Structural studies on enzymes of biotechnological and biomedical interest

Structural studies of proteins aim at elucidating the atomic details of molecular interactions in biological processes of living organisms. These studies are particularly important in understanding structure, function and evolution of proteins and in defining their roles in complex biological settings. Furthermore, structural studies can be used for the development of novel properties in biomolecules of environmental, industrial and medical importance. X-ray crystallography is an invaluable tool to obtain accurate and precise information about the structure of proteins at the atomic level. Glutathione transferases (GSTs) are amongst the most versatile enzymes in nature. They are able to catalyze a wide variety of conjugation reactions between glutathione (GSH) and non-polar components containing an electrophilic carbon, nitrogen or sulphur atom. Plant GSTs from the Tau class (a poorly characterized class) play an important role in the detoxification of xenobiotics and stress tolerance. Structural studies were performed on a Tau class fluorodifen-inducible glutathione transferase from Glycine max (GmGSTU4-4) complexed with GSH (2.7 Å) and a product analogue Nb-GSH (1.7 Å). The three-dimensional structure of the GmGSTU4-4-GSH complex revealed that GSH binds in different conformations in the two subunits of the dimer: in an ionized form in one subunit and a non-ionized form in the second subunit. Only the ionized form of the substrate may lead to the formation of a catalytically competent complex. Structural comparison between the GSH and Nb-GSH bound complexes revealed significant differences with respect to the hydrogen-bonding, electrostatic interaction pattern, the upper part of -helix H4 and the C-terminus of the enzyme. These differences indicate an intrasubunit modulation between the G-and Hsites suggesting an induced-fit mechanism of xenobiotic substrate binding. A novel binding site on the surface of the enzyme was also revealed. Bacterial type-II L-asparaginases are used in the treatment of haematopoietic diseases such as acute lymphoblastic leukaemia (ALL) and lymphomas due to their ability to catalyze the conversion of L-asparagine to L-aspartate and ammonia. Escherichia coli and Erwinia chrysanthemi asparaginases are employed for the treatment of ALL for over 30 years. However, serious side-effects affecting the liver and pancreas have been observed due to the intrinsic glutaminase activity of the administered enzymes. Structural studies on Helicobacter pylori L-asparaginase (HpA) were carried out in an effort to discover novel L-asparaginases with potential chemotherapeutic utility in ALL treatment. Detailed analysis of the active site geometry revealed structurally significant differences between HpA and other Lasparaginases that may be important for the biological activities of the enzyme and could be further exploited in protein engineering efforts.

Obstrução intestinal após uso de infliximab no tratamento de fístula êntero-cutânea na doença de Crohn

Crohn's disease is often complicated by the development of fistulas. Infliximab, a monoclonal antibody that binds tumor necrosis factor a have shown to be successful in the treatment of fistulizing Crohn's disease. It's possible complications and side effects have not been completely elucidated. Our objective is to report a case of a patient who developed intestinal obstruction after treatment of fistulizing Crohn's disease with infliximab. A 50 years-old female with Crohn's disease presented with spontaneous enterocutaneous fistula. She was successfully treated with the infusion of 5mg/kg infliximab at weeks 0, 2, and 6, with complete closure of the fistula after the first infusion. Eight weeks after treatment she developed small bowel obstruction secondary to stenosis of the ileum. She was subjected to exploratory laparotomy and resection of the stenotic ileum. The patient had good recovery, with no complications, and was discharged on the 5th postoperative day. Although a faster and complete healing of enterocutaneous fistula was induced by infliximab, this treatment may have caused intestinal obstruction in this case.

