Corticosteroid: A Review by Rahel Woldo, Pharm.D.

It is estimated that more than 5 million patients are treated with glucocorticosteroids annually. The anti-inflammatory and immunosuppressive effects of steroids have been exploited in the treatment of diseases such as; bronchial asthma, chronic obstructive pulmonary disease, rheumatoid arthritis, collagen vascular diseases, chronic active hepatitis and certain forms of nephritides. In many instances the use of glucocorticosteroids is for short-term purposes. However, in some patients, such as organ transplant patients, glucocorticoid use becomes of a chronic basis because of exacerbation of underlying disease upon withdrawal.
GCs have been known since the early 1920s to affect the immune system. As of the early days of renal transplantation in the 1960s, GCs remain a vital part of almost all chronic immunosuppressive organ transplant therapies due to their ability to significantly decrease rejection. The first successful use of steroids in organ transplant was in 1960, when it was used to reverse a renal rejection episode in a living-related donor transplant recipient who was immunosuppressed by whole body radiation. Since those early days of organ transplantation, even though the development of immunosuppressive agents has undergone substantial evolution, corticosteroids have been the mainstay of clinical immunosupression.
The principal circulating glucocorticoid in humans is cortisol (hydrocortisone). The physiological effects of GC include regulation of lipids, carbohydrates, protein and amino acids metabolism. GC increases blood glucose level by antagonizing insulin effects and decreasing insulin secretion, thereby decreasing peripheral glucose uptake, which it promotes hepatic gluconeogenesis and increases glycogen content of the liver
Cortisol levels respond to physical (trauma, surgery, exercise), psychological (anxiety, depression) and physiological (hypoglycemia, fever) stresses within minutes. The reason why elevated glucose level protects an organism is not clearly understood, however it is believed that in conditions of GC deficiency such stress might lead to hypotension, shock and death. Corticosteroids exhibit a wide range of effects on almost every phase of the immune and inflammatory responses in animals and humans. Corticosteroids dosed in various pharmacological levels play a major role in treating different diseases.
Although GCs have been used for a period of time and it is known to have effect on the immune system, cardiovascular, central nervous system, musculoskeletal, gastrointestine and bone, the exact mechanism of action of its effect is still a controversy. The purpose of this paper is to briefly discuss the different chemical structures of natural and synthetic steroids, their pharmacokinetics, half-life, potency, bioavailability, mechanism of action, and their effect on the immune system.

Cortisol contains a cyclopentenoperhydrophenanthrane nucleus, which is made up of three 6-carbon rings and a single 5-carbon pentane ring. Cortisol has 21 carbon atoms with 2-carbon side chains (C-20 and C-21) attached at position 17 and methyl groups at C-18 and C-19. Alteration at different position of the steroid molecule results in the formation of different synthetic analogs of cortisol.

The five important analogs of cortisol are cortisone, prednisone, dexamethasone, prednisolone and methylprednisolone. The minimal change in the molecular structure leads to diversity in the potency, half-life and duration of action. Cortisone is derived from cortisol by replacement of the hydroxy group at C-11 by a keto group. Prednisolone is also an 11-Keto compound, but contains a double bond between C-1 and C-2. Dexamethasone on the other hand, is an 11-hydroxy compound with a double bond between C-1 and C-2 but has the important characteristics of fluorination of the B rings and methylation at C-17. Prednisolone is derived from cortisol by double bonding between 1 and 2 positions, and methylation of these positions produces methylprednisolone.

Glucocorticoid Equivalent anti-inflammatory dose (mg) Plasma half-life (min) Biological half-life(hrs)
Cortisol                                                    20                          90         8-12
Cortisone                                                 25                          90         8-12
Prednisolone                                              5                         200        12-36
Methylprednisolone                                     5                         200        12-36
Trimcinolone                                              4                         200        12-36
Bethametasone                                          0.6                      300        36-54
Dexamethasone                                          0.75                    300        36-54

