Drug Targeting Organ-Specific Strategies

14	1 Drug Targeting: Basic Concepts and Novel Advances
Table 1.2. Overview of recently reported clinical studies exploiting antibodies.
Antibody	Antigen	Disease treateda	Clinical responseb	Remarks	Reference
Rituximab	CD20	NHL	21/39 OR	Similar results	[81]
(rituxan)			14/39 SD or MR	in follicular
				and small
				lymphocytic
				lymphoma
		PT-LPD after liver	2/2 CR: poly-		[82]
		Tx	morphic PT-LPD;
			1/1 NR: large cell NHL
		PT-LPD after solid	15/26 CR		[83]
		organ Tx	2/26 PR
		PT-LPD after BMT	5/6 CR		[83]
		Mantle cell lym-	36/120 OR (varied	Limited	[84]
		phoma (MCL), im-	with tumour type).	activity
		munocytoma, small	CR only seen	in SLL
		B cell lymphocytic	with MCL
		lymphoma (SLL)
Herceptin;	Her2/neu	Metastatic breast	1/43 CR	Responses	[85]
trastuzumab		carcinoma	4/43 PR	seen in
			2/43 MR	mediastinum,
			14/43 SD	lymph nodes,
				liver, chest
				wall lesions
MKC-454	Her2/neu	Metastatic breast	2/18 OR		[86]
		carcinoma
rhuMAb HER2	Her2/neu	Advanced breast	9/37 PR	Combination	[87]
		carcinoma	9/37 MR – SD	therapy with
			19/37 DP	cisplatin
rhuMab VEGF	VEGF	Metastatic renal cell	Phase III study in		(clinical-
		carcinoma	progress		trials.gov
					website)
hOKT3γ4	CD3	Various malignancies	3/24 responses		[58]
Infliximab	TNFα	Rheumatoid arthritis	428 patients: 20–50%	Concomitant	[71]
			improvement in ~80%	methotrexate
			of patients	therapy
		Psoriasis lesions	Improved appearance		[88]
			of a patient 2 weeks
			after treatment
Synagis;	RSV F -	RSV infection	35 children < 2 years		[89]
palivizumab	glyco-	in infants	of age: significant
	protein		reduction in tracheal
			RSV concentration,
			but not in nasal
			aspirate. No difference
			in disease severity
			compared to placebo
Abciximab	Platelet gp	Ischaemic complica-	Platelet aggregation	All patients	[90]
	IIb/IIIa	tions during balloon	inhibition with	received aspi-
	receptor	angioplasty or	abciximab provided	rin co-medi-
		atherectomy	long-term clinical	cation; pa-
			benefits after coronary	tients with
			intervention com-	stent implanta-
			pared to placebo	tion received
				ticlopidine co-
				medication

aBMT, bone marrow transplantation; NHL, non-Hodgkin lymphoma; PT-LPD, post-transplantation lymphoproliferative disorders; RSV, respiratory syncytial virus; Tx, transplantation.

bNumber of patients responding/total number of evaluable patients. CR, complete remission/response; PR, partial response; MR, minor response; NR, no response OR, objective response; SD, stable disease; DP, disease progression.

1.5 Vaccination Strategies for Enhanced Immunity

15

methotrexate, and the general aim of these conjugates is to lower the (dose-limiting cardio-) toxicity of these drugs. Enhanced permeability retention of the conjugates at the site of the tumour is most likely the basis of local accumulation. In the studies reported, some objective clinical responses were seen without concomitant toxicity of the drugs (Table 1.3). Although the clinical responses until now seem rather disappointing, one should realize that the patients who undergo clinical testing with these conjugates often carry large tumour burdens which have been non-responsive towards other therapies available.

Table 1.3. Overview of recently reported clinical studies with miscellaneous drug targeting strategies.

