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Page 190
is the reason for its success. For example, BR96 binds to a tumor-associated antigen that is abundantly (greater than 200000 molecules per cell) expressed on human tumors [209]. Second, the ability of the conjugate to bind antigen, i.e. its immunoreactivity, was tested and found to be as effective as that of unconjugated BR96 [210]. Third, it was shown that conjugate immunoreactivity was predictive of the antigen-specific cytotoxic potency [211]. Fourth, the BR96-DOX conjugate is rapidly endocytosed after binding to the cell receptors [209,212]. Fifth, a heterobifunctional linker, 6-maleimidocaproylhydrazone of Doxorubicin, was synthesized that imparts an acceptable stability at neutral pH but rapidly releases Doxorubicin under acidic conditions that mimic those of the lysosomal environment [213]. Human trials now in progress with this novel immunoconjugate, which if successful, would open up a new drug delivery approach of vast potential.
3. Regulated Delivery to Satisfy Chronobiology
Chronobiology deals with physiological functions that exhibit prominent rhythmic change. The daily rhythm, known as circadian, has been studied most extensively although high-frequency (ultradian, menstrual, and seasonal) rhythms have been well documented. Circadian rhythms have been demonstrated for over 100 variables in the blood, urine, and saliva [214]. Circadian rhythms also exist for body temperature, blood pressure [215], heart rate [216], and the plasma concentration of hormones [217]. The pulsed secretion of hormones has been described for insulin [218], somatostatin [219], luteinizing hormone [220], and several other peptides [221223]. Many studies in a variety of pharmacological fields have shown a strong dependency of the onset of disease on circadian temporal rhythm. In asthmatics, episodes of dyspnea take place mainly between 2 and 7 a.m. and are 50-fold higher between 4 and 5 a.m. than between 4 and 5 p.m. [224]. Digestive tract rhythmicity has been described for many functions of the gastrointestinal tract, e.g. high gastric acid secretion rates in the evening hours and low in the morning hours [225,226]. Myocardial infarction [227], sudden cardiac death [228], thrombotic stroke [229], and arterial embolism [230] show a strong circadian rhythm with a morning peak and an evening trough. A circadian rhythm in all blood cell types has been well documented [231] including cells of the immune system such as B and T lymphocytes and natural killer cells [232234]. Circadian rhythms have also been demonstrated for lymphocyte proliferation [235], as well as CD3 and CD4 epitope molecules at their surface [235], which may relate to cell reactivity to antigen exposure and may be critical to the fight against malignancies and other immune diseases [237238]. Therefore, better management of patients could be obtained by including in the clinical protocol the application of the chronobiological aspects of the pathophysiology of the disease [239].

 
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