Cholesterol derived from the diet and from endogenous synthesis are the substrates for steroidogenesis. Uptake of the cholesterol by adrenal cortex is mediated by the low-density lipoprotein (LDL) receptor. With long term stimulation of the adrenal cortex by adrenocorticotropic hormones (ACTH), the number of the LDL receptors increases. The three major adrenal biosynthesis pathways lead to the production of glucocorticoids (cortisol), mineralcorticoids (aldosterone) and adrenal androgens (dihydropiandrosterone). Separate zones of the adrenal cortex synthesize specific hormones. These separate zones express specific genes that encode unique enzymes for the synthesis of each type of steroids. The outer cell layer expresses gene for aldosterone synthase and 17-hydroxlase is expresses in the inner cell layer (faciculatareticularis), these sites synthesize cortisol and androgen respectively.
Stress leads to the release of and activation of the sympathetic nervous system. These changes in turn increase adrenocorticotropic hormone (ACTH) release by either acting individually or in concert. Further the release of ACTH leads to an increase of cortisol in the body.
ACTH is a 39-aminoacid peptide synthesized and stored in basophilic cells of the anterior pituitary. **The release of ACTH and related peptides is mediated by cortisol, cortisol releasing hormone (CRH), a 41-aminoacid peptide produced in the hypothalamus. The major factors controlling ACTH release include CRH, free cortisol concentrations in the plasma, stress (hypoglycemia, exercise, emotional trauma, surgery) and the sleep-wake cycle. The plasma ACTH level varies throughout the day as a result of its pulsatile secretion, and it follows a circadian pattern with a peak just to prior to waking and all-time low before sleeping. ACTH and cortisol levels also increase in response to eating. Cortisol has a negative feedback on its own synthesis and secretion. Cortisol exerts a negative feedback control over the HPA-axis system by acting on the hypothalamus to inhibit CRH secretion and the anterior pituitary to reduce responsiveness to CRH.
Cortisol exists in the plasma in three different forms; free cortisol, protein bound and cortisol metabolite. Free cortisol is physiologically active and can bind and act on different tissue sites. Normally about 5% of circulating cortisol is free. Only free cortisol and its metabolites are filtered in the glomerulus. Increased levels of free cortisol are excreted in the urine in a state of cortisol hypersecretion. Approximately about 80% of the circulating cortisol is bound to transcortin or cortisol binding protein (CBG) or albumin. Binding affinity of cortisol decreases during inflammation thus increases the concentration of free cortisol at area of inflammation. When concentration of cortisol exceeds 700nmol (25g/dL), part of the excess binds to albumin, and a greater amount of cortisol than usual circulates unbound.
Most synthetic glucocorticoid analogues bind less efficiently to CBG ( 70% binding). This may explain the propensity of synthetic analogues to produce cushingoid effects at low doses. Cortisol metabolites are biologically inactive and bind only weak to circulating plasma proteins.
The daily secretion of cortisol ranges between 40 and 80mol. The plasma concentration is determined by the rate of secretion, inactivation and excretion of free cortisol. The liver is the main organ responsible for steroid inactivation. The major enzyme regulating cortisol metabolism is 11-hydroxysteroid dehydrogenase (11-HSD). There are two isoforms:11-HSD I is primarily expressed in the liver and acts as a reductase, converting the inactive cortisone to the active glucocorticoid, cortisol. The 11-HSD II isoform is expressed in many tissues and converts the active cortisol to the inactive metabolite cortisone.

Cortisol has anti-inflammatory properties, which are probably related to effects on microvasculature and to suppression of inflammatory cytokines. GC cause decrease in circulating eosinophils and lymphoid tissue, especially T cells by promoting redistribution from circulation into tissue compartments. Thus cortisol impairs cell-mediated immunity. In addition to inhibition of T-cell mediated immunity, GCs inhibit production of lymphokines and prostaglandins, which are mediators of inflammation cascade. GCs inhibit the production and action of interferon by t-lymphocytes and production of IL-2 by T-lymphocytes and production of IL-1 and IL-6 by macrophages. The antipyretic action of GC is believed to be due to its effect on IL-1, which is an endogenous pyrogen.
Cortisol levels respond to physical (trauma, surgery, exercise), psychological (anxiety, depression) and physiological (hypoglycemia, fever) stresses within minutes. The reason why elevated glucose level protects an organism is not clearly understood, however it is believed that in conditions of GC deficiency such stress might lead to hypotension, shock and death.
Corticosteroids exhibit a wide range of effects on almost every phase of the immune and inflammatory responses in animals and humans. Corticosteroids dosed in various pharmacological levels play a major role in treating different diseases.

Immunosuppression: Mechanism of Action of Corticosteroids
Corticosteroids (CS) interrupt multiple steps in the activation of the immune system. This interruption is as a result of omnipresence of glucocorticoid receptors throughout the body. CSs inhibit antigen presentation, cytokine production, and proliferation of lymphocytes. CSs have a profound effect on the concentration of peripheral blood leuckocytes. Lymphocyte, monocyte, and basophil counts decrease in response to corticosteroid administration, while neutrophil counts increases. It is believed that CSs exert these effects by binding on the glucocorticoid receptor at the cytoplasm. After they bind to the receptor, the steroid-receptor complex is formed and it translocates into the nucleus of the cell, where it attaches to DNA and causes transcription of specific messenger RNA, which produces synthesis of proteins that mediate glucocorticoid activity.

The Lymphocyte Effects:
A single dose of CS produces lymphocytopenia within 4 hours. The peripheral lymphocyte count returns to normal within 24 to 48 hours. CS-induced lymphocytopenia occurs as a result of redistribution of circulating lymphocytes into other lymphoid compartments (e.g., lymph nodes, spleen, thoracic ducts and bone marrow).