Carrier	Drug	Disease	Clinical	Remarksc	Reference
	incorporated		responsea,b
Antibody	90Y	Metastatic	2/25 OR	Pre-targeted strategy:	[91]
		colon cancer	2/25 PR	mAb NR-LU-10 –
			4/25 SD	streptavidin, followed
				by biotin-gal-HSA
				clearing and biotin-
				DOTA-90Y
		Non-Hodgkin	3/7 CR	Pre-targeted strategy:	[92]
		lymphoma	1/7 PR	mAb Rituximab –
			2/7 OR	streptavidin, followed
				by biotin-N-acetyl-
				galactosamine clearing
				and biotin-DOTA90Y
HPMA	Doxorubicin	Various	2/36 PR	Clinical proof of
copolymer		malignancies	2/36 MR	principle of decrease	[15]
(PK1)				of doxorubicin toxicity
				when polymer bound
Poly(styrene-	Neocar-	Renal cell	Improved survival,	SMANCS dissolved in	[93]
co-maleic acid) zinostatin		carcinoma	depending on	lipiodol contrast
polymers		patients	tumour size	medium, administered
(SMANCS)				via the renal artery
Human serum Methotrexate Cancer patients			17 patients: 1 PR		[94]
albumin			and 1 MR in RCC
			patients; 1 MR in
			pleural mesothe-
			lioma patient

aNumber of patients responding/total number of evaluable patients. CR, complete remission/response; PR, partial response; MR, minor response; OR, objective response; SD, stable disease.

bRCC, renal cell carcinoma.

cmAb, monoclonal antibody.

1.5 Vaccination Strategies for Enhanced Immunity

Vaccination can be considered as a therapeutic modality that actively engages the immune system of the patient. It encompasses numerous principles derived from drug targeting research. Modification of the responses of the immune system may be an effective approach to improve the disease status of patients with a variety of diseases. Either prevention of au-

16 1 Drug Targeting: Basic Concepts and Novel Advances

toimmune diseases or allergic responses, or enhancement of immune responses against infectious agents and tumour growth can be induced by these strategies [95,96]. Vaccination strategies can be divided into gene-, peptide-, proteinand cell-based strategies.

Antigen presenting cells (APC), particularly dendritic cells (DC), play a central role in the induction of the desired immune response [97]. For successful (antitumour) vaccination therapy, either an in vitro or an in vivo approach can be followed. In the in vitro approach, DCs from an animal or a patient are isolated and manipulated by transfection with DNA or RNA encoding (tumour) antigens or pro-inflammatory factors, or by loading the cells with proteins or peptides. After transfer back into the animal or patient, the cells can evoke antigen-spe- cific immune responses [98]. Similarly, in vitro transfected tumour cells encoding a combination of genes involved in regulation of immune responses (e.g. MHC class II, a co-stimulato- ry molecule and a superantigen [96]) may serve as a vaccine.

The major drawback in the use of isolated DCs is the time-consuming isolation and culture methods required to obtain the cell type of interest. Therefore, in vivo vaccination strategies employing DCs have been developed. For this purpose, fusion proteins of e.g. tumour antigens and molecules that are specifically recognized by DCs are under investigation. The DC targeting molecules consist of e.g. DC-specific chemokines, mannose, the Fc moiety of immunoglobulin, cytotoxic T lymphocyte-associated antigen (CTLA-4) molecule [99–103]. They enable efficient uptake of the fusion protein by the DCs, induction of DC migratory capacity into the lympoid organs and maturation into a dedicated APC. As an example, studies by Biragyn et al. showed that active targeting of a tumour antigen into a receptor-mediated uptake route in APCs by the fusion of the antigen to APC-specific chemokines, elicited superior protection against a large tumour challenge in mice [99].

For a more detailed discussion of vaccines and vaccination strategies, the reader is referred to Volume 170 of Immunological Reviews (1999), in which various aspects of this area of research are discussed in greater detail.

