ALZET®胶囊渗透压泵
发布时间：2006-10-26
ALZET® Osmotic Pumps----植入式胶囊渗透压泵
ALZET® Osmotic Pumps----植入式胶囊渗透压泵
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持续長時間定量給药的好帮手--製造廠商：DURECT Corporation
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中国、香港地区独家代理商 --- 美国健康医疗仪器国际公司
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Rates & DurationsThere are 12 models of ALZET Osmotic Pumps in 3 physical sizes, and a variety of release rates and durations. |
Neuroscientists have used ALZET pumps to deliver compounds to the brain, the spinal cord, and nerves. The ability to infuse the brain directly circumvents the blood-brain barrier. Growth factor administration has allowed scientists to delve into potential treatments for neurodegenerative disorders such as Alzheimer’s and Parkinson’s diseases.
Alzheimer’s AD Animal Models:
AD 动物模型的制作方法
Okadaic acid 慢性损害AD 模型
Okadaic acid（OA）是一种丝氨酸／苏氨酸蛋白磷酸化酶（1A 和2A）的特异性抑制剂，OA 的长期
脑室投递可引起动物的记忆严重缺失，同时导致脑内Aβ 淀粉样沉积斑块形成以及NFT 样磷酸化
Tau 蛋白出现。
投递装置用ALZA 公司的ALZET 微渗透泵（Model 2004），使用前充填OA 溶液。
1. 实验动物：用SD 或Wistar 大鼠，体重250～300 克。
2. 材料和试剂：脑立体定位仪，微量注射器，颅钻。OA 选用Sigma 公司产品，产品编号是0-
1506，以pH7.4 的磷酸缓冲的人工脑脊液配制，置4℃冰箱备用。
3. 操作程序：动物麻醉后固定于脑立体定位仪。参照脑立体定位图谱的侧脑室座标，颅骨钻孔，装
置微渗透泵的投递针，使针管头端置于侧脑室，用牙科水泥将其柄部固定在颅骨上，泵体埋在动物
项部皮下，输送管经头皮下传送。平均投递速度0.25±0.02μl／小时，通常一次埋泵可维持4 周，
如需要长时间的投递，更换一次新充填的微渗透泵即可。2～3 周后进行行为试验，动物将出现工
作记忆和参考记忆的损害。术后6 周免疫组化分析可见经OA 处理的大鼠脑的纹状体、海马和皮质
等部位出现高磷酸化Tau 蛋白免疫阳性神经元、App 免疫阳性星形胶质细胞和Aβ 淀粉样蛋白免疫
阳性斑块。
Biotechnology researchers have used the pumps to deliver cytokines, growth factors, hormones, and newly-synthesized proteins. Continuous infusion can be advantageous when delivering such agents, as the short half-lives of proteins and peptides can render injections ineffective.
Pharmacologists have used the pumps to target delivery to specific organs and tissues. This decreases the amount of valuable compound which needs to be delivered, isolates compound effects, and avoids potential problems with systemic toxicity. Compounds that cannot be delivered orally can still be tested, and can drive development of more successful chemical structures. Many receptor characterization studies have been done using this method.
Cardiovascular researchers have used the pumps to deliver agents such as renin-angiotensin hormones, nitric oxide synthase inhibitors, and angiogenic factors. Endocrinologists and physiologists have used the pumps to deliver hormones, cytokines, and growth factors. Toxicologists and cancer researchers have used the pump to measure cell proliferation, to understand the process of carcinogenesis more fully, and to investigate the effects of teratogenic compounds when given prenatally. Researchers interested in immunology have infused immunosuppressants, cytokines, and antibodies.
In Vivo Pharmacology
"The majority of chemicals are eliminated considerably faster in small laboratory animals as compared with humans, and constant-rate infusion offers several advantages over conventional, bolus delivery regimens in compensating for this difference." 1
When testing a novel compound in vivo, rapid elimination can result in mistaken assessment of activity. Rats and mice generally eliminate test compounds more rapidly than humans. After a single injection, plasma concentration rises to a peak and then declines rapidly until the compound is eliminated from plasma and tissues. Often the duration of serum activity following a single injection is limited to several hours, hence biological effects either fail to develop or develop poorly:
If no effect is observed following injection, it is difficult to determine whether the compound is inactive or if it simply was not present in adequate concentration and for a sufficient duration to elicit an effect.
