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Mycophenolate mofetil (MMF) is widely used to minimise acute rejection following solid organ transplantation as it inhibits inosine monophosphate dehydrogenase (IMPDH) and thereby reduces lymphocyte activation. The effects of MMF and azathioprine on renal allograft outcome were examined by analysis of the national transplant database held at National Health Service (NHS) Blood and Transplant, Stoke Gifford, Bristol, UK. In a paired kidney analysis, MMF treated patients had a 3 year death censored graft survival of 91% (n=217) contrasts to 97% (n=231) in azathioprine treated patients (p=0.07) with an increased acute rejection rate in the first year after transplantation (44 v 31%, n=105 v 74, p<0.01). In a further study, 13% (n=71) of patients were found to be taking less than 1 g of MMF which was associated with a 3-fold increased risk of graft failure and inferior graft function up to 36 months. One strategy to improve graft outcome would entail targeting MMF dose according to pre-transplant IMPDH activity, which is known to display wide variability between patients, in order to maximize efficacy and minimize toxicity. Therefore, it was decided to measure pre-transplant IMPDH activity and to investigate associations with renal allograft outcome and MMF dose tolerated after transplantation. IMPDH activity was measured by detection of generated XMP by a validated HPLC method in the peripheral mononuclear cells of 55 patients waiting for renal transplantation and was found to exhibit a 4-fold variation of IMPDH activity. Black males had significantly increased IMPDH activity contrasts to Black females (p=0.01). Within the first year of transplantation, 71% (n=12) patients required a reduction in MMF dose. There was no association between pre-transplant IMPDH activity and MMF dose achieved at 1 year or MMF associated side effects or eGFR up to 36 months. It was proposed that the inter-individual variability of IMPDH activity may be associated with genetic polymorphisms and therefore sequencing of the exons of IMPDH I and II was undertaken. Two novel single nucleotide polymorphisms (SNPs), Leu244Leu and Ala285Thr, were identified in the IMPDH I gene. Patients with these variants did not exhibit differential IMPDH activity. Genotyping for established intronic SNPs was undertaken in our patient cohort as well as a random sample of 1040 recipients from the Collaborative Transplant Study DNA bank based at the University of Heidelberg, Germany. The presence of these SNPs did not increase the risk of rejection or affect graft function or MMF dose tolerated at 1 year after transplantation and there was no association between pre-transplant IMPDH activity, 5 year graft and patient survival and genotype. In our study, MMF treatment did not result in improved renal allograft outcomes in comparison to azathioprine therapy. Furthermore, we suggest that measurement of pre-transplant IMPDH activity or genotyping of the IMPDH enzymes is unlikely to assist in optimizing MMF dose and renal allograft outcome.

Background. Lupus nephritis (LN) can be complicated with requirement for kidney replacement therapy and death. Efficacy of induction therapies using mycophenolate mofetil (MMF) or intravenous cyclophosphamide (IVCYC) has been reported from studies, but there is limited data in Africans comparing both treatments in patients with proliferative LN. Methods. This was a retrospective study of patients with biopsy-proven proliferative LN diagnosed and treated with either MMF or IVCYC in a single centre in Cape Town, South Africa, over a 5-year period. *e primary outcome was attaining complete remission after completion of induction therapy. Results. Of the 84 patients included, mean age was 29.6 ± 10.4 years and there was a female preponderance (88.1%). At baseline, there were significant differences in estimated glomerular filtration rate (eGFR) and presence of glomerular crescents between both groups (p ≤ 0.05). After completion of induction therapy, there was no significant difference in remission status (76.0% versus 87.5%; p = 0.33) or relapse status (8.1% versus 10.3%; p = 0.22) for the IVCYC and MMF groups, respectively. Mortality rate for the IVCYC group was 5.5 per 10,000 person-days of follow-up compared to 1.5 per 10,000 person-days of follow-up for the MMF group (p = 0.11), and there was no significant difference in infection-related adverse events between both groups. Estimated GFR at baseline was the only predictor of death (OR: 1.0 [0.9–1.0]; p = 0.001). Conclusion. This study shows similar outcomes following induction treatment with MMF or IVCYC in patients with biopsy-proven proliferative LN in South Africa. However, a prospective and randomized study is needed to adequately assess these outcomes.