1.6 Drug Targeting as a Research Tool to Study Disease

From an historical point of view, ‘magic bullets’ were initially proposed as novel therapeutic tools for treatment of disease. Another, similarly attractive application of drug targeting is its use as a tool to study e.g. mechanisms of disease pathology. By selectively manipulating one specific cell type or one specific protein, the role of this cell type or protein in disease progression can be determined. The advent of knock-out and transgenic animal models has provided the researcher in the laboratory with a powerful tool to investigate the relative contribution of gene products of interest. Yet, counter-regulatory processes that compensate for the loss of a certain gene product may significantly hamper correct interpretation of the results obtained in these models. Manipulating a gene in an appropriately matured animal using drug targeting strategies can therefore be a valuable tool.

It is not always appreciated that the application of in vivo gene therapy actually represents a drug delivery protocol. By the incorporation of plasmids into e.g. adenoviral or liposomal coats they are protected from degradation. Subsequent interaction with the target cells leads to intracellular delivery and then to nuclear localization of the plasmid. Although transfec-

1.6 Drug Targeting as a Research Tool to Study Disease

17

tion efficiencies in in vivo gene therapy protocols are still not without problems, the transcription of genes of interest by transduced liver cells and muscle cells are quite satisfactory when using a strong promoter. Liver cells can be targeted by i.v. injection of the encapsulated plasmids, whereas skeletal muscle cells can be locally transfected by intramuscular injections. As an example, i.v. administration of recombinant adenovirus AdCMV-PDX-1 into mice resulted in specific transfection of liver cells. Expression of the PDX-1 gene, encoding a protein that regulates insulin gene expression, led to increased plasma levels of insulin and ameliorated hyperglycaemia in diabetic mice treated with streptozotocin [104]. Transduction of skeletal muscle with liposome formulated human hepatocyte growth factor (HGF) gene induced high plasma levels of protein, phosphorylation of the HGF target receptor, subsequent suppression of transforming growth factor β production and improved survival rates of animals suffering from severe liver cirrhosis [105].

Proteins have also been selectively targeted to study a disease process. Selective induction of oxidative vascular injury in the lungs was accomplished by targeting glucose oxidase (GOX) to the vasculature using anti-PECAM-1 (anti-CD31)-antibody–GOX conjugates. As the pulmonary vasculature represents approximately 30% of endothelial cells in the body and receives all the cardiac output, the majority of anti-PECAM-GOX conjugate accumulated in the endothelium in the lung. By generating H2O2 locally from glucose, severe endothelial cell damage was induced. The injury was associated with the production of the oxidative marker iPF2a-III isoprostane.This model of targeted induction of oxidative stress can now be applied to study the effects of pharmacological agents [106].

Inhibition of tumour blood flow or the outgrowth of solid tumour vasculature by angiogenesis inhibition has been proposed as a powerful strategy to eradicate cancer ever since the recognition of the importance of blood supply for tumour growth. Subsequent development of compounds which inhibited the formation of new tumour blood vessels, demonstrated the dependence of solid tumour growth on blood vessel formation. It was however the selective induction of blood coagulation in the vasculature of a well-developed tumour by a drug targeting strategy that really demonstrated the potency of instantaneous inhibition of blood flow as a therapy for solid tumours [107]. Based on these and other ‘proof of principle’ studies, enormous research effort is now put into the identification of target epitopes on human tumour vasculature to further develop this strategy for clinical application (see Chapter 9 and reference [57] for a more detailed discussion on tumour vasculature targeting).

The inhibition of TNFα by neutralizing antibodies in rheumatoid arthritis patients is one of the few examples of targeted interference with disease activity in humans that can provide us with new insights into the pathophysiology of the disease [108]. The concomitant reduction in systemic levels of acute phase proteins, endothelial cell adhesion molecule expression in synovial biopsies and inflamed joint-associated blood vessel density all suggest a central role for TNFα as a driving force in RA. Furthermore, it provides evidence for the existence of a relationship between pro-inflammatory activity and the occurrence of angiogenesis, although it is at present unfeasible to conclude on cause and effect relations due to limited knowledge on this subject.

