ANTECEDENTES

c 18 16 14 12 8 6 4 2 0 0 50 100 150 200 2 5 0 S iz e (nm )

Figure 4.1. A typical size distribution o f stable DNA complexes. The complexes were p rep a red using ctDNA com plexed to PLL 29300 at a charge ratio o f + 2 .0 in 20mM HEPES pH7.2. The size distribution was m easured imm ediately after mixing.

4.1 DNA Concentration

A lth o u g h g en e d eliv ery treatm en ts prove e ffec tiv e in sm all an im als th e m ag n itu d e o f

re sp o n se s in d u ced in p rim ates is g en erally lo w er (S elby et a l , 2000). T h is p ro b lem is

ag g rav ated by the fact th at there is a lim it to the v o lu m e that can b e ad m in iste red in trav e n o u sly , fu rth erm o re, since the in tera ctio n o f gene d eliv ery v ecto rs w ith pro tein s and cells is often no n -sp ecific a vast m ajo rity o f input d o se is lost d u e to un d esirab le

b in d in g to n o n -targ et ce lls and serum c o m p o n e n ts (Li et a l , 1999). It is th erefo re

g en e rally ag reed that the clinical ap p licatio n o f n on-viral d eliv ery v ecto rs to h u m an s is lik ely to re q u ire high co n c en tratio n s o f lip o p le x es o r p o ly p lex es c o n ta in in g h ig h er

c o n c e n tra tio n s o f D N A than are cu rren tly av a ila b le (D avis, 2002; S elb y et a l , 2000;

W o o d le et a l , 2001). In d icatio n s g en e rally su g g est th at th e do se w ill ra n g e from

m icro g ra m s to m illig ram s and ex p e rim en ts have su g g ested th at a m in im u m

co n c e n tra tio n o f D N A p e r dose will be a p p ro x im a te ly 1 5 0 |4 g m r’ (B ally et a l , 1999;

L e v y et a l , 2000). T h ere are tw o p ro b lem s stem m in g from th is re q u irem en t. F irst o f

all a strict control o f the final d o sag e o f fo rm u late d p ro d u c t w ill be critical and alth o u g h it has yet to be e stab lish ed how m any p lasm ids are re q u ire d to reach the

Chapter 4._____________________________________Formulation Optimisation: Results and Discussion

nucleus to achieve efficient transgene expression it is anticipated, especially in the case of nucleic acid vaccines, that the final dosage form may contain multiple plasmids (Bally et al., 1999; Marquet et a l, 1997b). Secondly, several authors have previously reported DNA concentration to have a negative influence on the aggregative characteristics of non-viral DNA complexes (Duguid et a l , 1998). At present the aggregating nature of non-viral complexes is aggravated by increases in the concentration of DNA in the formulation and the typical dose size is as yet only 0.3|Xg (Prazeres e t a l , 1999).

In order to achieve wide spread use and agency approval gene delivery systems should be prepared in a state which allows for the design of an administration strategy which meets patient and physician approval. The options, at present consist o f preparation of the gene delivery treatment, containing high DNA concentrations, by the physician at the bedside just prior to administration. Alternatively, aggregation has been shown to be noticeably reduced at DNA concentrations typically less than 0.5m gm f \ However, minimising aggregation by these methods is unsatisfactory clinically due to a number of issues. For instance, a complete treatment may require a large volume of material and repeated administration which is both costly, time consuming, and is likely to increase the toxicity of the treatment and cause physician compliance and regulatory approval to be hampered (Davis, 2002; Lee et a l , 2001; Prazeres et a l , 1999; Selby et a l, 2000). For these gene ‘drugs’ to be of any value to the pharmaceutical industry it is therefore essential that they be produced in a stable and easy to administer state, possess high DNA concentrations and can be stored for prolonged periods of up to two years without losing their therapeutic value or original physiochemical characteristics.

Chapter 4. Formulation Optimisation: Results and Discussion