Pharmaceutical Manufacturing Handbook: Production and

TOXICOLOGICAL CONSIDERATIONS 667
et al. [115] introduced a scale of irritation in the nose (0, no irritation; 1, slight irritation;
2, acceptable; 3, unwilling to accept the treatment again) that was later used to
evaluate the degree of nasal powder irritation after administration in human volunteers
[27] . Ugwoke et al. [82] classifi ed the degree of ciliary beat frequency change
caused by nasally applied liquid formulation as follows: no effect (less than 10%),
mild (10 – 20%), moderate (20 – 50%), and severe (more than 50%). Soon after, that
classifi cation was applied to a powder ciliotoxicity study [116] . Reversibility of ciliotoxic
effect after washing out the tested compound was classifi ed as well, resulting
in three categories: reversible, partially reversible, and irreversible effects [82] .
Merkus et al. [117] proposed the three categories of constituents of nasal formulations
based on the recovery of ciliary beat frequency after the tested compound was
washed out . The fi rst category is cilio friendly, with ciliary beat frequency recovery
of 75% or more; the second is cilio inhibiting, with recovery between 25 and 75%;
and the third category is ciliostatic, with recovery of 25% or less.
Methods to evaluate the possible toxicological effect of formulations on the nasal
mucosa described in the literature mainly refer to histopathological evaluation as a
standard method for cytotoxicity evaluation [64] and a study on the release of marker
enzymes [118] . In the work reported by Callens et al. [118] the possible toxicological
effects of multiple starch and carbopol powder nasal administration were evaluated
using rabbits by measuring the proteins and lactate dehyrogenase (LDH) release
from the nasal mucosa. Contrary to the invasive in situ perfusion method, performable
with anaesthetized animals [119] , this method has been shown to be noninvasive,
applicable to nonanaesthetized and nonsedated animals, and suitable for repeated
measurement of the marker protein release on the same animal during a long - term
administration study. A histopathological study has also been performed. In agreement
with attempting to replace the use of vertebrates in scientifi c experiments with
lower organisms such as invertebrates, plants, and microorganisms, Adriaens and
Remon [120] developed a new mucosal toxicity screening method using the slug
Arion lusitanicus as the model organism. The body wall of the slug resembles the nasal
mucosa since it consists of a single - layered epithelium containing both ciliated and
mucus - secreting cells. Callens et al. [118] successfully used that method for screening
the mucosal irritation potential of bioadhesive starch and carbopol powder formulations.
The possible toxicological effects of the powder formulations were evaluated
by measuring the proteins LDH and alkaline phosphatase (ALP) release from the
body wall of the slugs as well as the amount of mucus produced.
Among various nasal toxicity studies, ciliotoxicity studies are of special interest
due to the importance of maintaining optimal ciliary beating to protect the lower
respiratory system from infections. If the drug formulation inhibits the ciliary beating,
such inhibition needs to be completely reversible upon formulation removal [116] .
Methods to determine ciliary beat frequency and mucociliary transport in vitro
and in vivo have been extensively reviewed elsewhere [84, 121] . In general, in vivo
methods are more reliable for ciliotoxicity studies than in vitro methods and are
essential to confi rm the safety of nasal drug formulations. However, in vitro methods
are more suitable for the large number of screening studies required during formulation
development [122] .
In vitro methods to measure ciliary beat frequency can be performed on explants
of ciliated mucosa [64] or on different types of ciliated cells, such as ciliated chicken
embryo trachea cells [123] , nasal cell lines derived from carcinomas of epithelial
origin (RPMI 2650, BT, NAS 2BL), or human lung adenocarcinoma cell line Calu - 3