Bullous pemphigoid (BP) is a common autoimmune blistering disease in the adult population, but extremely rare in the pediatric population. Childhood BP usually has a favorable prognosis and responds well to topical and oral steroids. However, for patients that do not respond to corticosteroids, therapeutic alternatives are scarce. We report a case of a toddler with recalcitrant BP who was successfully treated with mycophenolate mofetil (MMF).

Mesenchymal stem cells (MSC) represent a heterogenous population of nonhematopoietic stem cells with multipotent differential potential. MSC can be isolated from various tissues of organism, the most common tissue are bone marrow or adipose tissue. MSC are good candidates for treatment of autoimmune diseases and possess the ability to prevent graft rejection or graft versus host disease. The immunosuppressive drugs are currently used for inhibition of unwanted immune reaction but they exhibit serious side effects. The use of MSC in therapy can reduce doses of immunosuppressive drugs and eliminate side effects. The study of MSC and immunosuppressant interactions should be detected before MSC can be used for clinical application. We aimed to analyze the interaction between MSC and immunosuppressive drugs and their effect on immune cells. Cyclosporine A and mycophenolate mofetil were used in our research. We demonstrated changes in the expression of surface molecules, production of interleukin 6 and in metabolic activity of MSC after treatment with immunosuppressive drugs. MSC are in organism, in cooperation with the number of other cells. Therefore we studied MSC cocultured with splenocytes in the presence of immunosuppressive drugs. Our results show the effect of MSC and immunosuppressive drugs on different...

The use of lung transplantation, a life saving intervention, has been increasing over the last thirty years with a disappointing median survival of only 4.8 years. Despite the progress made in immunosuppressive therapies, allograft rejection following transplantation is the leading cause of death. As part of the immunosuppressive therapy, mycophenolate mofetil (MMF), the ester prodrug of mycophenolic acid (MPA) has proven its efficacy among heart, liver, kidney as well as lung transplanted patients. However, due to its rapid excretion, high daily doses are necessary and lead to serious side effects, forcing the patient to stop and change their course of treatment. Administration of drugs to the lungs is known to minimize local and systemic side effects by employing a lower amount of drug, to increase patient compliance and to improve the efficacy of the treatment. Therefore, developing novel MMF formulations for targeted delivery to the lungs will broaden the therapeutic options against lung transplant rejection. Within the framework of this dissertation, the development of an inhaled formulation of MMF was investigated. MMF must be metabolized by carboxylesterases to become active and its metabolism suffers from high inter- and intra-patient variability. The first objective of this dissertation was to investigate the occurrence of MMF hydrolysis in the lung. The second objective was to study the in vivo deposition,metabolism and distribution in rats, of an inhaled micron-size MMF suspension in comparison to inhaled IV Cellcept® and oral Cellcept®, the currently marketed products. According to the in vitro results, MMF is metabolized in human lung cells by carboxylesterases. The in vivo results showed an incomplete metabolism of MMF when delivered as a suspension due to the limited dissolution of the drug in the lungs. Following inhalation, the MMF suspension achieved higher and more prolonged concentration of the total drug in the lungs and lymphoid tissues as compared to the inhaled IV Cellcept®. The pulmonary delivery of the MMF suspension was able to achieve similar levels of drug in the lungs, higher levels in the lymphoid tissues and significantly lower levels in the systemic circulation when compared to the levels obtained from the oral gavage of oral Cellcept®. Ultimately, this dissertation demonstrated that the administration of micron-size MMF suspension offers a great potential for pulmonary administration. / text

