasive buccal delivery may be
required for mucoadhesion but not controlled delivery. There is a well
established track record in the use of poly(lactic-co-glycolic acid) (PLGA)
microspheres for the controlled delivery of peptide hormones (Table 1.4) [30].
However, peptide encapsulation into PLGA microspheres using an emulsi-
ficationesolvent evaporation protocol continues to represent a technological
challenge in terms of manufacture and product quality. It is notable that
the transfer of microencapsulation technologies for proteins from the labo-
ratory bench to the clinic and market has proven far more difficult than
anticipated. Many research papers initially showed promising results for
protein encapsulation in PLGA microspheres, in part because model proteins
(namely lysozyme and albumins) were used that were unusually stable under
12 Peptide and Protein Delivery

TABLE 1.4 Commercially available biodegradable drug delivery systems

Biodegradable
Name of product Dosage form Active ingredient polymer

LupronÒ Depot Microspheres Leuprolide PLGA

Sandostatin LARÒ Microspheres Octreotide PLGA

Depot

NeutropinÒ Depot Microspheres Somatropin PLGA

TrelstarÒ Depot Microspheres Triptorelin PLGA

Ò
Decapeptyl Microspheres Triptorelin pamoate PLGA
Ò
Parlodel Depot Microspheres Bromocriptine mesylate PLGA
Ò
Enantone Depot Microspheres Leuprolide PLGA

ZoladexÒ Rod Goserelin PLGA

emulsion conditions on account of multiple disulphide bridges and strongly

amphipathic helices [5]. Optimization of an encapsulation protocol is there-
fore dependent on the physicochemical characteristics of the peptide/protein
and the polymer itself (monomer composition, molecular weight, glass tran-
sition, etc.).
The selection of the right encapsulation technique must ensure the satis-
faction of three main criteria:
(i) the structure of the peptide/protein remains intact, particularly with
respect to aggregation and neutralizing antibodies [31];
(ii) the size of the spheres has to support the administration route aimed for e
from microns for injectable and inhalable particles, to nanoparticles for
oral administration; and
(iii) the drug release profile must be appropriate to the intended medical
application (vaccine, long-term release, delayed release, etc.).
Fortunately, for short peptides, little consideration needs to be made of
secondary structure, and microencapsulation is now a standard route to
achieving a controlled or sustained delivery profile. Achieving an appropriate
release profile can be tricky since the so-called “burst release” is often observed
in initial encapsulation-release experiments, wherein the majority of the drug
entrapped in the microsphere is subsequently released within the first few hours
upon immersion in aqueous media. Similarly, maximizing the encapsulation
efficiency is essential to ensure the process remains cost-effective. Despite
these challenges, the field has progressed rapidly in the last decade, and this
Chapter j 1 Future Trends 13

method of release may one day become routine for proteins. Current research in
this area involves scale-up of organic solvent-free encapsulation technologies
[32] and encapsulation of complex, self-assembling systems such as bacte-
riophages, which are expected to play a role in anti-bacterial therapies [33].

1.5. FUTURE TRENDS

The case studies described above outline future opportunities, and one
objective of each of the following chapters is to describe or predict future
trends. It is useful to broadly categorize the strategies into recombinant
approaches and materials technologies. The former includes the engineering of
protein/peptide “pro-drugs” and/or “chimeras” which may involve appending
a peptide or protein domain to a therapeutic protein in order to target and bind
a particular receptor. Bacterial exotoxins (as distinct from endotoxins) are
useful examples, since they have a general AB (or AB5) structure; the A
subunit brings about the enzymatic activity of the toxin while the B subunit
binds to a specific cell surface receptor, triggering endocytosis and internal-
ization. The key to drug delivery therefore is to isolate the B subunits to bring
about internalization without toxic effects. Another example of chimeric
proteins is to fuse a protein drug to the end of the neonatal Fc domain (cf.
antibody structure, Figure 1.1). The intention is for these chimeras to bind the
neonatal Fc receptor (FcRn), which remains expressed in adults in the lung
epithelium, in order for FcRn-mediated endocytosis to internalize the bound
drug, intact. Similarly, the humanization of murine mAbs (those originally
raised in mice) to reduce immunogenicity and prolong blood levels similarly
involves complete substitution of the Fc and partial substitution of the Fab
domains; Trastuzumab (HerceptinÒ) is an example of this technology (Table
1.1) [34]. Site directed mutagenesis is a central, simple tool in molecular
biology but very effective in protein engineering since specific amino acids can
be targeted to alter protein pharmacokinetics and physical/chemical/enzymatic
stability. One such example is VitatropinÔ (human growth hormone), but the
many recombinant mAbs are also modified in this manner. Of particular
interest to the area of protein engineering will be the incorporation of artificial
amino acids into peptides and proteins, possibly expanding their function or
improving bioprocessing. Recently, the tools required to efficiently encode and
direct the incorporation of unnatural amino acids into proteins were developed,
yielding a novel protein cross-link [35]. This is an exciting development which
will drive protein engineering towards custom-designed protein function.
Materials technology will become an increasingly important area for the
development of particulates. Their utility in the delivery of biomolecules has
already been noted above with respect to invasive (e.g. subcutaneous) and non-
invasive (e.g. pulmonary and oral) routes. It should be remembered that these
same particulate technologies will also play a role in the bioprocessing of
proteins. This represents a slight departure from the focus on delivery strategies
14 Peptide and Protein Delivery

