A L Z A SPECIAL DELIVERY ¨ VOLUME 18, NO. 1 ALZA CORPORATION LORRI PERKINS, EDITOR MARCH 1996 Manipulating the Local L-NAME Elucidates the Role of Nitric Nerve Environment through Oxide

Targeted Delivery Nitric oxide (NO) is a free-radical gas that acts as a vasodilator. It is derived from L-arginine in endothe- Targeted delivery is especially stumps of a sectioned sciatic nerve lial cells, macrophages, neutrophils, useful when the desired drug effect is and to guide regeneration (Figure 1). platelets, and neurons. A shortage dependent on local events. Peripheral Via a catheter, neurotrophic agents of nitric oxide may result in arterial nerve regeneration following injury were delivered from an osmotic pump hypertension and atherosclerosis. is one process where events at the into the lumen of the nerve chamber. Abnormal nitric oxide levels can also injury site are critical to successful A second catheter functioned as a 180 regeneration. Recent publications vent to prevent pressure accumula- Day 17 (Before L-NAME treatment) in the neuroscience literature demon- tion. Using this device, Hekimian et Day 18 Day 22 strate techniques for targeting peri- al. confirmed that continuous four- 160 pheral nerves with neurotrophins. week delivery of drug solutions can Targeted delivery can also be used to be achieved in an environment which block nerve conductivity temporarily, is conducive to nerve regeneration. 140 a technique which may have utility The authors concluded that “the suc- in the study of neuromuscular devel- cess of this model opens the door for opment and reinnervation. a multitude of studies. With relative 120 ease, any stable solution can be Systolic Blood Pressure (mmHg) infused continuously into a nerve- A Novel Method for Targeting 1000 Peripheral Nerves guide chamber at a steady rate over Control 25 mg L-NAME 50 mg L-NAME a period of time.” 1 Several neurotrophic factors stimulate nerve regeneration in vitro, Figure 2: Changes in systolic blood pres- sure before L-NAME treatment (day 17), but these agents have proven difficult IGF-I Protects Against Diabetic Neuropathy after 1 day of treatment (day 18), and after to study in vivo. Hekimian et al. five days of treatment (day 22). Control from the Lahey Clinic developed a Neuropathy is a common compli- animals received saline. Changes in blood method for manipulating the nerve cation of diabetes. It is accompanied pressure after 25 mg and 50 mg L-NAME environment during regeneration.1 by impaired nerve regeneration in infusion were significantly different from This group constructed a chamber experimental diabetes, a condition controls (p < 0.01). to contain the distal and proximal associated with reduced levels of Reprinted with permission from Yallampalli C, Garfield RE. (Continued on Page 3, Column 1) AM J Obstet Gynecol 1993; 169:1316-1320.

contribute to infection and inflam- mation, neurodegenerative Osmotic disorders, and the brain damage Pump Figure 1: Schematic diagram seen with stroke.1 of a nerve regeneration cham- ber. Drug solution from the Nitric Oxide Inhibition pump continuously bathes the Simulates Preeclampsia Inlet Catheter lumen of the nerve chamber, Exhaust Preeclampsia, or pregnancy- Catheter allowing the effect of neuro- trophic agents on nerve regen- induced hypertension, is a significant eration to be determined. health problem which affects up to Sciatic Nerve 30% of all women. It is the leading Nerve Guide Reprinted with permission from Hekimian cause of fetal growth retardation, Chamber KJ et al. J Reconstruct Microsurg 1995; 11(2):93-98. infant morbidity, and mortality asso- ciated with premature delivery and maternal death. Yallampalli and Garfield hypothesized that a causal factor in preeclampsia might be 1 (Continued on Page 4, Column 1) A L Z A Very Special Delivery