L’immunosuppression optimale après greffe d’organe solide est une balance délicate et propre à chaque individu entre le risque de rejet et les risques liés à une surexposition au traitement immunosuppresseur. L’évaluation de la fonction résiduelle des lymphocytes T après stimulation par un mitogène (pharmacodynamie effective) devrait permettre de mesurer l’effet direct des médicaments immunosuppresseurs sur leur cible. Nous avons étudié différents paramètres de pharmacodynamie effective chez 34 receveurs pédiatriques de greffe d’organes solides traités par tacrolimus et mycophénolate. Les tests proposés dans ce travail sont adaptés au milieu pédiatrique et à une réalisation en temps réel. La quantification du CD25 parmi les CD4 activés par l’OKT3 permet de distinguer deux groupes de patients selon leur degré d’immunosuppression. L’âge médian est plus bas et la concentration plasmatique médiane en MPA plus élevée dans le groupe de patients plus fortement immunosupprimés. L’étude des paramètres immunologiques pouvant influencer la réponse (sécrétion des interleukines, proportion des sous-populations lymphocytaires CD4, CD8, T naïfs et Trég) ainsi que l’étude du pouvoir de restauration de la fonction lymphocytaire par l’Il-2, la guanosine ou la xanthosine, ne permettent pas de mieux comprendre les variabilités interindividuelles observées. Ces résultats devront être confirmés sur une cohorte plus grande de patients afin de juger de leur intérêt en pratique clinique. / Optimal immunosuppression following solid organ transplantation is unique to each individual and requires a balance between risks of rejection and overexposure to immunosuppressive therapy. The evaluation of residual function of T lymphocytes after mitogen stimulation (effective pharmacodynamic monitoring) should allow measurement of the direct effect of immunosuppressive drugs on their target. We studied various parameters of effective pharmacodynamic monitoring in 34 paediatric patients receiving solid organ transplants and treated with tacrolimus and mycophenolate (MPA). The tests proposed in this work are adapted to the paediatric setting in real time. Quantification of CD25 among CD4 cells activated by OKT3 can differentiate two groups of patients according to their degree of immunosuppression. Median values for age and MPA plasma concentration are lower and higher, respectively, in the patient group most heavily immunosuppressed. Neither study of the parameters that may influence the response (secretion of interleukins, proportion of lymphocyte subpopulations CD4, CD8, naive and regulatory T cells) nor study of the restoration of basal cell function brought about by Il2, guanosine or xanthosine, helped to explain the observed inter-individual variability. These results should be confirmed in a larger cohort of patients in order to test their relevance in clinical practice.

Da die Arzneistoffpermeation durch okulare Gewebe einen entscheidenden Einfluss auf die Heilung vieler Augenleiden hat, wurde die in vitro-Permeation hydrophiler und lipophiler Arzneistoffe durch okulare Gewebe und Zellkulturen in dieser Arbeit untersucht. Die Dissertation befasst sich vorrangig mit der Permeation des hydrophilen Modellarzneistoffs Pilocarpinhydrochlorid (P-HCl) durch isolierte Schweinecornea (SC), Schweinesklera, Kaninchenkonjunktiva und corneale bzw. konjunktivale Kaninchenepithelzellkultur. Der Einfluss verschiedener Formulierungsparameter wie Benzalkoniumchlorid (BAC), Natriumedetat, pH-Wert und Tonizität auf die P-HCl-Permeation wurde untersucht. Dabei konnte eine gute Korrelation zwischen isolierten Geweben und Zellkulturen in Reaktion auf die Variation der