but it is important to remember the downstream processes that must occur

before a pure product can be formulated, especially steps involving purifica-
tion, concentration and storage. A new technology which straddles both
delivery and bioprocessing is Protein Coated MicroCrystals (PCMCs). This
involves coprecipitation of an aqueous solution of protein in near saturated
concentrations of a solute (e.g. an amino acid) mixed rapidly with a non-
solvent. This promotes very rapid protein dehydration and, as the two
components coprecipitate, the protein (or other biomolecule) is immobilized
onto the surface of the crystalline excipient support. Many proteins are
compatible with the PCMC technique, irrespective of size and pI, e.g.
hormones, cytokines, mAbs and antigens. The microcrystals can be engineered
to be of a diameter consistent with inhalation but the process is also suitable for
rapid concentration of dilute protein solutions and subsequent storage as a dry
powder at standard temperature [36]. These stable powders can be rapidly
redissolved back into aqueous media to release the protein in a native form.
Protein stability and aggregation are major, and still largely unsolved, issues
affecting the development and production of biopharmaceuticals. Beside their
impact on development costs, the safety of biopharmaceuticals is also signifi-
cantly affected by these issues. Protein aggregation is a major element behind
the immunogenicity, and perhaps also the toxicity, of many bioactive
polypeptides.

1.6. TOXICITY PROFILES

The complex nature of proteins, peptides and other compounds derived through
biotechnological processes present many challenges when it comes to under-
standing their physicochemical and therapeutic behavior. Their specific
primary, secondary, tertiary and quaternary structures, as previously mentioned,
play key roles in defining the integrity and biological activity of bio-
macromolecules. This is the case whether they are directly incorporated into
simple, formulated systems, or use more sophisticated delivery platforms.
Either way their safety and toxicity must be evaluated through the use of
scientifically sound methods as a matter of paramount importance and neces-
sity. It does not mean that a routine study design is adopted. In fact, the
intricacies of these compounds require a customized and question based
approach [37]. Even after implementing a well designed approach questions
remain; are the safety margins sufficient, are the risk-to-benefit ratios accept-
able? A level of uncertainty will always exist e as exemplified by a life-
threatening experience involving TGN1412, a compound which was investi-
gated for the treatment of chronic inflammatory conditions [38]. This was
a fully humanized monoclonal antibody (mAb) with activity directed against
the CD28 costimulatory receptor on T-cells. No adverse effects were expected,
especially since genetically engineered monoclonal antibodies were considered
not only to be an exciting class of compounds for their potential to target
Chapter j 1 Toxicity Profiles 15

diseases, but also because they would not harm healthy cells. After all,
numerous mAb drug products are approved e such as HerceptinÒ for treating
breast cancer (including its new indication for advanced HER2-positive
stomach cancer) and RemicadeÒ for treating inflammatory disorders, providing
treatment benefit to patients. There are still many more mAbs undergoing
clinical trials. What separated TGN1412 from the mode of action of other
antibody treatments was that, in all other cases, biological reactions were
inhibited and shut down, but TGN1412 was designed to do the opposite. The
intent was to activate CD4þ effector memory T-cells which would in turn over-
stimulate any rogue T-cells, causing them to apoptose. Although the evidence is
inconclusive, it was believed that activation of the T-cells by TGN1412 was
responsible for eliciting a “cytokine storm” which produced serious adverse
effects in the human volunteers [39]. Absence of CD28 expression on the
CD4þ effector memory T-cells in the species used for non-clinical safety
testing provided something of an explanation for the failure to predict why
a cytokine storm occurred in humans. From a rational perspective, this expe-
rience underscores the need for an integrated safety assessment through
appropriate species selection and relevant animal models, including appropriate
use of biomarkers.
In stark contrast to the investigation of an unintentionally toxic compound,
it is interesting to review the anecdotal history of the discovery and intentional
development of a known toxic biomacromolecule. It was the 19th century
German poet and physician Justinus Kerner (1786e1862) who first provided
comprehensive details of the clinical symptoms of botulism associated with
ingestion of unhygienically produced sausages [40]. Kerner’s description of
what he termed “sausage poison” covered symptoms, duration, and other
findings, and was written decades before the causative botulinum toxin was
identified. His somewhat heroic, and extremely risky, scientific approach was to
self-ingest extracts of tainted sausages to gain direct knowledge and experience
of the symptoms. This provided from his perspective the most accurate
assessment of the effects of the “sausage poison”. Although not advocated here,
Kerner’s unorthodox approach led him to insights concerning the aetiology and
pathophysiology of the poison. He deduced that the toxic substance acted by
interfering with the signal transmission within the peripheral, sympathetic and
parasympathetic nervous system, while leaving the sensory signal transmission
intact. He further developed ideas about using botulinum toxin as a remedy for
various diseases that involve an overactive nervous system such as chorea, as
well as speculating on its use in other disorders.
Unbeknownst to Kerner, he was clearly dealing with Clostridium botulinum,
an anaerobic, gram positive bacterium which produced highly potent protein
complexes called neurotoxins. These toxins were responsible for the serious
food poisoning observed and the neuroparalytic effects of botulism. Seven
generally immunologically distinct botulinum neurotoxins have been charac-
terized from different strains of C. botulinum, these being respectively
16 Peptide and Protein Delivery