In summary it can be said that besides being important in the development of therapeutic strategies to combat disease with minimal toxicity and maximal effects, drug targeting may be of interest for more basic studies on the mechanistic background of diseases and the identification of new molecules as targets for therapeutic intervention.

18 1 Drug Targeting: Basic Concepts and Novel Advances

1.7 Challenges in Drug Targeting Research

In 1984, Poznansky and Juliano published a critical review on the biological approaches to the controlled delivery of drugs [109]. Already at that time, they had envisioned the use of molecular biology and molecular immunology in the delineation of structure and function of important proteins and nucleic acids as being the true frontier in the drug delivery field. The elucidation of signal transduction routes in cells responding to a variety of environmental factors of disease has indeed led to the development of numerous NCEs to be used in future therapies. Furthermore, the understanding of protein folding and minimal amino acid sequences required for receptor recognition provided significant added value in the development of better carriers and ligands for drug targeting.

Numerous problems in the construction of clinically applicable drug targeting moieties still need to be solved. Of these issues, immunogenicity after repeated administration, counterproductive liver clearance, and production yields are the most important. Although the problem of immunogenicity is believed to have been solved for monoclonal antibody therapy by the development of humanized and fully human antibodies [110], for other carrier systems such as modified plasma proteins and peptide modified polymers, this remains an important issue.

For many drug targeting approaches, extensive uptake by the liver is undesirable when the target cells are of non-hepatic origin. In this respect, defining the optimal physicochemical characteristics that would circumvent extensive hepatic clearance of the drug targeting preparation would be of great value. This may, however, be wishful thinking more than a realistic aim, as hepatic elimination of (slightly) modified proteins is one method of protecting the body from non-functional or aged proteins. Furthermore, drug targeting preparations have to be eliminated from the body, as do regular drugs, so that their pharmacological activity can be controlled.

In general, validation of the targeting concept (therapeutic effectiveness, lack of toxicity even after prolonged periods of time) for each drug targeting conjugate that is under development should be performed in vivo in animal models of disease. Not only is this the only unambiguous approach for showing proof of the targeting principle, but it also takes into account the pharmacokinetic characteristics of the conjugate which determine the added value of the targeting strategy compared to non-targeted therapy (see Chapter 13 for pharmacokinetic considerations in drug targeting).

Most of the drug targeting strategies explored so far have been aimed at tumour cell killing or targeting of drugs and genes to the liver (i.e. Kupffer cell or hepatocyte). More recently the potential of targeting other cells of interest has been put forward, e.g. endothelial cells lining tumour blood vessels (Chapter 9) or the vasculature in chronic inflamed tissue (Chapter 7), and hepatic stellate cells in the fibrotic liver (Chapter 4) [10,57].As the number of target cells in these cases is often relatively low and at present little knowledge exists regarding basic cell regulatory processes such as responses to drugs or endocytotic activity, all aspects of drug targeting (pharmacological activity of the drug in the target cell, cellular handling and kinetics of release of the drug) need to be addressed in full.

One of the most frequently heard criticisms is that drug targeting preparations cannot be administered orally and are therefore either of secondary or no interest for development into therapeutics. Although significant research effort has been put into the development of oral-

1.8 Conclusions

19

ly available protein therapeutics [111], there is a good chance that these preparations will not be feasible because of the inherent limitations in manipulating the physiological barriers in humans. Possibly, the understanding of the structural requirements for drug targeting preparations may lead to the development of minimized proteins that may be orally administered or become systemically available via e.g. pulmonary delivery (see Chapter 3). Furthermore, major efforts are being invested in the development of transdermal administration and programmable infusion systems. These systems may lower the barriers for long-term parenteral administration of drugs and may become common practice in pharmacotherapy. It is noteworthy that if a single dose of a targeted protein is potent enough to silence a disease for a prolonged period of time, the requirement of parenteral administration will not hamper its development. This is exemplified by the development of the TNFα neutralizing antibodies and receptors in the therapy of chronic inflammatory diseases such as rheumatoid arthritis and inflammatory bowel disease.