Formulierungsparameter festgestellt werden. Unter den getesteten Parametern zeigte BAC den größten Enhancereffekt. Weiterhin wurden vergleichende Permeationsstudien an gelaserter SC mit P-HCl und dem relativ lipophilen Diclofenac-Natrium (D-Na) durchgeführt. Das Entfernen von Epithelschichten der SC mittels Excimer-Laser sollte den Heilungsverlauf, vor allem aber die zeitabhängige Reepithelisierung der Cornea nach erfolgter Photorefraktiver Keratektomie, simulieren. Die Ergebnisse dieser Studie zeigten, dass unterschiedliche Epitheldicken einen signifikanten Einfluss auf die P-HCl-Permeation haben. Im Gegensatz dazu blieb die D-Na-Permeation nahezu unbeeinflusst. Ein weiteres Anliegen dieser Arbeit bestand darin, eine okular applizierbare Formulierung des Immunsuppressivums Mycophenolatmofetil (MMF) zu entwickeln. Sowohl für das Prodrug MMF als auch für dessen aktiven Metaboliten Mycophenolsäure (MPA) wurde die Permeabilität von SC getestet. Ausgewählt wurde eine Zubereitung, die 1% MMF in Glutathion-Bicarbonat-Ringerlösung enthält und mit 10% Hydroxypropyl-beta-Cyclodextrin versetzt ist. Diese Suspension wurde bei 121° C und 200 kPa für 15 min autoklaviert, um das schwerlösliche MMF in Lösung zu bringen. Da der Ester MMF bei der Herstellung der Testlösung einer teilweisen Hydrolyse zu MPA unterliegt, außerdem eine minimale in vitro-Verfügbarkeit aufweist und sehr schwer wasserlöslich ist, sollte MPA in Augentropfenformulierungen der Vorzug gegeben werden. Die Korrelierbarkeit mit in vivo-Resultaten ist jedoch im Rahmen dieser Arbeit nicht untersucht worden, so dass die Ergebnisse als Grundlage für Permeationsstudien am in vivo-Modell zu bewerten sind. / The permeation of drugs through ocular tissues plays an important role in healing of various eye diseases. The objective of this work was to investigate the in vitro permeability of hydrophilic and lipophilic drugs through ocular tissues and cell cultures. Mainly, the permeability of the hydrophilic model drug pilocarpine hydrochloride (P-HCl) through isolated pig cornea (PCr) and sclera, rabbit conjunctiva, and rabbit conjunctival or corneal epithelial cell culture was compared. Additionally, the study included investigations about the influence of the formulation parameters benzalkonium chloride (BAC), ethylene diamine tetra acetic acid disodium salt, pH value and tonicity on the permeability of the small drug. In summary, a good correlation between the isolated tissues and cell cultures with regard to P-HCl transport could be observed. In general, BAC caused the most facilitated drug transport within the formulation parameters. Furthermore, the permeation of P-HCl and the lipophilic diclofenac-sodium (D-Na) through lasered PCr was studied. To investigate the effects of photorefractive keratectomy on drug permeation, excimer laser ablations with varying depths were performed on isolated pig eyes. As a result, P-HCl demonstrated a significant enhancement of permeation in relation to the ablation depth. In contrast, corneal epithelial thickness scarcely influenced the permeation rate of D-Na. Another aim of this work was to develop an appropriate application form for topical ocular use of the immunomodulating substance mycophenolate mofetil (MMF) and to investigate the in vitro permeability of PCr for the prodrug MMF and its parent substance mycophenolic acid (MPA). The test formulation consisted of Glutathion-bicarbonate-Ringer's solution, 10% hydroxypropyl- beta-cyclodextrin and 1% MMF. To reach a concentration of 1% of the poor soluble MMF a treatment under autoclaving conditions at 121° C and 200 kPa for 15 min was needed. MPA should be preferred in eye drops because of an higher in vitro availability compared to MMF, an hydrolysis of MMF to MPA in the cornea during permeation and the poor water solubility of MMF. The correlation with in vivo results was not studied in this work but the findings can be assumed as basis for investigations on in vivo models.