Internet-based breast cancer patient’s pathway as an empowering patient educational tool

This three-phase study was conducted to examine the effect of the Breast Cancer Patient’s Pathway program (BCPP) on breast cancer patients’ empowering process from the viewpoint of the difference between knowledge expectations and perceptions of received knowledge, knowledge level, quality of life, anxiety and treatment-related side effects during the breast cancer treatment process. The BCPP is an Internet-based patient education tool describing a flow chart of the patient pathway during the breast treatment process, from breast cancer diagnostic tests to the follow-up after treatments. The ultimate goal of this study was to evaluate the effect of the BCPP to the breast cancer patient’s empowerment by using the patient pathway as a patient education tool. In phase I, a systematic literature review was carried out to chart the solutions and outcomes of Internet-based educational programs for breast cancer patients. In phase II, a Delphi study was conducted to evaluate the usability of web pages and adequacy of their content. In phase III, the BCPP program was piloted with 10 patients and patients were randomised to an intervention group (n=50) and control group (n=48). According to the results of this study, the Internet is an effective patient education tool for increasing knowledge, and BCPP can be used as a patient education method supporting other education methods. However, breast cancer patients’ perceptions of received knowledge were not fulfilled; their knowledge expectations exceed the perceived amount of received knowledge. Although control group patients’ knowledge expectations were met better with the knowledge they received in hospital compared to the patients in the intervention group, no statistical differences were found between the groups in terms of quality of life, anxiety and treatment-related side effects. However, anxiety decreased faster in the intervention group when looking at internal differences between the groups at different measurement times. In the intervention group the relationship between the difference between knowledge expectations and perceptions of received knowledge correlated significantly with quality of life and anxiety. Their knowledge level was also significant higher than in the control group. These results support the theory that the empowering process requires patient’s awareness of knowledge expectations and perceptions of received knowledge. There is a need to develop patient education to meet patients’ perceptions of received knowledge, including oral and written education and BCPP, to fulfil patient’s knowledge expectations and facilitate the empowering process. Further research is needed on the process of cognitive empowerment with breast cancer patients. There is a need for new patient education methods to increase breast cancer patients’ awareness of knowing.

Treatment of low-risk gestational trophoblastic neoplasia comparing biweekly eight-day Methotrexate with folinic acid versus bolus-dose Actinomycin-D, among Brazilian women

PURPOSE: To compare two single-agent chemotherapy (ChT) regimens evaluating, in first-line treatment, response and side effects and, in final single-agent treatment, the outcomes, among Brazilian patients with low-risk gestational trophoblastic neoplasia (GTN), according to International Federation of Gynecology and Obstetrics (FIGO) 2002. METHODS: Retrospective analysis of two concurrent cohorts with 194 low-risk GTN patients: from 1992 to 2012, as first-line treatment, 115 patients received 4 intramuscular doses of methotrexate alternated with 4 oral doses of folinic acid (MTX/FA) repetead every 14 days and, since 1996, 79 patients received an endovenous bolus-dose of actinomycin D (Act-D), biweekly. At GTN diagnosis, patient opinion was taken into consideration when defining the initial single-agent ChT regimen, and when there was resistance or toxicity to one regimen, the other drug was used preferentially. This study was approved by the Irmandade da Santa Casa de Misericórdia de Porto Alegre Ethical Committee. RESULTS: Both groups were clinically similar (p>0.05). In first-line treatments, frequency of complete response was similar (75.7% with MTX/FA and 67.1% with bolus Act-D); the number of ChT courses -median 3 (range: 1-10) with MTX/FA and 2 (range: 1-6) with bolus Act-D - and the time to remission -median 9 weeks (range: 2-16) with MTX/FA and 10 weeks (range: 2-16) with bolus Act-D) - were not different between the groups. In both groups, first-line side effects frequency were high but intensity was low; stomatitis was higher with MTX/FA (p<0.01) and nausea and vomit with Act-D (p<0.01). Final single-agent ChT responses were high in both groups (94.8% with MTX/FA and 83.5% with bolus Act-D; p<0.01) and 13% higher in the group initially treated with MTX/FA. Rates of hysterectomy and of GTN recurrence were low and similar. No patient died due to GTN. CONCLUSION: The two regimens had similar first-line ChT response. Final single-agent response rates were high and similar in both groups but the final single-agent remission rate was higher in the MTX/FA group.