Chronic Heavy For example, Cuevas et al. used basic Battler et al. also increased micro- fibroblast growth factor (bFGF), a vessel counts with bFGF in a porcine Metal Exposure peptide with a very short half-life, ischemia model.6 Stimulation of to induce angiogenesis in the arterial angiogenesis by locally-administered While the teratogenicity of cad- adventitia.1 Hayek et al. relied on bFGF bears promise for ischemic 2+ mium (Cd ) exposure has been well intrasplenic FGF infusion to promote disease treatment. documented, investigation into the the angiogenesis needed to maintain A wealth of knowledge about mechanism of action has been im- pancreatic islet grafts.2 Eppley et al. bFGF and other growth factors con- peded by difficulty in administering applied FGF directly to a bone graft 2+ tinues to accumulate as researchers precise amounts of Cd throughout to advance healing through angio- systematically deliver individual fac- gestation. Administration of cad- genesis.3 tors to various tissues. To draw upon mium in food or drinking water is More recently, Landau et al. published research in these areas, complicated by a lack of control over capitalized on bFGF’s angiogenic request an updated literature search dose. Gavage and injection offer enhancement of myocardial perfusion from ALZET Technical Services at greater control, but are stressful to in a rabbit model of ischemia.4 (800) 692-2990 or (415) 962-2251. animals, and hence can skew results. Increased coronary collateral vascu- Mahalik et al. used ALZET lature might rescue jeopardized pumps to surmount some of these tissue in conditions of decreased per- 1. Cuevas P, Gonzalez AM, Carceller F & Baird A. 2+ Circ Res 1991; 69:360-369. obstacles and to deliver Cd success- fusion. Landau et al. induced left 2. Hayek A, Lopez AD & Beattie GM. 1 fully to pregnant mice for 14 days. ventricular hypertrophy (LVH) by Transplantation 1990; 50(6):931-933. Their model resulted in a pattern of infusing intravenous angiotensin II 3. Eppley BL, Doucet M, Connolly DT, & Feder J. 2+ J Oral Maxillofac Surg 1988; 46:391-398. fetal anomalies and Cd distribution (AII). Rapidly-acquired LVH results 4. Landau C, Jacobs AK, & Haudenschild CC. which closely mirrored results in myocardial oxygen deficit due to Am Heart J 1995; 129:924-931. obtained using other dosing routes. 5. Yanagisawa-Miwa A, Uchida Y, Nakamura F, inadequate capillary density to sup- Tomaru T, Kido H, Kamijo T, Sugimoto T, Kaji Mahalik et al. stated that “chronic port hypertrophic muscle. Landau K, Utsuyama M, Kurashima C, & Ito H. Science teratogenic studies are often compli- et al. hoped to offset this deficit by 1992; 257:1401-1403. cated by the requirement for daily 6. Battler A, Scheinowitz M, Bor A, Hasdai D, stimulating new capillary formation. Vered Z, Segni E, Varda-Bloom N, Nass D, drug administration and animal After one week of AII treatment, Engelberg S, Eldar M, Belkin M & Savion N. handling, which have been shown to bFGF was infused by ALZET pump J Am Coll Cardiol 1993; 