Zusammenfassung der Habilitationsschrift: Untersuchungen zum Einfluss von Mycophenolat Mofetil auf die Transplantat-Vaskulopathie nach allogener Aorten-Transplantation im Primaten-Modell Ein Hauptmerkmal der chronischen Rejektion nach allogener Organtransplantation ist die Transplantat-Vaskulopathie. Durch eine konzentrische Intimahyperplasie in den Arterien und Arteriolen des Transplantates, hervorgerufen durch eine Proliferation von glatten Muskelzellen und Fibroblasten, kommt es zu einer Minderperfusion des Organs und letztendlich zu einem chronisch fortschreitenden Transplantatversagen. Mycophenolat Mofetil (MMF) zeigt neben seiner Eigenschaft akute Rejektionen zu verhindern, auch eine antiproliferative Wirksamkeit auf glatte Muskelzellen, die eine Schlüsselrolle in der Entwicklung der chronischen Rejektion spielen. In mehreren Tierexperimenten konnte im Rattenmodell gezeigt werden, dass MMF die Entwicklung der Intimaproliferation hemmen kann. In der hier vorgestellten Studie wurde nun der Effekt von MMF auf eine fortgeschrittene, bereits etablierte Transplantat-Vaskulopathie im Primatenmodell geprüft. Nachdem in mehreren Vorstudien die Dosis, das Dosierungsintervall und der Applikationsweg getestet wurde, konnte MMF in einer maximal tolerierten Dosis Cynomolgus Affen verabreicht werden. Diese Tiere dienten jeweils als Spender und Empfänger eines 3 cm langen, infrarenalen Aortensegmentes. Um sicher zu stellen, dass sich eine Transplantat-Vaskulopathie etablieren konnte, erhielten die Tiere in den ersten 6 Wochen nach Transplantation keinerlei Immunsuppression. Erst ab Tag 45 wurde mit der MMF Therapie begonnen. Die Entwicklung der Intimahyperplasie wurde mit intravaskulären Ultraschalluntersuchungen dokumentiert und mit einer unbehandelten Kontrollgruppe verglichen. Während sich in der Kontrollgruppe die Intimahyperplasie ungebremst entwickelte, kam es in der Therapiegruppe zu einer Verlangsamung des Intimawachstums. Auch wenn der Unterschied zwischen den Gruppen am Versuchsende nicht signifikant war, so zeigte sich eine hohe Korrelation zwischen der verabreichten MMF Dosis und der sich entwickelten Transplantat-Vaskulopathie: Tiere, welche die MMF Therapie gut tolerierten, zeigten eine signifikant geringer Intimahyperplasie als Tiere der Kontrollgruppe. Bei den Tieren, bei denen die MMF Dosis aufgrund von Nebenwirkungen reduziert werden musste, entwickelte sich die Intimaproliferation ungehindert. Ferner konnte gezeigt werden, dass pharmakodynamische Messungen, welche die unterschiedlichen Medikamenten-Sensibilität der Tiere widerspiegelten, ebenfalls mit der Transplantat-Vaskulopathie korrelierten. / Evaluation of the influence of mycophenolate mofetil on graft vascular disease after allogenic aortic transplantation in non-human primates Graft vascular disease is pathognomonic for chronic rejection after solid organ transplantation. By inhibiting smooth muscle cell proliferation Mycophenolate Mofetil (MMF) has theoretically a beneficial effect on graft vascular disease and in rodent models MMF was able to halt graft vascular disease progress. To evaluate the efficacy of MMF on advanced graft vascular disease, a study was performed in non-human primates. Aortic allografts were exchanged between MLR mismatched, blood group compatible cynomolgus monkeys. 6 control animals received no immunosuppression, 6 animals were treated with MMF from day 45 after transplantation on in an individual maximal tolerated dose, which was determined in elaborative pre-studies in rodents and non-human primates. Until day 45 the animals did not receive any immunosuppressive treatment. The progression of graft vascular disease was quantified by intravascular ultrasound as changes in intimal area in the midsegments of all grafts every 3 weeks until day 105 when the animals were euthanized and the grafts have been harvested for histopathological analysis. Pharmacokinetik and pharmacodynamic monitoring was used to optimize the immunosuppressive efficacy. While in grafts from the control animals intimal hyperplasia developed unhindered, the increase of intimal areas over time was attenuated in the treatment group. Although this effect was not statistically significant, there was a high correlation between the daily MMF dose administered and the intimal proliferation in the treated animals. Animals which tolerated high doses of MMF showed significant lower graft vascular disease than animals of the control group. In two animals with MMF toxicity and dose reduction, high intimal hyperplasia was observed. In this demanding model evaluating advanced graft vascular disease in non-human primates MMF was able to halt GVD when given in a high maximal tolerated dose. In case of toxicity and individual necessary dose reduction, progress of GVD was not altered.