botulinum neurotoxin (BoNT) serotypes A, B, C.sub.1, D, E, F and G, each of

which is distinguished by neutralization with type-specific antibodies [41]. The
different serotypes of botulinum toxin vary in the animal species that they affect
and in the severity and duration of the paralysis they evoke. For example, it has
been determined that botulinum toxin type A is 500 times more potent, as
measured by the rate of paralysis produced in the rat, than is botulinum toxin
type B. Additionally, botulinum toxin type B has been determined to be non-
toxic in primates at a dose of 480 U/kg, which is about 12 times the primate
LD50 for botulinum toxin type A. Notwithstanding, it is botulinum toxin type A
which is considered to be the most lethal natural biological agent known to
humankind. To put this into perspective, on a molar basis, botulinum toxin type
A is about 1.8 billion times more lethal than diphtheria, about 600 million times
more lethal than sodium cyanide, about 30 million times more lethal than cobra
toxin and about 12 million times more lethal than cholera. Rhetorically, one
wonders whether, had Kerner been privy to this information, would he still have
pursued a path of self-experimentation?
Therapeutic proteins inherently have the potential to elicit antibody
production leading to an immunogenic response. Antibody responses can vary
appreciably across a range of biopharmaceutical products in both incidence and
clinical sequelae [42]. Detectable antibodies from less than 1% (e.g.
ActimmuneÒ, RituxanÒ) to over 80% (e.g. OKT3, FabrazymeÒ) show the
disparity of incidence between products. For clinical sequelae the variability can
be from no effect or reduced efficacy to allergic reactions, thrombocytopenia
and anemia. The serious adverse events causing these unintended consequences
are a constant area of concern, which have resulted in FDA mandated “black box
warnings” for certain products that were originally deemed very safe; such as
RemicadeÒ. The infliximab product was reported to contribute to tuberculosis
and other serious opportunistic infections including histoplasmosis, listeriosis
and pneumocystosis, after findings in both the clinical research and post-
marketing surveillance settings. Despite advances in gene sequencing, expres-
sion systems and post-expression techniques such as PEGylation, the worry
surrounding the clinical immunogenicity of therapeutic proteins has not gone
away [43]. Moreover, it raises the question about the usefulness and ability of
non-clinical data in predicting human immunogenicity.