Depending on the rate of elimination and the frequency of dosing, injections can result in periods during which drug is absent from plasma and tissues. Such extreme variability in compound exposure over time can influence the expression of drug action.
Thus, the data from such experiments can be misleading as to the nature of compound effects and the dose required to elicit them. Additionally, repeated injections are stressful to the animal and difficult to maintain around the clock.
ALZET pumps are drug discovery tools that offer researchers enhanced control over test compound levels in plasma and tissues. Through continuous infusion, these pumps maintain a well-defined, consistent pattern of drug exposure throughout the duration of the experiment. ALZET pumps ensure that test compounds are present in plasma and tissues for a sufficient duration to allow their biological effects to develop fully and reproducibly. The scientific literature contains many examples where ALZET pumps have facilitated full development of drug effects, including those of proteins, peptides, and other rapidly-eliminated compounds. For more information on in vivo pharmacology, consult our bibliography.
動物腦部給藥導管組(ALZET Brain Infusion Kits)
Brain Kit Downloads
The following are a list of PDFs that provide more details on the ALZET Brain Kits:
Many agents do not cross the blood-brain barrier sufficiently to evaluate their effects on the brain without delivering them locally. Cerebral injection is one local delivery method, but it can be challenging to deliver an effective dose in a physiologically-compatible volume. In addition, the agent may not remain in the target location long enough to see an effect. For many compounds, local infusion directly into the brain is the only way to generate reliable data. ALZET pumps have been used in hundreds of published neuroscience studies to infuse agents, from growth factors to siRNA to psychoactive drugs and more, to the central nervous system.
The ALZET Brain Infusion Kits are designed specifically for use with ALZET pumps for targeted delivery to the central nervous system.
They can be used in two ways:
1. Infusion into the cerebral ventricles exposes a wide variety of brain regions to the infusate via the cerebrospinal fluid which bathes the brain.
2. Direct microperfusion of discrete brain structures results in localized distribution of infusate in the target tissue.
Each ALZET Brain Infusion Kit includes materials for 10 brain infusions:
‧ 10 Brain Infusion Cannulae
‧ 10 Vinyl Catheter Tubes
‧ 40 Depth-Adjustment Spacers
‧ 1 Instruction Sheet
‧ 10 Vinyl Catheter Tubes
‧ 40 Depth-Adjustment Spacers
‧ 1 Instruction Sheet
Features of Brain Kits
- Compatible with all ALZET pumps models. (Pumps and kits are sold separately.)
- Targets lateral ventricles: Without modification, Brain Kits 1 & 2 will penetrate 5 mm below the surface of the skull. When affixed to the skull in the stereotaxically correct location, this will put the tip of the cannula in the region of the cerebral ventricles of a 250-300 g rat. Brain Kit 3 will penetrate 3 mm below the skull surface, which is appropriate for targeting the lateral ventricles in an adult mouse.
- Easily customized to target different brain regions or adjust for differences in animal size. Uniquely designed depth adjustment spacers allow the depth of the cannula tip within the brain to be adjusted in 0.5 mm increments. Note that the cannula can easily be cut to target more superficial structures.
- Two specialized cannula designs: The original Brain Infusion Kit 1 has a taller base with a narrower diameter and grooves which are ideal for anchoring sutures in certain applications. The Brain Infusion Kits 2 and 3 feature a wide base pedestal which have been designed for greater stability, and may obviate the need for anchor screws. The Brain Kits 2 and 3 have a lower profile pedestal, which facilitates closure of the skin after placement.
- Design minimizes local trauma: Fine gauge stainless steel cannula minimizes trauma to the brain during cannula placement. (Brain Kits 1 & 2 are 28 gauge. Brain Kit 3 is 30 gauge)
- Flexible vinyl tubing that is well-suited for brain infusion.
- All components are provided sterile.