22:2001-2006. produce significant fetal and/or post- for 11 to 28 days via a catheter natal effects. The osmotic minipump advanced into the pericardial space provides a convenient and depend- using a transphrenic approach. able route of drug or toxicant admin- Controls received albumin in place istration that minimizes animal of bFGF or intravenous AII alone. Direct Delivery to the CNS handling and maternal stress. Non-ischemic controls received saline Surgical implantation of the pumps instead of AII. In a recently-published review, is also well-tolerated and does not Histological analysis of subepi- Theo Hagg provides a comprehensive typically cause infection or other cardial myocardium graded capillary protocol for the infusion of agents observable complications.” 1 For ref- density, ischemic necrosis, and fibro- into the brain using ALZET osmotic erences on using ALZET pumps to sis. Greater fibrosis in AII-treated pumps.1 Dr. Hagg outlines a variety administer cadmium and other heavy animals was consistent with microin- of procedures and recommendations, metals, contact ALZET Technical farcts resulting from LVH-induced including the construction of coiled Services at (800) 692-2990. oxygen deficit. Capillary density tubing to serve as an external reser- was increased markedly in animals voir, preparation of a dental acrylic 1. Mahalik MP, Hitner HW, and Prozialeck WC. platform for securing the cannula to Toxicol Letters 1995; 76:195-202. treated with both bFGF and AII, as compared with normal epicardial the skull, preimplantation advice, vascularity in animals receiving surgical tips, and post vivo analysis. Intrapericardial FGF AII alone. Hagg concludes that “the ALZET May Salvage Ischemic Myocardial angiogenesis has osmotic pump has clearly facilitated been demonstrated in other animal and expanded the possibilities of Myocardium models. Yanagisawa et al. produced experimental approaches in the a canine model of ischemia caused investigation of the CNS”.1 For a In numerous instances, by coronary artery stenosis and an copy of this review, contact ALZET researchers have used ingenious artificial thrombus.5 Slow injection Technical Services at (800) 692-2990 surgery and an ALZET pump to of bFGF after occlusion increased or at (415) 962-2251. deliver growth factors directly to a the number of capillaries and arteri- specific tissue. Targeted delivery 1. Hagg T. Continuous Central Nervous System oles versus controls. Left ventricular Infusion with ALZET Osmotic Pumps. In can avoid confusing or toxic systemic ejection fraction, a measure of car- Flanagan TR, Emerich DF, and Winn SR (eds). effects, while ensuring that the diac performance, decreased post- Methods in Neurosciences, Volume 21: growth factor reaches the target tis- Providing Pharmacological Access to the Brain: occlusion, but rebounded significant- Alternate Approaches. Academic Press, 1994, sue and is maintained there at suffi- ly after one week of bFGF treatment. pp. 201-213. cient concentration to exert its effects. 2 A L Z A