Finally, drug targeting technology should be integrated in the earliest phases of drug development. Many drugs that are pharmacologically active at low concentrations are withdrawn from the R & D pipeline due to toxicity in non-target cells. Would it be possible during an early phase in the development of a drug to construct cell-specific targeting conjugates a large number of drugs could still be candidates for development into clinical therapeutic strategies.

1.8 Conclusions

At present, many of the problems encountered during the development of drug targeting strategies for clinical application, especially for cancer therapy, have been identified, analysed and solved. Several drug targeting preparations have entered the phases of clinical testing and/or have now been marketed. However, these strategies should be subjected to continuous evaluation in the light of advances in the understanding of the numerous processes occuring in response to administration of the carriers and/or the drugs. New strategies under investigation will need to be optimized and extensively evaluated, taking advantage of the ‘bench to bed-side’ experience available today. Furthermore, in the coming years, combining expertise in the drug targeting field with the technological developments in molecular biology and molecular medicine will facilitate the elucidation of the cellular and molecular processes underlying disease.

Acknowledgements

Drs E. S. Vitetta, D. K. F. Meijer and M. Everts are acknowledged for critically reading the manuscript. Drs G. Storm, R. J. Kok and L. Beljaars are thanked for their help with the preparation of the figures.

20 1 Drug Targeting: Basic Concepts and Novel Advances

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Drug Targeting Organ-Specific Strategies. Edited by G. Molema, D. K. F. Meijer Copyright © 2001 Wiley-VCH Verlag GmbH ISBNs: 3-527-29989-0 (Hardcover); 3-527-60006-X (Electronic)

2 Brain-Specific Drug Targeting Strategies

Ulrich Bickel, Young-Sook Kang, Jörg Huwyler

2.1 Introduction

The brain is unique as a target organ for drug delivery: while it ranks amongst organs with the greatest blood supply and receives about 20% of the cardiac output in humans, access to the tissue is highly restricted by a tight vascular barrier, the blood–brain barrier (BBB). Due to the existence of the BBB, the transport of potentially neuroactive drugs from blood into brain is rarely blood-flow limited (for example for highly diffusible drugs like diazepam), but is in many cases extraction-limited. Therefore, drug delivery/targeting to the brain is primarily a permeability problem.

The major neurological diseases affecting the brain may be categorized as neurodegenerative, cerebrovascular, inflammatory (infectious or autoimmune) and brain tumours. Progress is being made for most of these disorders in terms of identification of epidemiological risk factors and of the aetiology and pathophysiological mechanisms, as will be briefly discussed. This information will facilitate the development of more efficient pharmacotherapy [1]. Solving the BBB delivery problem must be an integral part of these efforts.

The present chapter discusses approaches to brain drug delivery for small molecular weight drugs and the macromolecular drugs which become available as a result of advances in molecular biology and biotechnology, including peptides, monoclonal antibodies (mAb), and DNAor RNA-based therapeutics (oligonucleotides, genes).

2.2 Overview of Central Nervous System Diseases

2.2.1 Neurodegenerative Diseases

2.2.1.1 Alzheimer Disease (AD)

AD affects an increasing number of elderly people. Its prevalence is currently estimated at about 4 million in the USA alone [2]. The lack of an early diagnosis is by itself impeding progress in the therapy of the disease. Although the aetiology is still not fully elucidated, overwhelming evidence has accumulated for the crucial involvement of the Aβ-peptide [3] in the pathophysiological process. Aβ deposits occur as cerebrovascular amyloid or as parenchymal plaques (senile plaques). The 39–43-amino acid peptide is derived from the amyloid precursor protein (APP) [4], an ubiquitous cellular transmembrane protein of unknown function which is present in several splice variants. APP mutations have been identified as rare causes of familial AD, but mutations in other proteins, the presenilins PS-1 and