1.7. REGULATORY MATTERS

The regulation of biotechnology, from research through to product approval
and eventual marketing of the product, is not a simple process. There are
several series of complex laws and guidance documents. It can be confidently
stated that the regulations will continue to evolve and no doubt increase as
scientific knowledge advances, novel technologies are developed and new
questions are asked. Unequivocally, the progression to product licensure must
rely heavily on sound scientific approaches, comprehensive data and analysis,
Other documents randomly have
different content
 Nils Lykke.
God be thanked! Know you not that a good conscience
is the best pillow?
Nils Stensson.
Ay, it must be even so; for with all my rattling and
thundering, I——
Nils Lykke.
——You won not in?
Nils Stensson.
You have hit it. So I said to myself: As you are bidden
to be in Östråt to-night, if you have to go through fire
and water, you may surely make free to creep through a
window.
Nils Lykke.
[Aside.] Ah, if it should be——!
[Moves a step or two nearer.
Was it, then, of the last necessity that you should
reach Östråt to-night?
Nils Stensson.
Was it? Ay, faith but it was. I love not to keep folk
waiting, I can tell you.
Nils Lykke.
Aha,—then Lady Inger Gyldenlöve looks for your
coming?
Nils Stensson.
Lady Inger Gyldenlöve? Nay, that I can scarce say for
certain; [with a sly smile] but there might be some one
else——
Nils Lykke.
 [Smiles in answer.] Ah, so there might be some one
else—?
Nils Stensson.
Tell me—are you of the house?
Nils Lykke.
I? Well, in so far that I am Lady Inger’s guest this
evening.
Nils Stensson.
A guest?—Is not to-night the third night after
Martinmas?
Nils Lykke.
The third night after—? Ay, right enough.—Would you
seek the lady of the house at once? I think she is not yet
gone to rest. But might not y o u sit down and rest
awhile, dear young Sir? See, here is yet a flagon of wine
remaining, and doubtless you will find some food. Come,
fall to; you will do wisely to refresh your strength.
Nils Stensson.
You are right, Sir; ’twere not amiss.
[Sits down by the table and eats and drinks.
Both roast meat and sweet cakes! Why, you live like
lords here! When one has slept, as I have, on the naked
ground, and lived on bread and water for four or five
days——
Nils Lykke.
[Looks at him with a smile.] Ay, such a life must be
hard for one that is wont to sit at the high-table in noble
halls——
Nils Stensson.
Noble halls——?
 Nils Lykke.
But now can you take your ease at Östråt, as long as it
likes you.
Nils Stensson.
[Pleased.] Ay? Can I truly? Then I am not to begone
again so soon?
Nils Lykke.
Nay, that I know not. Sure you yourself can best say
that.
Nils Stensson.
[Softly.] Oh, the devil! [Stretches himself in the chair.]
Well, you see—’tis not yet certain. I, for my part, were
nothing loath to stay quiet here awhile; but——
Nils Lykke.
——But you are not in all points your own master?
There be other duties and other affairs——?
Nils Stensson.
Ay, that is just the rub. Were I to choose, I would rest
me at Östråt at least the winter through; I have for the
most part led a soldier’s life, and——
[Interrupts himself suddenly, fills a goblet, and
drinks.
Your health, Sir!
Nils Lykke.
A soldier’s life? H’m!
Nils Stensson.
Nay, what I would have said is this: I have long been
eager to see Lady Inger Gyldenlöve, whose fame has
spread so wide. She must be a queenly woman,—is’t not
so?——The one thing I like not in her, is that she is so
cursedly slow to take open action.
Nils Lykke.
Open action?
Nils Stensson.
Ay, ay, you understand me; I mean she is so loath to
take a hand in driving the foreign masters out of the
land.
Nils Lykke.
Ay, there you are right. But if now you do what you
can, you will doubtless move her.
Nils Stensson.
I? God knows ’twould but little serve if I——
Nils Lykke.
Yet ’tis strange you should seek her here if you have so
little hope.
Nils Stensson.
What mean you?—Tell me, know you Lady Inger?
Nils Lykke.
Surely; since I am her guest——
Nils Stensson.
Ay, but it in nowise follows that you know her. I too am
her guest, yet have I never seen so much as her shadow.
Nils Lykke.
Yet did you speak of her——
Nils Stensson.
 ——as all folk speak. Why should I not? And besides, I
have often enough heard from Peter Kanzler——
[Stops in confusion, and falls to eating busily.
Nils Lykke.
You would have said——?
Nils Stensson.
[Eating.] I? Nay, ’tis all one.
[Nils Lykke laughs.
Nils Stensson.
Why laugh you, Sir?
Nils Lykke.
At nothing, Sir!
Nils Stensson.
[Drinks.] A pretty vintage ye have in this house.
Nils Lykke.
[Approaches him confidentially.] Listen—were it not
time now to throw off the mask?
Nils Stensson.
[Smiling.] The mask? Why, do as seems best to you.
Nils Lykke.
Then off with all disguise. You are known, Count Sture!
Nils Stensson.
[Bursts out laughing.] Count Sture? Do you too take
me for Count Sture?
[Rises from the table.
You mistake, Sir! I am not Count Sture.
Nils Lykke.
 You are not? Then who are you?
Nils Stensson.
My name is Nils Stensson.
Nils Lykke.
[Looks at him with a smile.] H’m! Nils Stensson? But
you are not Sten Sture’s son Nils? The name chimes at
least.
Nils Stensson.
True enough; but God knows what right I have to bear
it. My father I never knew; my mother was a poor
peasant-woman, that was robbed and murdered in one
of the old feuds. Peter Kanzler chanced to be on the
spot; he took me into his care, brought me up, and
taught me the trade of arms. As you know, King Gustav
has been hunting him this many a year; and I have
followed him faithfully, wherever he went.
Nils Lykke.
Peter Kanzler has taught you more than the trade of
arms, meseems.——Well, well; then you are not Nils
Sture. But at least you come from Sweden. Peter Kanzler
has sent you hither to find a stranger, who——
Nils Stensson.
[Nods cunningly.]——who is found already.
Nils Lykke.
[Somewhat uncertain.] And whom you do not know?
Nils Stensson.
As little as you know me; for I swear to you by God
himself: I am not Count Sture!
Nils Lykke.
 In sober earnest, Sir?
Nils Stensson.
As truly as I live! Wherefore should I deny it, if I were?
Nils Lykke.
But where, then, is Count Sture?
Nils Stensson.