- Biocompatible: All materials in the Kits meet U.S. Pharmacopoeia (USP) Class VI standards for the biocompatibility of medical plastics.
Neuroscience Research Applications
Neuroscientists were among the very first to develop research methods incorporating ALZET® osmotic pumps. Publishing in Science in 1976, Wei and Loh at UC Berkeley and UC San Francisco demonstrated dependence on opiate peptides in rats following chronic infusion targeted to the frontal cortex and periaqueductal gray.1
ALZET pumps quickly became a mainstay in neuroscience work for their ability to provide precise delivery, either systemically or locally targeted, without interference in elaborate behavioral testing, and without the animal stress inherent in other methods requiring frequent animal handling. The result is a large and rapidly growing body of neuroscience work employing ALZET pumps in the forefront of research on spinal cord repair, neuropathic pain, stroke & ischemia, neurodegenerative diseases, and much more. Included here are brief introductions to a few of these applications. The full array of neuroscience research incorporating ALZET pumps is available in the neuroscience section of our website (www.alzet.com>Research Applications> Neuroscience). Or, request a reference search customized to your research area from ALZET Technical Support.
1Wei E, Loh H. Physical dependence on opiate-like peptides. Science 1976; 193:1262-1263.
Case study: siRNA Infusion by ALZET Pumps Attenuates Neuropathic Pain
The discovery that short, double-stranded RNA fragments could directly
induce post-transcriptional gene silencing in vivo has opened
the door to enormous possibilities at the intersection of neuroscience
and functional genomics. For the first time, siRNA has been demonstrated
to have therapeutic application for a pathophysiological phenotype
in the CNS. Dorn et al. at Novartis (2004) used ALZET pumps
to chronically administer a 21-nucleotide siRNA complementary to
P2X3, a pain-implicated receptor expressed in dorsal root ganglia
and spinal cord.1
This receptor target was selected based on prior work by Barclay
et al. at Novartis (2002), in which an antisense oligonucleotide
against the P2X3 receptor, administered chronically and intrathecally
via ALZET pump, elicited a partial, functional down-regulation of the
receptor along with a significant, although partial, relief of hyperalgesia
in rat models of inflammatory and neuropathic pain.2 The ALZET
pumps enabled intrathecal delivery of the oligonucleotide via a PE-10
catheter inserted through a guide needle into the intrathecal space
at the level of L1. The PE-10 catheter was connected to each pump
presumably via a segment of PE-60 or similar tubing. A control group
was similarly cannulated but received only saline. The authors reportedly
“observed no signs of neurotoxicity from the surgical procedure
such as paralysis, vocalization, or gross anatomical changes in the
spinal cord.” 2 (p. 8144)
Dorn et al. continued the above work, further postulating that siRNA
could be used to selectively block the P2X3 receptor in an effective,
and potentially safer, treatment strategy for neuropathic pain. Indeed,
siRNA treatment significantly diminished mechanical hyperalgesia
and tactile allodynia compared to control animals receiving either vehicle
or missense siRNA. This correlated with a downregulation of
P2X3 mRNA in dorsal root ganglia, and protein levels in the dorsal
horn of the spinal cord. Dorn et al. concluded that “these observations
open a path toward use of siRNA as a genetic tool for drug target
validation in the mammalian CNS, as well as for proof of concept
studies and as therapeutic agents in man.” 1 (p. 1)
For more information on the use of ALZET pumps to deliver genetic
material, please contact ALZET Technical Support.
1Dorn G, Patel S, Wotherspoon G, Hemmings-Mieszczak M, Barclay J, Natt FJC, Martin
P, Bevan S, Fox A, Ganju P, Wishart W, & Hall J. siRNA relieves chronic neuropathic pain.
NUCLEIC ACIDS RESEARCH 2004; 32(5):U11-U16.
2Barclay J, Patel S, Dorn G, Wotherspoon G, Moffatt S, Eunson L, Abdel'al S, Natt F, Hall
J, Winter J, Bevan S, Wishart W, Fox A & Ganju P. J Neurosci 2002;22(18):8139-8147.
Case study: Local & Systemic Infusion
Contribute to Spinal Cord Injury Research
Spinal cord regeneration following injury remains an elusive research goal.