During reinnervation, Barry and Ribchester blocked conduction in the 11 Figure 3: Effects of locally- sciatic nerve by infusing tetrodotoxin infused aFGF on motor axon Untreated Groups (TTX) via a silastic cuff connected to regeneration distance. Filled 10 Received Heparin Only Received aFGF an osmotic pump. Hind-limb para- squares correspond to groups lysis confirmed effective nerve block which received aFGF while 9 open squares indicate groups by TTX. After two weeks of paraly- which were given heparin only. sis, the level of dual innervation in 8 Open circle indicates untreated the 4LM was assessed. Dual inner- groups. P < 0.05 (compared to vation was found in 50% of 4LM untreated controls) for groups 7 muscle fibers in the limb subjected to which were given 36 and 360 Regeneration Distance (mm) temporary nerve block, compared ng/day aFGF. 6 with 20% in untreated, contralateral Untreated 0.36 3.6 36 360 Reprinted with permission from Laird J.M.A. controls. Upon removal of the nerve et al. Neurosci 1995; 65(1):209-216. aFGF or Heparin (ng/day) block, conduction in the sciatic nerve resumed, as evidenced by normal use of the previously paralyzed hind limb. After 8 weeks of recovery, the Nerve infusion (Cont. from Page 1, Achieving Nerve Block limb subjected to temporary paraly- Column 2) Through Targeted Delivery sis retained a greater level of dual insulin-like growth factors (IGFs). Targeted delivery can also be innervation (35%) than the untreat- IGFs stimulate neurite survival and used to block conductivity in peri- ed, contralateral control (17%). outgrowth in vitro, and may play a pheral nerves. Barry and Ribchester Barry and Ribchester concluded that role in peripheral nerve regeneration. used a temporary nerve block to “paralysis produced an enduring, study the effect of activity on reinner- stable pattern of polyneuronal inner- In a rat model of diabetic neuro- 4 pathy, Ishii and Lupien compared the vation.4 Partial denervation of the vation.” These findings have thera- effects of systemic and targeted deliv- fourth deep lumbrical muscle (4LM) peutic implications because muscles ery of insulin-like growth factor-I was created in the rat by crushing rarely recover full innervation (IGF-I) on sensory nerve regenera- the lateral plantar nerve (LPN). following injury. Improvements in tion.2 Following crush injury of the Normally, muscle fibers in the 4LM the extent of reinnervation may be are mononeuronally innervated by achieved by prolonging the period sciatic nerve, rats made diabetic with 4 streptozotocin were implanted with the LPN or the sural nerve (SN). of muscle disuse after injury. When either nerve is injured, axons ALZET pumps containing IGF-I, 1. Hekimian KJ, Seckel BR, Bryan DJ, Wang KK, IGF-II, or vehicle. IGF-I was deliv- from the uninjured nerve sprout to Chakalis DP, & Bailey A. J Reconstruct ered directly to the injured nerve innervate the denervated muscle Microsurg 1995; 11(2):93-98. fibers. During reinnervation, axons 2. Ishii DN and Lupien SB. J Neurosci Res by connecting the pumps to a fene- 1995; 40:138-144. strated catheter, the tip of which may establish connections with mus- 3. Laird JMA, Mason GS, Thomas KA, Hargreaves was sutured to the epineurium and cle fibers which are already inner- RJ, & Hill RG. Neurosci 1995; 65(1):209-216. vated, resulting in dual innervation. 4. Barry JA & Ribchester RR. J Neurosci underlying muscle. For comparison, 1995; 15(10):6327-6339. IGF-I and IGF-II were delivered The competitive elimination of these systemically at a subcutaneous site superfluous connections resembles distant from the nerve injury. events in neonatal muscle develop- Ishii and Lupien concluded that ment and provides a model for “locally infused IGF-I significantly understanding this process. ameliorated the impairment of nerve regeneration in diabetic rats.” 2 Similar results were achieved with Introducing the New systemic administration, but a 20-fold increase in the dose of IGF-I Model 2004 ALZET Osmotic Pump was required. Laird et al. used a similar experimental model to determine A new ALZET osmotic the effects of local and systemic pump, the Model 2004, is delivery of acidic fibroblast growth factor (aFGF) on regeneration follow- now available. With a reser- ing nerve crush in normoglycemic voir volume of 200 µl, this pump 3 rats. The administration of aFGF delivers agents continuously for 4 at the site of injury resulted in a 47% increase in regeneration distance weeks at 0.25 µl/hr. Its small size (just (Figure 3). Similar results were 3 cm long, and 0.7 cm in diameter) makes achieved with systemic administra- tion, but the dose required was 250- it ideal for subcutaneous implantation in fold higher. These results may have mice or rats. For more information on the new therapeutic implications for the Model 2004, call (800) 692-2990 or (415) 962-2251. treatment of diabetic neuropathy.