[In a low voice.] Ay, t h a t is just the secret.
Nils Lykke.
[Whispers.] Which is known to you? Is’t not so?
Nils Stensson.
[Nods.] And which I am to tell you.
Nils Lykke.
To tell me? Well then,—where is he?
[Nils Stensson points upwards.
Nils Lykke.
Up there? Lady Inger holds him hidden in the loft-
room?
Nils Stensson.
Nay, nay; you mistake me.
[Looks round cautiously.
Nils Sture is in Heaven!
Nils Lykke.
Dead? And where?
Nils Stensson.
In his mother’s castle,—three weeks since.
Nils Lykke.
 Ah, you are deceiving me! ’Tis but five or six days
since he crossed the frontier into Norway.
Nils Stensson.
Oh, that was I.
Nils Lykke.
But just before that the Count had appeared in the
Dales. The people, who were restless already, broke out
openly and would have chosen him for king.
Nils Stensson.
Ha-ha-ha; that was me too!
Nils Lykke.
You?
Nils Stensson.
I will tell you how it came about. One day Peter
Kanzler called me to him and gave me to know that great
things were preparing. He bade me set out for Norway
and fare to Östråt, where I must be on a certain fixed
day——
Nils Lykke.
[Nods.] The third night after Martinmas.
Nils Stensson.
There I was to meet a stranger——
Nils Lykke.
Ay, right; I am he.
Nils Stensson.
From him I should learn what more I had to do.
Moreover, I was to let him know that the Count was dead
of a sudden, but that as yet ’twas known to no one save
to his mother the Countess, together with Peter Kanzler
and a few old servants of the Stures.
Nils Lykke.
I understand. The Count was the peasants’ rallying-
point. Were the tidings of his death to spread, they
would fall asunder,—and ’twould all come to nought.
Nils Stensson.
Ay, maybe so; I know little of such matters.
Nils Lykke.
But how came you to give yourself out for the Count?
Nils Stensson.
How came I to——? Nay, what know I? Many’s the
mad prank I have hit on in my day. And yet ’twas not I
hit on it neither; for whereever I appeared in the Dales,
the people crowded round me and hailed me as Count
Sture. Deny it as I pleased, ’twas wasted breath. The
Count had been there two years before, they said—and
the veriest child knew me again. Well, so be it, thought I;
never again will you be a Count in this life; why not try
what ’tis like for once?
Nils Lykke.
Well,—and what did you more?
Nils Stensson.
I? I ate and drank and took my ease. The only pity
was that I had to take the road again so soon. But when
I set forth across the frontier—ha-ha-ha—I promised
them I would soon be back with three or four thousand
men—I know not how many I said—and then we would
lay on in earnest.
Nils Lykke.
 And you did not bethink you that you were acting
rashly?
Nils Stensson.
Ay, afterwards; but then, to be sure, ’twas too late.
Nils Lykke.
I grieve for you, my young friend; but you will soon
come to feel the effects of your folly. Let me tell you that
you are pursued. A troop of Swedish men-at-arms is out
after you.
Nils Stensson.
After me? Ha-ha-ha! Nay, that is rare! And when they
come and think they have Count Sture in their clutches—
ha-ha-ha!
Nils Lykke.
[Gravely.]——Then ’tis all over with you.
Nils Stensson.
All over——? But I am not Count Sture.
Nils Lykke.
You have called the people to arms. You have given
seditious promises, and raised troubles in the land.
Nils Stensson.
Ay, but ’twas only in jest!
Nils Lykke.
King Gustav will scarce take that view of the affair.
Nils Stensson.
Truly, there is something in what you say. To think I
could be so featherwitted——Well, well, I’m not a dead
man yet! You will protect me; and besides—-the men-at-
arms can scarce be at my heels yet.
Nils Lykke.
But what else have you to tell me?
Nils Stensson.
I? Nothing. When once I have given you the packet
——
Nils Lykke.
[Off his guard.] The packet?
Nils Stensson.
Ay, sure you know——
Nils Lykke.
Ah, right, right; the papers from Peter Kanzler——
Nils Stensson.
See, here they all are.
[Takes out a packet from inside his doublet, and
hands it to Nils Lykke.
Nils Lykke.
[Aside.] Letters and papers for Olaf Skaktavl.
The packet is open, I see. ’Tis like [To Nils Stensson.
you know what it contains?
Nils Stensson.
No, good sir; I love not to read writing; and for reason
good.
Nils Lykke.
I understand; you have given most care to the trade of
arms.
 [Sits down by the table on the right, and runs
through the papers.
Aha! Here is light enough and to spare on what is
brewing.
This small letter tied with a silken thread—[Examines
the address.] This too for Olaf Skaktavl. [Opens the
letter, and glances through its contents.] From Peter
Kanzler. I thought as much. [Reads under his breath.] “I
am hard bested, for—”; ay, sure enough; here it stands,
—“Young Count Sture has been gathered to his fathers,
even at the time fixed for the revolt to break forth”—“—
but all may yet be made good—” What now? [Reads on
in astonishment.] “You must know, then, Olaf Skaktavl,
that the young man who brings you this letter is a son of
—” Heaven and earth—can it be so?—Ay, by the cross of
Christ, even so ’tis written! [Glances at Nils Stensson.]
Can he be—? Ah, if it were so! [Reads on.] “I have
nurtured him since he was a year old; but up to this day
I have ever refused to give him back, trusting to have in
him a sure hostage for Inger Gyldenlöve’s faithfulness to
us and to our friends. Yet in that respect he has but little
availed us. You may marvel that I told you not this secret
when you were with me here of late; therefore will I
confess freely that I feared you might seize upon him,
even as I had done, and to the same intent. But now,
when you have seen Lady Inger, and have doubtless
assured yourself how loath she is to have a hand in our
undertaking, you will see that ’tis wisest to give her back
her own as soon as may be. Well might it come to pass
that in her joy and security and thankfulness—” —— “—
that is now our last hope.”
[Sits for a while as though struck dumb with
surprise; then exclaims in a low voice:
Aha,—what a letter! Gold would not buy it!
Nils Stensson.
 ’Tis plain I have brought you weighty tidings. Ay, ay,—
Peter Kanzler has many irons in the fire, folk say.
Nils Lykke.
[To himself.] What to do with all this? A thousand
paths are open to me—What if I were—? No, ’twere to
