Estructura de la matriz
Apéndice 5. Indicadores de la circuncisión masculina
Phosphorothioate oligonucleotides are taken up by a wide range of cells in vitro
188, In fact, uptake of phosphorothioate oligonucleotides into a Vibrio parahaemoyticus, has been reported as has up- take into Schistosoma Mansoni (190, 191). Uptake is time and temperature dependent. It is also influenced by cell type, cell-culture con- ditions, media and sequence, and length of the oligonucleotide No obvious correlation between the lineage of cells, whether the cells are transformed or are virally infected, and uptake has been identified (93); nor are the factors that result in differences in uptake of different sequences of oligonucleotide under- stood. Although several studies have sug- gested that receptor-mediated endocytosis may be a mechanism of cellular up- take, the data are not yet compelling enough to conclude that receptor-mediated
sis accounts for a significant portion of the up take in most cells (87).
Numerous studies have shown that phorothioate oligonucleotides distribute broadly in most cells once taken up (93, 192). Again, however, significant differences in subcellular distribution between various types of have been noted.
lipids and other approaches have been used to enhance uptake of phosphoro- thioate oligonucleotides in cells that take up little oligonucleotide in vitro (193-195). Again, however, there are substantial varia- tions from cell type to cell type. Other ap- proaches to enhanced intracellular uptake
in
vitro have included streptolysin D treatment of cells and the use of dextran sulfate and other liposome formulations as well as physical means such as microinjections (93, 196, 197).
Characteristics of Phosphorothioate Oligonucleotides
In Pharmacokinetics
osphorothioate oligonucleotides bind to se-
m
albumin and a-2 macroglobulin. The affinity for albumin is quite lowand comparable to the low affinity binding observed for a number of drugs
pirin, penicillin) (167, 168, 182). Serum in binding, therefore, provides a repository
these drugs and prevents rapid renal
on. Because serum protein binding is satura-
ble, at higher doses, intact oligomer may be und in urine (174,198). Studies in our
suggest that in rats, oligonucleotides intravenously a t doses of 15-20 saturate the serum protein binding Phosphorothioate oligonucleotides are
and extensively absorbed after
inistration. For example, in rats, after an radermal dose 3.6 of 2105, a phosphorothioate, approximately 70% f the dose was absorbed within 4 h and total stemic bioavailability was in excess of 90% 00). After intradermal injection in humans, absorption of 2105 was similar to that ob- served in rats (201). Subcutaneous administra-
tion to rats and monkeys results in somewhat lower bioavailability and greater distribution to
nodes, as would be expected
Distribution of phosphorothioate oligonu- cleotides from blood after absorption or administration is extremely rapid. We have re- ported distribution half-lives of less than 1 h, and similar data have been reported by others (174, 198, 200, 203). Blood and plasma clear- ance is multiexponential, with a terminal elimination half-life from 40 to 60 h in all species except humans, where the terminal elimination half-life may be somewhat longer (201).
Phosphorothioates distribute broadly to all peripheral tissues. Liver, kidney, bone mar- row, skeletal muscle, and skin accumulate the highest percentage of a dose, but other tissues display small quantities of drug (200,203). No evidence of significant penetration of the blood-brain barrier has been reported. The rates of incorporation and clearance from tis- sues vary as a function of the organ studied, with liver accumulating drug most rapidly (20% of a dose within 1-2 and other tissues accumulating drug more slowly, and similarly,
elimination of drug relatively rapidly from liver compared to that from many other tis- sues terminal half-life from liver, 62 h; from renal medulla, 156 The distribution into the kidney has been studied more exten- sively and drug was shown to be present in Bowman's capsule, the proximal convoluted tubule, the brush border membrane, and within renal tubular epithelial cells (204). The data suggested that the oligonucleotides are filtered by the glomerulus, then reabsorbed by the proximal convoluted tubule epithelial cells. Moreover, the authors suggested that absorption might be mediated by interactions with specific proteins in the brush border membranes. In addition, the oligonucleotide is accumulated in a nonfiltering kidney, suggest- ing that there is uptake from the basal side also.
Clearance of phosphorothioate oligonucle- otides is attributed primarily to metabolism (200, 203, 205). Metabolism is mediated by exo- and endonucleases that result in shorter oligonucleotides and, ultimately, nucleosides are degraded by normal metabolic path- ways. Although no direct evidence of base ex- cision or modification has been reported, these are theoretical possibilities that may occur. In one study, a larger molecular weight radioac- tive material was observed in urine, but not fully characterized (174). Clearly, the poten- tial for conjugation reactions and extension of oligonucleotides by these drugs serving as primers for polymerases must be explored in more detail. In a very thorough study, 20-nu- cleotide phosphodiester and phosphorothioate oligonucleotides were administered at a dose of 6 to mice. The oligonucleotides were internally labeled with by
ylation of an internal deoxycytidine residue using Hhal methylase and
methionine (181). The pharmacokinetic prop- erties observed were consistent with our re- sults as described above. Additionally, in this report, autoradiographic analyses showed drug in renal cortical cells (181).
