Protein stability during freezing: separation of stresses and mechanisms of protein stabilization

Although proteins are often frozen during processing or freeze-dried after formulation to improve their stability, they can undergo degradation leading to losses in biological activity during the process. During freezing, the physical environment of a protein changes dramatically leading to the development of stresses that impact protein stability. Low temperature, freeze-concentration, and ice formation are the three chief stresses resulting during cooling and freezing. Because of the increase in solute concentrations, freeze-concentration could also facilitate second order reactions, crystallization of buffer or non-buffer components, phase separation, and redistribution of solutes. An understanding of these stresses is critical to the determination of when during freezing a protein suffers degradation and therefore important in the design of stabilizer systems. With the exception of a few studies, the relative contribution of various stresses to the instability of frozen proteins has not been addressed in the freeze-drying literature. The purpose of this review is to describe the various stages of freezing and examine the consequences of the various stresses developing during freezing on protein stability and to assess their relative contribution to the destabilization process. The ongoing debate on thermodynamic versus kinetic mechanisms of stabilization in frozen environments and the current state of knowledge concerning those mechanisms are also reviewed in this publication. An understanding of the relative contributions of freezing stresses coupled with the knowledge of cryoprotection mechanisms is central to the development of more rational formulation and process design of stable lyophilized proteins.     

Development of New Localized Drug Delivery System Based on Ceftriaxone-Sulbactam Composite Drug Impregnated Porous Hydroxyapatite: A Systematic Approach for <Emphasis Type="Italic">In Vitro</Emphasis> and <Emphasis Type="Italic">In Vivo</Emphasis> Animal Trial

Purpose  

Present investigation deals with an extensive approach incorporating in vitro and in vivo experimentation to treat chronic osteomyelitis, using hydroxyapatite porous scaffolds.     

Fabrication and Evaluation of Bi-layer Tablet Containing Conventional Paracetamol and Modified Release Diclofenac Sodium

The objectives of present investigation were to achieve immediate release of paracetamol and tailored release of diclofenac sodium from bi-layer tablets. A 2³ full factorial design was adopted using the amount of polyethylene glycol, microcrystalline cellulose and crospovidone as independent variables for fabricating paracetamol tablets. Diclofenac sodium tablets were prepared using hydroxypropyl methylcellulose as a matrixing agent. The results of analysis of variance showed that the friability of paracetamol was distinctly influenced by the formulation variables. The in vitro drug release behaviour of diclofenac tablets was compared with a marketed formulation. The optimized formulations of paracetamol and diclofenac sodium were used for manufacturing of bi-layer tablets. The bi-layer tablets showed immediate release of paracetamol and modified release of diclofenac.     

Comparative evaluation of aqueous and plasma concentration of topical moxifloxacin alone and with flurbiprofen in patients of cataract surgery

Objectives:

To determine the aqueous and plasma concentrations of moxifloxacin administered topically alone and with flurbiprofen in patients undergoing cataract surgery.

Materials and Methods:

A total of 50 subjects scheduled for routine cataract surgery were randomly allocated to two groups (n = 25 each). Group-1 patients were treated with topical moxifloxacin alone: One drop 6 times/day for 3 days before surgery and one drop 4 times on the day of surgery: Group-2 patients were treated with topical moxifloxacin as in Group-1 and with topical flurbiprofen: One drop 4 times/day for 3 days before and on the day of surgery. The interval between two drugs was 30 min for last 3 days and 15 min on the day of surgery. Last dose was administered 1 h before aqueous humor and blood sampling for both the groups. The antibiotic concentration in aqueous humor and plasma were determined by using high performance liquid chromatography.

Results:

The mean concentration of moxifloxacin in aqueous humor was 1.71 ± 0.82 mg/ml in Group-1 and 2.39 ± 1.34 mg/ml in Group-2. Concentrations of moxifloxacin in aqueous humor were significantly higher in Group-2 than that of Group-1.

Conclusion:

Flurbiprofen may increase the concentration of moxifloxacin in aqueous humor.KEY WORDS: Aqueous humor, cataract surgery, flurbiprofen, moxifloxacin     

Intravenous subdissociative-dose ketamine versus morphine for acute geriatric pain in the Emergency Department: A randomized controlled trial

Study objective

We compare the analgesic efficacy and safety of subdissociative intravenous-dose ketamine (SDK) versus morphine in geriatric Emergency Department (ED) patients.

Azathioprine-associated acute myeloid leukemia in a patient with Crohn's disease and thiopurine S-methyltransferase deficiency

Immunosuppressive thiopurines like azathioprine, 6-mercaptopurine, and thioguanine are commonly used in inflammatory and neoplastic disorders. A subset of these patients are genetically slow metabolizers due to point-mutations in enzyme thiopurine S-methyltransferase (TPMT), and are at a higher risk of hematologic toxicity and leukemogenesis. We present such a patient who was a slow metabolizer for azathioprine, and developed a rapidly lethal form acute myeloid leukemia after relatively low dose exposure to the drug. There was prominent hemophagocytic activity in the bone marrow, and cytogenetic analysis showed a complex karyotype with monosomy 7, but no involvement of chromosome 8.     