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L-NAME (Cont. from Page 1, Column 3) fetal growth retardation. As these to atherosclerotic neointima forma- impaired vascular nitric oxide effects are similar to those of pre- tion. Indeed, administration of synthesis.2 ALZET pumps were eclampsia, a change in NO synthesis the NO precursor L-arginine limits implanted subcutaneously in preg- may be a factor in this disorder.2 the development of aortic athero- nant rats to infuse two different sclerosis and improves endothelial doses of the NO synthase inhibitor L-NAME Increases cell function.3 NG-nitro-L-arginine methyl ester Neointimal Formation Cayatte et al. examined the (L-NAME) or nitroglycerin (a nitric Atherosclerosis can be induced effects of NO inhibition on neointima oxide donor) from day 17 of gestation by hypercholesteremia. Endothelium- formation in hypercholesteremic to term. While nitroglycerin had no dependent vasodilation is impaired rabbits.3 After five weeks of a high effect, L-NAME was found to elevate in both hypercholesteremic animals cholesterol diet, rabbits were treated blood pressure in a dose-dependent and in those which suffer from athero- with L-NAME for 4 weeks using sub- fashion (Figure 2, Page 1). It also sclerosis. This decreased vasodilation cutaneous ALZET pumps. Since decreased pup weight, increased may be related to reduced NO syn- increases in blood pressure can also pup mortality, and caused maternal thesis, as NO is thought to act as an accelerate atherosclerosis, the dose proteinuria. However, length of “endothelium-derived relaxing fac- of L-NAME was adjusted so that it gestation was not affected. These tor”. Since NO also has antiprolifera- would not affect blood pressure. researchers concluded that NO syn- tive effects on vascular cells, the Compared to untreated hypercholes- thesis inhibition during pregnancy endothelial cell dysfunction seen with teremic animals, L-NAME-treated caused maternal hypertension and hypercholesteremia could contribute animals had significant increases in neointimal lesion area and impaired endothelial function. L-NAME did not induce atherosclerosis in rabbits fed a normal diet, indicating that New Agents in the ALZET Literature it accelerates the atherosclerosis 3 With more than 4,500 publications from the scientific literature, the ALZET induced by hypercholesteremia. pump bibliography is a valuable source of information on the controlled delivery NO is involved in a wide range of proteins, peptides, and other agents. Updates are frequent, and the most of pathophysiological processes, recent includes references on the delivery of: including many of cardiovascular origin. As these articles suggest, ABT-418 (cholinergic channel activator) H7 (PKC inhibitor) chronic modulation of NO synthesis Alaproclate LY-274614 (NMDA receptor antagonist) in vivo may elucidate the role of NO and be used to create novel disease AMPA (excitatory amino acid) Memantine (NMDA receptor antagonist) models. For a complete list of refer- Barium Hydroxide Neomycin ences on ALZET pump applications CGS 22652 (thromboxane A2 synthase inhibitor) Pertussis Toxin in NO research, contact the ALZET Fluvoxamine Venlafaxine (5-HT reuptake inhibitor) Technical Services desk at (800) 692- Harman (ß-carboline) 2990 or (415) 962-2251. 1. Chustecka Z. Scrip Magazine 1995; July/ References on these and other agents are available to you through ALZET August:39-41. 2. Yallampalli C, Garfield RE. AM J Obstet Technical Services at (800) 692-2990 or (415) 962-2251. A bibliography search Gynecol 1993; 169:1316-1320. customized to your research interests can be mailed or faxed to you at no charge 3. Cayatte AJ, Palacino JJ, Horten K, Cohen RA. (see attached reply card). Arterioscler Thromb 1994; 14:753-759.

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1996 Scientific Meetings at Which We Will Exhibit

Experimental Biology Washington, DC April 15-17 American Association of Washington, DC April 21-23 Cancer Research ASBMB New Orleans, LA June 3-6 Endocrine Society San Francisco, CA June 12-14 American Association of Seattle, WA October 28-30 Pharmaceutical Sciences Society for Neuroscience Washington, DC November 17-20

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I am interested in receiving more information about ALZET ® osmotic pumps. Please send me: ❑ General information on ALZET osmotic pumps. (TIM)

❑ Information on using ALZET pumps for delivering proteins and peptides. (BB)

❑ Information on using ALZET pumps for brain infusion. (BRAI, CNSI)

❑ A videotape of surgical techniques for implanting ALZET pumps. (VHS VIDEO)

❑ A reprint of the Hagg review on CNS infusion. (CNSI)

I would like to receive references on the use of ALZET pumps in the following areas: ❑ Antisense Oligodeoxynucleotides (ANTS)

❑ Cytokines (CYTO) ❑ Metal Infusion (META)

❑ Peptide Growth ❑ Nerve Infusion (NERV) Factors (GF) ❑ Nitric Oxide Research (NOSI) ❑ ICV Delivery (ICV) ❑ Targeted Delivery (TARG)

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