One study of prolonged infusions of a
oligonucleotide to humans has been reported (206). In this study, five pa- tients with leukemia were given 10-day in- fusions at a dose of mg Elimi- nation half-lives reportedly varied from 5.9 to
Oligonucleotide Therapeutics
14.7 days. Urinary recovery of radioactivity was reported to be 30-60% of the total dose, with 30% of the radioactivity being intact drug. Metabolites in urine included both higher and lower molecular weight com- pounds. In contrast, when (a 25-mer phosphorothioate oligodeoxynucleotide) was administered to humans as a 2-h infusion at a dose of 0.1 a peak plasma concen- tration of 295.8 was observed at the cessation of the infusion. Plasma clearance of total radioactivity was biexponential with ini- tial and terminal elimination half-lives of 0.18 and 26.71 h, respectively. However, degrada- tion was extensive and intact drug
kinetic models were not presented. Nearly 50% of the administered radioactivity was re- covered in urine, but most of the radioactivity represented degrades. In fact, no intact drug was found in the urine at any time (207).
In a more recent study in which the level of intact drug was carefully evaluated using cap- illary gel electrophoresis, the
of 2302, a 20-mer phosphorothioate oligodeoxynucleotide, after a 2-h infusion, were determined. Doses from 0.06 to 2.0 mgkg were studied and the peak plasma con- centrations were shown to increase linearly with dose, with the 2 dose resulting in peak plasma concentrations of intact drug of about 9.5 Clearance from plasma, however, was dose dependent, with the 2
dose having a clearance of 1.28 kg-', whereas that of 0.5 was 2.07 kg-'. Essentially, no intact drug was found in urine.
Clearly, the two most recent studies differ from the initial report in several facets. Al- though a number of factors may explain the discrepancies, the most likely explanation is related to the evolution of assay methodology, not differences between compounds. Overall, the behavior of phosphorothioates in the plasma of humans appears to be similar to that in other species.
In addition to the pharmacological effects that have been observed after
ate oligonucleotides have been administered to animals (and humans), a number of other lines of evidence show that these drugs enter cells in organs. Autoradiographic, fluorescent, and immunohistochemical approaches have
shown that these drugs are localized in endo- promal convoluted tubular cells, various bone marrow cells, and cells in the skin and liver (204, 208,209).
Perhaps more compelling and of more long- term value are studies recently reported show- ing the distribution of phosphorothioate oli- gonucleotides in the liver of rats treated intravenously with the drugs at various doses This study showed that the and extent of the accumulation into Kupffer, endo- and hepatocyte cell population varied and that as doses were increased, the distribu- tion changed. Moreover, the study showed that subcellular distribution also varied.
6.5.1 Aerosol Administration. Phosphoro- thioate oligodeoxynucleotides have been shown to be attractive for inhalation delivery to the lung and upper airway (211-213). Tar- get reduction in the lung has been demon- strated (214) and these drugs have been shown to distribute broadly to all cell types in the lung after aerosol administration. Fur- ther, these drugs were shown to be well toler- ated at doses up to 12 (212).
6.5.2 Topical Administration. Phosphoro- thioate oligonucleotides formulated in a very cream formulation have been shown to penetrate normal mouse, pig, and human and to penetrate and accumulate in human psoriatic grown on nude mice. Further, in a phase study in patients with plaque pso- riasis, 2302, in ICAM-1 inhibitor, was shown to accumulate throughout the dermis and epidermis after topical administration and to result in a positive trend in the primary endpoint, that is, induration unpub- lished observations, 2000). Thus, studies have demonstrated a substantial accumulation of drug throughout the dermis and epidermis and reduction of targets such as ICAM-1, B71, and B72 (for review, see Ref. 215).
6.5.3 Summary. In summary,
netic studies of several phosphorothioates demonstrate that they are well absorbed from sites, distribute broadly to many peripheral tissues, do not cross the
brain barrier, and are eliminated primarily by nuclease metabolism. In short, once daily or
6 Characteristics of Phosphorothioate Oligonucleotides
every other day systemic dosing is feasible. Al- though the similarities between oligonucleo- tides of different sequences are far greater than the differences, additional studies are re- quired before determining whether there are subtle effects of sequence on the
netic profile of this class of Moreover, they can be delivered to the lung by aerosol and topically administered, and progress in achieving acceptable oral bioavailability con- tinues.