Scrambling to understand the molecular contributors to axonal regrowth, researchers
are using ALZET pumps both to help identify these contributors,
and to test agents capable of promoting regrowth. In recent work in several
murine models of spinal cord injury (SCI), researchers used ALZET pumps to
infuse agents either intrathecally near the trauma, or systemically via subcutaneously-
implanted pumps.
In one example, Faulkner et al. at UCLA used a transgenic mouse model to
demonstrate that reactive astrocytes protect against tissue degeneration and
functional impairment following SCI.1 Prior research has shown that reactive
astrocytes contribute to gliosis, forming a scar which impedes axonal repair
and functional recovery. Faulkner and colleagues used mice in which thymidine
kinase from HSV was targeted to reactive astrocytes using the mouse glial fibrillary
acidic protein promoter. In this model, the anti-viral agent ganciclovir
selectively ablates reactive astrocytes expressing this transgene product after
CNS injury. Following either a crush or stab injury at the level of L1-L2, ALZET
pumps were implanted subcutaneously to deliver ganciclovir for 7 days. Animals
treated with ganciclovir exhibited demyelination, oligodendrocyte degeneration,
persistent leakage of the blood-brain barrier, and significant deterioration
in hindlimb function relative to vehicle-infused controls.
While the subcutaneous infusion route can be used effectively to deliver potential
therapeutics, since the blood-brain barrier loses its integrity for a time following
injury, ALZET pumps also allow researchers to target agents directly to
the injury site. Sivasankaran et al. from Boston Children's Hospital, Harvard
Medical School and University of Louisville, used ALZET pumps to show that the
protein kinase C inhibitor, Gö6976, promotes regeneration of dorsal column axons
across and beyond the lesion site following injury.2 Following hemisection
at C3-4, ALZET pumps attached to intrathecal catheters delivered Gö6976 or
vehicle directly to the lesion site for 14 days. Treated animals were significantly
more likely to have regenerating axons beyond the lesion site, and also had significantly
higher numbers of axons at various distances from the lesion relative
to vehicle-infused controls.
For a complete list of references on the study of spinal cord injury using ALZET
pumps, please contact ALZET Technical Support.
1Faulkner JR, Herrmann JE, Woo MJ, Tansey KE, Doan NB & Sofroniew MV. J Neurosci
2004:24(9):2143-2155.
2Sivasankaran R, Pei J, Wang KC, Zhang YP, Shields CB, Xu X-M & He Z. Nature Neuro
2004;7(3);261-268.
Benefits of ALZET Pumps in Neuroscience Research
· Controlled delivery of neuroactive compounds
· Ideal for studies involving behavioral testing – no animal handling required during infusion
· Easily attached to a catheter for delivery to the brain, spinal cord, peripheral nerve, tumor or wound
· Over 27 years of published neuroscience research — well-established methods for many animal models
· Improved bioavailability of short half-life peptides and proteins
· Convenient & cost-effective for chronic treatment in lab animals
· Reproducible, consistent results
· Automatic nighttime and weekend dosing
· , Small enough for use in mice and very young rats
Agents Recently Infused by ALZET Pump
Continuous administration continues to be popular in neuroscience studies designed to understand neuropathological processes, or to identify agents with therapeutic potential. ALZET pump infusion of the following agents has recently been reported in the literature for the first time:
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Agent
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Action
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8-Bromo-cGMP
10,17S-Docosatriene
AR-R15896 AR
CGS12970
Decorin
MRZ 2/570
Neostigmine methylsulfate
PACEP (Pituitary Adenylate Cyclase-Activating
Polypeptide)
PP2
U-46619
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Cyclic GMP analog
Neuroprotectant with potential for brain ischemia-reperfusion
Non-competitive NMDA antagonist
Thromboxane A2 synthase inhibitor
Proteoglycan, natural antagonist of scar formation
NMDA receptor antagonist
Anticholinesterase
Neuroprotectant
Tyrosine kinase inhibitor
Thromboxane A2 mimetic
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Case study: Parkinson's Disease Models
Studied Using ALZET Pumps
An animal model of Parkinson's Disease (PD) was developed by Betarbet
et al.1 This model uses ALZET osmotic pumps to chronically infuse rats with
rotenone, a mitochondrial toxin and common pesticide. ALZET pumps ensure
that sufficient amounts of the neurotoxin are present to effectively establish
and sustain the neurodegenerative state. Studies using this model were successful
at reproducing the key neuropathological effects characteristic of PD:
nigrostriatial neuronal degeneration, formation of Lewi-body inclusions in the
substantia nigra, and motor dysfunction. These effects are believed to be the
direct result of mitochondrial dysfunction, via complex I inhibition, leading to
neuronal cell death.