risk too much. But if—ah, if I—? I will venture it!
[Tears the letter across, crumples up the pieces,
and hides them inside his doublet; puts back
the other papers into the packet, which he
thrusts inside his belt; rises and says:
A word, my young friend!
Nils Stensson.
[Approaching him.] Well—your looks say that the game
goes bravely.
Nils Lykke.
Ay, by my soul it does. You have given me a hand of
nought but court cards,—queens and knaves——
Nils Stensson.
But what of me, that have brought all these good
tidings? Have I nought more to do?
Nils Lykke.
You? Ay, that have you. You belong to the game. You
are a king—and king of trumps too.
Nils Stensson.
I a king? Oh, now I understand; you are thinking of
my exaltation——
Nils Lykke.
Your exaltation?
Nils Stensson.
 Ay; that which you foretold for me, if King Gustav’s
men got me in their clutches——
[Makes a motion to indicate hanging.
Nils Lykke.
True enough;—but let that trouble you no more. It
now lies with yourself alone whether within a month you
shall have the hempen noose or a chain of gold about
your neck.
Nils Stensson.
A chain of gold? And it lies with me?
[Nils Lykke nods.
Nils Stensson.
Why then, the devil take doubting! Do you but tell me
what I am to do.
Nils Lykke.
I will. But first you must swear me a solemn oath that
no living creature in the wide world shall know what I
confide to you.
Nils Stensson.
Is that all? You shall have ten oaths, if you will.
Nils Lykke.
Not so lightly, young Sir! ’Tis no jesting matter.
Nils Stensson.
Well, well; I am grave enough.
Nils Lykke.
In the Dales you called yourself a Count’s son;—is’t not
so?
Nils Stensson.
 Nay—begin you now on t h a t again? Have I not made
free confession——
Nils Lykke.
You mistake me. What you said in the Dales was the
truth.
Nils Stensson.
The truth? What mean you by that? Tell me but——!
Nils Lykke.
First your oath! The holiest, the most inviolable you
can swear.
Nils Stensson.
That you shall have. Yonder on the wall hangs the
picture of the Holy Virgin——
Nils Lykke.
The Holy Virgin has grown infirm of late. Know you not
what the monk of Wittenberg maintains?
Nils Stensson.
Fie! how can you heed the monk of Wittenberg? Peter
Kanzler says he is a heretic.
Nils Lykke.
Well, let us not dispute the matter. Here can I show
you a saint will serve full well to make oath by.
[Points to a picture hanging on one of the panels.
Come hither,—swear that you will be silent till I myself
release your tongue—silent, as you hope for Heaven’s
salvation for yourself and for the man whose picture
hangs there.
Nils Stensson.
 [Approaching the picture.] I swear it—so help me
God’s holy word!
[Falls back a step in amazement.
But—Christ save me——!
Nils Lykke.
What now?
Nils Stensson.
The picture—! Sure ’tis I myself!
Nils Lykke.
’Tis old Sten Sture, even as he lived and moved in his
youthful years.
Nils Stensson.
Sten Sture!—And the likeness—? And—said you not I
spoke the truth, when I called myself a Count’s son?
Was’t not so?
Nils Lykke.
So it was.
Nils Stensson.
Ah, I have it, I have it! I am——
Nils Lykke.
You are Sten Sture’s son, good Sir!
Nils Stensson.
[With the quiet of amazement.] I Sten Sture’s son!
Nils Lykke.
On the mother’s side too your blood is noble. Peter
Kanzler spoke not the truth, if he said that a poor
peasant woman was your mother.
Nils Stensson.
 Oh strange! oh marvellous!—But can I believe——?
Nils Lykke.
You may believe all that I tell you. But remember, all
this will be merely your ruin, if you should forget what
you swore to me by your father’s salvation.
Nils Stensson.
Forget it? Nay, t h a t you may be sure I never shall.—
But you, to whom I have given my word,—tell me—who
are you?
Nils Lykke.
My name is Nils Lykke.
Nils Stensson.
[Surprised.] Nils Lykke? Surely not the Danish
Councillor?
Nils Lykke.
Even so.
Nils Stensson.
And it was you—? ’Tis strange. How come you——?
Nils Lykke.
——to be receiving missives from Peter Kanzler? You
marvel at that?
Nils Stensson.
I cannot deny it. He has ever named you as our
bitterest foe——
Nils Lykke.
And therefore you mistrust me?
Nils Stensson.
 Nay, not wholly that; but—well, the devil take musing!
Nils Lykke.
Well said. Go but your own way, and you are as sure of
the halter as you are of a Count’s title and a chain of
gold if you trust to me.
Nils Stensson.
That will I. My hand upon it, dear Sir! Do you but help
me with good counsel as long as there is need; when
counsel gives place to blows, I shall look to myself.
Nils Lykke.
’Tis well. Come with me now into yonder chamber, and
I will tell you how all these matters stand, and what you
have still to do.
[Goes out to the right.
Nils Stensson.
[With a glance at the picture.] I Sten Sture’s son! Oh,
marvellous as a dream——!
[Goes out after Nils Lykke.
 ACT FOURTH
The Banquet Hall, as before, but without the supper-
table.
Biörn, the majordomo, enters carrying a lighted branch-
candlestick, and lighting in Lady Inger and Olaf
Skaktavl by the second door on the left. Lady Inger has
a bundle of papers in her hand.
Lady Inger.
[To Biörn.] And you are sure my daughter had speech
with the knight, here in the hall?
Biörn.
[Putting down the branch-candlestick on the table on
the left.] Sure as may be. I met her even as she stepped
into the passage.
Lady Inger.
And she seemed greatly moved? Said you not so?
Biörn.
She looked all pale and disturbed. I asked if she were
sick; she answered not, but said: “Go to my mother and
tell her the knight sets forth from here ere daybreak; if
she have letters or messages for him, beg her not to
delay him needlessly.” And then she added somewhat
that I heard not rightly.
Lady Inger.
Did you not hear it at all?
Biörn.
 It sounded to me as though she said:—“Almost I fear
he has already tarried too long at Östråt.”
Lady Inger.
And the knight? Where is he?
Biörn.
In his chamber belike, in the gate-wing.
Lady Inger.
It is well. What I have to send by him is ready. Go to
him and say I await him here in the hall. [Biörn goes out
to the right.
Olaf Skaktavl.
Know you, Lady Inger,—’tis true that in such things I
am blind as a mole; yet seems it to me as though——
h’m!
Lady Inger.
Well?
Olaf Skaktavl.
——as though Nils Lykke bore a mind to your daughter.
Lady Inger.
Then ’twould seem you are not so blind after all; for I