Mucormycosis at a tertiary care centre in Gujarat,India

The prevalence of mucormycosis is reportedly high in India, although the studies are mainly from north and south India only. We analysed the mucormycosis cases at tertiary care centres of West India. We retrieved the clinical details of all the patients with probable and proven mucormycosis diagnosed at Sterling Hospital and ID clinic at Ahmedabad, Gujarat over the period from 1 January 2013 through 30 April 2015. The data were analysed to determine demography, risk factors, underlying diseases, site of infection and outcome of these patients. A total of 27 patients with the median age of 50 (16‐65) years were diagnosed with mucormycosis during the period. Rhino‐orbital‐cerebral mucormycosis was the most common (51.9%) presentation. Majority (55.6%) of the patients had uncontrolled diabetes with or without ketoacidosis; 25.9% patients had no underlying disease and most of them (85.7%) had cutaneous mucormycosis. In this group, the mortality was 25.9% and an equal percentage of patients were lost to follow up; 14 (51.9%) patients could complete 6 weeks of amphotericin B therapy. All patients who completed antifungal therapy survived except one. Like other parts of India, uncontrolled diabetes was the predominant risk factor for mucormycosis in our group. Patients completing 6 weeks of amphotericin B treatment were likely to survive.     

The risk of intra-urethral Foley catheter balloon inflation in spinal cord-injured patients: Lessons learned from a retrospective case series

Background

Inflating the balloon of Foley catheter in urethra is a complication of urethral catheterisation. We report five patients in whom this complication occurred because of unskilled catheterisation. Due to lack of awareness, the problem was not recognised promptly and patients came to harm.

Case series

1. 1.
   A tetraplegic patient developed pain in lower abdomen and became unwell after transurethral catheterisation. CT pelvis revealed full bladder with balloon of Foley catheter in dilated urethra.
    
2. 2.
   Routine ultrasound examination in an asymptomatic tetraplegic patient with urethral catheter drainage, revealed Foley balloon in the urethra. He was advised to get catheterisations done by senior health professionals.
    
3. 3.
   A paraplegic patient developed bleeding and bypassing after transurethral catheterisation. X-ray revealed Foley balloon in urethra; urethral catheter was changed ensuring its correct placement in urinary bladder. Subsequently, balloon of Foley catheter was inflated in urethra several times by community nurses, which resulted in erosion of bulbous urethra and urinary fistula. Suprapubic cystostomy was performed.
    
4. 4.
   A tetraplegic patient developed sweating and increased spasms following urethral catheterisations. CT of abdomen revealed distended bladder with the balloon of Foley catheter located in urethra. Flexible cystoscopy and transurethral catheterisation over a guide-wire were performed. Patient noticed decrease in sweating and spasms.
    
5. 5.
   A paraplegic patient developed lower abdominal pain and nausea following catheterisation. CT abdomen revealed bilateral hydronephrosis and hydroureter and Foley balloon located in urethra. Urehral catheterisation was performed over a guide-wire after cystoscopy. Subsequently suprapubic cystostomy was done.
    

Conclusion

Spinal cord injury patients are at increased risk for intra-urethral Foley catheter balloon inflation because of lack of sensation in urethra, urethral sphincter spasm, and false passage due to previous urethral trauma. Education and training of doctors and nurses in proper technique of catheterisation in spinal cord injury patients is vital to prevent intra-urethral inflation of Foley catheter balloon. If a spinal cord injury patient develops bypassing or symptoms of autonomic dysreflexia following catheterisation, incorrect placement of urethral catheter should be suspected.

     

Intra-operative imprint cytology for assessing the sentinel node in breast cancer – Results of its routine use over 8 years

Introduction

Intra-operative imprint cytology (IIC) for analysing sentinel lymph node/s (SLN) in breast cancer allows definitive axillary surgery as a one-step procedure. Most reported studies are research oriented. This study reports long-term results of IIC done as routine clinical practice.

Materials and methods

Eight hundred ninety-six female, operable breast cancer patients underwent SLN biopsy over an 8-year period (January 1999–December 2006). Data were extracted retrospectively from medical records. SLNs were sent intra-operatively to the laboratory where they were bisected, touch imprinted and stained with Hematoxylin & Eosin. Patients with positive IIC had axillary clearance. Formal histological analyses of SLNs were compared with IIC findings. The impact of routine pre-operative axillary ultrasound (introduced in 2003) on IIC sensitivity and specificity was also assessed.

Results

Median age was 61 years (26–89) and median tumour size was 18 mm (2–100). A total of 244/896 patients had SLN metastases on final paraffin histology of which 177 were correctly detected by IIC (67 false negatives). 39/67 false negatives could be attributed to sampling error. The overall sensitivity and specificity of IIC for the identification of SLN metastases was 73% and 100%, respectively. The sensitivity of IIC after introduction of pre-operative axillary ultrasound decreased from 75% to 71%.

Discussion

Routine use of IIC for analysis of the SLN in breast cancer allows complete axillary surgery during a single anaesthetic for a majority of patients undergoing SLN biopsy. Almost two thirds of positive axillae were spared a second operation. False negative results are frequent and patients should be warned about the potential need for further axillary surgery.