Increased acceptance of the rotenone infusion model has provided further
insight into the pathogenesis of PD, and the mechanisms involved in rotenone
neurotoxicity. Sherer et al. from Emory University recently established that activated
microglia, the brain's immune cell defense, is another pathological feature
of PD.2 Compared to vehicle-infused animals (50% dimethylsulfoxide; 50%
polyethylene glycol), Lewis rats treated with subcutaneous rotenone were
found to have extensive microglial activation in the striatum and substantia
nigra. Interestingly, microglial activation was prominent even in the absence
of detectable nigrostriatal dopaminergic lesions, implying that microglial activation
can be used as an earlier marker of rotenone neurotoxicity in PD.
A separate study by Sherer et al. suggests that rotenone neurotoxicity may be
the result of oxidative stress.3 In vivo data demonstrated evidence of oxidative
damage, indicated by high levels of soluble protein carbonyls, in brains of rats
treated with rotenone during a period of 5 weeks, but not in vehicle infused animals.
The midbrain and olfactory bulb, dopaminergic brain regions commonly
affected by PD, showed higher protein carbonyl levels compared to other brain
regions. In vitro assays showed that pre-treatment with a -tocopherol, an antioxidant,
effectively reduced rotenone-induced cell death in a dose-dependent
manner. These experiments position antioxidant therapy as a novel therapeutic
approach for PD.
For a complete list of references on research on Parkinson's Disease, or other
neurodegenerative diseases, using ALZET pumps, please contact ALZET
Technical Support.
1Betarbet R, Sherer TB, MacKenzie G, Garcia-Osuna M, Panov AV & Greenamyre JT. Nat Neurosci
2000:3;1301-1306.
2Sherer TB, Betarbet R, Kim JH & Greenamyre JT. Neurosci Letters 2003:341;87-90.
3Sherer TB, Betarbet R, Testa CM, Seo BB, Richardson JR, Kim JH, Miller GW, Matsuno-Yagi &
Greenamyre JT. J Neurosci 2003:23(34);10756-10764.
Case study: Local Infusion Produces Central Neu, roprotective Effects in Cerebral Ischemia
Stroke is the third most common cause of death in the U.S., with ischemic stroke
being the most frequent type. Hypertension is a known contributor, which is why
neuroprotective effects seen with antihypertensive agents were attributed to peripheral
control of blood flow. However, research using ALZET pumps to chronically
administer angiotensin II receptor antagonists has demonstrated a role for
central AT1 receptors in cerebral ischemia. In addition, a recent follow-on study
has explored the temporal relationship , between AT1 antagonism and injury, resulting
in valuable information which could have clinical implications.
Dai et al. at the University of Kiel in Germany used local administration to eliminate
any confounding influence from systemic AT1 receptor blockade.1 They found that
controlled infusion directly to the brain of irbesartan, an agent indicated for hypertension,
selectively elicited central neuroprotective effects without impacting either
mean arterial blood pressure or the pressor response to angiotensin II challenge,
both of which are affected by peripheral AT1 inhibition. In this model, ALZET pumps
were used to infuse irbesartan intraventricularly in normotensive Wistar rats for 5
days, followed by focal cerebral ischemia induced by middle cerebral artery occlusion.