am the more deceived if you be not right. Marked you
not at the supper-board how eagerly he listened to the
least word I let fall concerning Elina?
Olaf Skaktavl.
He forgot both food and drink.
Lady Inger.
And our secret affairs as well.
Olaf Skaktavl.
 Ay, and what is more—the papers from Peter Kanzler.
Lady Inger.
And from all this you conclude——?
Olaf Skaktavl.
From all this I chiefly conclude that, as you know Nils
Lykke and the name he bears, especially in all that
touches women——
Lady Inger.
——I should be right glad to know him outside my
gates?
Olaf Skaktavl.
Ay; and that as soon as may be.
Lady Inger.
[Smiling.] Nay—the case is just the contrary, Olaf
Skaktavl!
Olaf Skaktavl.
How mean you?
Lady Inger.
If things be as we both think, Nils Lykke must in
nowise depart from Östråt yet awhile.
Olaf Skaktavl.
[Looks at her with disapproval.] Are you again
embarked on crooked courses, Lady Inger? What guile
are you now devising? Something that may increase your
own power at the cost of our——
Lady Inger.
Oh this blindness, that makes you all do me such
wrong! I see well you think I purpose to make Nils Lykke
my daughter’s husband. Were such a thought in my
mind, why had I refused to take part in what is afoot in
Sweden, when Nils Lykke and all the Danish crew seem
willing to support it?
Olaf Skaktavl.
Then if it be not your wish to win him and bind him to
you—what would you with him?
Lady Inger.
I will tell you in few words. In a letter to me, Nils Lykke
has spoken of the high fortune it were to be allied to our
house; and I do not say but, for a moment, I let myself
think of the matter.
Olaf Skaktavl.
Ay, see you!
Lady Inger.
To wed Nils Lykke to one of my house were doubtless
a great step toward stanching many discords in our land.
Olaf Skaktavl.
Meseems your daughter Merete’s marriage with
Vinzents Lunge might have taught you what comes of
such a step. Scarce had my lord gained firm footing
among us, when he began to make free with both our
goods and our rights——
Lady Inger.
I know it even too well, Olaf Skaktavl! But times there
be when my thoughts are manifold and strange. I cannot
impart them fully either to you or to any one else. Often
I know not the right course to choose. And yet—a second
time to make a Danish lord my son-in-law,—nought but
the uttermost need could drive me to that resource; and
heaven be praised—things have not yet come to t h a t!
 Olaf Skaktavl.
I am no wiser than before, Lady Inger;—why would
you keep Nils Lykke at Östråt?
Lady Inger.
[In a low voice.] Because I owe him an undying hate.
Nils Lykke has done me deadlier wrong than any other
man. I cannot tell you wherein it lies; but never shall I
rest till I am avenged on him. See you not now? Say that
Nils Lykke were to love my daughter—as meseems were
like enough. I will persuade him to tarry here; he shall
learn to know Elina well. She is both fair and wise.—Ah, if
he should one day come before me, with hot love in his
heart, to beg for her hand! Then—to chase him away like
a dog; to drive him off with jibes and scorn; to make it
known over all the land that Nils Lykke had come a-
wooing to Östråt in vain—! I tell you I would give ten
years of my life but to see that day!
Olaf Skaktavl.
In faith and truth, Inger Gyldenlöve—is t h i s your
purpose towards him?
Lady Inger.
This and nought else, as sure as God lives! Trust me,
Olaf Skaktavl, I mean honestly by my countrymen; but I
am in nowise my own mistress. Things there be that
must be kept hidden, or ’twere my death-blow. But let
me once be secure on t h a t side, and you shall see if I
have forgotten the oath I swore by Knut Alfson’s bier.
Olaf Skaktavl.
[Shakes her by the hand.] Thanks for those words! I
am loath indeed to think evil of you.—Yet, touching your
design towards this knight, methinks ’tis a venturesome
game you would play. What if you had misreckoned?
What if your daughter—? ’Tis said no woman can stand
against this subtle devil.
Lady Inger.
My daughter? Think you that she—? Nay, have no fear
of that; I know Elina better. All she has heard of his
renown has but made her hate him the more. You saw
with your own eyes——
Olaf Skaktavl.
Ay, but—a woman’s mind is shifting ground to build on.
’Twere best you looked well before you.
Lady Inger.
That will I, be sure; I will watch them narrowly. But
even were he to succeed in luring her into his toils, I
have but to whisper two words in her ear, and——
Olaf Skaktavl.
What then?
Lady Inger.
——She will shrink from him as though he came
straight from the foul Tempter himself.
Hist, Olaf Skaktavl! Here he comes. Now be cautious.
[Nils Lykke enters by the foremost door on the right.
Nils Lykke.
[Approaches Lady Inger courteously.] My noble hostess
has summoned me.
Lady Inger.
I have learned through my daughter that you are
minded to leave us to-night.
Nils Lykke.
 Even so, to my sorrow;—since my business at Östråt is
over.
Olaf Skaktavl.
Not before I have the papers.
Nils Lykke.
True, true. I had well-nigh forgot the weightiest part of
my errand. ’Twas the fault of our noble hostess. With
such gracious skill did she keep her guests in talk at table
——
Lady Inger.
That you no longer remembered what had brought you
hither? I rejoice to hear it; for that was my design.
Methought that if my guest, Nils Lykke, were to feel at
his ease in Östråt, he must forget——
Nils Lykke.
What, lady?
Lady Inger.
——First of all his errand—and then all that had gone
before it.
Nils Lykke.
[To Olaf Skaktavl, as he takes out the packet and hands
it to him.] The papers from Peter Kanzler. You will find in
them a full account of our partizans in Sweden.
Olaf Skaktavl.
It is well.
[Sits down by the table on the left, where he
opens the packet and examines its contents.
Nils Lykke.
 And now, Lady Inger Gyldenlöve,—I know not that
there is aught else for me to do here.
Lady Inger.
Had it been things of state alone that brought us
together, you might be right. But I should be loath to
think so.
Nils Lykke.
You would say——?
Lady Inger.
I would say that ’twas not alone as a Danish Councillor
or as the ally of Peter Kanzler that Nils Lykke came to be
my guest.—Do I err in fancying that somewhat you may
have heard down in Denmark may have made you