A sobrecarga de proteínas nas células tubulares proximais pode levar a lesão intersticial por mecanismos não claros que podem envolver a ativação da imunidade inata. Nós investigamos a hipótese de que a exposição prolongada de células tubulares a altas concentrações de proteínas estimula a imunidade inata, desencadeando inflamação intersticial progressiva e lesão renal. Além disso, investigamos se a inibição específica da imunidade inata ou adaptativa proporcionaria renoproteção em um modelo estabelecido de proteinúria maciça, nefropatia por adriamicina (ADR). Os ratos adultos Munich-Wistar receberam uma dose única de ADR (5 mg / kg iv), sendo acompanhados por 2, 4 e 20 semanas. A albuminúria maciça foi associada à ativação precoce das vias da imunidade inata NF-?B e NLRP3, cuja intensidade correlacionou-se fortemente com a densidade da infiltração de linfócitos. Além disso, os ratos ADR exibiram sinais claros de estresse oxidativo renal. Vinte semanas após a administração de ADR, observaram-se fibrose intersticial intensa, glomerulosclerose e perda da função renal. A administração de micofenolato de mofetil (MMF), 10 mg / Kg / dia, impediu a ativação da imunidade inata e adaptativa, bem como do estresse oxidativo renal e fibrose renal. Além disso, o tratamento MMF foi associado com a mudança de MØ do tipo M1 para o fenótipo M2. Nas células cultivadas de NRK52-E, o excesso de albumina aumentou o teor de proteína de TLR4, NLRP3, Caspase-1, IL6, IL1-beta, MCP-1, alfa-SMA e COLL-1. O silenciamento do TLR4 e / ou NLRP3 mRNA atenuou esse comportamento proinflamatório / profibrótico. A ativação simultânea de imunidade inata e adaptativa podem ser fundamentais para o desenvolvimento de lesão renal em doenças altamente proteinúricas. A inibição da imunidade inata e/ou adaptativa podem constituir uma estratégia para prevenir a DRC nesse contexto / Protein overload of proximal tubular cells can promote interstitial injury by unclear mechanisms that may involve activation of innate immunity. We investigated the hypothesis that prolonged exposure of tubular cells to high protein concentrations stimulates innate immunity, triggering progressive interstitial inflammation and renal injury. In addition, we investigated whether specific inhibition of innate or adaptive immunity would provide renoprotection in an established model of massive proteinuria, adriamycin (ADR) nephropathy. Adult male Munich-Wistar rats received a single dose of ADR (5 mg/kg iv), being followed for 2, 4 or 20 weeks. Massive albuminuria was associated with early activation of both the NF-kB and NLRP3 innate immunity pathways, whose intensity correlated strongly with the density of lymphocyte infiltration. In addition, ADR rats exhibited clear signs of renal oxidative stress. Twenty weeks after ADR administration, marked interstitial fibrosis, glomerulosclerosis and renal functional loss were observed. Administration of mycophenolate mofetil (MMF), 10 mg/Kg/day, prevented activation of both innate and adaptive immunity, as well as renal oxidative stress and renal fibrosis. Moreover, MMF treatment was associated with shifting of M0 from the M1 to the M2 phenotype. In cultivated NRK52-E cells, excess albumin increased the protein content of TLR4, NLRP3, Caspase-1, IL6, IL- 1beta, MCP-1, alpha-actin and collagen-1. Silencing of TLR4 and/or NLRP3 mRNA abrogated this proinflammatory/profibrotic behavior. Simultaneous activation of innate and adaptive immunity may be key to the development of renal injury in heavily proteinuric disease. Inhibition of innate and/or adaptive immunity may constitute a strategy to prevent CKD in this setting