Alueellinen energiatehokkuusmalli

Energian kulutus ja siitä aiheutuvat haittavaikutukset lisääntyvät globaalisti, minkä johdosta tarve uusille toimintamalleille ja ajatustavoille on merkittävä. Yksi mahdollisuus vastata energian kulutuksen kasvuun on lisätä energiatehokkuutta. Energiatehokkuudella voidaan vähentää energian käytöstä aiheutuvia päästöjä kustannustehokkaasti. Tässä työssä tarkastellaan energiatehokkuutta ja sen lisäämistä alueellisella tasolla. Työssä käsitellään menetelmiä ja keinoja alueellisen energiatehokkuuden lisäämiseksi esimerkiksi kaupungeissa ja yhteisöissä. Työssä esitetään alueellisen energiatehokkuuden arviointimalli, jolla voidaan määrittää alueellisen energiatehokkuuden toteutumista ja energiaratkaisujen kannattavuutta ympäristön kannalta. Malli sisältää matemaattisesti määritettäviä tunnuslukuja, jotka kuvaavat ympäristövaikutuksia. Mallia sovelletaan Lohjan alue-tarkasteluun, jossa mallin pohjalta esitetään vaihtoehtoja energiatehokkuuden lisäämiseksi. Voidaan todeta, että arviointimalli on käyttökelpoinen ja sillä voidaan havainnol-listaa alueellista energian käyttöä ja sen energiatehokkuutta. On kuitenkin huomattava, että malli huomioi lähinnä vain ympäristövaikutuksia ja sisältää virheläh-teitä johtuen esimerkiksi käytettävissä olevan tiedon määrästä.

Complement activation-related pseudoallergy in dogs following intravenous administration of a liposomal formulation of meglumine antimoniate

The increasing use of nanotechnologies in advanced therapies has allowed the observation of specific adverse reactions related to nanostructures. The toxicity of a novel liposome formulation of meglumine antimoniate in dogs with visceral leishmaniasis after single dose has been investigated. Groups of 12 animals received by the intravenous route a single dose of liposomal meglumine antimoniate (group I [GI], 6.5 mg Sb/kg), empty liposomes (GII) or isotonic saline (GIII). Evaluation of hematological and biochemical parameters showed no significant changes 4 days after administration. No undesired effects were registered in the GIII. However, adverse reactions were observed in 67.7% of dogs from both groups that received liposomal formulations. The side effects began moments after bolus administration and disappeared during the first 15 minutes after treatment. Prostation, sialorrhea and defecation were the most frequent clinical signs, registered in 33.3% and 41.6 % of animals from the groups GI and GII, respectively. Tachypnea, mydriasis, miosis, vomiting and cyanosis were also registered in both groups. The adverse reactions observed in this study were attributed to the activation of the complement system by lipid vesicles in a phenomenon known as Complement Activation-Related Pseudoallergy (CARPA). The influence of the physical-chemical characteristics of liposomal formulation in the triggering of CARPA is discussed.

Influence of surface functionalization on the behavior of silica nanoparticles in biological systems