curious to know more of the Lady of Östråt.
Nils Lykke.
Far be it from me to deny——
Olaf Skaktavl.
[Turning over the papers.] Strange. No letter.
Nils Lykke.
——Lady Inger Gyldenlöve’s fame is all too widely
spread that I should not long have been eager to see her
face to face.
Lady Inger.
So I thought. But what, then, is an hour’s jesting talk
at the supper-table? Let us try to sweep away all that
has till now lain between us; it may well come to pass
that the Nils Lykke I know may wipe out the grudge I
bore the one I knew not. Prolong your stay here but a
few days, Sir Councillor! I dare not persuade Olaf
Skaktavl thereto, since his secret charge in Sweden calls
him hence. But as for you, doubtless your sagacity has
placed all things beforehand in such train that your
presence can scarce be needed. Trust me, your time shall
not pass tediously with us; at least you will find both me
and my daughter heartily disposed to do all in our power
to pleasure you.
Nils Lykke.
I doubt neither your goodwill towards me nor your
daughter’s; of that I have had ample proof. And I trust
you will not doubt that my presence elsewhere must be
vitally needful, since, despite of all, I must declare my
longer stay at Östråt impossible.
Lady Inger.
Is it even so!—Know you, Sir Councillor, were I evilly
minded, I might fancy you had come to Östråt to try a
fall with me, and that, having lost, you cared not to
linger on the battlefield among the witnesses of your
defeat.
Nils Lykke.
[Smiling.] There might be some show of reason for
such a reading of the case; but sure it is that as yet I
hold not the battle lost.
Lady Inger.
However that may be, it might at any rate be
retrieved, if you would tarry some days with us. You see
yourself, I am still halting and wavering at the parting of
the ways,—persuading my redoubtable assailant not to
quit the field.—Well, to speak plainly, the thing is this:
your alliance with the disaffected in Sweden still seems
to me somewhat—how shall I call it?—somewhat
miraculous, Sir Councillor! I tell you this frankly, dear Sir!
The thought that has moved the King’s Council to this
secret step is in truth most politic; but ’tis strangely at
variance with the deeds of certain of your countrymen in
bygone years. Be not offended, then, if my trust in your
fair promises needs to be somewhat strengthened ere I
can place my whole welfare in your hands.
Nils Lykke.
A longer stay at Östråt would scarce help towards that
end; since I purpose not to make any further effort to
shake your resolve.
Lady Inger.
Then must I pity you from my heart. Ay, Sir Councillor
—’tis true I stand here an unfriended widow; yet may
you trust my word when I foretell that this visit to Östråt
will strew your future path with thorns.
Nils Lykke.
[With a smile.] Is that your forecast, Lady Inger?
Lady Inger.
Truly it is! What can one say, dear Sir? ’Tis an age of
tattling tongues. Many a scurril knave will make jeering
rhymes at your expense. Ere half a year is out, you will
be all men’s fable; people will stop and gaze after you on
the high roads; ’twill be: “Look, look; there rides Sir Nils
Lykke, that fared north to Östråt to trap Inger
Gyldenlöve, and was caught in his own nets.”—Softly,
softly, Sir Knight, why so impatient! ’Tis not that I think
so; I do but forecast the thoughts of the malicious and
evil-minded; and of them, alas! there are many.— Ay, ’tis
shame; but so it is—you will reap nought but mockery—
mockery, because a woman was craftier than you. “Like a
cunning fox,” men will say, “he crept into Östråt; like a
beaten hound he slunk away.”—And one thing more:
think you not that Peter Kanzler and his friends will
forswear your alliance, when ’tis known that I venture
not to fight under a standard borne by you?
Nils Lykke.
You speak wisely, lady! Wherefore to secure me from
mockery—and not to endanger the alliance with all our
dear friends in Sweden—I must needs——
Lady Inger.
[Hastily.] ——prolong your stay at Östråt.
Olaf Skaktavl.
[Who has been listening.] He is in the trap!
Nils Lykke.
No, my noble lady;—I must needs bring you to terms
within this hour.
Lady Inger.
But what if you should fail?
Nils Lykke.
I shall n o t fail.
Lady Inger.
You lack not confidence, it seems.
Nils Lykke.
What shall be the wager that you make not common
cause with myself and Peter Kanzler?
Lady Inger.
Östråt Castle against your knee-buckles!
Nils Lykke.
[Slaps his breast and cries:] Olaf Skaktavl—here stands
the master of Östråt!
 Lady Inger.
Sir Councillor——!
Olaf Skaktavl.
[Rises from the table.] What now?
Nils Lykke.
[To Lady Inger.] I accept not the wager; for in a
moment you will gladly give Östråt Castle, and more to
boot, to be freed from the snare wherein not I but you
are tangled.
Lady Inger.
Your jest, Sir, grows a vastly merry one.
Nils Lykke.
’Twill be merrier yet—at least for me. You boast that
you have overreached me. You threaten to heap on me
all men’s scorn and mockery. Ah, beware that you stir not
up my vengefulness; for with two words I can bring you
to your knees at my feet.
Lady Inger.
Ha-ha——!
[Stops suddenly, as if struck by a foreboding.
And these two words, Nils Lykke?—these two words
——?
Nils Lykke.
——The secret of Sten Sture’s son and yours.
Lady Inger.
[With a shriek.] Oh, God in heaven——!
Olaf Skaktavl.
Inger Gyldenlöve’s son! What say you?
Welcome to our website – the ideal destination for book lovers and
knowledge seekers. With a mission to inspire endlessly, we offer a
vast collection of books, ranging from classic literary works to
specialized publications, self-development books, and children's
literature. Each book is a new journey of discovery, expanding
knowledge and enriching the soul of the reade

Our website is not just a platform for buying books, but a bridge
connecting readers to the timeless values of culture and wisdom. With
an elegant, user-friendly interface and an intelligent search system,
we are committed to providing a quick and convenient shopping
experience. Additionally, our special promotions and home delivery
services ensure that you save time and fully enjoy the joy of reading.

Let us accompany you on the journey of exploring knowledge and

personal growth!

ebookultra.com