After a series of neurological assessments, the research group reported that
“this study demonstrates for the first time that inhibition of central AT1 receptors
improves the neurological outcome of focal brain ischemia.”1 (pg. 2395)
In a more recent study, a combined team from University of Kiel and Zhejiang
University in China employed ALZET pumps to study the effects of sustained AT1
antagonism before and after ischemia.2 Using a Model 2002 ALZET pump, this
group infused irbesartan intraventricularly for 5 days prior to ischemic injury, and
then continued for another 3 or 7 days. At the end of the treatment period, the
brains of rats were analyzed for evidence of neuroprotective effects of irbesartan
on infarct size and volume, inflammation and apoptosis.
Lou et al. found that treatment with irbesartan before ischemic injury improved
recovery of motor function, but continued treatment post-injury did not increase
this effect. In contrast, irbesartan pretreatment did not improve sensory impairments,
but treatment in the post-ischemic period was beneficial.2 In addition,
irbesartan infusion significantly reduced the infarct volume by as much as 42%
compared to vehicle-treated animals. Brain edema, apoptosis and inflammation
were also decreased in rats treated with irbesartan compared to vehicle-treated
rats. These studies stress the importance of localized, long-term AT1 receptor
blockade before and after ischemia.
The ALZET database includes more than 50 studies on the use of ALZET pumps
in stroke research. For additional information on the use of ALZET pumps for
localized delivery of neuroactive agents to the cerebral ventricles or solid brain
tissue, or for additional references on cerebral ischemia research using ALZET
pumps, please contact ALZET Technical Support.
1Dai W-J, Funk A, Herdegen T, Unger T, & Culman J. Stroke 1999;30:2391-2399.
2Lou M, Blume A, Zhao Y, Gohlke P, Deuschl G, Herdegen T & Culman J.
Journal of Cerebral Blood Flow & Metabolism 2004;24(5):536-546.
Tips on Using ALZET® Osmotic Pumps
The following topics are covered:
- Formulating the test solution to be administered.
- Maintaining pump sterility.
- Ensuring the sterility of test solutions.
- Working with peptides and proteins.
- Filling the pump's reservoir.
- Removing spent osmotic pumps.
- Verifying delivery of agents from ALZET pumps.
- Defining residual volume.
- Assessing stability of test solutions.
- Priming pumps in vitro before using in vivo.
The first step in formulating a test solution to be administered is to decide on the hourly mass flow of test material you wish to deliver. The fluid flow rate of the pump is fixed. To cut the mass flow rate in half, use half the concentration; to double the mass flow rate, use twice the concentration, etc. The highest mass flow of test material that the pump can deliver is set by the maximum solubility of the test agent in its vehicle (saturated solution) at the temperature of loading (usually room temperature, at about 22°C).
To determine the concentration of ADH (lysine vasopressin) for subcutaneous delivery into rats with diabetes insipidus, the following steps were undertaken:
A search of the literature indicates that the normal secretion of ADH in the rat is 30 ng/day, or 1.25 ng/hr. We next calculate the concentration of ADH in the infusion medium (saline) that would be necessary to accomplish the desired infusion of 1.25 ng/hr:
Mass flow = concentration x volume flow rate
The volume flow rate of the Model 2001 pump is 1 μl/hr, so the required concentration (C) is:
C = (1.25 ng/hr) / 1 μl/hr = 1.25 ng/μl = 1.25 x 10-3 μg/μl
The Model 2001 pump has a nominal reservoir volume of 200 μl. In order to give each rat 1.25 x 10-3 μg/μl, the amount of ADH which should be dissolved in 200 μl is:
(1.25 x 10-3 μg/μl) x 200 μl = 0.25 μg
In summary, in order to mimic the normal secretion of ADH in the rat of 30 ng/day (or 1.25 ng/hr) one must make up a concentration of 1.25 x 10-3 mg/ml. To achieve this concentration in the model 2001 pump, 0.25 μg of ADH must be dissolved in 200 μl of solvent. Please note that excess solution should be made up to allow for a safety margin during pump filling.
The delivery rate of some marginally soluble agents is limited by their solubility in aqueous media. Solutions to this problem include:
· Choose a vehicle which provides greater solubility for the agent in question.
· Implant a higher flow rate pump (e.g., Model 2ML1), replacing it periodically to achieve longer durations.
· If the animal is of sufficient size (a very large rat or larger), more than one pump can be implanted at one time and the dose divided between the pumps.