Personalized nanomedicine has been shown to provide advantages over traditional clinical imaging, diagnosis, and conventional medical treatment. Using nanoparticles can enhance and clarify the clinical targeting and imaging, and lead them exactly to the place in the body that is the goal of treatment. At the same time, one can reduce the side effects that usually occur in the parts of the body that are not targets for treatment. Nanoparticles are of a size that can penetrate into cells. Their surface functionalization offers a way to increase their sensitivity when detecting target molecules. In addition, it increases the potential for flexibility in particle design, their therapeutic function, and variation possibilities in diagnostics. Mesoporous nanoparticles of amorphous silica have attractive physical and chemical characteristics such as particle morphology, controllable pore size, and high surface area and pore volume. Additionally, the surface functionalization of silica nanoparticles is relatively straightforward, which enables optimization of the interaction between the particles and the biological system. The main goal of this study was to prepare traceable and targetable silica nanoparticles for medical applications with a special focus on particle dispersion stability, biocompatibility, and targeting capabilities. Nanoparticle properties are highly particle-size dependent and a good dispersion stability is a prerequisite for active therapeutic and diagnostic agents. In the study it was shown that traceable streptavidin-conjugated silica nanoparticles which exhibit a good dispersibility could be obtained by the suitable choice of a proper surface functionalization route. Theranostic nanoparticles should exhibit sufficient hydrolytic stability to effectively carry the medicine to the target cells after which they should disintegrate and dissolve. Furthermore, the surface groups should stay at the particle surface until the particle has been internalized by the cell in order to optimize cell specificity. Model particles with fluorescently-labeled regions were tested in vitro using light microscopy and image processing technology, which allowed a detailed study of the disintegration and dissolution process. The study showed that nanoparticles degrade more slowly outside, as compared to inside the cell. The main advantage of theranostic agents is their successful targeting in vitro and in vivo. Non-porous nanoparticles using monoclonal antibodies as guiding ligands were tested in vitro in order to follow their targeting ability and internalization. In addition to the targeting that was found successful, a specific internalization route for the particles could be detected. In the last part of the study, the objective was to clarify the feasibility of traceable mesoporous silica nanoparticles, loaded with a hydrophobic cancer drug, being applied for targeted drug delivery in vitro and in vivo. Particles were provided with a small molecular targeting ligand. In the study a significantly higher therapeutic effect could be achieved with nanoparticles compared to free drug. The nanoparticles were biocompatible and stayed in the tumor for a longer time than a free medicine did, before being eliminated by renal excretion. Overall, the results showed that mesoporous silica nanoparticles are biocompatible, biodegradable drug carriers and that cell specificity can be achieved both in vitro and in vivo.

Cyclosporin inhibits hyperalgesia and edema in arthritic rats: role of the central nervous system

Since arthritis induced by Mycobacterium products (adjuvant) in rats is considered to be immunologically driven, the objective of the present study was to determine if the immunosuppressor drug cyclosporin could affect hindpaw edema and joint hyperalgesia simultaneously. Female Holtzman rats (140-170 g) presented hyperalgesia and edema on the 8th and 12th day following adjuvant injection. Daily systemic (oral or intramuscular) administration of cyclosporin (0.5-5.0 mg kg-1 day-1) or dexamethasone (0.01-0.1 mg kg-1 day-1) for 15 days starting on day zero dose-dependently inhibited the hindpaw edema and hyperalgesia in arthritic rats. However, hyperalgesia but not edema could be detected two days after cyclosporin withdrawal. We concluded that a) the continuous presence of cyclosporin is essential to reduce the development of joint hyperalgesia and that b) different mechanisms underlie the appearance of hyperalgesia and edema in this model. The intracerebroventricular (icv) administration of 5-50-fold smaller doses of cyclosporin (1.5-150 µg/day) or dexamethasone (15 µg/day) also reduced the arthritic hindpaw edema and hyperalgesia. Peripheral blood from animals injected with effective systemic cyclosporin doses showed detectable levels of the drug, whereas peripheral blood from those injected with icv cyclosporin did not, as measured by specific RIA. Our results indicate that cyclosporin administered by the central route is as effective as by the systemic route to reduce joint hyperalgesia and hindpaw edema in arthritic rats. The antiarthritic effect induced by low doses of cyclosporin in the central nervous system (CNS) could be explored to avoid its often associated systemic side effects during chronic therapy. However, the mechanism(s) involved in the antiarthritic response to cyclosporin in the CNS remain to be elucidated