The Monocyte Effect
Monocytes(macrophages) play a major role in the induction and regulation of immune activity. Macrophages are involved in the presentation of antigens to lymphocytes and in the subsequent removal of immune complexes. Therefore pharmacologic effects of CS on these cell directly and indirectly impair the immune responses. CSs cause a profound depletion of monocytes. CS-induced monocytopenia also appears to be due to inhibition if inflammation by blocking responses to chemotactic factors and macrophage activation factor, phygocytosis, pyrogen production, and secretion of collagenase, elastase and plasminogen activator. In addition CS induce monocytopenia due to the redistribution phenomenon.

The Neutrophil Effects:
CSs induce neutrophillia by increasing the neutrophil count by 2000 to 5000 cells/mm3. This increases causes an accelerated release of neutrophils from the bone marrow into the circulation and a reduction in the migration of neutrophils out of the circulation. CS also inhibit the ability of neutrophils to adhere to vessel walls, which is an essential step in the migration of cells from the circulation into the tissue. The net effect of these phenomena is reduced number of neutrophils available to accumulate at the inflammatory site.

The Eosinophil Effect:
CS cause a profound eosinophilia manifested by a decrease in the eosinophil count to less than 25 cells/mm3. CS-induced eosinophilia is also caused by redistribution phenomenon. Chemotaxis is also affected and may result from the inhibition of responses to chemotactic factors.

Other Effects:
CSs have a negative effect on prostaglandin, probably as a result of reduction in the fatty acid precursor necessary for prostaglandin (PG) production. CSs block production of IL-1, which normally stimulates PG synthesis. Another effect of CSs is the inhibition of T-cell growth factor or IL-2. IL-2 is produced by activated T-cells and promotes proliferation of other T-cells. Therefore, T-cells under the influence of CS lose their ability to proliferate and react to antigens. Antibody production is not suppressed commonly at conventional doses in humans, however, high doses of CSs guven for long periods of time may lead to a decreased in antibody formation, particularly IgG.

Insights into the Mechanism of Glucocorticosteroid – Induced Apoptosis

Apoptosis is a form of programmed cell death that occurs under numerous developmental and physiological conditions that require the selective elimination of cells from tissues and organs without the production of an inflammatory response. The initiation of apoptosis is controlled by a regulation of the balance between death and life signals perceived by the cell. A typical response of cells to an apoptotic stimulus includes a reduction in cell volume, compaction of intracellular organelles, chromatin condensation, and the generation of apoptotic bodies which contain degraded cellular components. Apoptotic bodies are often engulfed by neighboring cells or macrophages, preventing the occurrence of an inflammatory response in the region of the dying cells. Although the molecular basis for this cellular suicide is poorly understood, evidence indicates that apoptosis is an active process, requiring energy for its effective completion.
One of the earliest models to gain insight into the regulation of the biochemical mechanism of apoptosis has been the death of lymphocytes by gluocortiocsteroids. These hormones as mentioned above are essential for to life and they also have a antiproliferative effects in many cell types. The effects of glucocorticosteroids on lymphoid cells are dramatic and include the induction of the G1 cell cycle arrest and the programmed cell death of immature thymocytes, pre B lymphoma cells, mature peripheral T lymphocytes and several leukemic cell lines. This has lead to their extensive use and efficacy in the treatment of corresponding leukemias and lymphomas.
The biological effects of GCs are mediated by the GR, a ligand-binding transcription factor, which upon activation can regulate the transcription of responsive genes. Despite what is known about the effects of GCs on tissues of the body and their mediation by the GR, the molecular mechanisms leading to GC-induced apoptosis remains poorly understood. What does seem clear is that functional GR is required for the process, and that the signal transduction pathway is initiated by binding of GC to GR. Furthermore, the final steps of GC-induced apoptosis have shown to involve the loss of mitochondrial potential and the subsequent activation of capsases. It is theorized that GR may transmit the death signal through activation of supposed lysis genes, repression of gene expression required for proliferation and growth or perhaps by both mechanism.
GC induced apoptosis has been under extensive scrutiny for the past 20 –40 years, however there are still contradicting evidences on the induction of apoptosis. GC induced apoptosis is divided into three stages, which are the initiation stage, the decision making stage and the execution stage.

1. Initiation stage:
In the initiation stage of CS-induced apoptosis several candidate genes that are up or down regulated in response to GC have been identified. However, it is not still clear whether repression or expression of certain genes by GC to be responsible for induction of programmed cell death.