After packaging, ALZET pumps are irradiated by a sterilizing dose of 60Co. During use, however, exterior contamination from handling may occur. Therefore, we recommend using sterile technique whenever handling the pumps. After loading, the surface of the pump can be cleaned by wiping it with isopropyl alcohol (70% in water). If this optional step is taken, avoid getting alcohol into the delivery port of the pump.
Do not use Zephiran® in any concentration to clean the pumps. We have found that it remains on the surface of the pump and provokes a serious inflammatory reaction in subcutaneous tissues.
Note: Do not autoclave ALZET pumps, flow moderators, or filling tubes. ALZET pumps cannot be gas sterilized either. Pumps and flow moderators can be reirradiated with 2.5 Mrad of gamma irradiation from a 60Co source, though this should not be necessary if sterile technique is maintained. This additional sterilization can cause pump discoloration.
Because the ALZET pump is designed to contain solutions for as long as four weeks (depending on the model) at 37°C, any microorganisms present in the loading solution may grow, metabolize, and alter the composition of the test solution. This risk is higher if the solution contains materials such as proteins that are potential substrates for microorganisms. For this reason, all solutions loaded into the pumps should be sterile.
Use the following guidelines to minimize interior contamination of the pumps:
· Prepare solutions using sterile laboratory ware.
· Prevent particulate contamination during loading of test solutions by using a bacterial filter attached to the filling syringe. The Millex-GV syringe-end filter is the standard low protein binding filter (0.22 μm pore size), and it is available from:
· The filling needles supplied with the pump are sterile, and care should be taken not to contaminate them.
Customers have reported instances in which the biological activity of an agent delivered by the pump seemed to fade or disappear as the experiment progressed. While this observation could be a real and important aspect of the pharmacology of the substance in some cases, in other cases it suggests an inoculation of the test solution with microorganisms.
For example, one laboratory delivered insulin subcutaneously to diabetic dogs using ALZET pumps, changing spent pumps for fresh ones on a weekly basis. Plasma insulin levels always peaked just after a new pump was placed each week, and then faded rapidly. Because the pumps deliver at a constant rate, it was thought that these peculiar results may have been due to bacterial contamination, with degradation of the insulin within the pumps as the infusion time progressed. When we repeated the work, using sterile solutions, insulin levels did not peak and fade, but remained steady.
Generally, peptides do not adsorb to the interior reservoir of ALZET pumps. When working with those which do, the addition of 1-5% serum albumin (usually bovine serum albumin, BSA) will prevent nonspecific adsorption.
Some peptides and proteins are susceptible to contaminants in plastics (residual monomers or plasticizers) or impure water (metal ions). Glassware that is chemically clean and thoroughly rinsed in metal-free deionized water should be used whenever solutions of peptides or proteins are being prepared. This precaution is well known. Less familiar is the fact that plastic laboratory ware may contain residual plasticizers, antioxidants, or monomers that can leach. Antioxidants (reducing agents) have the potential for inactivating molecules with disulfide bonds (e.g., vasopressin or insulin) or other structural features susceptible to reduction.
If a filled pump has an air bubble trapped within, its delivery rate (specified for aqueous solutions) may fall when the bubble enters the delivery port or the flow moderator. If there is a chance that an air bubble is in the pump reservoir, the fluid in the reservoir should be aspirated using the filling tube attached to a syringe. Then, reload the pump. This procedure applies to all models of pumps.
After its pumping lifetime has ended, an ALZET pump becomes an inert object for a period of time lasting about half again longer than its specified pumping duration. Even after the reservoir is depleted, however, the salt layer's osmotic attraction of water into the pump continues. If left in place, the pump may become damaged by hydrostatic forces and begin to leak some of its salt layer. This hypertonic solution can be irritating to local tissue.
Therefore, it is recommended that ALZET pumps be removed prior to reaching 1.5 times their stated duration. Removal is accomplished by a simple operation after the animal is lightly anesthetized. Use the following equation to determine explanation times for all pump durations:
1.5 x duration of pump (days) = maximum length of implantation (days)
At the end of an experiment, some users may want to check that the pumps delivered the solution loaded into them. Weighing the pumps will not give this information, because the pumps imbibe water through their outer membrane while delivering the fluid contained in the internal reservoir. Weight change therefore would be misleading, rather than verifying delivery.