Minimal doses of hydroxyurea for sickle cell disease

The use of hydroxyurea (HU) can improve the clinical course of sickle cell disease. However, several features of HU treatment remain unclear, including the predictability of drug response and determination of adequate doses, considering positive responses and minimal side effects. In order to identify adequate doses of HU for treatment of sickle cell disease, 10 patients, 8 with sickle cell anemia and 2 with Sß thalassemia (8SS, 2Sß), were studied for a period of 6 to 19 months in an open label dose escalation trial (10 to 20 mg kg-1 day-1). Hemoglobin (Hb), fetal hemoglobin (Hb F) and mean corpuscular volume (MCV) values and reticulocyte, neutrophil and platelet counts were performed every two weeks during the increase of the HU dose and every 4 weeks when the maximum HU dose was established. Reduction in the number of vasoocclusive episodes was also considered in order to evaluate the efficiency of the treatment. The final Hb and Hb F concentrations, and MCV values were significantly higher than the initial values, while the final reticulocyte and neutrophil counts were significantly lower. There was an improvement in the concentration of Hb (range: 0.7-2.0 g/dl) at 15 mg HU kg-1 day-1, but this concentration did not increase significantly when the HU dose was raised to 20 mg kg-1 day-1. The concentration of Hb F increased significantly (range: 1.0-18.1%) when 15 mg HU was used, and continued to increase when the dose was raised to 20 mg kg-1 day-1. The final MCV values increased 11-28 fl (femtoliters). However, reticulocyte (range: 51-205 x 109/l) and neutrophil counts (range: 9.5-1.3 x 109/l) obtained at this dose were significantly lower than those obtained with 15 mg kg-1 day-1. All patients reported a decrease in frequency or severity of vasoocclusive episodes. These results suggest that a hydroxyurea dose of 15 mg kg-1 day-1 seems to be adequate for treatment of sickle cell disease in view of the minimal side effects observed and the improvement in laboratory and clinical parameters.

Effect of all-trans retinoic acid on newly diagnosed acute promyelocytic leukemia patients: results of a Brazilian center

Thirty-seven patients with acute promyelocytic leukemia (APL) were treated with all-trans retinoic acid (ATRA). Patients received 45 mg m-2 day-1 po of ATRA until complete remission (CR) was achieved, defined as: a) presence of less than 5% blasts in the bone marrow, with b) white blood cells >103/mm3, c) platelets >105/mm3 and d) hemoglobin concentration >8 g/dl, with no blood or platelet transfusions. Thirty-one (83.7%) patients achieved CR by day 50, and 75% of these before day 30. Correction of the coagulopathy, achieved between days 2 and 10 (mean, 3 days), was the first evidence of response to treatment. Only one patient had been previously treated with chemotherapy and three had the microgranular variant M3 form. Dryness of skin and mucosae was the most common side effect observed in 82% of the patients. Thrombosis, hepatotoxicity and retinoid acid syndrome (RAS) were observed in 7 (19%), 6 (16%) and 4 (11%) patients, respectively. Thirteen (35%) patients had to be submitted to chemotherapy due to hyperleukocytosis (above 40 x 103/mm3) and six of these presented with new signs of coagulopathy after chemotherapy. Four (11%) patients died secondarily to intracerebral hemorrhage (IH) and two (5.4%) dropped out of the protocol due to severe ATRA side effects (one RAS and one hepatotoxicity). RAS and IH were related strictly to hyperleukocytosis. The reduced use of platelets and fresh frozen plasma probably lowered the total cost of treatment. We conclude that ATRA is an effective agent for inducing complete remission in APL patients.

Efficacy, safety, quality control, marketing and regulatory guidelines for herbal medicines (phytotherapeutic agents)

This review highlights the current advances in knowledge about the safety, efficacy, quality control, marketing and regulatory aspects of botanical medicines. Phytotherapeutic agents are standardized herbal preparations consisting of complex mixtures of one or more plants which contain as active ingredients plant parts or plant material in the crude or processed state. A marked growth in the worldwide phytotherapeutic market has occurred over the last 15 years. For the European and USA markets alone, this will reach about $7 billion and $5 billion per annum, respectively, in 1999, and has thus attracted the interest of most large pharmaceutical companies. Insufficient data exist for most plants to guarantee their quality, efficacy and safety. The idea that herbal drugs are safe and free from side effects is false. Plants contain hundreds of constituents and some of them are very toxic, such as the most cytotoxic anti-cancer plant-derived drugs, digitalis and the pyrrolizidine alkaloids, etc. However, the adverse effects of phytotherapeutic agents are less frequent compared with synthetic drugs, but well-controlled clinical trials have now confirmed that such effects really exist. Several regulatory models for herbal medicines are currently available including prescription drugs, over-the-counter substances, traditional medicines and dietary supplements. Harmonization and improvement in the processes of regulation is needed, and the general tendency is to perpetuate the German Commission E experience, which combines scientific studies and traditional knowledge (monographs). Finally, the trend in the domestication, production and biotechnological studies and genetic improvement of medicinal plants, instead of the use of plants harvested in the wild, will offer great advantages, since it will be possible to obtain uniform and high quality raw materials which are fundamental to the efficacy and safety of herbal drugs.