Repression Hypothesis:
The repression hypothesis states that activation protein –1 transcription factor regulates expression of genes involved in cell growth, differentiation and transformation. This finding has lead to the suggestion that interference with transcription factors (such as AP-1) is required for cell survival and may play a role in induction of apoptosis. The repression of AP-1 transcription factor activity in corticosteroids -treated lymphocytes is accompanied by repression of other pro-survival transcription factors including the nuclear factor of activated T- lymphocytes (NFAT). It is believed that the GC induced decrease in NFAT is due to decrease in AP-1 activity. Additionally, repression of the oncogene c-myc following treatment by GC has been demonstrated in T-lymphocytes suggesting that c-myc down regulation may be directly involved in the inhibition of cellular proliferation and in the apoptosis of lymphocytes. In other words, over expression of c-myc can inhibit GC-induced apoptosis.

Expression Hypothesis:
Some studies suggest that the activation GR is required to mediate CS-induced apoptosis. It was observed that mutation of GR gene lead to failure in mediating CS-induced apoptosis, indicating that activation function of the GR is essential in GC-induced apoptosis.

2. Decision Stage:
Proteosome is a malticatalytic protease complex located in the cytoplasm and the nucleus that degrades proteins targeted for destruction by polyubiquitination. Although not much is known about the role of proteosome, it is believed that it is involved in apoptosis. In laboratory studies it was shown that proteosome inhibitors block CS induced cleavage of poly ADP ribose polymerase, which is a downstream target of caspase and apoptosis in thymocytes. This finding has lead to the conclusion that the proteosome may either degrade regulatory proteins that normally inhibit the apoptotic pathway, or may proteolytically activate proteins that promote cell death.
Factors produced by the thylamic cells suppress GC induced apoptosis. Specifically, INF- and IL-6 inhibit GC-induced apoptosis in plasma and myeloma cells. IL- 4, IL-6 and IL-9 block apoptosis induced by GC in thymocytes and thymoma cells and IL-15 inhibits GC-induced apoptosis in activated T and B cells. Also insulin like growth factor protects mylemos cells from GC-induced cell death. Based on this finding one can say that cytokines do counteract GC induction of apoptosis. It is thought that the mechanism of action of interleukins in counterinteracting GC induced apoptosis could be by increasing expression of survival genes.
The balance between prosurvival and prodeath signal is based in NF-B. The NF-B transcription factor family required for expression of many cytokines and the immunosuppressive action of GC is mediated in part through interference with NF-B activity and inhibition of the cytokines production.
Bottom line: Cytokines increase AP-1 and NF-B activities. Cytokines (IL-2, 4, 6,9, 15) counteract GC-induced apoptosis by inducing expression of transcription factors that regulate genes involved in cell proliferation. Likewise, GC can induce expression of IB, which inhibits the function of NF-B, a transcription factor necessary for the prosurvival cytokines. It is also hypothesized that GC might directly repress NF-B activity independent of IB.
Based on these mechanisms, the counterbalance of the GC and the NF-B effects in induction and inhibition of cytokines may play a key role in the prosurvival or prodeath processe.

3. Execution stage:
Apoptosis has been shown to occur in numerous cell types and in response to a variety of signals; the execution of cell death is uniformly mediated through the activation of caspases. The aspartate-specific cysteine protease is central to the apoptotic machinery, which is conserved from nematodes to mammals. Caspases are comprised of three domains: an amino terminal prodomain, a large subunit and a small subunit. Caspases are characterized based on the length of their domains. Caspases 2, 8,9 and 10 are activators, while caspases 3, 6 and 7 are considered effectors. The remaining caspases 1, 4,5,11,12, 13 and 14 are characterized as cytokine processors with majority having long prodomains.
Caspases are grouped into the initiator and effector caspases. Caspase-8 activates effector caspase via mitochondria- dependent or mitochondria independent pathways. In mitochondria dependent pathway, caspase-8 mediates cleavage of Bid. Bid is a proapoptotic enzyme member of the Bcl-2 family. After cleavage by caspase-8, Bid is converted to the active form, which triggers the release of cytochrome C from the mitochondria. Cytochrome C binds APAF-1, there by activating another initiator caspase, caspase-9, which in turn activates effector caspases. In the mitochondria independent pathway, caspase-8 initially activated by the same mechanism, and then directly cleaves and activates caspase-3 bypassing mitochondria and cytochrome C release.
Experiments have indicated that both APAF-1 and caspase-9 are essential for CS-induced apoptosis. This statement was endorsed when mice deficient in either APAF-1 or caspase-9 were found to be resistant to CS-induced apoptosis.
It is important to keep in perspective that caspase activation and caspase-mediated protein cleavage are components of the execution stage of programmed cell death. In the case CS-induced apoptosis critical events proceeding caspase activation determine whether the cell is to live or to die. Works showing that caspase inhibitors block proteolysis of endogenous substrates and reduce nuclear condensation emphasizes this point.
In summary, based on collection of findings, inhibitor studies and caspase assays, the prevailing pathway to CS-induced apoptosis appears to be caspase dependent and caspase-3 independent.

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