There are two techniques which can provide some information about the contents of a spent pump's reservoir, with varying levels of reliability and accuracy.
· Plasma levels provide the most reliable and quantitative information for verifying pump functionality. This method involves measuring the in vivo plasma levels of the compound delivered by the implanted pumps.
· Using a filling tube attached to a syringe, insert the filling tube to the bottom of the drug reservoir and aspirate the residual volume. Subtract the volume removed from the initial loading volume. This net amount of solution, divided by the elapsed time, provides a measure of the average release rate. This method is not as quantitative as measuring plasma levels of the compound. Additionally, the pump's reservoir sometimes collapses, and therefore it can be difficult to extract solution from its interior.
At the completion of its period of pumping at its nominal rate (from o, ne day to four weeks depending upon the model), the pump reservoir will still contain some of the solution originally loaded into it. This residual volume (approximately 5% of the filled volume of the reservoir) represents a safety margin in the design o, f these devices. The reservoirs are deliberately made slightly larger than is necessary for the specified duration of pumping. Because of this feature, on the last day of the nominal period of pu, mping, there is enough fluid left in the reservoir to support the specific constant pumping rate. This safety margin also allows for time to prime the pumps, and provides some flexibility in case the experiment cannot be terminated immediately. The residual volume can essentially be ignored as it has no effect on the experiment except to give additional security to its design.
Long-term, constant delivery of test agents can be obtained only if the test agents are stable at body temperature for the duration of infusion. While it is important to use sterile solutions, especially if test materials are pote, ntial substrates for microorganisms, it is equally important to choose a vehicle of the right characteristics. Some considerations are pH, ionic strength, redox potential, etc. Some antioxidants, buffers, and similar compounds may suffice to stabilize, but the choice may have to be mad, e empirically, rather than on theoretical grounds. It may be helpful to consult with a, n industrial or hospital pharmacist, or with the manufacturer of the test agent, to obtain assistance with regard to optimum formulations.
As a preliminary method for assessing the stability of a test material at 37°C, first fill a small sterile test tube with the sterile material and cap tightly. Incubate at 37°C for the expected duration of the planned experiment and analyze for specific activity. Compare the activity to that of a freshly made sample. If the respective activities are equivalent, the test material is stable in the above conditions. Providing the vehicle is compatible with the pump reservoir, then positive results for an agent's stability using the above test will probably be indicative of stability within the pump. However, it is recommended that stability testing include an in vitro pumping rate test to ultimately ascertain compatibility of the test material inside the pump.
All ALZET pumps have a start-up gradient during which the pump, , s soak up fluid and come to temperature. When usi, ng a catheter, delivering viscous solutions, or if immediate delivery is needed, in vitro priming prior to implantation is essential. The length of pr, iming time v, aries according to mod, el and is detailed on the package insert.
· Fill the pump (and catheter if applicable) in the usual way.
· Place the pump (and catheter if applicable) in a beaker of sterile, 0.9% saline, and heat in an oven or water bath at 37°C for four to six hours. (Note: the model 2004 pump should be primed for at least 40 hours.) If using a catheter, it is possible to drape the end of the catheter outside the beaker to avoid any mixing of solutions. Do not be concerned if due to evaporation, fluid is not observed dripping from the end of the catheter, as evaporative loss can occur.
· Remove the pump from the saline and implant immediately.
· Fill the pump (and catheter if applicable) in the usual way.
· Place the pump (and catheter if applicable) in a beaker of sterile, 0.9% saline, and heat in an oven or water bath at 37°C for four to six hours. (Note: the model 2004 pump should be primed for at least 40 hours.) If using a catheter, it is possible to drape the end of the catheter outside the beaker to avoid any mixing of solutions. Do not be concerned if due to evaporation, fluid is not observed dripping from the end of the catheter, as evaporative loss can occur.
· Remove the pump from the saline and implant immediately.
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