Quality of radiotherapy care by development of e-feedback knowledge

The aim of this three phase study was to develop quality of radiotherapy care by the e-Feedback knowledge of radiotherapy -intervention (e-Re-Know). In Phase I, the purpose was to describe the quality of radiotherapy care and its deficits experienced by cancer patients. Based on the deficits in patient education in Phase II, the purpose was to describe cancer patients’ e-knowledge expectations in radiotherapy. In Phase III, the purpose was to develop and evaluate the outcomes of the e-Re-Know among breast cancer patients. The ultimate aim was to develop radiotherapy care to support patients’ empowerment with patient e-education. In Phase I (2004-2005), the descriptive design was used, and 134 radiotherapy patients evaluated their experiences by Good Nursing Care Scale for Patients (GNCS-P) in the middle of RT period. In Phase II (2006-2008), the descriptive longitudinal design was used and 100 radiotherapy patients’ e-knowledge expectations of RT were evaluated using open-ended questionnaire developed for this study before commencing first RT, in the middle of the treatment, and concluding RT period. In Phase III, firstly (2009-2010), the e-Re-Know intervention, i.e. knowledge test and feedback, was developed in terms of empowering knowledge and implemented with e-feedback approach based on literature and expert reviews. Secondly (2011-2014), the randomized controlled study was used to evaluate the e-Re-Know. Breast cancer patients randomized to either the intervention group (n=65) receiving the e-Re-Know by e-mail before commencing first RT and standard education or the control group (n=63) receiving standard education. The data were collected before commencing first RT, concluding last RT and 3 months after last RT using RT Knowledge Test, Spielberger’s State Trait Inventory (STAI) and Functional Assessment of Cancer Therapy - Breast (FACT-B) –instruments. Data were analyzed using statistical methods and content analysis. The study showed radiotherapy patients experienced quality of care high. However, there were deficits in patient education. Furthermore, radiotherapy patients’ multidimensional e-knowledge expectations through Internet covered mainly bio-physiological and functional knowledge. Thus, the e-Re-Know was developed and evaluated. The study showed when breast cancer patients’ carried out the e-Re-Know their knowledge of side effects self-care was significantly increased and quality of life (QOL) significantly improved in line with decrease in anxiety from time before radiotherapy period to three months after. In addition, the e-Re-Know has potential to have positive effects on anxiety and QOL, regardless of patient characteristics or knowledge level. The results support the theory of empowering patient education suggesting that empowerment can be supported by confirming patients’ understanding of own knowledge level. In summary, the e-Feedback knowledge of radiotherapy (e-Re-Know) intervention can be recommended in development of quality of radiotherapy care experienced by breast cancer patients. Further research is needed to assess and develop patient-centred quality of care by patient education among cancer patients.

Somatic gene therapy for hypertension

Gene therapy for hypertension is needed for the next generation of antihypertensive drugs. Current drugs, although effective, have poor compliance, are expensive and short-lasting (hours or one day). Gene therapy offers a way to produce long-lasting antihypertensive effects (weeks, months or years). We are currently using two strategies: a) antisense oligodeoxynucleotides (AS-ODN) and b) antisense DNA delivered in viral vectors to inhibit genes associated with vasoconstrictive properties. It is not necessary to know all the genes involved in hypertension, since many years of experience with drugs show which genes need to be controlled. AS-ODN are short, single-stranded DNA that can be injected in naked form or in liposomes. AS-ODN, targeted to angiotensin type 1 receptors (AT1-R), angiotensinogen (AGT), angiotensin converting enzyme, and ß1-adrenergic receptors effectively reduce hypertension in rat models (SHR, 2K-1C) and cold-induced hypertension. A single dose is effective up to one month when delivered with liposomes. No side effects or toxic effects have been detected, and repeated injections can be given. For the vector, adeno-associated virus (AAV) is used with a construct to include a CMV promoter, antisense DNA to AGT or AT1-R and a reporter gene. Results in SHR demonstrate reduction and slowing of development of hypertension, with a single dose administration. Left ventricular hypertrophy is also reduced by AAV-AGT-AS treatment. Double transgenic mice (human renin plus human AGT) with high angiotensin II causing high blood pressure, treated with AAV-AT1-R-AS, show a normalization of blood pressure for over six months with a single injection of vector. We conclude that ODNs will probably be developed first because they can be treated like drugs for the treatment of hypertension with long-term effects. Viral vector delivery needs more engineering to be certain of its safety, but one day may be used for a very prolonged control of blood pressure.

Treatment of cytomegalovirus retinitis with an intraocular sustained-release ganciclovir implant

The objective of this prospective study was to evaluate the efficacy and complications of the use of an intraocular sustained-release ganciclovir implant for the treatment of active cytomegalovirus (CMV) retinitis in AIDS patients. Thirty-nine eyes of 26 patients were submitted to ocular surgery. All patients underwent complete ocular examination before and after surgery. The surgical procedure was always done under local anesthesia using the same technique. The mean time for the surgical procedure was 20 min (range, 15 to 30 min). The average follow-up period was 3.7 months. Of all patient, only 4 presented recurrence of retinitis after 8, 8, 9 and 2 months, respectively. Three of them received a successful second implant. All 39 eyes of the 26 patients presented healing of retinitis as shown by clinical improvement evaluated by indirect binocular ophthalmoscopy and retinography. Retinitis healed within a period of 4 to 6 weeks in all patients, with clinical regression signs from the third week on. Six (15.4%) eyes developed retinal detachment. None of the patients developed CMV retinitis in the contralateral eye. The intraocular implant proved to be effective in controlling the progression of retinitis for a period of up to 8 months even in patients for whom systemic therapy with either ganciclovir or foscarnet or both had failed. The intraocular sustained-release ganciclovir implant proved to be a safe new procedure for the treatment of CMV retinitis, avoiding the systemic side effects caused by the intravenous medications and improving the quality of life of the patients.

Amifostine (WR-2721), a cytoprotective agent during high-dose cyclophosphamide treatment of non-Hodgkin's lymphomas: a phase II study

Clinical trials indicate that amifostine may confer protection on various normal tissues without attenuating anti-tumor response. When administered prior to chemotherapy or radiotherapy, it may provide a broad spectrum of cytoprotection including against alkylating drugs. The mechanism of protection resides in the metabolism at normal tissue site by membrane-bound alkaline phosphatase. Toxicity of this drug is moderate with hypotension, nausea and vomiting, and hypocalcemia being observed. We report a phase II study using amifostine as a protective drug against high-dose cyclophosphamide (HDCY) (7 g/m2), used to mobilize peripheral blood progenitor cells (PBPC) and to reduce tumor burden. We enrolled 29 patients, 22 (75.9%) affected by aggressive and 7 (24.1%) by indolent non-Hodgkin's lymphoma (NHL), who were submitted to 58 infusions of amifostine and compared them with a historical group (33 patients) affected by aggressive NHL and treated with VACOP-B followed by HDCY. The most important results in favor of amifostine were the reduction of intensity of cardiac, pulmonary and hepatic toxicity, and a significant reduction of frequency and severity of mucositis (P = 0.04). None of the 29 patients died in the protected group, while in the historical group 2/33 patients died because of cardiac or pulmonary toxicity and 2 patients stopped therapy due to toxicity. Amifostine did not prevent the aplastic phase following HDCY. PBPC collection and hematological recovery were adequate in both groups. The number of CFU-GM (colony-forming units-granulocyte/macrophage) colonies and mononuclear cells in the apheresis products was significantly higher in the amifostine group (P = 0.02 and 0.01, respectively). Side effects were mild and easily controlled. We conclude that amifostine protection should be useful in HDCY to protect normal tissues, with acceptable side effects.